- Email: [email protected]
Enzymatic hydrolysis of cellulose using extracts from insects
Carbohydrate Research 485 (2019) 107811
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Enzymatic hydrolysis of cellulose using extracts from insects a,∗
a
c
b
Kinga Szentner , Agnieszka Waśkiewicz , Sandra Kaźmierczak , Tatiana Wojciechowicz , Piotr Golińskia, Elżbieta Lewandowskaa, Oskar Wasielewskic a b c
T
Department of Chemistry, Poznan University of Life Sciences, Poznan, Poland Department of Animal Physiology and Biochemistry, Poznan University of Life Sciences, Poznan, Poland Institute of Zoology, Poznan University of Life Sciences, Poznan, Poland
ARTICLE INFO
ABSTRACT
Keywords: Midgut insect digestion Enzymatic hydrolysis of cellulose FTIR GPC HPLC analysis
The use of Zophobas morio extracts in the aspect of cellulose hydrolysis is presented for the first time. The aim of this study was to investigate the action of enzymes obtained from Z. morio on cellulose hydrolysis and to determine their influence on the structural properties of cellulose with use the Fourier transform infrared spectroscopy (FTIR) and gel permeation chromatography (GPC). Cellulose hydrolysis products were analyzed by high performance liquid chromatography (HPLC). This analysis indicated that microcrystalline cellulose with smaller particle size was more susceptible to enzymatically treatment. Moreover, our investigation of cellulase activity showed a different profile of the used enzyme during particular developmental stages of Z. morio. Midgut extracts obtained from adult insects are more effective in degrading cellulose than extracts from larvae. The analysis of cellulose hydrolysis confirms that the efficiency of this reaction also depends on the structure of cellulosic materials and internal conditions of enzymatic reaction. In this study the cellulolytic activity of Z. morio midgut extracts showed that these insects could be valuable sources of cellulases.
1. Introduction Cellulose is one of the most abundant polysaccharides on Earth and it is composed of D-anhydroglucopyranose units linked by β-1,4-glycosidic bonds [1]. It consists of both crystalline and amorphous components, each of which shows different digestibility in enzymatic hydrolysis [2]. Crystalline regions in cellulose are more stable because of hydrogen bonds found between adjacent hydroxyl groups [3]. They are formed between the oxygen atom in the pyranose ring in one polymer chain and the hydrogen atoms from the hydroxyl group on C3 in the adjacent polymer chain, as well as between the hydroxyls on C2 and C6 in the other adjacent polymer chain. In contrast, the amorphous areas contain fewer hydrogen bonds among the polymer chains. The proportions of crystalline and amorphous areas in cellulose materials, referred to as crystallinity, vary depending on the source and the method of isolation from a particular material. The amounts of these forms have a significant effect on the chemical properties of cellulose and make cellulose a highly attractive raw material that has diverse applications in research and industrial practice. In view of the considerable applicability of this polymer researchers are searching for new sources
∗
capable of hydrolyzing cellulose. Recently we have been observing increased interest in biological methods of cellulose hydrolysis which in contrast to chemical methods (e.g. acid hydrolysis) are run under milder reaction conditions, generating lesser amounts of by-products [4,5]. Biological methods are based on enzymes - cellulases capable of hydrolyzing diverse cellulose products. The sources of enzymes for cellulose hydrolysis include protozoa, bacteria, fungi (e.g. the genera Trichoderma and Aspergillus) [6,7] and currently also some insects and mollusks [8]. Enzymatic hydrolysis of cellulose requires the synergistic action of three main classes of cellulases: endo-β-1,4-glucanases (EC 3.2.1.4), exo-β-1,4-glucanases (or cellobiohydrolases) (EC 3.2.1.91, EC 3.2.1.176), and β-glucosidases (EC 3.2.1.21) [9–12]. Endo-β-1,4-glucanases provide random cleavage of β-1,4 glycosidic bonds in the internal (amorphous) areas of cellulose. In this way, new oligosaccharide chains of different lengths are formed. In turn, the action of exo-β-1,4glucanases involves the removal of subunits from both reducing and non-reducing ends of the cellulose chain. Consequently, progressively shorter polymer chains are formed, while cellobiose and glucose units are released as major products [13] and subsequently β-glucosidases degrade cellobiose molecules [14]. The final effect of the cellulose degradation process is connected with the formation of glucose units, used
Corresponding author. E-mail address: [email protected] (K. Szentner).
https://doi.org/10.1016/j.carres.2019.107811 Received 12 April 2019; Received in revised form 2 September 2019; Accepted 8 September 2019 Available online 09 September 2019 0008-6215/ © 2019 Elsevier Ltd. All rights reserved.
Carbohydrate Research 485 (2019) 107811
K. Szentner, et al.
in subsequent biochemical processes [1]. Next to the synergistic action of the enzymes, the physical availability of cellulose and their susceptibility to enzymatic degradation are also affected by the material's structure, particle size, porosity, degree of polymerization and crystallinity [2,15–27]. Cellulose polymorphism [28,29] and the dispersive-morphological properties of cellulose [30,31] are extremely important during chemical and enzymatic reactions of cellulose. Based on literature reports, it is known that the supermolecular structure determines and affects the efficiency of enzymatic processes. Cellulose particle size as well as the specific surface area and pore size determine the susceptibility to reactants and, as a consequence, to hydrolysis reactions. Polymorphic cellulose, which also affects the efficiency of the cellulose modification reaction, is also extremely important, which is explained by matching at the crystallographic level between the appropriate supermolecular structure of cellulose and the reaction medium. In the hydrolysis process a significant role is played by proper reaction conditions (temperature, pH, the composition and concentration of enzymes as well as the type of cellulosic materials). The best known enzymes of bacteria and fungi are those that are more extensively used. Due to the significant interest in such studies novel sources for cellulose degrading enzymes, i.e. cellulases, are required. Insects are interesting sources for the production of novel cellulases [13,32,33]. In fact, bacteria colonizing the digestive system of insects and living in symbiosis with their hosts form biocatalyst complexes with cellulolytic activity [34,35]. Some of the insects are also known to be capable of producing enzymes. For many years it has been generally known that cellulose is digested by enzymes produced by symbiotic gut microbes in insects. Recently some studies have shown that insects' digestive fluid also exhibits an endogenous cellulolytic activity [36,37]. Insects are equipped with endogenous and symbiotic enzymes that efficiently utilize lignocellulosic materials as a source for glucose [36]. Examples of highly effective bioreactors include termites [38]. This is because of the symbiotic interactions between insects and various microorganisms, which result in potent hydrolytic activities decomposing cellulose to simple sugars within the insects’ digestive track [39]. Upgrading the process of cellulose hydrolysis using insect enzymes is of great importance for our understanding of the chemistry of polysaccharides, as well as industrial and research applications [40–44]. The Biological methods are widely used and may be applied e.g. in bioconversion of lignocellulose materials. Hydrolysis of the cellulose fraction is a key stage in the enzymatic degradation of biomass used in industry, e.g. in the production of biofuels (2G - second generation bioethanol) [45]. It is crucial for the reduction of greenhouse gas emissions and constitutes an alternative in the solution of environmental problems. However, there are certain factors limiting the biological degradation of carbohydrates within the biomass. In the lignocellulose material the presence of lignins and xylanes, the primary components of hemicelluloses, constitutes a barrier in the process of hydrolysis limiting enzyme access to the surface of cellulose. For this reason in order to enhance hydrolysis efficiency a common approach is to subject the input material to chemical, physical, physicochemical or biological treatment [46]. In this process changes take place in the cell wall, the crystalline structure is disturbed, the porosity in the substrate is increased and the particle size is reduced. An adequately selected and performed stage of processing enhances enzyme availability to the polysaccharide fraction and improves hydrolysis. As a consequence, the efficiency of bioconversion is increased and the formation of reaction inhibitors in the next stage (fermentation) is reduced. Theoretically it is assumed that after preliminary treatment the rate of hydrolysis, as a key stage of bioconversion, increases by as much as 90% [47]. A considerable advantage of enzymatic hydrolysis is connected with the environmental aspect thanks to the use of enzymes, which are biodegradable [48]. Moreover, no toxic compounds such as furfural,
phenolics or weak acids are formed in this process. Another advantage is also connected with mild operation conditions [49]. Thanks to the reaction conditions there are no problems with corrosion of equipment, which is frequently observed in chemical methods using acids in the hydrolysis process. In turn, a drawback of biological hydrolysis of the lignocellulose material is connected first of all with the high cost of cellulolytic compounds [45]. For this reason from the economic point of view the application of enzymatic hydrolysis for commercial purposes is limited. In view of the above diverse strategies are being developed in order to minimise drawbacks of this process. One of them is connected with the recycling of enzymes, which is effective on condition of maintenance of stability of the enzymes, which during the process may undergo deactivation. Several studies concern the addition of surfactants, e.g. Tween 20, 80 or non-catalytic proteins in order to improve bioconversion [48]. Moreover, a strategy increasing the economics of cellulose hydrolysis involves integration of enzymatic and fermentation processes in one stage (SSF). A necessary element improving the efficiency at the stage of saccharification is also an adequately designed bioreactor. In the process of hydrolysis stirred tank bioreactors (STBR) and membrane bioreactors (MBR) are used [50]. The latter are considered to be a promising alternative to decrease the feedback inhibition effect in order to enhance cellulose conversion and improve enzyme efficiency. Enzymatic hydrolysis of both pure cellulose and cellulose present in lignocellulose materials is a highly complex process, determined by many factors. In view of the above intensive studies are being conducted on natural biocatalyst systems, which are a promising prospect for the development of a preliminary biological treatment or consolidated biomass processing. Available – up to the date – literature data and information indicate on possibilities to apply larvae of insects as a rich source of lipids in biodiesel production [51], as well as interesting supplier of proteins in animals production (valuable, in nutritive point of view, components of feeds [52]. It is also worth to underline that insects form peptides with anticancer potential and abilities, what is of great interest in medical applications [53]. Insects might be possibly used as an alternative source of material for chitin and chitosan production [54]. It should be stressed that – in contrary to the other representatives of Coleoptera or Tenebrionidae – no or limited very much information is available in updated literature on analysis, chemical composition, catalytic and kinetic data of extracts obtained from genus Z. morio, especially in aspects of their use and application in cellulose hydrolysis (cellulolytic activity). Taking the above into consideration, the mean aim of our study was to collect experimental data on hydrolytic ability of an extract isolated from the midgut of a tenebrionid beetle, Z. morio. According to our knowledge, for the first time extracts obtained from Z. morio were used in cellulose hydrolysis process to analyze, describe, elucidate and discuss potential changes in the structure of this material. We believe that obtained results of our study will significantly broaden knowledge on novel, potential sources of cellulases with important indication on their practical use and application. Since till now - preferences for hydrolysis of various cellulose materials and their digestion by Z. morio was not studied, analyzed and discussed, our achievements will obviously broaden current knowledge on bioconversion of cellulose and provide basis, grounds and valuable indications for the process further modifications to improve the efficiency and velocity of this reaction. Moreover we expect that obtained information concerning novel sources for enzymatic hydrolysis should interestingly contribute to the preparation and production of biodegradable materials, what is of prime concern for the ecosystem. 2
Carbohydrate Research 485 (2019) 107811
K. Szentner, et al.
2. Materials and methods
50 mM sodium citrate buffer at 6.0 pH. Reaction mixtures placed in closed vials were incubated (Incubated Shaker, Lab Companion, Korea) at 40 °C for the larvae and at 50 °C for imagoes (selected based on earlier analysis of their activity – 2.3) for the time periods of 60 and 120 minutes. The rotation speed was 250 rpm. The hydrolysis reaction was stopped by boiling the hydrolysis residue for 5 min. The samples were stored at −20 °C until HPLC analysis could be performed.
2.1. Insects A laboratory culture of Zophobas morio (Coleoptera: Tenebrionidae) was maintained as described by Quennedey and Quennedey [55]. Larvae and adults were reared at 28 °C in plastic boxes. Cardboard eggpacking was used as shelter and breeding-places for the adults. The insect diet was composed of wheat bran and freshly sliced carrot (administered three times per week). Pupae from the stock culture were collected and kept separately on the turf to ensure higher humidity until adults emerged. Last instar larvae (with a body length of about 5 cm) and adults were used in the experiments for extract preparation. All insects were obtained from the stock culture of Z. morio maintained at the Institute of Zoology, the Poznan University of Life Sciences (Poznan, Poland).
2.5.1. Cellulose depolymerization analysis Samples after enzymatic hydrolysis were centrifuged at 10 500 g for 15 min, and the resulting supernatant was filtered through a 0.20 μm filter (Chromafil Pet 20/15 MS, Macherey-Nagel, Steinheim, Germany) prior to chromatographic analysis. Sugar concentrations were analyzed using a 2695 Waters high-performance liquid chromatograph (HPLC) system with a 2414 refractive index (RI) detector (Waters, Milford, MA, USA). A Bio-Rad Aminex HPX-87H column (Bio-Rad, Woodinville, WA, USA) was used with the HPLC system operating at a column temperature of 65 °C and applying the Empower™ 1 software (Waters, Milford MA, USA). The mobile phase was 0.5 mM H2SO4 with a flow rate of 0.6 mL/min. The quantification of sugars was performed by measuring the peak areas at a specified retention time according to a relevant calibration curve.
2.2. Preparation of crude extract The midguts from Z. morio larvae and adults were separated and homogenized by sonication on ice using a citrate buffer (50 mM; pH 6.0) that contained a protease inhibitor - cOmplete Protease (200 μl inhibitor/5 ml extract). The homogenates were centrifuged at 12 000 G for 10 min at 4 °C and the resulting supernatants were transferred into a test tube that was stored at −80 °C until needed.
2.5.2. Analysis of structural changes in cellulose after enzymatic treatment 2.5.2.1. FTIR spectroscopy. The cellulose residue after the enzymatic reaction was washed with Milli-Q water and filtrated. Next cellulose was dried and analyzed using the pellet technique of Fourier transform infrared spectroscopy (FTIR). The spectra were obtained using bromide potassium (KBr, Sigma-Aldrich, Steinheim, Germany) at 1 mg cellulose/ 200 mg KBr). Spectra were recorded using a FTIR Nicolet iS5 spectrometer (Thermo Fisher Scientific, Madison, USA) over the range from 4000 to 450 cm−1 at a resolution of 4 cm−1 recording 16 scans. The spectra were baseline corrected and normalized using the OMNIC 9 software (Thermo Fisher Scientific, Madison, USA) and next used for the calculation of the lateral order index (LOI, A1429/A897) [58], total crystalline index (TCI, H1372/H2900) [59] and hydrogen bond intensity (HBI, A3400/A1320) [60].
2.3. Determination of cellulolytic activity Cellulase activity was assayed by the modified dinitrosalicylic acid (DNS) procedure (Miller) [56], using carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC). Absorbance was measured at 540 nm using a Synergy2 Multi-Mode Microplate Reader (BioTek Instruments, Winooski, USA). One unit of cellulolytic activity was defined as the amount of the enzyme required to produce 1 μmol of reducing sugars (equivalent to glucose) per minute. Specific activities were reported as units per mg of protein. A standard curve of absorbance against the amount of glucose released was constructed to calculate the amount of glucose released during the cellulase assay. Series of glucose dilutions using biologygrade water were made to obtain the following concentrations: 0.125, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5 and 2.0 mg/mL. A blank without the substrates, but with the cellulase extract and the control containing no cellulose extract, but with the substrate were run simultaneously with the reaction mixture. All specific activities represent averages from triplicate measurements of at least three independent biological replicates. In order to compare cellulase activity at different temperatures and substrate concentrations, assays were performed at temperatures of 40, 50 and 70 °C, while the effect of substrate concentration was studied applying the 0.5, 2 and 3% CMC and MCC concentration.
Each insect gut was homogenized (as described above) and the extracts were analyzed. The proteins in insect fluid samples were assayed using a bicinchoninic acid (BCA) Protein Assay Kit (Pierce, Rockford, IL, USA) with bovine serum albumin (BSA) as a standard [57]. Solution absorbance was measured at 562 nm using a Synergy2 Multi-Mode Microplate Reader (BioTek Instruments).
2.5.2.2. Gel permeation chromatography (GPC) analysis of cellulose. Gel permeation chromatography (GPC) analysis was used to determine weight-average (Mw) and number-average (Mn) molecular weight, polydispersity (PD) and degree of polymerization (DP) after enzymatic hydrolysis of cellulose. It was decided to analyze the cellulose material after the maximum time (120 min) of the hydrolysis process. Cellulose was dried at a temperature of 60 °C. Such prepared cellulose was analyzed using gel chromatography to determine the degree of polymerization according to the method proposed by Ekmanis. Analyses were conducted in an Agilent gel chromatograph with an RI Wyatt detector (column 3xPLgel MixedA, 300 mm, 20 μm (Agilent), temperature of column 80 °C, flow rate 1.00 ml/min; calibration – polystyrene standards; solvent 0.5% LiCl/DMAc (dimethylacetamide); 1260 Iso Pump (Agilent Technologies). The polystyrene standards were used to calculate the molar mass of cellulose according to MarkHouwink and Sakurada. Parameters for polystyrene were α = 0.642, K = 17.35 × 10−5 cm3/g [61]. Parameters for cellulose were α = 0.957, K = 2.78 × 10−5 cm3/g [62]. All the presented values of the degree of polymerization (DP) and the polydispersity index were averages of three replications.
2.5. Enzymatic hydrolysis of cellulose
2.6. Statistical analysis
A 1.0 mL sample of the extract obtained from Z. morio was mixed with 2% (w/v) microcrystalline cellulose (MCC 50) 50 μm particle size (Avicel PH-101, Sigma Aldrich)or microcrystalline cellulose (MCC 20) 20 μm particle size (Sigmacell, Type 20, Sigma Aldrich) suspended in
Statistical analyses were conducted using the software package Statistica (Statsoft, V. 12.0). Data were analyzed by one way-ANOVA with Tukey's post hoc tests. In the Figures the parameters are presented as means ± standard deviations.
2.4. Protein concentration
3
Carbohydrate Research 485 (2019) 107811
K. Szentner, et al.
3. Results and discussion
insects from three different orders with different diets (woodborer, grasshopper and silkworm). In order to examine the concentration effect on cellulase activity, the enzyme activities with 1, 3, 5 mg/ml CMC were measured. In silkworm cellulase activities decreased with an increasing substrate concentration, while in grasshopper the enzymatic activity increased greatly. Cellulase activities are also affected by temperature. In adults and larvae the midgut cellulase activity was substantially different at various incubation temperatures (p < 0.05; Fig. 2A). In adults the activity was clearly highest at 50 °C compared with other temperatures (50 °C–0.517 U/mg protein; 40 °C–0.396 U/mg protein; 70 °C–0.26 U/mg protein). In contrast to the adults, in larvae at a temperature of 40 and 70 °C the effectiveness of CMC cellulose degradation was highest. MCC cellulose as a substrate generated different profile of enzyme activity (Figs. 1 and 2 B). In this case the midgut extracts from larvae were more effective towards the cellulose degrading (one way-ANOVA F1,18 = 29.24, p < 0.05). Similar to the chart of CMC enzymatic degradation also for MCC we recorded identical optimal enzymatic conditions for larvae and adults (2% substrate concentration and 50 °C). Data are means ± SD of 80 individuals samples. Bars with different superscripts denote significant differences (p < 0.05) identified using One-Way ANOVA followed by Tukey's multiple comparison test. Data are expressed in activity units per milligram protein. The pattern of cellulolytic activity varies between two development stages of Z. morio, larvae and adults. The differences in the decomposition of cellulose substrates indicate different activities of endo- and exocellulases in the Z. morio midgut. Larvae are well-adapted to collect and store food for further utilization in the moulting process. Therefore it is expected that larval enzyme activity will be higher as compared to that of imagoes. Scrivener et al. [68] showed that Psacothea hilaris larvae have an approximately two-fold higher enzymes activity comparing with adults. Other studies indicated that expression of mRNA encoding cellulolytic enzymes varies between the larval and adult stages during the development of Mesosa myops [69]. Expression levels of six cellulase genes were totally different between larvae and adults (high in larvae and low in adults). The different expression of cellulosedigesting enzymes in Z. morio larvae and imagoes is the result of adaptation to the type of food consumed. Larvae, during their intensive growth, absorb easy-degradable and assimilable food to increase their mass in a short time. In contrast, adults consume mainly wood or woodderived material. A similar adaptation occurs in other species from the Tenebrionidae family [13]. A number of studies have found that 50 °C is the optimum temperature for cellulase activity [33,70]. Studies performed on a closely related species (Tenebrio molitor) reported that the optimum
The present section is divided into two parts. The first subchapter (3.1) analyses the extract obtained from the culture of Z. morio run by our team. In turn, the second part (3.2) concerns the effect of applied enzymatic extracts on bioconversion of cellulose. This subchapter focuses both on soluble products of cellulose hydrolysis and on structural changes occurring after enzymatic treatment of the cellulose material. In our study the enzymatic hydrolysis of cellulose using extracts from Z. morio is reported for the first time. So far, research has focused mainly on the evaluation of enzyme activity in this family of insects, but not from the genus of Z. morio. 3.1. Activity of extract from Z. morio Activities of insect gut extracts (adults and larvae) were assayed using UV–Vis. Activities of extracts from Z. morio were evaluated using carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC). The substrates were selected based on earlier literature reports [28] indicating that CMC may reflect endoglucanase activity, while microcrystalline cellulose may reflect the activity of cellobiohydrolases (exoglucanases) [63] or assess the synergism of endo-, exo- and betaglucosidases [13]. Data for are means ± SD of 80 individuals sampled. Bars with different superscripts denote significant differences (p < 0.05) using one-way ANOVA followed by Tukey's multiple comparison test. Data are expressed in activity units per milligram protein. Our investigation of cellulase activity showed a different profile of this enzyme during particular developmental stages. Midgut extracts obtained from the adults, as shown by the estimated cellulase activity, are more effective in degrading CMC cellulose than extracts from larvae (one way-ANOVA F1,18 = 28.25, p < 0.05). In adults of Z. morio the highest cellulase activity was recorded at 2% substrate and 50 °C (Figs. 1A and 2A). The cellulase activity from midguts of adults was 2.5fold greater than in larvae (adults: 0.517 U/mg protein versus larvae: 0.222 U/mg protein). Using the CMC substrate at different concentrations it was found that 2% CMC is more effective in terms of enzyme activity, especially for adults. A slightly different profile was observed for larvae, similarly for 0.5 and 3% CMC (Tukey's post hoc test, P < 0.05) (Fig. 1A). Previous research estimating cellulase activity in beetle species was conducted with 2% CMC concentration [64,65], as it was assumed to be optimal for insect cellulases. Studies on cellulase from Tribolium castaneum showed that the activity was increasing to 2% (of CMC concentration), then a slight decline was observed due to the product inhibition [66]. Shi et al. [67] compared cellulase activities in
Fig. 1. Cellulase activity in midgut of Z. morio larvae or adults at different substrate concentrations A) CMC cellulose and B) MCC cellulose. 4
Carbohydrate Research 485 (2019) 107811
K. Szentner, et al.
Fig. 2. Cellulase activity in midguts of Z. morio larvae or adults at different incubation temperatures A) CMC cellulose and B) MCC cellulose.
temperatures for endogenous cellulases were 40 °C (for exo-β-1,4-glucanase) and 50 °C (for endo-β1,4-glucanase) [71]. Other studies showed that the optimum temperature was 50 °C, almost half of activity was maintained at 60 °C, whereas at 80 °C no enzymatic activity was observed. Investigations concerning endo-β-1,4-D-glucanase of Podontia quatuordecimpunctata (Coleoptera) revealed the highest activity at 35–45 °C and 60 °C [50]. It is supposed that this wide range of optimum temperatures for this enzyme is due to the disablement of some multiple forms of proteins at different temperatures. The UV–Vis spectroscopic data from the current study provided important information on the activities of extracts from Z. morio.
incubation, glucose concentration was determined (Fig. 3). The two microcrystalline cellulosic substrates (MCC 50 and MCC 20) and the two insect gut extracts (from larvae and imagoes) were used in these reactions. For each of the cellulosic materials, glucose concentration at two time points was higher when using adult midgut extracts than in the case of extracts from larvae (Fig. 3), which corresponds to the activity of extracts (Fig. 1). After 2 h of reaction it was found that glucose content [in mg/ml] was higher for the cellulose preparation MCC 20 (2.480) compared to cellulose MCC 50 (1.134). When using larval extracts, the level of monosaccharide was comparable and changed only slightly during the reaction (0.595–0.491 and 0.399–0.383 mg/ml for MCC 50 and MCC 20, respectively). These differences between Z. morio developmental stages can be explained as a substrate specifically expressing different activity in relation to the MCC substrate.
3.2. Analysis of cellulose after enzymatic hydrolysis 3.2.1. HPLC analysis The effect of enzymes obtained from Z. morio on depolimerisation of cellulose is revealed e.g. by HPLC/RI analysis. This chromatographic technique allows the monitoring of cellulose hydrolysis products in terms of both quality and quantity. After the first and second hour of
3.2.2. FTIR analysis The FTIR spectroscopy was used to elucidate the structure of the cellulose substrates after enzymatic reactions. This analysis will also be
Fig. 3. Glucose contents [mg/ml] under cellulose hydrolysis. 5
Carbohydrate Research 485 (2019) 107811
K. Szentner, et al.
Fig. 4. FTIR spectra of cellulose after enzymatic reaction: A) MCC 20 cellulose control; B) MCC 20 cellulose after hydrolysis using larvae; C) MCC 20 cellulose after hydrolysis using imagoes; D)MCC 50 control; E) MCC 50 after hydrolysis using larvae; F) MCC 50 after hydrolysis using imagoes.
used to explain the action of cellulases found in extracts on crystalline and amorphous regions of cellulose. Changes in the spectra at 4000 to 2700 cm−1 as well as the “fingerprint region” in the range of 1800 to 800 cm−1 (Fig. 4) were analyzed. The FTIR spectra of MCC 50 and MCC 20 cellulose (Fig. 4) indicated that enzymatic reactions had effect on the structure of cellulose. It needs to be remembered that changes are more evident in the reaction systems using enzymes obtained from imagoes. The relative absorbance of the band at 3400 cm−1 (vibrations of the hydrogen bonded OH) for cellulose following hydrolysis decreased in comparison to the band for non-hydrolyzed cellulose. This absorption band was observed to be slightly narrower after enzymatic hydrolysis. The spectra were recorded for cellulose samples after 120 minutes of hydrolysis. The results of FTIR analyses showed that hydrogen bonding energy changes after enzymatic hydrolysis. Similar dependencies were observed by Cao [34] in experiments on lignocellulose materials and enzymes from Aspergillus niger and Trichoderma reesei. More pronounced changes in band intensity were observed for MCC 20 in comparison to MCC 50, which suggested greater substrate susceptibility to the enzymes. The above observations correspond to the HPLC analyses of the hydrolysates, where greater glucose concentrations [in mg/ml] were found for MCC 20 versus MCC 50. These changes were recorded for samples of cellulose hydrolyzed using extracts from imagoes. The cellulose material subjected to bioconversion from larvae remained practically unchanged, as presented by the obtained spectra (Fig. 4). After enzymatic treatment the FTIR spectra for MCC 20 cellulose exhibited an intensity decrease in the band for the C–O–C antisymmetric bridge stretching at the β(1,4) glucosidic bond (1165 cm−1) for the deformation vibrations (δ) in the plane at C6. Moreover, a decrease in band intensity for MCC 20 cellulose was observed for vibrations in the amorphous areas at the 897 cm−1 band, which is characteristic of βlinkages, whereas for MCC 50 these changes in the band were slight. Similar trends were observed for changes at the 1058 cm−1 band, which represents stretching vibrations ν(CO) at C-3, ν(C–C). The
reduction in intensity of the peaks for cellulose materials after enzymatic hydrolysis indicated that some cellulose was degraded. Analogous dependencies were observed in studies using enzyme from Aspergillus niger [72]. In turn, FTIR analyses of the obtained spectra showed slight changes in the amounts of crystalline and amorphous areas and in the values of the cellulose crystallinity index. The changes both for microcrystalline - MCC 50 and microcrystalline - MCC 20 cellulose were recorded for bands for the crystalline areas at 1429 cm−1. This band indicates deformation of vibrations δ-for the CH2 groups at C6, at C6 δ OCH. Moreover, vibrations of this band show the rotational energy of hydroxyls located at C3 and C6 3 and 6 (C3–O3 and C6–O6) [73]. Changes in that band affected the crystallinity index of cellulose LOI. Observed changes are explained by the two-stage process of hydrolysis. In the first stage, more accessible amorphous areas of cellulose are degraded. In turn, in the second stage crystalline areas of cellulose start to be digested. As a consequence the ordering of cellulose structure is reduced, which contributes to further bioconversion of the material. In the initial period of the reaction after 60 minutes the values of TCI, LOI and HBI were comparable. In contrast, significant differences were observed after 120 minutes of hydrolysis in the case of MCC 20 when compared to microcrystalline MCC 50. These changes were recorded mainly in the post-reaction cellulose at the use of the imago extracts. The greatest decrease in the crystallinity index TCI from 1.057 to 0.608 and LOI from 2.134 to 1.898 was observed for the MCC 20 cellulose. In turn, for the MCC 50 these values were practically almost identical during all the stages of the reaction. The obtained results are presented in Table 1. Similarly as in our experiments, Shi et al. [74] also stated that the index of crystallinity for microcrystalline cellulose MCC 50 at 2h reaction remained practically the same. Some scientific reports indicate a slight increase in the index of crystallinity of cellulose from 2 to 3% [75,76]. The slight increase index crystallinity TCI and LOI after hydrolysis suggests a preferential hydrolysis of amorphous cellulose [3]. This study was conducted on other materials exhibiting different 6
Carbohydrate Research 485 (2019) 107811
K. Szentner, et al.
Table 1 Infrared crystallinity indexes (TCI and LOI) and hydrogen bond intensity (HBI) of the studied cellulose after reaction with the use of larval and imago extracts. Time of treatment [min]
MCC 50 0 60 120 MCC 20 0 60 120
IR crystallinity ratio
Hydrogen bond intensity
H 1372/H2900 (TCI)
A 1429/A897 (LOI)
A3400/A1320 (HBI)
imago larval
imago larval
imago larval
0.927 ± 0.011 0.941 ± 0.018 0.937 ± 0.015 0,940 ± 0.010 0.957 ± 0.028
1.828 ± 0.025 1.859 ± 0.028 1.875 ± 0.025 1.821 ± 0.037 1.896 ± 0.050
1.525 ± 0.011 1.520 ± 0.010 1.561 ± 0.025 1.466 ± 0.011 1.589 ± 0.025
1.057 ± 0.031 1.088 ± 0.011 1.061 ± 0.010 0.608 ± 0.010 1.069 ± 0.050
2.134 ± 0.028 2.144 ± 0.014 2.039 ± 0.150 1.898 ± 0.015 2.019 ± 0.110
1.874 ± 0.012 1.929 ± 0.010 1.848 ± 0.055 1.559 ± 0.013 1.820 ± 0.075
porosity and crystallinity, which obviously affects bioconversion. The decrease in TCI indicates a disturbance of the crystalline structure during enzymatic treatment of cellulose. This was also confirmed in our results, as the greatest LOI reduction was recorded in for MCC 20 cellulose, which showed greater susceptibility to hydrolysis compared to MCC 50. The action of cellulases found in the applied extracts is also confirmed based on changes in the hydrogen bond intensity (HBI). Similarly as for the crystallinity ratio, MCC 20 cellulose is more susceptible to the action of imago extract cellulases. The hydrogen bond intensity (HBI) in cellulose ranged from 1.87 to 1.58. The chain mobility and binding distance, as well as hydrogen bonding (HBI) of cellulose are dependent on the crystalline system and the degree of intermolecular regularity [60]. These changes were also found in the case of decreases of the degree of polymerization under the influence of enzymes. The structural changes in cellulose after the enzymatic reaction were more precisely characterized by the degree of polymerization (DP), the average molecular weight and the degree of polydispersity. These parameters, similarly as crystallinity, determine physical and chemical properties of cellulose. The results of our study are presented in Table 2. A decrease in the degree of polymerization from 329 for the control sample to 308 was observed for MCC 20 cellulose in the case of imago extracts used in the hydrolysis reaction. In contrast, for MCC 50 cellulose a less marked DP reduction was found from 341 to 338. The GPC analysis (Table 2) shows that the greatest changes for this cellulose were also observed for the average molecular mass (Mn). The degree of polydispersion (Mw/Mn) was comparable at 2.3. In the case of larval extracts used in hydrolysis the DP values were similar for MCC 50, while for MCC 20 cellulose a lesser decrease in DP was recorded from 329 to 317 when compared with the imago extracts, for which the degree of polymerization decreased from 329 to 309. A similar dependence was reported by Pala et al. [77] and Park et al. [3] in their experiments on identical cellulose material but fungi as the source of enzymes. The higher rate of enzymatic hydrolysis for MCC 20 cellulose compared to MCC 50 was also recorded in studies using enzymes from
Trichoderma reesei and Trichoderma veride and similar cellulose materials conducted by Pala et al. [77], Kim et al. [78] and Dourado et al. [79] Those authors explained the greater accessibility of microcrystalline cellulose MCC 20 by the specific surface area of microcrystalline cellulose. The observed changes in cellulose, which is a material containing both crystalline and amorphous areas, may indicate that the complex of enzymes obtained from Z. morio, apart from endoglucanases, also contains active exoglucanases that act on the terminal ends (fragments) of the crystalline cellulose polymer chains to cleave them into monomeric glucose units. Results of HPLC analyses (Fig. 3), showing no contents of cellobiose or other shorter chain saccharides in the hydrolysate, suggest the presence of β-glucosidase responsible for the degradation of cellobiose to glucose. Slightly lower glucose levels were obtained in the enzymatic reaction with MCC 50 cellulose, which indicates lower accessibility of this substrate. Moreover, certain endoglucanases present in the gut of Z. morio may hydrolyze crystalline cellulose, resulting in the production of the main reducing sugar, i.e. glucose [2]. Further analysis connected with the purification and modification of the hydrolysis reaction involving the proposed complex will definitely increase the efficiency of the reaction, thus enhancing its further applicability. 4. Conclusions The recorded results of bioconversion of microcrystalline cellulose indicate that the imago extract is a more efficient system than the larval extract. The optimum cellulase activity from midguts of Z. morio recorded towards 2% cellulose substrate and at 50 °C. Additionally, microcrystalline cellulose MCC 20 is more susceptible to hydrolysis in comparison to MCC 50. This confirms the observation that a material of smaller particle size and lower crystallinity shows greater susceptibility to hydrolysis. For the microcrystalline cellulose MCC 20 the values of TCI, LOI, HBI and the degree of polymerization decreased, while hydrolysis efficiency improved. In the case of a more crystalline cellulose MCC 50 the values of these parameters remained unchanged, while no major changes were observed in the process of hydrolysis. The more dynamic hydrolysis of MCC 20 cellulose observed in our study confirms
Table 2 Molecular parameters of cellulose after hydrolysis (120 minutes treatment with cellulases). Cellulose MCC 50 before hydrolysis after hydrolysis (larval extract) after hydrolysis (imago extract) MCC 20 before hydrolysis after hydrolysis (larval extract) after hydrolysis (imago extract)
Mn [g/mol]
Mw [g/mol]
Mw/Mn
DP
25 150 23 593 23 673
55 970 55 187 54 763
2.2 ± 0.1 2.3 ± 0.2 2.3 ± 0.1
345 ± 2 341 ± 3 338 ± 2
23 160 22 510 21 503
53 275 51 410 49 970
2.3 ± 0.1 2.3 ± 0.1 2.3 ± 0.1
329 ± 2 317 ± 4 308 ± 2
7
Carbohydrate Research 485 (2019) 107811
K. Szentner, et al.
that the extract produced from the insect contains endocellulases required for cellulose hydrolysis. Thanks to the presence of other exocellulases and cellobiohydrolases we observe the formation of glucose as the final effect. The presence of endogenous cellulases in this insect is a valuable finding in terms of its further use for biofuel production. This study along with results of its continuation will definitely find applications for the enzymatic modification of cellulose and lignocellulose materials commonly used in industry.
[31] S. Borysiak, A. Grząbka-Zasadzińska, J. Appl. Polym. Sci. 133 (2016) 42864–42873. [32] S.A. Lucena, L. Lima, L.S.A. Cordeiro, C. Sant'Anna, R. Constantino, P. Azambuja, W. de Souza, E.S. Garcia, F.A. Genta, Biotechnol. Biofuels 4 (2011) 1–9, https://doi.org/10. 1186/1754-6834-4-51. [33] L.-J. Su, H.-F. Zhang, X.-M. Yin, M. Chen, F.-Q. Wang, H. Xie, G.-Z. Zhang, A.-D. Song, Genet. Mol. Res. 12 (2013) 2432–2441, https://doi.org/10.4238/2013. January.4.11. [34] Y. Cao, H. Tan, J. Mol. Struct. 705 (2004) 189–193, https://doi.org/10.1016/j.molstruc. 2004.07.010. [35] R. Fischer, R. Ostafe, R.M. Twyman, Yellow Biotechnol. II 136 (2013) 51–64, https://doi. org/10.1007/10_2013_206. [36] J.D. Willis, C. Oppert, J.L. Jurat-Fuentes, Insect Sci. 17 (2010) 184–198, https://doi.org/ 10.1111/j.1744-4917.2010.01322.x. [37] D. Zhang, A.R. Lax, A.K. Raina, J.M. Bland, Insect Biochem. Mol. Biol. 39 (2009) 516–522, https://doi.org/10.1016/j.ibmb.2009.03.006. [38] X. Zhou, J.A. Smith, F.M. Oi, P.G. Koehler, G.W. Bennett, M.E. Scharf, Gene 395 (2007) 29–39, https://doi.org/10.1016/j.gene.2007.01.004. [39] R.J. Ma, C.Y. Wang, Y.W. Liu, T.R. Sivakumar, Z.X. Ren, Y. Fang, J.Q. Jia, Z.Z. Gui, J. Asia Pac. Entomol. 17 (2014) 67–71, https://doi.org/10.1016/j.aspen.2013.10.007. [40] S. Jonardhnan, M.M. Sain, BioRes 1 (2006) 176–188, https://doi.org/10.15376/biores.1. 2.176-188. [41] M.G. Adsul, K.B. Bastwade, A.J. Varma, D.V. Goghale, Bioresour. Technol. 98 (2007) 1467–1473, https://doi.org/10.1016/j.biortech.2006.02.036. [42] S.W. Huang, H.-Y. Zhang, S. Marshall, T.A. Jackson, Insect Sci. 17 (2010) 175–183, https://doi.org/10.1111/j.1744-7917.2010.01320.x. [43] M.S. Taggar, J. Appl. Nat. Sci. 7 (2015) 625–630, https://doi.org/10.31018/jans.v7i2. 656. [44] K. Poszytek, Postępy Mikrobiologii [Adv. Microbiol.] 55 (2016) 132–146. [45] D.L. Aguilar, R.M. Rodríguez-Jasso, E. Zanuso, A.A. Lara-Flores, C.N. Aguilar, A. Sanchez, H.A. Ruiz, S. Kumar, R.K. Sani (Eds.), Biorefining of Biomas to Biofuels, Biofuels and Biorefinery Technologies, vol. 4, Springer, Cham, 2018, pp. 223–248 https://doi.org/10. 1007/978-3-319-67678-4_10. [46] A. Singh, R.M. Rodríguez-Jasso, K.D. Gonzalez-Gloria, M. Rosales, R. Belmares-Cerda, C.N. Aquilar, R.R. Singhania, H.A. Ruiz, Bioresour. Technol. Rep. 7 (2019) 100257 https://dx.doi.org/10.1016/j.biteb.2019.100257. [47] J.K. Saini, R. Saini, L. Tewari, 3 Biotech 5 (2015) 337–353. [48] V. Brummer, P. Skryja, T. Jurena, V. Hlawacek, P. Stehlik, Appl. Biochem. Biotechnol. 174 (2014) 1299–1308. [49] S. Sasmal, K. Mohanty, S. Kumar, R.K. Sani (Eds.), Biorefining of Biomas to Biofuels, Biofuels and Biorefinery Technologies, vol. 4, Springer, Cham, 2018, pp. 203–221 https://doi.org/10.1007/978-3-319-67678-4_9. [50] M.S. Pino, R.M. Rodrίquez-Jasso, M. Michelin, A.C. Flores-Gallegos, R. MoralesRodrίquez, J.A. Teixeira, H.A. Ruiz, Chem. Eng. J. 347 (2018) 119–136. [51] D. Leung, D. Yang, Z. Li, Z. Zhao, J. Chen, L. Zhu, Ind. Eng. Chem. Res. 51 (2012) 1036–1040. [52] J.X. Zhang, Q. Guo, G.M. He, N. Liu, Food Sci. (N. Y.) 18 (2011) 006. [53] F. Yusof, S. Chowdhury, M.O. Faruck, N. Sulaiman, IFRJ 24 (2017) 446–451. [54] Ch.Y. Soon, Y.B. Tee, Ch.H. Tan, A.T. Rosnita, Int. J. Biol. Macromol. 108 (2018) 135–142. [55] A. Quennedey, B. Quennedey, Cell Tissue Res. 296 (1999) 619–634. [56] G.L. Miller, Anal. Chem. 31 (1959) 426–428, https://doi.org/10.1021/ac60147a030. [57] P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson, D.C. Klenk, Ann. Biochem. 150 (1985) 76–85, https://doi.org/10.1016/0003-2697(85)90442-7. [58] F. Carrilo, X. Colom, J.J. Suñol, J. Saurina, Eur. Polym. J. 40 (2004) 2229–2234. [59] M.L. Nelson, R.T.J. O′Connor, Appl. Polym. Sci. 8 (1964) 1325–1341, https://doi.org/10. 1002/app.1964.070080323. [60] S.Y. Oh, D.I. Yoo, Y. Shin, H.C. Kim, H.Y. Kim, Y.S. Chung, W.H. Park, J.H. Youk, Carbohydr. Res. 340 (2005) 2376–2391, https://doi.org/10.1016/j.carres.2005.08.007. [61] J.D. Timpa, Agric. Food Chem. 39 (1991) 270–275. [62] T. Bikova, A. Treimanis, Carbohydr. Polym. 48 (2002) 23–28. [63] S.M. Geib, M. Jimenez-Gasco, M. Del, J.E. Carlson, M. Tien, K. Hoover, Environ. Entomol. 38 (2009) 686–699, https://doi.org/10.1603/022.038.0320. [64] M.M. Uddin, M.H. Chowdhury, S. Mojumder, D. Skidar, Pak. J. Biol. Sci. 15 (2012) 333–340. [65] B. Dojnova, R. Pavlović, N. Božić, A. Margetić, V.J. Nenadović, Vujčić Z. Ivanović, Comp. Biochem. Physiol. B 164 (2013) 259–267. [66] F.U. Rehman, M. Aslam, M.I. Tariq, A. Shaheen, A.J. Sami, N.H. Naveed, I. Batool, Afr. J. Biotechnol. 8 (2009) 6710–6715. [67] W. Shi, S.-Y. Ding, J.S. Yuan, BioEnergy Res. 4 (2011) 1–10. [68] A.M. Scrivener, H. Watanabe, H. Noda, J. Insect Physiol. 43 (1997) 1039–1052. [69] J. Liu, K. Song, H. Teng, B. Zhang, W. Li, H. Xue, X. Yang, Acta Biochim. Biophys. Sin. 47 (2015) 741–748, https://doi.org/10.1093/abbs/gmv070. [70] A.J. Sami, M.A. Anwar, F.U. Rehman, A.R. Shakoori, Pak. J. Zool. 43 (2011) 393–401. [71] X. Huang, G. Wang, Biol. Disaster Sci. 2 (2008) 67–70. [72] I. Spridon, C.-A. Teacӑ, R. Bodîrlӑu, Biores.com 6 (2011) 400–413. [73] R.G. Zhbankov, S.P. Firsov, D.K. Buslov, N.A. Nikonenko, M.K. Marchewka, H. Ratajczak, J. Mol. Struct. 614 (2002) 117–125, https://doi.org/10.1016/S0022-2860(02)00252-1. [74] J. Shi, D. Wu, L. Zhang, B.A. Simmons, S. Singh, B. Yang, C.E. Wyman, Biotechnol. Bioeng. 114 (2016) 503–515, https://doi.org/10.1002/bit.26180. [75] Y. Chen, A.J. Stipanovic, W.T. Winter, D.B. Wilson, Y.J. Kim, Cellulose 14 (2007) 283–293. [76] L. Wang, Y. Zhang, P. Gao, D. Shi, H. Liu, H. Gao, Biotechnol. Bioeng. 93 (2006) 443–456. [77] H. Pala, M. Mota, F.M. Gama, Carbohydr. Polym. 68 (2007) 101–108. [78] D.W. Kim, J.H. Yang, Y.K. Jeong, Appl. Microbiol. Biotechnol. 28 (1988) 148–154. [79] F. Dourado, M. Mota, H. Pala, F.M. Gama, Cellulose 6 (1999) 265–282.
Funding This research was funded by the NATIONAL SCIENCE CENTRE, Poland (Grant number 2014/13/B/NZ9/02442). Conflicts of interest The authors declare no conflict of interest. Acknowledgments We would like to thank Agnieszka Graclik for her technical support in Z. morio laboratory culture and maintenance. References [1] T.T. Teeri, Trends Biotechnol. 15 (1997) 160–167, https://doi.org/10.1016/S01677799(97)01032. [2] Y.-H. Zhang, L.R. Lynd, Biotechnol. Bioeng. 88 (2004) 797–824, https://doi.org/10. 1002/bit.20282. [3] S. Park, J.O. Baker, M.E. Himmel, P.A. Parilla, D.K. Johnson, Biotechnol. Biofuels 3 (2010) 1–10, https://doi.org/10.1186/1754-6834-3-10. [4] S. Al-Zuhair, Bioresour. Technol. 99 (2007) 4078–4085. [5] S. Marques, L. Alves, J.C. Roseiro, F.M. Gırio, Biomass Bioenergy 32 (2008) 400–406. [6] R.E. Quiroz-Castaneda, J.L. Folch-Mallol, Intech 6 (2013) 119–155, https://doi.org/10. 5772/53719. [7] E.J.P. Kouadio, K.H. Konan, K.M. Djѐ, E.A. Duѐ, L.P. Kouamѐ, Int. J. Entomol. Res. 4 (2016) 67–86. [8] H. Watanabe, G. Tokuda, Cell. Mol. Life Sci. 58 (2001) 1167–1178. [9] E.A. Bayer, H. Chanzy, R. Lamed, Shoham, Curr. Opin. Struct. Biol. 8 (1998) 548–557, https://doi.org/10.1016/S0959-440x(98)80143-7. [10] L.R. Lynd, P.J. Weiner, W.H. Van Zyl, I.S. Pretorius, Microbiol. Mol. Biol. Rev. 66 (2002) 506–577, https://doi.org/10.1128/MMBR.66.3.506-577.2002. [11] P.C. Ozioko, A.P. Ikeyi, O.P.C. Ugwu, The experiment, Int. J. Sci. Technol. 12 (2013) 778–785. [12] H. Watanabe, G. Tokuda, Annu. Rev. Entomol. 55 (2010) 609–632. [13] C. Oppert, W.E. Klingeman, J.D. Willis, B. Oppert, J.L. Jurat-Fuentes, Comp. Biochem. Physiol. B 155 (2010) 145–154, https://doi.org/10.1016/j.cbpb.2009.10.014. [14] J. Pѐrez, J. Muñoz-Dorado, T. De la Rubia, J. Martinez, Int. Microbiol. 5 (2002) 53–63, https://doi.org/10.1007/s10123-002-0062-3. [15] S.D. Mansfield, C. Mooney, J.N. Saddler, Biotechnol. Prog. 15 (1999) 804–816, https:// doi.org/10.1021/bp9900864. [16] Y. Cao, H. Tan, Carbohydr. Res. 337 (2002) 1291–1296, https://doi.org/10.1016/S00086215(02)00134-9. [17] Y. Cao, H. Tan, Enzym. Microb. Technol. 36 (2005) 314–317, https://doi.org/10.1016/j. enzmictec.2004.09.002. [18] A.V. Gusakov, T. Salanovich, A.I. Antonov, B.B. Ustinov, O.N. Okunev, R. Burlingame, M. Emalfarb, M. Baez, A.P. Sinitsyn, Biotechnol. Bioeng. 97 (2007) 1028–1038, https:// doi.org/10.1002/bit.21329. [19] A.S. Bommarius, A. Katona, S.E. Cheben, A.S. Patel, A.J. Ragauskas, K. Knudson, Y. Pu, Metab. Eng. 10 (2008) 370–381, https://doi.org/10.1016/j.ymben.2008.06.008. [20] K. Clarke, X. Li, K. Li, Biomass Bioenergy 35 (2011) 3943–3950, https://doi.org/10. 1016/j.biombioe.2011.06.007. [21] M. Ioelovich, E. Morag, BioRes 6 (2011) 2818–2835, https://doi.org/10.15376/biores.6. 3.2818-2835. [22] M. Wiman, D. Dienes, M.A.T. Hansen, T. Meulen, G. Zacchi, G. Liden, Bioresour. Technol. 126 (2012) 208–215, https://doi.org/10.1016/j.biortech.2012.08.082. [23] M. Hall, P. Bansal, J.H. Lee, M.J. Realff, A.S. Bommarius, FEBS J. 277 (2010) 1571–1582, https://doi.org/10.1111/j.1742-4658.2010.07585.x. [24] D. Ciolacu, S. Gorgieva, D. Tampu, V. Kokol, Cellulose 18 (2011) 1527–1541. [25] Yi-H. Zhang, L.R. Lynd, Biotechnol. Bioeng. 88 (2004) 797–824. [26] An-I. Yeh, Y-Ch. Huang, S.H. Chen, Carbohydr. Polym. 79 (2010) 192–199. [27] Y. Horikawa, N. Konakahara, T. Imai, A. Kentaro, Y. Kobayashi, J. Sugiyama, Polym. Degrad. Stab. 98 (2013) 2351–2356. [28] S. Borysiak, J. Therm. Anal. Calorim. 113 (2013) 281–289. [29] S. Borysiak, J. Therm. Anal. Calorim. 109 (2012) 595–603. [30] A. Grząbka-Zasadzińska, W. Smułek, E. Kaczorek, S. Borysiak, Polym. Compos. 39 (2018) 448–456.
8
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Enzymatic hydrolysis of cellulose using extracts from insects a,∗
a
c
b
Kinga Szentner , Agnieszka Waśkiewicz , Sandra Kaźmierczak , Tatiana Wojciechowicz , Piotr Golińskia, Elżbieta Lewandowskaa, Oskar Wasielewskic a b c
T
Department of Chemistry, Poznan University of Life Sciences, Poznan, Poland Department of Animal Physiology and Biochemistry, Poznan University of Life Sciences, Poznan, Poland Institute of Zoology, Poznan University of Life Sciences, Poznan, Poland
ARTICLE INFO
ABSTRACT
Keywords: Midgut insect digestion Enzymatic hydrolysis of cellulose FTIR GPC HPLC analysis
The use of Zophobas morio extracts in the aspect of cellulose hydrolysis is presented for the first time. The aim of this study was to investigate the action of enzymes obtained from Z. morio on cellulose hydrolysis and to determine their influence on the structural properties of cellulose with use the Fourier transform infrared spectroscopy (FTIR) and gel permeation chromatography (GPC). Cellulose hydrolysis products were analyzed by high performance liquid chromatography (HPLC). This analysis indicated that microcrystalline cellulose with smaller particle size was more susceptible to enzymatically treatment. Moreover, our investigation of cellulase activity showed a different profile of the used enzyme during particular developmental stages of Z. morio. Midgut extracts obtained from adult insects are more effective in degrading cellulose than extracts from larvae. The analysis of cellulose hydrolysis confirms that the efficiency of this reaction also depends on the structure of cellulosic materials and internal conditions of enzymatic reaction. In this study the cellulolytic activity of Z. morio midgut extracts showed that these insects could be valuable sources of cellulases.
1. Introduction Cellulose is one of the most abundant polysaccharides on Earth and it is composed of D-anhydroglucopyranose units linked by β-1,4-glycosidic bonds [1]. It consists of both crystalline and amorphous components, each of which shows different digestibility in enzymatic hydrolysis [2]. Crystalline regions in cellulose are more stable because of hydrogen bonds found between adjacent hydroxyl groups [3]. They are formed between the oxygen atom in the pyranose ring in one polymer chain and the hydrogen atoms from the hydroxyl group on C3 in the adjacent polymer chain, as well as between the hydroxyls on C2 and C6 in the other adjacent polymer chain. In contrast, the amorphous areas contain fewer hydrogen bonds among the polymer chains. The proportions of crystalline and amorphous areas in cellulose materials, referred to as crystallinity, vary depending on the source and the method of isolation from a particular material. The amounts of these forms have a significant effect on the chemical properties of cellulose and make cellulose a highly attractive raw material that has diverse applications in research and industrial practice. In view of the considerable applicability of this polymer researchers are searching for new sources
∗
capable of hydrolyzing cellulose. Recently we have been observing increased interest in biological methods of cellulose hydrolysis which in contrast to chemical methods (e.g. acid hydrolysis) are run under milder reaction conditions, generating lesser amounts of by-products [4,5]. Biological methods are based on enzymes - cellulases capable of hydrolyzing diverse cellulose products. The sources of enzymes for cellulose hydrolysis include protozoa, bacteria, fungi (e.g. the genera Trichoderma and Aspergillus) [6,7] and currently also some insects and mollusks [8]. Enzymatic hydrolysis of cellulose requires the synergistic action of three main classes of cellulases: endo-β-1,4-glucanases (EC 3.2.1.4), exo-β-1,4-glucanases (or cellobiohydrolases) (EC 3.2.1.91, EC 3.2.1.176), and β-glucosidases (EC 3.2.1.21) [9–12]. Endo-β-1,4-glucanases provide random cleavage of β-1,4 glycosidic bonds in the internal (amorphous) areas of cellulose. In this way, new oligosaccharide chains of different lengths are formed. In turn, the action of exo-β-1,4glucanases involves the removal of subunits from both reducing and non-reducing ends of the cellulose chain. Consequently, progressively shorter polymer chains are formed, while cellobiose and glucose units are released as major products [13] and subsequently β-glucosidases degrade cellobiose molecules [14]. The final effect of the cellulose degradation process is connected with the formation of glucose units, used
Corresponding author. E-mail address: [email protected] (K. Szentner).
https://doi.org/10.1016/j.carres.2019.107811 Received 12 April 2019; Received in revised form 2 September 2019; Accepted 8 September 2019 Available online 09 September 2019 0008-6215/ © 2019 Elsevier Ltd. All rights reserved.
Carbohydrate Research 485 (2019) 107811
K. Szentner, et al.
in subsequent biochemical processes [1]. Next to the synergistic action of the enzymes, the physical availability of cellulose and their susceptibility to enzymatic degradation are also affected by the material's structure, particle size, porosity, degree of polymerization and crystallinity [2,15–27]. Cellulose polymorphism [28,29] and the dispersive-morphological properties of cellulose [30,31] are extremely important during chemical and enzymatic reactions of cellulose. Based on literature reports, it is known that the supermolecular structure determines and affects the efficiency of enzymatic processes. Cellulose particle size as well as the specific surface area and pore size determine the susceptibility to reactants and, as a consequence, to hydrolysis reactions. Polymorphic cellulose, which also affects the efficiency of the cellulose modification reaction, is also extremely important, which is explained by matching at the crystallographic level between the appropriate supermolecular structure of cellulose and the reaction medium. In the hydrolysis process a significant role is played by proper reaction conditions (temperature, pH, the composition and concentration of enzymes as well as the type of cellulosic materials). The best known enzymes of bacteria and fungi are those that are more extensively used. Due to the significant interest in such studies novel sources for cellulose degrading enzymes, i.e. cellulases, are required. Insects are interesting sources for the production of novel cellulases [13,32,33]. In fact, bacteria colonizing the digestive system of insects and living in symbiosis with their hosts form biocatalyst complexes with cellulolytic activity [34,35]. Some of the insects are also known to be capable of producing enzymes. For many years it has been generally known that cellulose is digested by enzymes produced by symbiotic gut microbes in insects. Recently some studies have shown that insects' digestive fluid also exhibits an endogenous cellulolytic activity [36,37]. Insects are equipped with endogenous and symbiotic enzymes that efficiently utilize lignocellulosic materials as a source for glucose [36]. Examples of highly effective bioreactors include termites [38]. This is because of the symbiotic interactions between insects and various microorganisms, which result in potent hydrolytic activities decomposing cellulose to simple sugars within the insects’ digestive track [39]. Upgrading the process of cellulose hydrolysis using insect enzymes is of great importance for our understanding of the chemistry of polysaccharides, as well as industrial and research applications [40–44]. The Biological methods are widely used and may be applied e.g. in bioconversion of lignocellulose materials. Hydrolysis of the cellulose fraction is a key stage in the enzymatic degradation of biomass used in industry, e.g. in the production of biofuels (2G - second generation bioethanol) [45]. It is crucial for the reduction of greenhouse gas emissions and constitutes an alternative in the solution of environmental problems. However, there are certain factors limiting the biological degradation of carbohydrates within the biomass. In the lignocellulose material the presence of lignins and xylanes, the primary components of hemicelluloses, constitutes a barrier in the process of hydrolysis limiting enzyme access to the surface of cellulose. For this reason in order to enhance hydrolysis efficiency a common approach is to subject the input material to chemical, physical, physicochemical or biological treatment [46]. In this process changes take place in the cell wall, the crystalline structure is disturbed, the porosity in the substrate is increased and the particle size is reduced. An adequately selected and performed stage of processing enhances enzyme availability to the polysaccharide fraction and improves hydrolysis. As a consequence, the efficiency of bioconversion is increased and the formation of reaction inhibitors in the next stage (fermentation) is reduced. Theoretically it is assumed that after preliminary treatment the rate of hydrolysis, as a key stage of bioconversion, increases by as much as 90% [47]. A considerable advantage of enzymatic hydrolysis is connected with the environmental aspect thanks to the use of enzymes, which are biodegradable [48]. Moreover, no toxic compounds such as furfural,
phenolics or weak acids are formed in this process. Another advantage is also connected with mild operation conditions [49]. Thanks to the reaction conditions there are no problems with corrosion of equipment, which is frequently observed in chemical methods using acids in the hydrolysis process. In turn, a drawback of biological hydrolysis of the lignocellulose material is connected first of all with the high cost of cellulolytic compounds [45]. For this reason from the economic point of view the application of enzymatic hydrolysis for commercial purposes is limited. In view of the above diverse strategies are being developed in order to minimise drawbacks of this process. One of them is connected with the recycling of enzymes, which is effective on condition of maintenance of stability of the enzymes, which during the process may undergo deactivation. Several studies concern the addition of surfactants, e.g. Tween 20, 80 or non-catalytic proteins in order to improve bioconversion [48]. Moreover, a strategy increasing the economics of cellulose hydrolysis involves integration of enzymatic and fermentation processes in one stage (SSF). A necessary element improving the efficiency at the stage of saccharification is also an adequately designed bioreactor. In the process of hydrolysis stirred tank bioreactors (STBR) and membrane bioreactors (MBR) are used [50]. The latter are considered to be a promising alternative to decrease the feedback inhibition effect in order to enhance cellulose conversion and improve enzyme efficiency. Enzymatic hydrolysis of both pure cellulose and cellulose present in lignocellulose materials is a highly complex process, determined by many factors. In view of the above intensive studies are being conducted on natural biocatalyst systems, which are a promising prospect for the development of a preliminary biological treatment or consolidated biomass processing. Available – up to the date – literature data and information indicate on possibilities to apply larvae of insects as a rich source of lipids in biodiesel production [51], as well as interesting supplier of proteins in animals production (valuable, in nutritive point of view, components of feeds [52]. It is also worth to underline that insects form peptides with anticancer potential and abilities, what is of great interest in medical applications [53]. Insects might be possibly used as an alternative source of material for chitin and chitosan production [54]. It should be stressed that – in contrary to the other representatives of Coleoptera or Tenebrionidae – no or limited very much information is available in updated literature on analysis, chemical composition, catalytic and kinetic data of extracts obtained from genus Z. morio, especially in aspects of their use and application in cellulose hydrolysis (cellulolytic activity). Taking the above into consideration, the mean aim of our study was to collect experimental data on hydrolytic ability of an extract isolated from the midgut of a tenebrionid beetle, Z. morio. According to our knowledge, for the first time extracts obtained from Z. morio were used in cellulose hydrolysis process to analyze, describe, elucidate and discuss potential changes in the structure of this material. We believe that obtained results of our study will significantly broaden knowledge on novel, potential sources of cellulases with important indication on their practical use and application. Since till now - preferences for hydrolysis of various cellulose materials and their digestion by Z. morio was not studied, analyzed and discussed, our achievements will obviously broaden current knowledge on bioconversion of cellulose and provide basis, grounds and valuable indications for the process further modifications to improve the efficiency and velocity of this reaction. Moreover we expect that obtained information concerning novel sources for enzymatic hydrolysis should interestingly contribute to the preparation and production of biodegradable materials, what is of prime concern for the ecosystem. 2
Carbohydrate Research 485 (2019) 107811
K. Szentner, et al.
2. Materials and methods
50 mM sodium citrate buffer at 6.0 pH. Reaction mixtures placed in closed vials were incubated (Incubated Shaker, Lab Companion, Korea) at 40 °C for the larvae and at 50 °C for imagoes (selected based on earlier analysis of their activity – 2.3) for the time periods of 60 and 120 minutes. The rotation speed was 250 rpm. The hydrolysis reaction was stopped by boiling the hydrolysis residue for 5 min. The samples were stored at −20 °C until HPLC analysis could be performed.
2.1. Insects A laboratory culture of Zophobas morio (Coleoptera: Tenebrionidae) was maintained as described by Quennedey and Quennedey [55]. Larvae and adults were reared at 28 °C in plastic boxes. Cardboard eggpacking was used as shelter and breeding-places for the adults. The insect diet was composed of wheat bran and freshly sliced carrot (administered three times per week). Pupae from the stock culture were collected and kept separately on the turf to ensure higher humidity until adults emerged. Last instar larvae (with a body length of about 5 cm) and adults were used in the experiments for extract preparation. All insects were obtained from the stock culture of Z. morio maintained at the Institute of Zoology, the Poznan University of Life Sciences (Poznan, Poland).
2.5.1. Cellulose depolymerization analysis Samples after enzymatic hydrolysis were centrifuged at 10 500 g for 15 min, and the resulting supernatant was filtered through a 0.20 μm filter (Chromafil Pet 20/15 MS, Macherey-Nagel, Steinheim, Germany) prior to chromatographic analysis. Sugar concentrations were analyzed using a 2695 Waters high-performance liquid chromatograph (HPLC) system with a 2414 refractive index (RI) detector (Waters, Milford, MA, USA). A Bio-Rad Aminex HPX-87H column (Bio-Rad, Woodinville, WA, USA) was used with the HPLC system operating at a column temperature of 65 °C and applying the Empower™ 1 software (Waters, Milford MA, USA). The mobile phase was 0.5 mM H2SO4 with a flow rate of 0.6 mL/min. The quantification of sugars was performed by measuring the peak areas at a specified retention time according to a relevant calibration curve.
2.2. Preparation of crude extract The midguts from Z. morio larvae and adults were separated and homogenized by sonication on ice using a citrate buffer (50 mM; pH 6.0) that contained a protease inhibitor - cOmplete Protease (200 μl inhibitor/5 ml extract). The homogenates were centrifuged at 12 000 G for 10 min at 4 °C and the resulting supernatants were transferred into a test tube that was stored at −80 °C until needed.
2.5.2. Analysis of structural changes in cellulose after enzymatic treatment 2.5.2.1. FTIR spectroscopy. The cellulose residue after the enzymatic reaction was washed with Milli-Q water and filtrated. Next cellulose was dried and analyzed using the pellet technique of Fourier transform infrared spectroscopy (FTIR). The spectra were obtained using bromide potassium (KBr, Sigma-Aldrich, Steinheim, Germany) at 1 mg cellulose/ 200 mg KBr). Spectra were recorded using a FTIR Nicolet iS5 spectrometer (Thermo Fisher Scientific, Madison, USA) over the range from 4000 to 450 cm−1 at a resolution of 4 cm−1 recording 16 scans. The spectra were baseline corrected and normalized using the OMNIC 9 software (Thermo Fisher Scientific, Madison, USA) and next used for the calculation of the lateral order index (LOI, A1429/A897) [58], total crystalline index (TCI, H1372/H2900) [59] and hydrogen bond intensity (HBI, A3400/A1320) [60].
2.3. Determination of cellulolytic activity Cellulase activity was assayed by the modified dinitrosalicylic acid (DNS) procedure (Miller) [56], using carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC). Absorbance was measured at 540 nm using a Synergy2 Multi-Mode Microplate Reader (BioTek Instruments, Winooski, USA). One unit of cellulolytic activity was defined as the amount of the enzyme required to produce 1 μmol of reducing sugars (equivalent to glucose) per minute. Specific activities were reported as units per mg of protein. A standard curve of absorbance against the amount of glucose released was constructed to calculate the amount of glucose released during the cellulase assay. Series of glucose dilutions using biologygrade water were made to obtain the following concentrations: 0.125, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5 and 2.0 mg/mL. A blank without the substrates, but with the cellulase extract and the control containing no cellulose extract, but with the substrate were run simultaneously with the reaction mixture. All specific activities represent averages from triplicate measurements of at least three independent biological replicates. In order to compare cellulase activity at different temperatures and substrate concentrations, assays were performed at temperatures of 40, 50 and 70 °C, while the effect of substrate concentration was studied applying the 0.5, 2 and 3% CMC and MCC concentration.
Each insect gut was homogenized (as described above) and the extracts were analyzed. The proteins in insect fluid samples were assayed using a bicinchoninic acid (BCA) Protein Assay Kit (Pierce, Rockford, IL, USA) with bovine serum albumin (BSA) as a standard [57]. Solution absorbance was measured at 562 nm using a Synergy2 Multi-Mode Microplate Reader (BioTek Instruments).
2.5.2.2. Gel permeation chromatography (GPC) analysis of cellulose. Gel permeation chromatography (GPC) analysis was used to determine weight-average (Mw) and number-average (Mn) molecular weight, polydispersity (PD) and degree of polymerization (DP) after enzymatic hydrolysis of cellulose. It was decided to analyze the cellulose material after the maximum time (120 min) of the hydrolysis process. Cellulose was dried at a temperature of 60 °C. Such prepared cellulose was analyzed using gel chromatography to determine the degree of polymerization according to the method proposed by Ekmanis. Analyses were conducted in an Agilent gel chromatograph with an RI Wyatt detector (column 3xPLgel MixedA, 300 mm, 20 μm (Agilent), temperature of column 80 °C, flow rate 1.00 ml/min; calibration – polystyrene standards; solvent 0.5% LiCl/DMAc (dimethylacetamide); 1260 Iso Pump (Agilent Technologies). The polystyrene standards were used to calculate the molar mass of cellulose according to MarkHouwink and Sakurada. Parameters for polystyrene were α = 0.642, K = 17.35 × 10−5 cm3/g [61]. Parameters for cellulose were α = 0.957, K = 2.78 × 10−5 cm3/g [62]. All the presented values of the degree of polymerization (DP) and the polydispersity index were averages of three replications.
2.5. Enzymatic hydrolysis of cellulose
2.6. Statistical analysis
A 1.0 mL sample of the extract obtained from Z. morio was mixed with 2% (w/v) microcrystalline cellulose (MCC 50) 50 μm particle size (Avicel PH-101, Sigma Aldrich)or microcrystalline cellulose (MCC 20) 20 μm particle size (Sigmacell, Type 20, Sigma Aldrich) suspended in
Statistical analyses were conducted using the software package Statistica (Statsoft, V. 12.0). Data were analyzed by one way-ANOVA with Tukey's post hoc tests. In the Figures the parameters are presented as means ± standard deviations.
2.4. Protein concentration
3
Carbohydrate Research 485 (2019) 107811
K. Szentner, et al.
3. Results and discussion
insects from three different orders with different diets (woodborer, grasshopper and silkworm). In order to examine the concentration effect on cellulase activity, the enzyme activities with 1, 3, 5 mg/ml CMC were measured. In silkworm cellulase activities decreased with an increasing substrate concentration, while in grasshopper the enzymatic activity increased greatly. Cellulase activities are also affected by temperature. In adults and larvae the midgut cellulase activity was substantially different at various incubation temperatures (p < 0.05; Fig. 2A). In adults the activity was clearly highest at 50 °C compared with other temperatures (50 °C–0.517 U/mg protein; 40 °C–0.396 U/mg protein; 70 °C–0.26 U/mg protein). In contrast to the adults, in larvae at a temperature of 40 and 70 °C the effectiveness of CMC cellulose degradation was highest. MCC cellulose as a substrate generated different profile of enzyme activity (Figs. 1 and 2 B). In this case the midgut extracts from larvae were more effective towards the cellulose degrading (one way-ANOVA F1,18 = 29.24, p < 0.05). Similar to the chart of CMC enzymatic degradation also for MCC we recorded identical optimal enzymatic conditions for larvae and adults (2% substrate concentration and 50 °C). Data are means ± SD of 80 individuals samples. Bars with different superscripts denote significant differences (p < 0.05) identified using One-Way ANOVA followed by Tukey's multiple comparison test. Data are expressed in activity units per milligram protein. The pattern of cellulolytic activity varies between two development stages of Z. morio, larvae and adults. The differences in the decomposition of cellulose substrates indicate different activities of endo- and exocellulases in the Z. morio midgut. Larvae are well-adapted to collect and store food for further utilization in the moulting process. Therefore it is expected that larval enzyme activity will be higher as compared to that of imagoes. Scrivener et al. [68] showed that Psacothea hilaris larvae have an approximately two-fold higher enzymes activity comparing with adults. Other studies indicated that expression of mRNA encoding cellulolytic enzymes varies between the larval and adult stages during the development of Mesosa myops [69]. Expression levels of six cellulase genes were totally different between larvae and adults (high in larvae and low in adults). The different expression of cellulosedigesting enzymes in Z. morio larvae and imagoes is the result of adaptation to the type of food consumed. Larvae, during their intensive growth, absorb easy-degradable and assimilable food to increase their mass in a short time. In contrast, adults consume mainly wood or woodderived material. A similar adaptation occurs in other species from the Tenebrionidae family [13]. A number of studies have found that 50 °C is the optimum temperature for cellulase activity [33,70]. Studies performed on a closely related species (Tenebrio molitor) reported that the optimum
The present section is divided into two parts. The first subchapter (3.1) analyses the extract obtained from the culture of Z. morio run by our team. In turn, the second part (3.2) concerns the effect of applied enzymatic extracts on bioconversion of cellulose. This subchapter focuses both on soluble products of cellulose hydrolysis and on structural changes occurring after enzymatic treatment of the cellulose material. In our study the enzymatic hydrolysis of cellulose using extracts from Z. morio is reported for the first time. So far, research has focused mainly on the evaluation of enzyme activity in this family of insects, but not from the genus of Z. morio. 3.1. Activity of extract from Z. morio Activities of insect gut extracts (adults and larvae) were assayed using UV–Vis. Activities of extracts from Z. morio were evaluated using carboxymethyl cellulose (CMC) and microcrystalline cellulose (MCC). The substrates were selected based on earlier literature reports [28] indicating that CMC may reflect endoglucanase activity, while microcrystalline cellulose may reflect the activity of cellobiohydrolases (exoglucanases) [63] or assess the synergism of endo-, exo- and betaglucosidases [13]. Data for are means ± SD of 80 individuals sampled. Bars with different superscripts denote significant differences (p < 0.05) using one-way ANOVA followed by Tukey's multiple comparison test. Data are expressed in activity units per milligram protein. Our investigation of cellulase activity showed a different profile of this enzyme during particular developmental stages. Midgut extracts obtained from the adults, as shown by the estimated cellulase activity, are more effective in degrading CMC cellulose than extracts from larvae (one way-ANOVA F1,18 = 28.25, p < 0.05). In adults of Z. morio the highest cellulase activity was recorded at 2% substrate and 50 °C (Figs. 1A and 2A). The cellulase activity from midguts of adults was 2.5fold greater than in larvae (adults: 0.517 U/mg protein versus larvae: 0.222 U/mg protein). Using the CMC substrate at different concentrations it was found that 2% CMC is more effective in terms of enzyme activity, especially for adults. A slightly different profile was observed for larvae, similarly for 0.5 and 3% CMC (Tukey's post hoc test, P < 0.05) (Fig. 1A). Previous research estimating cellulase activity in beetle species was conducted with 2% CMC concentration [64,65], as it was assumed to be optimal for insect cellulases. Studies on cellulase from Tribolium castaneum showed that the activity was increasing to 2% (of CMC concentration), then a slight decline was observed due to the product inhibition [66]. Shi et al. [67] compared cellulase activities in
Fig. 1. Cellulase activity in midgut of Z. morio larvae or adults at different substrate concentrations A) CMC cellulose and B) MCC cellulose. 4
Carbohydrate Research 485 (2019) 107811
K. Szentner, et al.
Fig. 2. Cellulase activity in midguts of Z. morio larvae or adults at different incubation temperatures A) CMC cellulose and B) MCC cellulose.
temperatures for endogenous cellulases were 40 °C (for exo-β-1,4-glucanase) and 50 °C (for endo-β1,4-glucanase) [71]. Other studies showed that the optimum temperature was 50 °C, almost half of activity was maintained at 60 °C, whereas at 80 °C no enzymatic activity was observed. Investigations concerning endo-β-1,4-D-glucanase of Podontia quatuordecimpunctata (Coleoptera) revealed the highest activity at 35–45 °C and 60 °C [50]. It is supposed that this wide range of optimum temperatures for this enzyme is due to the disablement of some multiple forms of proteins at different temperatures. The UV–Vis spectroscopic data from the current study provided important information on the activities of extracts from Z. morio.
incubation, glucose concentration was determined (Fig. 3). The two microcrystalline cellulosic substrates (MCC 50 and MCC 20) and the two insect gut extracts (from larvae and imagoes) were used in these reactions. For each of the cellulosic materials, glucose concentration at two time points was higher when using adult midgut extracts than in the case of extracts from larvae (Fig. 3), which corresponds to the activity of extracts (Fig. 1). After 2 h of reaction it was found that glucose content [in mg/ml] was higher for the cellulose preparation MCC 20 (2.480) compared to cellulose MCC 50 (1.134). When using larval extracts, the level of monosaccharide was comparable and changed only slightly during the reaction (0.595–0.491 and 0.399–0.383 mg/ml for MCC 50 and MCC 20, respectively). These differences between Z. morio developmental stages can be explained as a substrate specifically expressing different activity in relation to the MCC substrate.
3.2. Analysis of cellulose after enzymatic hydrolysis 3.2.1. HPLC analysis The effect of enzymes obtained from Z. morio on depolimerisation of cellulose is revealed e.g. by HPLC/RI analysis. This chromatographic technique allows the monitoring of cellulose hydrolysis products in terms of both quality and quantity. After the first and second hour of
3.2.2. FTIR analysis The FTIR spectroscopy was used to elucidate the structure of the cellulose substrates after enzymatic reactions. This analysis will also be
Fig. 3. Glucose contents [mg/ml] under cellulose hydrolysis. 5
Carbohydrate Research 485 (2019) 107811
K. Szentner, et al.
Fig. 4. FTIR spectra of cellulose after enzymatic reaction: A) MCC 20 cellulose control; B) MCC 20 cellulose after hydrolysis using larvae; C) MCC 20 cellulose after hydrolysis using imagoes; D)MCC 50 control; E) MCC 50 after hydrolysis using larvae; F) MCC 50 after hydrolysis using imagoes.
used to explain the action of cellulases found in extracts on crystalline and amorphous regions of cellulose. Changes in the spectra at 4000 to 2700 cm−1 as well as the “fingerprint region” in the range of 1800 to 800 cm−1 (Fig. 4) were analyzed. The FTIR spectra of MCC 50 and MCC 20 cellulose (Fig. 4) indicated that enzymatic reactions had effect on the structure of cellulose. It needs to be remembered that changes are more evident in the reaction systems using enzymes obtained from imagoes. The relative absorbance of the band at 3400 cm−1 (vibrations of the hydrogen bonded OH) for cellulose following hydrolysis decreased in comparison to the band for non-hydrolyzed cellulose. This absorption band was observed to be slightly narrower after enzymatic hydrolysis. The spectra were recorded for cellulose samples after 120 minutes of hydrolysis. The results of FTIR analyses showed that hydrogen bonding energy changes after enzymatic hydrolysis. Similar dependencies were observed by Cao [34] in experiments on lignocellulose materials and enzymes from Aspergillus niger and Trichoderma reesei. More pronounced changes in band intensity were observed for MCC 20 in comparison to MCC 50, which suggested greater substrate susceptibility to the enzymes. The above observations correspond to the HPLC analyses of the hydrolysates, where greater glucose concentrations [in mg/ml] were found for MCC 20 versus MCC 50. These changes were recorded for samples of cellulose hydrolyzed using extracts from imagoes. The cellulose material subjected to bioconversion from larvae remained practically unchanged, as presented by the obtained spectra (Fig. 4). After enzymatic treatment the FTIR spectra for MCC 20 cellulose exhibited an intensity decrease in the band for the C–O–C antisymmetric bridge stretching at the β(1,4) glucosidic bond (1165 cm−1) for the deformation vibrations (δ) in the plane at C6. Moreover, a decrease in band intensity for MCC 20 cellulose was observed for vibrations in the amorphous areas at the 897 cm−1 band, which is characteristic of βlinkages, whereas for MCC 50 these changes in the band were slight. Similar trends were observed for changes at the 1058 cm−1 band, which represents stretching vibrations ν(CO) at C-3, ν(C–C). The
reduction in intensity of the peaks for cellulose materials after enzymatic hydrolysis indicated that some cellulose was degraded. Analogous dependencies were observed in studies using enzyme from Aspergillus niger [72]. In turn, FTIR analyses of the obtained spectra showed slight changes in the amounts of crystalline and amorphous areas and in the values of the cellulose crystallinity index. The changes both for microcrystalline - MCC 50 and microcrystalline - MCC 20 cellulose were recorded for bands for the crystalline areas at 1429 cm−1. This band indicates deformation of vibrations δ-for the CH2 groups at C6, at C6 δ OCH. Moreover, vibrations of this band show the rotational energy of hydroxyls located at C3 and C6 3 and 6 (C3–O3 and C6–O6) [73]. Changes in that band affected the crystallinity index of cellulose LOI. Observed changes are explained by the two-stage process of hydrolysis. In the first stage, more accessible amorphous areas of cellulose are degraded. In turn, in the second stage crystalline areas of cellulose start to be digested. As a consequence the ordering of cellulose structure is reduced, which contributes to further bioconversion of the material. In the initial period of the reaction after 60 minutes the values of TCI, LOI and HBI were comparable. In contrast, significant differences were observed after 120 minutes of hydrolysis in the case of MCC 20 when compared to microcrystalline MCC 50. These changes were recorded mainly in the post-reaction cellulose at the use of the imago extracts. The greatest decrease in the crystallinity index TCI from 1.057 to 0.608 and LOI from 2.134 to 1.898 was observed for the MCC 20 cellulose. In turn, for the MCC 50 these values were practically almost identical during all the stages of the reaction. The obtained results are presented in Table 1. Similarly as in our experiments, Shi et al. [74] also stated that the index of crystallinity for microcrystalline cellulose MCC 50 at 2h reaction remained practically the same. Some scientific reports indicate a slight increase in the index of crystallinity of cellulose from 2 to 3% [75,76]. The slight increase index crystallinity TCI and LOI after hydrolysis suggests a preferential hydrolysis of amorphous cellulose [3]. This study was conducted on other materials exhibiting different 6
Carbohydrate Research 485 (2019) 107811
K. Szentner, et al.
Table 1 Infrared crystallinity indexes (TCI and LOI) and hydrogen bond intensity (HBI) of the studied cellulose after reaction with the use of larval and imago extracts. Time of treatment [min]
MCC 50 0 60 120 MCC 20 0 60 120
IR crystallinity ratio
Hydrogen bond intensity
H 1372/H2900 (TCI)
A 1429/A897 (LOI)
A3400/A1320 (HBI)
imago larval
imago larval
imago larval
0.927 ± 0.011 0.941 ± 0.018 0.937 ± 0.015 0,940 ± 0.010 0.957 ± 0.028
1.828 ± 0.025 1.859 ± 0.028 1.875 ± 0.025 1.821 ± 0.037 1.896 ± 0.050
1.525 ± 0.011 1.520 ± 0.010 1.561 ± 0.025 1.466 ± 0.011 1.589 ± 0.025
1.057 ± 0.031 1.088 ± 0.011 1.061 ± 0.010 0.608 ± 0.010 1.069 ± 0.050
2.134 ± 0.028 2.144 ± 0.014 2.039 ± 0.150 1.898 ± 0.015 2.019 ± 0.110
1.874 ± 0.012 1.929 ± 0.010 1.848 ± 0.055 1.559 ± 0.013 1.820 ± 0.075
porosity and crystallinity, which obviously affects bioconversion. The decrease in TCI indicates a disturbance of the crystalline structure during enzymatic treatment of cellulose. This was also confirmed in our results, as the greatest LOI reduction was recorded in for MCC 20 cellulose, which showed greater susceptibility to hydrolysis compared to MCC 50. The action of cellulases found in the applied extracts is also confirmed based on changes in the hydrogen bond intensity (HBI). Similarly as for the crystallinity ratio, MCC 20 cellulose is more susceptible to the action of imago extract cellulases. The hydrogen bond intensity (HBI) in cellulose ranged from 1.87 to 1.58. The chain mobility and binding distance, as well as hydrogen bonding (HBI) of cellulose are dependent on the crystalline system and the degree of intermolecular regularity [60]. These changes were also found in the case of decreases of the degree of polymerization under the influence of enzymes. The structural changes in cellulose after the enzymatic reaction were more precisely characterized by the degree of polymerization (DP), the average molecular weight and the degree of polydispersity. These parameters, similarly as crystallinity, determine physical and chemical properties of cellulose. The results of our study are presented in Table 2. A decrease in the degree of polymerization from 329 for the control sample to 308 was observed for MCC 20 cellulose in the case of imago extracts used in the hydrolysis reaction. In contrast, for MCC 50 cellulose a less marked DP reduction was found from 341 to 338. The GPC analysis (Table 2) shows that the greatest changes for this cellulose were also observed for the average molecular mass (Mn). The degree of polydispersion (Mw/Mn) was comparable at 2.3. In the case of larval extracts used in hydrolysis the DP values were similar for MCC 50, while for MCC 20 cellulose a lesser decrease in DP was recorded from 329 to 317 when compared with the imago extracts, for which the degree of polymerization decreased from 329 to 309. A similar dependence was reported by Pala et al. [77] and Park et al. [3] in their experiments on identical cellulose material but fungi as the source of enzymes. The higher rate of enzymatic hydrolysis for MCC 20 cellulose compared to MCC 50 was also recorded in studies using enzymes from
Trichoderma reesei and Trichoderma veride and similar cellulose materials conducted by Pala et al. [77], Kim et al. [78] and Dourado et al. [79] Those authors explained the greater accessibility of microcrystalline cellulose MCC 20 by the specific surface area of microcrystalline cellulose. The observed changes in cellulose, which is a material containing both crystalline and amorphous areas, may indicate that the complex of enzymes obtained from Z. morio, apart from endoglucanases, also contains active exoglucanases that act on the terminal ends (fragments) of the crystalline cellulose polymer chains to cleave them into monomeric glucose units. Results of HPLC analyses (Fig. 3), showing no contents of cellobiose or other shorter chain saccharides in the hydrolysate, suggest the presence of β-glucosidase responsible for the degradation of cellobiose to glucose. Slightly lower glucose levels were obtained in the enzymatic reaction with MCC 50 cellulose, which indicates lower accessibility of this substrate. Moreover, certain endoglucanases present in the gut of Z. morio may hydrolyze crystalline cellulose, resulting in the production of the main reducing sugar, i.e. glucose [2]. Further analysis connected with the purification and modification of the hydrolysis reaction involving the proposed complex will definitely increase the efficiency of the reaction, thus enhancing its further applicability. 4. Conclusions The recorded results of bioconversion of microcrystalline cellulose indicate that the imago extract is a more efficient system than the larval extract. The optimum cellulase activity from midguts of Z. morio recorded towards 2% cellulose substrate and at 50 °C. Additionally, microcrystalline cellulose MCC 20 is more susceptible to hydrolysis in comparison to MCC 50. This confirms the observation that a material of smaller particle size and lower crystallinity shows greater susceptibility to hydrolysis. For the microcrystalline cellulose MCC 20 the values of TCI, LOI, HBI and the degree of polymerization decreased, while hydrolysis efficiency improved. In the case of a more crystalline cellulose MCC 50 the values of these parameters remained unchanged, while no major changes were observed in the process of hydrolysis. The more dynamic hydrolysis of MCC 20 cellulose observed in our study confirms
Table 2 Molecular parameters of cellulose after hydrolysis (120 minutes treatment with cellulases). Cellulose MCC 50 before hydrolysis after hydrolysis (larval extract) after hydrolysis (imago extract) MCC 20 before hydrolysis after hydrolysis (larval extract) after hydrolysis (imago extract)
Mn [g/mol]
Mw [g/mol]
Mw/Mn
DP
25 150 23 593 23 673
55 970 55 187 54 763
2.2 ± 0.1 2.3 ± 0.2 2.3 ± 0.1
345 ± 2 341 ± 3 338 ± 2
23 160 22 510 21 503
53 275 51 410 49 970
2.3 ± 0.1 2.3 ± 0.1 2.3 ± 0.1
329 ± 2 317 ± 4 308 ± 2
7
Carbohydrate Research 485 (2019) 107811
K. Szentner, et al.
that the extract produced from the insect contains endocellulases required for cellulose hydrolysis. Thanks to the presence of other exocellulases and cellobiohydrolases we observe the formation of glucose as the final effect. The presence of endogenous cellulases in this insect is a valuable finding in terms of its further use for biofuel production. This study along with results of its continuation will definitely find applications for the enzymatic modification of cellulose and lignocellulose materials commonly used in industry.
[31] S. Borysiak, A. Grząbka-Zasadzińska, J. Appl. Polym. Sci. 133 (2016) 42864–42873. [32] S.A. Lucena, L. Lima, L.S.A. Cordeiro, C. Sant'Anna, R. Constantino, P. Azambuja, W. de Souza, E.S. Garcia, F.A. Genta, Biotechnol. Biofuels 4 (2011) 1–9, https://doi.org/10. 1186/1754-6834-4-51. [33] L.-J. Su, H.-F. Zhang, X.-M. Yin, M. Chen, F.-Q. Wang, H. Xie, G.-Z. Zhang, A.-D. Song, Genet. Mol. Res. 12 (2013) 2432–2441, https://doi.org/10.4238/2013. January.4.11. [34] Y. Cao, H. Tan, J. Mol. Struct. 705 (2004) 189–193, https://doi.org/10.1016/j.molstruc. 2004.07.010. [35] R. Fischer, R. Ostafe, R.M. Twyman, Yellow Biotechnol. II 136 (2013) 51–64, https://doi. org/10.1007/10_2013_206. [36] J.D. Willis, C. Oppert, J.L. Jurat-Fuentes, Insect Sci. 17 (2010) 184–198, https://doi.org/ 10.1111/j.1744-4917.2010.01322.x. [37] D. Zhang, A.R. Lax, A.K. Raina, J.M. Bland, Insect Biochem. Mol. Biol. 39 (2009) 516–522, https://doi.org/10.1016/j.ibmb.2009.03.006. [38] X. Zhou, J.A. Smith, F.M. Oi, P.G. Koehler, G.W. Bennett, M.E. Scharf, Gene 395 (2007) 29–39, https://doi.org/10.1016/j.gene.2007.01.004. [39] R.J. Ma, C.Y. Wang, Y.W. Liu, T.R. Sivakumar, Z.X. Ren, Y. Fang, J.Q. Jia, Z.Z. Gui, J. Asia Pac. Entomol. 17 (2014) 67–71, https://doi.org/10.1016/j.aspen.2013.10.007. [40] S. Jonardhnan, M.M. Sain, BioRes 1 (2006) 176–188, https://doi.org/10.15376/biores.1. 2.176-188. [41] M.G. Adsul, K.B. Bastwade, A.J. Varma, D.V. Goghale, Bioresour. Technol. 98 (2007) 1467–1473, https://doi.org/10.1016/j.biortech.2006.02.036. [42] S.W. Huang, H.-Y. Zhang, S. Marshall, T.A. Jackson, Insect Sci. 17 (2010) 175–183, https://doi.org/10.1111/j.1744-7917.2010.01320.x. [43] M.S. Taggar, J. Appl. Nat. Sci. 7 (2015) 625–630, https://doi.org/10.31018/jans.v7i2. 656. [44] K. Poszytek, Postępy Mikrobiologii [Adv. Microbiol.] 55 (2016) 132–146. [45] D.L. Aguilar, R.M. Rodríguez-Jasso, E. Zanuso, A.A. Lara-Flores, C.N. Aguilar, A. Sanchez, H.A. Ruiz, S. Kumar, R.K. Sani (Eds.), Biorefining of Biomas to Biofuels, Biofuels and Biorefinery Technologies, vol. 4, Springer, Cham, 2018, pp. 223–248 https://doi.org/10. 1007/978-3-319-67678-4_10. [46] A. Singh, R.M. Rodríguez-Jasso, K.D. Gonzalez-Gloria, M. Rosales, R. Belmares-Cerda, C.N. Aquilar, R.R. Singhania, H.A. Ruiz, Bioresour. Technol. Rep. 7 (2019) 100257 https://dx.doi.org/10.1016/j.biteb.2019.100257. [47] J.K. Saini, R. Saini, L. Tewari, 3 Biotech 5 (2015) 337–353. [48] V. Brummer, P. Skryja, T. Jurena, V. Hlawacek, P. Stehlik, Appl. Biochem. Biotechnol. 174 (2014) 1299–1308. [49] S. Sasmal, K. Mohanty, S. Kumar, R.K. Sani (Eds.), Biorefining of Biomas to Biofuels, Biofuels and Biorefinery Technologies, vol. 4, Springer, Cham, 2018, pp. 203–221 https://doi.org/10.1007/978-3-319-67678-4_9. [50] M.S. Pino, R.M. Rodrίquez-Jasso, M. Michelin, A.C. Flores-Gallegos, R. MoralesRodrίquez, J.A. Teixeira, H.A. Ruiz, Chem. Eng. J. 347 (2018) 119–136. [51] D. Leung, D. Yang, Z. Li, Z. Zhao, J. Chen, L. Zhu, Ind. Eng. Chem. Res. 51 (2012) 1036–1040. [52] J.X. Zhang, Q. Guo, G.M. He, N. Liu, Food Sci. (N. Y.) 18 (2011) 006. [53] F. Yusof, S. Chowdhury, M.O. Faruck, N. Sulaiman, IFRJ 24 (2017) 446–451. [54] Ch.Y. Soon, Y.B. Tee, Ch.H. Tan, A.T. Rosnita, Int. J. Biol. Macromol. 108 (2018) 135–142. [55] A. Quennedey, B. Quennedey, Cell Tissue Res. 296 (1999) 619–634. [56] G.L. Miller, Anal. Chem. 31 (1959) 426–428, https://doi.org/10.1021/ac60147a030. [57] P.K. Smith, R.I. Krohn, G.T. Hermanson, A.K. Mallia, F.H. Gartner, M.D. Provenzano, E.K. Fujimoto, N.M. Goeke, B.J. Olson, D.C. Klenk, Ann. Biochem. 150 (1985) 76–85, https://doi.org/10.1016/0003-2697(85)90442-7. [58] F. Carrilo, X. Colom, J.J. Suñol, J. Saurina, Eur. Polym. J. 40 (2004) 2229–2234. [59] M.L. Nelson, R.T.J. O′Connor, Appl. Polym. Sci. 8 (1964) 1325–1341, https://doi.org/10. 1002/app.1964.070080323. [60] S.Y. Oh, D.I. Yoo, Y. Shin, H.C. Kim, H.Y. Kim, Y.S. Chung, W.H. Park, J.H. Youk, Carbohydr. Res. 340 (2005) 2376–2391, https://doi.org/10.1016/j.carres.2005.08.007. [61] J.D. Timpa, Agric. Food Chem. 39 (1991) 270–275. [62] T. Bikova, A. Treimanis, Carbohydr. Polym. 48 (2002) 23–28. [63] S.M. Geib, M. Jimenez-Gasco, M. Del, J.E. Carlson, M. Tien, K. Hoover, Environ. Entomol. 38 (2009) 686–699, https://doi.org/10.1603/022.038.0320. [64] M.M. Uddin, M.H. Chowdhury, S. Mojumder, D. Skidar, Pak. J. Biol. Sci. 15 (2012) 333–340. [65] B. Dojnova, R. Pavlović, N. Božić, A. Margetić, V.J. Nenadović, Vujčić Z. Ivanović, Comp. Biochem. Physiol. B 164 (2013) 259–267. [66] F.U. Rehman, M. Aslam, M.I. Tariq, A. Shaheen, A.J. Sami, N.H. Naveed, I. Batool, Afr. J. Biotechnol. 8 (2009) 6710–6715. [67] W. Shi, S.-Y. Ding, J.S. Yuan, BioEnergy Res. 4 (2011) 1–10. [68] A.M. Scrivener, H. Watanabe, H. Noda, J. Insect Physiol. 43 (1997) 1039–1052. [69] J. Liu, K. Song, H. Teng, B. Zhang, W. Li, H. Xue, X. Yang, Acta Biochim. Biophys. Sin. 47 (2015) 741–748, https://doi.org/10.1093/abbs/gmv070. [70] A.J. Sami, M.A. Anwar, F.U. Rehman, A.R. Shakoori, Pak. J. Zool. 43 (2011) 393–401. [71] X. Huang, G. Wang, Biol. Disaster Sci. 2 (2008) 67–70. [72] I. Spridon, C.-A. Teacӑ, R. Bodîrlӑu, Biores.com 6 (2011) 400–413. [73] R.G. Zhbankov, S.P. Firsov, D.K. Buslov, N.A. Nikonenko, M.K. Marchewka, H. Ratajczak, J. Mol. Struct. 614 (2002) 117–125, https://doi.org/10.1016/S0022-2860(02)00252-1. [74] J. Shi, D. Wu, L. Zhang, B.A. Simmons, S. Singh, B. Yang, C.E. Wyman, Biotechnol. Bioeng. 114 (2016) 503–515, https://doi.org/10.1002/bit.26180. [75] Y. Chen, A.J. Stipanovic, W.T. Winter, D.B. Wilson, Y.J. Kim, Cellulose 14 (2007) 283–293. [76] L. Wang, Y. Zhang, P. Gao, D. Shi, H. Liu, H. Gao, Biotechnol. Bioeng. 93 (2006) 443–456. [77] H. Pala, M. Mota, F.M. Gama, Carbohydr. Polym. 68 (2007) 101–108. [78] D.W. Kim, J.H. Yang, Y.K. Jeong, Appl. Microbiol. Biotechnol. 28 (1988) 148–154. [79] F. Dourado, M. Mota, H. Pala, F.M. Gama, Cellulose 6 (1999) 265–282.
Funding This research was funded by the NATIONAL SCIENCE CENTRE, Poland (Grant number 2014/13/B/NZ9/02442). Conflicts of interest The authors declare no conflict of interest. Acknowledgments We would like to thank Agnieszka Graclik for her technical support in Z. morio laboratory culture and maintenance. References [1] T.T. Teeri, Trends Biotechnol. 15 (1997) 160–167, https://doi.org/10.1016/S01677799(97)01032. [2] Y.-H. Zhang, L.R. Lynd, Biotechnol. Bioeng. 88 (2004) 797–824, https://doi.org/10. 1002/bit.20282. [3] S. Park, J.O. Baker, M.E. Himmel, P.A. Parilla, D.K. Johnson, Biotechnol. Biofuels 3 (2010) 1–10, https://doi.org/10.1186/1754-6834-3-10. [4] S. Al-Zuhair, Bioresour. Technol. 99 (2007) 4078–4085. [5] S. Marques, L. Alves, J.C. Roseiro, F.M. Gırio, Biomass Bioenergy 32 (2008) 400–406. [6] R.E. Quiroz-Castaneda, J.L. Folch-Mallol, Intech 6 (2013) 119–155, https://doi.org/10. 5772/53719. [7] E.J.P. Kouadio, K.H. Konan, K.M. Djѐ, E.A. Duѐ, L.P. Kouamѐ, Int. J. Entomol. Res. 4 (2016) 67–86. [8] H. Watanabe, G. Tokuda, Cell. Mol. Life Sci. 58 (2001) 1167–1178. [9] E.A. Bayer, H. Chanzy, R. Lamed, Shoham, Curr. Opin. Struct. Biol. 8 (1998) 548–557, https://doi.org/10.1016/S0959-440x(98)80143-7. [10] L.R. Lynd, P.J. Weiner, W.H. Van Zyl, I.S. Pretorius, Microbiol. Mol. Biol. Rev. 66 (2002) 506–577, https://doi.org/10.1128/MMBR.66.3.506-577.2002. [11] P.C. Ozioko, A.P. Ikeyi, O.P.C. Ugwu, The experiment, Int. J. Sci. Technol. 12 (2013) 778–785. [12] H. Watanabe, G. Tokuda, Annu. Rev. Entomol. 55 (2010) 609–632. [13] C. Oppert, W.E. Klingeman, J.D. Willis, B. Oppert, J.L. Jurat-Fuentes, Comp. Biochem. Physiol. B 155 (2010) 145–154, https://doi.org/10.1016/j.cbpb.2009.10.014. [14] J. Pѐrez, J. Muñoz-Dorado, T. De la Rubia, J. Martinez, Int. Microbiol. 5 (2002) 53–63, https://doi.org/10.1007/s10123-002-0062-3. [15] S.D. Mansfield, C. Mooney, J.N. Saddler, Biotechnol. Prog. 15 (1999) 804–816, https:// doi.org/10.1021/bp9900864. [16] Y. Cao, H. Tan, Carbohydr. Res. 337 (2002) 1291–1296, https://doi.org/10.1016/S00086215(02)00134-9. [17] Y. Cao, H. Tan, Enzym. Microb. Technol. 36 (2005) 314–317, https://doi.org/10.1016/j. enzmictec.2004.09.002. [18] A.V. Gusakov, T. Salanovich, A.I. Antonov, B.B. Ustinov, O.N. Okunev, R. Burlingame, M. Emalfarb, M. Baez, A.P. Sinitsyn, Biotechnol. Bioeng. 97 (2007) 1028–1038, https:// doi.org/10.1002/bit.21329. [19] A.S. Bommarius, A. Katona, S.E. Cheben, A.S. Patel, A.J. Ragauskas, K. Knudson, Y. Pu, Metab. Eng. 10 (2008) 370–381, https://doi.org/10.1016/j.ymben.2008.06.008. [20] K. Clarke, X. Li, K. Li, Biomass Bioenergy 35 (2011) 3943–3950, https://doi.org/10. 1016/j.biombioe.2011.06.007. [21] M. Ioelovich, E. Morag, BioRes 6 (2011) 2818–2835, https://doi.org/10.15376/biores.6. 3.2818-2835. [22] M. Wiman, D. Dienes, M.A.T. Hansen, T. Meulen, G. Zacchi, G. Liden, Bioresour. Technol. 126 (2012) 208–215, https://doi.org/10.1016/j.biortech.2012.08.082. [23] M. Hall, P. Bansal, J.H. Lee, M.J. Realff, A.S. Bommarius, FEBS J. 277 (2010) 1571–1582, https://doi.org/10.1111/j.1742-4658.2010.07585.x. [24] D. Ciolacu, S. Gorgieva, D. Tampu, V. Kokol, Cellulose 18 (2011) 1527–1541. [25] Yi-H. Zhang, L.R. Lynd, Biotechnol. Bioeng. 88 (2004) 797–824. [26] An-I. Yeh, Y-Ch. Huang, S.H. Chen, Carbohydr. Polym. 79 (2010) 192–199. [27] Y. Horikawa, N. Konakahara, T. Imai, A. Kentaro, Y. Kobayashi, J. Sugiyama, Polym. Degrad. Stab. 98 (2013) 2351–2356. [28] S. Borysiak, J. Therm. Anal. Calorim. 113 (2013) 281–289. [29] S. Borysiak, J. Therm. Anal. Calorim. 109 (2012) 595–603. [30] A. Grząbka-Zasadzińska, W. Smułek, E. Kaczorek, S. Borysiak, Polym. Compos. 39 (2018) 448–456.
8