Detection of ionic liquid stable cellulase produced by the marine bacterium Pseudoalteromonas sp. isolated from brown alga Sargassum polycystum C. Agardh

Bioresource Technology 132 (2013) 313–319

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Detection of ionic liquid stable cellulase produced by the marine bacterium Pseudoalteromonas sp. isolated from brown alga Sargassum polycystum C. Agardh Nitin Trivedi, Vishal Gupta, C.R.K. Reddy ⇑, Bhavanath Jha Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, Bhavnagar 364002, India

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

" Characterized an ionic liquid stable

"

"

"

"

cellulase from marine Pseudoalteromonas sp. Activation and stabilization of cellulase in the presence of ionic liquids. Retained catalytic activity even at ionic liquid concentration of 20% (v/ v). Enhanced activity and stability than the commercial cellulase in ionic liquids. Offers a single step continuous saccharification process in ionic liquids.

a r t i c l e

i n f o

Article history: Received 6 September 2012 Received in revised form 6 January 2013 Accepted 7 January 2013 Available online 22 January 2013 Keywords: Cellulase Cellulose Energy Enzyme stability Ionic liquid

a b s t r a c t An extracellular cellulase produced by marine bacterium Pseudoalteromonas sp. was studied for its activity and stability in six different ionic liquids (ILs) over a wide range of concentrations (1–20% v/v) and compared with aqueous medium as control. Enzyme showed its optimal activity at 45 °C and at pH 5 in control. Although the activity varied with the type of IL and its concentration used, the activity measured at 5% (v/v) was maximum with [EMIM]Br followed by [EMIM]Ac, [BMIM]Cl, [C2MIM][CH3SO3], [BMIM][OTF] and [BMPL][OTF] with 115%, 104.7%, 102.2%, 98.33%, 93.84% and 92.67%, respectively, and >80% activity at 15% (v/v) in all ILs. The enzyme stability at 5% (v/v) IL concentration for 36 h was superior to commercial cellulase. The cellulase activity enhanced by 1.35- to 1.72-fold over control when 5% (v/v) IL based reaction medium with algal biomass was used and thus showed potentials for saccharification of biomass in a single step process. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Cellulose is the most abundant renewable carbon source available on the earth for production of feedstock chemicals of commercial value, if converted efficiently to monomeric D-glucose units (Klemm et al., 2005). Both chemical and enzymatic hydrolysis ⇑ Corresponding author. Tel.: +91 278 256 5801/256 3805x6140; fax: +91 278 256 6970/256 7562. E-mail addresses: [email protected], [email protected] (C.R.K. Reddy). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.01.040

has been employed for effective conversion of cellulosic biomass into fermentable sugar. The former method is currently being employed as most common means for producing mono-saccharides from cellulose. Nevertheless, chemical hydrolysis often leads to the accumulation of undesired non-sugar byproducts that poses considerable problems in recovery of resultant products (Sasaki et al., 1998). On the contrary, enzymatic hydrolysis overrides all such hindrances and has been, thus, preferred over chemical hydrolysis. However, the enzymatic hydrolysis of cellulose is largely regarded as counterproductive due to its lower solubility in

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aqueous medium. As a result, cellulose is being pre-treated with various chemicals or solvent fractionations prior to enzymatic hydrolysis. Recently, ionic liquids (ILs) have been utilized as pre-treatment solvent because of their higher cellulose dissolution potential over other solvents. The recyclability of IL is another addendum to their utility as pre-treatment solvent. Although ILs have better cellulose dissolution potential, their inhibitory effects on enzyme activity has been the major concern (Turner et al., 2003). Therefore, considerable efforts have been directed to explore the IL stable cellulolytic enzymes from various sources. Such enzymes not only allow elimination of cellulose regeneration step but also enable to carry out downstream conversion in a single-step continuous process. In earlier studies, commercial cellulases were mainly investigated for IL stability (Turner et al., 2003; Lozano et al., 2011; Wang et al., 2011). Also, a few IL stable cellulases derived from metagenome (Pottkamper et al., 2009) as well as from culturable microbes such as Penicillium janthinellum (Adsul et al., 2009), Thermatoga maritime and Pyrococcus horikoshii, (Datta et al., 2010), and Halorhabdus utahensis (Zhang et al., 2011) have been reported. IL induced partitioning of protein from surrounding hydration shell and disrupts the hydrogen and hydrophobic interactions leading to aggregation of protein that inhibits the enzyme activity (Bose et al., 2010; Moniruzzaman et al., 2010; Constatinescu et al., 2010). The extracellular enzymes produced under high salt concentrations showed modifications such as excessive number of charged acidic amino acids on their surface which prevent the formation of protein aggregation through electrostatic repulsive charges at protein surface. Such adaptations are presumed to make the enzyme stable in IL. The present study was therefore undertaken to find out an IL stable cellulase from a marine bacterium isolated from brown seaweed Sargassum polycystum C. Agardh. The bacterial cellulase obtained was first investigated for optimization of assay conditions in aqueous medium and then tested for its activity and stability in the presence of different ILs at various concentrations ranging from 1% to 20% (v/v). Further, the salt tolerance of the enzyme was also investigated under different concentration of NaCl ranging from 1% to 20% w/v. Subsequently, the activation and stabilization efficiency of bacterial cellulase in ILs has also been validated against commercial cellulase from Trichoderma viride (cellulase Onozuka – R-10, Yakult Pharmaceutical Ind. Co. Ltd, Japan).

The morphological and biochemical characterization of the strain was performed and compared with the Bergey’s Manual of Systematic Bacteriology. The molecular identification of the bacterial strain was carried out by 16S rDNA sequencing following the method described by Trivedi et al. (2011).

2.2. Cellulase production and activity assay A bacterial suspension (2  107 CFU/mL) was inoculated in the enzyme production medium containing, Artificial Seawater Salt (ASW): 3.5% w/v; CMC-Na: 2% w/v; and Yeast extract: 0.5% w/v. The flask was incubated at 30 ± 2 °C for 72 h in a shaker incubator with shaking speed of 150 rpm. Following incubation, the bacterial culture medium was centrifuged at 12,000g for 20 min at 4 °C. The obtained clear supernatant was considered as crude enzyme mix and used for cellulolytic assay. Cellulase activity was determined by 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959). The assay mixture contained enzyme: substrate CMC-Na (1%) (1:2) and pH of the mix was adjusted to 5.0 using sodium acetate buffer (50 mM). The reaction mixture was incubated at 45 °C for 1 h. After incubation equal volume of DNS reagent was added and the mixture was heated to 99 °C for 15 min in a boiling water bath. The release of reducing sugar was estimated spectrophotometrically by measuring the absorbance at 546 nm. One unit of enzymatic activity was defined as the amount of enzyme that released 1 lmol of reducing sugars per minute in glucose equivalents. The protein content was estimated by Bradford method using BSA as standard (Bradford, 1976). Total cellulase activity was also determined by FPase assay using filter paper (Whatman No. 1) as substrate. Cellulolytic activity of the crude enzyme mix was further confirmed by monitoring the change in molecular weight of the enzymatically hydrolysed CMC-Na using Gel permeation chromatography (GPC) (Water Allaince, model 2695) equipped with GPC column ultra hydrogel 120 and 500 and refractive index detector (Waters 2414). The molecular mass distribution of hydrolysed product was also determined by mass spectrometry (Waters Q-Tof) equipped with an electrospray ionization interface, MCP detector and Waters MassLynx software (version 4). The mass spectrometer was run employing direct flow injection technique and the mass fragmentation was recorded on ESI positive mode (ESI+).

2.3. Effect of pH and temperature on cellulase activity and stability 2. Methods 2.1. Isolation, screening and identification of cellulose degrading bacteria Bacterial strains were isolated from partially deteriorated marine macrophytic alga S. polycystum C. Agardh collected from Veraval coast (N 20° 54.870 ; E 70° 20.830 ), India in the month of February 2011. The deteriorated thallus was rinsed with sterile distilled water and the wash-off water was spread on Zobell marine agar plates (Zobell marine agar 2216, Hi-media Labs Pvt. Ltd., India). Petri plates were then incubated at 30 ± 2 °C for 48 h and different bacterial colonies obtained were maintained as pure cultures. Cellulolytic potential of the isolates were determined by streaking each strain on agar plates enriched with 2% carboxymethyl cellulose (CMC-Na) as a sole carbon source. After an incubation of 48 h at 30 ± 2 °C, plates were stained with Lugol’s iodine solution and the zone of clearance was considered as qualitative measure of extracellular cellulase activity (Kasana et al., 2008). The strain showing highest zone of clearance on CMC-agar plates was considered for further analysis.

The pH and temperature optima for enzyme activity was assayed at different pH ranging from 2 and 3 (50 mM Glycine–HCl buffer), 4, 5 and 6 (50 mM Sodium acetate buffer), to 7 and 8 (50 mM Phosphate buffer) and at different temperatures ranging from 15 to 75 °C with increments of 15 degrees unit. Further, the stability of enzyme was investigated by estimating the residual enzyme activity after pre-incubation of 1 h at aforementioned pH and temperature ranges.

2.4. Effect of various additives on enzyme activity The effect of various additives on the enzyme activity was determined in the presence of metal ions and other reagents. The additives used in this study were CoCl2, PbCl2, FeCl2, MgCl2, MnCl2, ZnCl2, EDTA and PMSF (5 mM each) and relative activity (%) was estimated against control (without additive). Further, salt tolerance of enzyme was investigated by estimating the residual activity in the presence of different concentrations of NaCl ranging from 1% to 20% w/v against control (without NaCl) under optimized assay conditions.

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2.5. Effect of ionic liquids on cellulase enzyme The ILs used in this study includes 1-ethyl-3-methylimidazolium methanesulfonate (IL1); 1-ethyl-3-methylimidazolium bromide (IL2); 1-ethyl-3-methylimidazolium acetate (IL3); 1-butyl1-methylpyrrolidinium trifluromethanesulfonate (IL4); 1-butyl-3methylimidazolium trifluoromethanesulfonate (IL5); 1-butyl-3methylimidazolium chloride (IL6). The residual enzyme activity was determined at different ILs concentrations (1%, 3%, 5%, 10%, 15% and 20% v/v) under optimized assay conditions and was compared with the control reaction carried out in aqueous medium. The ILs did not affect the pH values in the enzyme assay mix and retained to pH around 5.0. The IL tolerance of the bacterial cellulase was further validated against the commercial cellulase derived from T. viride (Cellulase Onozuka R-10, Yakult, Japan). Both bacterial and commercial cellulase were pre-incubated in ILs (5% v/v) for different time intervals ranging from 12 to 48 h. Thereafter, the residual activities for both the enzymes were determined under the optimized assay conditions. The ILs in which enzyme showed the highest activity and stability were then investigated for their cellulose dissolution potential according to the method described by Dadi et al. (2006). Cellulose (50 mg) was mixed with 450 ll of each IL in a separate glass vials and incubated for 1 h at 45 °C in inert atmosphere of nitrogen to prevent the water uptake by IL. After incubation, cellulose was precipitated by washing the reaction medium with water as an antisolvent. The regenerated cellulose was then analyzed for loosening of crystalline structure by Scanning Electron Microscope (LEO 1430VP, UK). The thermal stability of enzyme in the presence of ILs (5% v/v), wherein highest tolerance was observed, was investigated with a pre-incubation of 1 h at different temperatures ranging from 15 to 60 °C with increments of 15 degrees unit under optimized assay conditions. The effect of various additives such as PMSF, ZnCl2, CoCl2, and FeCl2 (5 mM each) on the stability of enzyme was carried out with a pre-incubation of 1 h in the same set of ILs (5% v/v) under optimized assay conditions. The enzyme activity in the presence of different ILs was also assayed against cellulose (cotton linter) and natural algal biomass (Ulva lactuca) in addition to CMC-Na. 2.6. Cellulase production using different substrates Fresh bacterial culture was inoculated into the enzyme production medium containing 0.5% w/v Yeast extract, 3.5% w/v ASW and different carbon source (1.5% w/v) viz green seaweed (U. lactuca), Brown seaweed (S. polycystum) and CMC-Na. The medium was incubated at 30 ± 2 °C for 5 days in a shaker incubator at150 rpm. The supernatant from the production medium was taken periodically after every 12 h and assayed for cellulase activity using DNS method under optimized assay conditions.

315

otide sequences homology match within the NCBI GenBank. The DNA sequence for the isolated strain was submitted to NCBI GenBank with Accession No. HQ156241. 3.2. Effect of pH and temperature on cellulase activity and stability The enzyme showed optimum activity at pH 5 and temperature 45 °C. The cellulase (CMCase) and total cellulase (FPase) activity in the crude enzyme mix was found to be 6.04 and 2.11 U/mL, respectively. The GPC spectrum for enzymatic hydrolyzed products confirmed the degradation of polymeric cellulose into low molecular weight fractions. The GPC chromatogram showed peaks corresponding to molecular weights of 1049 and 156 indicative for oligosaccharide and monosaccharide, respectively (Supplementary Fig. 2). Further, LC–MS analysis confirmed that the liberated monosaccharide was glucose. The peaks obtained at 223.19, 245.03 and 269.09 have matched well with the calculated m/z peaks 224.03 [(D-Glucose) + 2Na+], 246.01 [(D-Glucose) + 3Na+] and 267.99 [(DGlucose) + 4Na+]. Hence the formation of monomeric unit (Glucose) is confirmed in the hydrolysate. These results provide clear evidence for the existence of functionally active cellulase mixture of all the three units in crude enzyme mix. Interestingly, the enzyme was found to be active over a broad range of temperatures from 15 to 75 °C with marginal decline in activity by 2% at 60 °C and 10% at 75 °C. The thermal stability analysis of enzyme showed 100% activity at 4 °C whereas activity at 15, 30 and 45 °C was found to be 90%, 86% and 80%, respectively, after a pre-incubation of 1 h. Higher temperatures are generally detrimental to enzyme activity but the enzyme in this study retained its activity even at 60 and 75 °C with 66% and 57% activity, respectively. The thermal stability of cellulase in this study was found similar to the cellulolytic activity (57%) reported from Geobacillus sp. T1 at 80 °C (Assareh et al., 2012). However, cellulase from Aspergillus fumigates Z5 has been shown to have an enzyme activity <50% at 70 °C (Liu et al., 2012). It has been reported that cellulases originated from Bacillus strains were stable at 0–50 °C while at higher temperature (>70 °C) the enzyme activity declined to <50% (Lin et al., 2012). The bacterial cellulase with higher thermal stability as described in this study would find wider applications particularly in the bio-refineries and food processing industries. The enzyme activity showed linear increase with increasing pH from 2.0 to 5.0 and found to be optimum at pH 5.0 indicating the acidic nature of the enzyme. These results are in accordance with those reported from the genus Bacillus and Aspergillus having an optimum enzyme activity in acidic range (pH 3.5–6.5) and at temperature 40–60 °C (Assareh et al., 2012: Lin et al., 2012: Liu et al., 2012). Nevertheless, the enzyme activity marginally declined with increasing pH from 6.0 to 8.0 and ranged between 83% and 85%. The findings of pH stability experiments showed retention of more than 80% of the enzyme activity at pH 6.0–8.0. In contrast, the cellulase from bacterial strain Geobacillus sp. T1 (Assareh et al., 2012) and fungal strain Chrysosporium lucknow (Dotsenko et al., 2012) showed decreased enzyme activity of about 60% at pH >7 and 50% at pH >5, respectively.

3. Results and discussion 3.3. Effects of additives on enzyme activity 3.1. Screening and identification of cellulose degrading bacteria Of the total 12 bacterial strains isolated from S. polycystum, four isolates were positive for cellulase activity with Lugol’s iodine test. Among these four isolates, the one showed highest zone of clearance on CMC agar plate was selected for further studies (Supplementary Fig. 1). The morphological and biochemical analysis revealed that the strain with maximum cellulase activity was a gram negative, aerobic and short rods. The identity of the strain was confirmed as Pseudoalteromonas sp. based on 16S rDNA nucle-

The influence of various metal ions and additives on cellulase activity was studied at 5 mM concentration each. The enzyme activity increased to 139%, 150% and 157% in the presence of Zn+2, Co+2 and Fe+2, respectively. However, slight decline in the enzyme activity was found in the presence of Mg+2 (95%), PMSF (93%), Pb+2 (82%), Mn+2 (78%) and metal chelator EDTA (75%). Li and Yu, 2012 also reported inhibitory effect of EDTA, PMSF and Mn+2 on cellulase isolated from halotolerant Bacillus sp. L1. In another study on soil metagenome-derived endoglucanase Cel5A, the inhibitory

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Fig. 1. Effect of different ionic liquids on cellulase (a) activity, (b) stability at 5% IL concentration and (c) stability of commercial cellulase at 5% IL concentration.

effect of Zn+2, Co+2, Fe+2 has been reported (Voget et al., 2006). In addition to metal ions, effect of salt on enzyme activity has also been investigated. Enzyme could retain 95.46% and 88.45% activity at salt concentration 10% and 20% w/v, respectively. Such salt tolerance revealed for the halophilic nature of the studied enzyme.

3.4. Effect of ionic liquids on cellulase enzyme The enzyme activity and stability was found to vary across the investigated IL types and their concentrations. The enzymatic activity was found >90% for all the ILs when used at concentration 5% v/v. The maximum activity was measured with [EMIM]Br (IL2) followed by [EMIM]Ac (IL3), [BMIM]Cl (IL6), [C2MIM][CH3SO3] (IL1), [BMIM][OTF] (IL5) and [BMPL][OTF] (IL4) with 115%, 104.7%, 102.2%, 98.33%, 93.84% and 92.67%, respectively. The enzyme activity at 20% (v/v) IL concentration was in the following order of IL3 (94.37%) > IL4 (80.2%) > IL5 (74.69%) > IL6 (73.2%) > IL2 (67%) > IL1 (59%) (Fig. 1a). The cellulase activity in [EMIM]Ac, regarded as gold standard for biomass treatment, at 15% v/v was 97.22% and was substantially higher than that of 50% reported previously for recombinant cellulase (Tma Cel5A) from bacterium T. maritime but was similar to the Pho EG from P. horikoshii (Datta et al., 2010). Furthermore, the residual activity of the studied enzyme at higher concentration (20% v/v) of [EMIM]Ac was 94.37% which was also superior to that of the same reported as 55% and 43% from fungal strain Trichoderma reesei (Zhang et al., 2011) and Aspergillus fumigatus (Singer et al., 2011), respectively. With other ILs at higher concentration of 20% v/v, the residual activity estimated was 80.2%, 74.69% and 73.2% for IL4, IL5 and IL6, respectively. These values were comparatively superior to that of the same reported from metagenome derived cellulases (pFosCelA2 and pFosCelA84) but similar to pFosCelA3 (Ilmberger et al., 2012).

Fig. 2. Effect of (a) temperature and (b) additives on enzyme stability in the presence of ionic liquids. The values represent averages from triplicate experiments. Error bars represent the standard deviation.

In addition to functional activity of the enzyme, its stability with ILs (5% v/v) was also studied for different pre-incubation

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N. Trivedi et al. / Bioresource Technology 132 (2013) 313–319 Table 1 Recently reported ionic liquid stable cellulase from different sources. Cellulase

Optimum pH

Cellulase

5.0

CelA2

6.5

CelA3 CelA84

Optimum Temperature (°C) 45

Specific activity (U/mg) 49.21

55

11.9

70 46

1.9 7.5

Cel5A

6.0–6.5

Hu-CBH1

9.5

37

–

5.0

50

–

4.8

45

Cellulase

4.5

37

11

Tma Cel5a

4.8

80

30

PhoEG

6.4

>95

1.9

CellA10

7.5

55

2.4

CelA24

7

55

23.3

Endoglucanase Celluclast

Ò

75–80

294

0.096

Rel. activity (%) in (%) IL

Rel. enzyme activity in (%) IL (stability)

Organism

References

115% in 5% and 59% in 20% [EMIM]Br 98% in 5% and 67% in 20% [C2MIM] [CH3SO3] 105% in 5% and 94% in 20% [EMIM]Ac 93% in 5% and 80.2% in 20% [BMPL] [OTF] 94% in 5% and 74.69% in 20% [BMIM] [OTF] 102% in 5% and 73.2% in 20% [BMIM]Cl 54% in 30% [EMIM] [OTF]

92.16% (48 h) in 5% [EMIM]Br 102% (48 h) in 5% [C2MIM] [CH3SO3] 97% (48 h) in 5% [EMIM]Ac

Pdeudoalteromonas sp.

Present study

Metagenome

Ilmberger et al. (2012)

Thermoanaerobacter tengcongensis Halorhabdus utahensis

Liang et al. (2011) Zhang et al. (2011) Singer et al. (2011) Engel et al. (2010) Datta et al. (2010)

68% in 30% [EMIM] [OTF] 8% in 30% [BMPL] [OTF] 65% in 20% [BMIM]Cl 60% in 30% [Amim]Cl at 2 M NaCl 43% in 20% [C2mim][OAc] 40% in 10% [MMIM] [DMP] 40% in 5% [EMIM]Ac 40% in 20% [EMIM]Ac 95% in 20% [EMIM]Ac 74% in 30% [BMPL] [OTF] 2% in 30% [BMPL] [OTF]

intervals from 12 to 48 h. An increase in enzyme activity was measured for all the ILs after a pre-incubation of 24 h. The enzyme activity after 24 h of pre-incubation was highest with IL2 (130.86%) followed by IL1 (122.9%), IL6 (113%), IL3 (102%), IL4 and IL5 (both 101.7%). Further, an increase in pre-incubation period to 36 h the resultant activity was in the order of IL2 (121%) > IL6 (119%) > IL1 (108%) > IL3 (100%) > IL5 (99.51%) > IL4 (98%) (Fig. 1b). The stability of enzyme was then evaluated against commercial cellulase. The residual activity of commercial enzyme after 24 and 36 h were lower than bacterial cellulase. The residual activity was found in the following order of 101.46 (IL1) > 99.5% (IL5) > 95.75% (IL3) > 94.22% (IL6) > 93.13% (IL4) > 91.84% (IL2) after 24 h and 82.56% (IL2) > 91.38% (IL6) > 91% (IL1) > 96% (IL3) > 96% (IL5) > 92% (IL4) after 36 h (Fig. 1c). This study further analyzed the thermal stability of the enzyme with ILs. The ILs (IL1, IL2, IL3 and IL6) where enzyme showed comparatively higher activity were selected. The enzyme showed activity of 136% (IL1), 110% (IL2), 101.7% (IL6) and 97.2% (IL3) at 15 °C after a pre-incubation of 1 h. At higher temperature of 60 °C, partial decline in activity was observed with IL3 (82.93%) and IL6 (81.49%) whereas activity got declined to 68% and 51% for IL1 and IL2, respectively (Fig. 2a). The earlier studies reported that the activity of cellulases of fungal origin has been highly inhibited by ILs than the same from bacterial origin (Datta et al., 2010; Singer et al., 2011; Zhang et al., 2011). The disruption of hydrogen and hydrophobic interactions of protein along with the partitioning of surrounding hydration shell could be some of the common attributes for IL induced inhibition of enzyme activity (Bose et al., 2010; Moniruzzaman et al., 2010; Constatinescu et al., 2010). Similar disruption mechanisms have also been caused by

89% (48 h) in 5% [BMPL] [OTF] 93% (48 h) in 5% [BMIM] [OTF] 83% (38 h) in 5% [BMIM]Cl 11% (5 days) 60% [BMPL] [OTF] 79% (4 days) 60% [BMIM]Cl 81% (4 days) 60% [BMPL] [OTF] 80% (5 h) 40% [BMIM]Cl 100% (1 h) in 20% [Amim]Cl at 2 M Nacl 10% (12 h) in 10% [C2mim][OAc] 40% (11 days) 10% [MMIM] [DMP] 0% (15 h) 15% [EMIM]Ac 44% (15 h) 15% [EMIM]Ac 79% (15 h) 15% [EMIM]Ac 0.8% (17 h) 60% [BMPL] [OTF] 0.02% (17 h) 60% [BMIM] [OTF]

Aspergillus fumigatus JF1 Trichoderma reesei Trichoderma viride Thermotoga maritima Pyrococcus horikoshii Metagenome

Pottkamper et al. (2009)

Metagenome

Fig. 3. Effect of different substrates (a) CMC (b) Ulva lactuca (c) Sargassum polycystum on cellulase production.

high salt concentration as well. Microbes dwelling in marine environment (>3 M salt) develop adaptive mechanisms to survive in high salt conditions. Proteins of such microbes contain an excessive number of charged acidic amino acids on their surface which allow protein to get solubilise even in high salt concentration either by forming a hydrated ion network with cations/anions or by preventing the formation of protein aggregation through electrostatic repulsive charges at protein surface (Fukuchi et al., 2003; Paul et al., 2008; Tadeo et al., 2009). The isolated bacterial strain Pseudoalteromonas sp. in this study is also of marine origin and produced a cellulase of high salt tolerance (up to 20% w/v) thereby gave circumstantial evidence for aforementioned adaptations which led to IL tolerance.

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The additives which are inducers for cellulolytic activity in aqueous medium were further studied for their effect on enzyme stability in association with ILs. In the presence of PMSF, the enzyme could retain its functional activity with IL1 (108%) and IL3 (115%) whereas Zn+2 was found to induce activity in IL1 (105%), IL2 (112%) and IL3 (103%). However, the enzyme was found active only in IL3 (100%) and IL6 (102%) in the presence of Fe+2 and Co+2, respectively (Fig. 2b). The activation and stabilization of the cellulase in the presence of conventionally defined cellulose dissolving ionic liquids (Freire et al., 2011; Liu et al., 2011) offers its potential for efficient hydrolysis of cellulosic biomass in a one step continuous process. Consequently, the steps involved in regeneration of cellulose followed in conventional strategies can be bypassed. Recently reported cellulases capable of tolerating ILs are summarized in Table 1. Additionally, an attempt was made in this study to evaluated the industrial applicability of the cellulase enzyme using the algal substrate (U. lactuca) and cellulose (Cotton linter) in a medium containing ILs (IL1, IL2, IL3 and IL6) (5% v/v). The specific activity of the enzyme against cellulose (Cotton linter) was found in the following order of IL1 (138.46 U/mg) > IL2 (102.44 U/mg) > IL6 (94.6 U/mg) and IL3 (91.76 U/mg) while the same in aqueous medium was comparatively low as 61.11 U/mg. In the presence of natural biomass (U. lactuca) as substrate, the specific activity was found in the order of IL1 (112 U/mg) > IL6 (98 U/mg) > IL2 (91 U/mg) and IL3 (88 U/mg) while in aqueous medium registered specific activity was as low as 64.82 U/mg. The overall results showed that the activity of enzyme is being induced by ILs. The higher cellulolytic activity with ILs is mostly due to increased accessibility of enzyme to cellulose because of its higher dissolution. The loosening of rigid crystalline structure of cellulose is generally attributed to its solubility in ILs and the same is confirmed through SEM analysis. 3.5. Cellulase production using different substrates The effect of different naturally available carbon sources particularly algal biomass from marine origin on the production of cellulase enzyme was also investigated to increase the scope of industrial application. The marine macroalgal species belonging to chlorophyta (U. lactuca) and phaeophyta (S. polycystum), rich in cellulosic content, were employed as a sole carbon source in enzyme production medium. The production medium supplemented with CMC-Na as carbon source was considered as control. The maximum enzyme production was recorded on second day for medium supplemented with seaweed biomass and on third day with CMC. The specific activity was found to be highest with U. lactuca (73.84 U/mg) followed by S. polycystum (49.21 U/mg) and CMC (42.82 U/mg) after their respective optimal incubation period (Fig. 3). However in the earlier report, highest cellulase production from Thermoascus aurantiacus and Thielavia terrestris was found from artificial substrate than the natural substrate (McClendon et al., 2012). 4. Conclusion This study reports the ionic liquid stable cellulase from marine bacterium Pseudoalteromonas sp. The cellulase had activity over a broad range of pH, temperature and salt concentrations. The activation and stabilization of cellulase in ILs including in [EMIM]Ac, regarded as gold standard for biomass treatment, substantiate its utility for simultaneous IL treatment (for cellulose solubilisation) and saccharification of cellulose in a single step continuous process. This enzyme therefore holds merit for the development of efficient biomass hydrolysis process provided the production of the enzyme is substantially enhanced using the synthetic and systems biology approach.

Acknowledgements The financial support received from CSIR, New Delhi (Biomass to chemicals) is gratefully acknowledged. Mr. Nitin Trivedi and Mr. Vishal Gupta gratefully acknowledge the CSIR for the award of Senior Research Fellowships.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2013. 01.040.

References Adsul, M.G., Terwadkar, A.P., Varma, A.J., Gokhale, D.V., 2009. Cellulases from Penicillium janthinellum mutants: solid-state production and their stability in ionic liquids. Bioresour. Technol. 4, 1670–1681. Assareh, R., Zahiri, S.H., Noghabi, K.A., Aminzadeh, S., Khaniki, G.B., 2012. Characterization of the newly isolated Geobacillus sp. T1, the efficient cellulase-producer on untreated barley and wheat straws. Bioresour. Technol. 120, 99–105. Bose, S., Armstrong, D.W., Petrich, J.W., 2010. Enzyme-catalyzed hydrolysis of cellulose in ionic liquids: a Green approach toward the production of biofuels. J. Phys. Chem. B 114, 8221–8227. Bradford, M.M., 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Constatinescu, C., Herrmann, C., Weingartner, H., 2010. Patterns of protein unfolding and protein aggregation in ionic liquids. Phys. Chem. Chem. Phys. 12, 1756. Dadi, A.P., Varanasi, S., Schall, C.A., 2006. Enhancement of cellulose saccharification kinetics using an ionic liquid pretreatment step. Biotechnol. Bioeng. 95, 904– 910. Datta, S., Holmes, B., Park, J.I., Chen, Z.W., Dibble, D.C., Hadi, M., Blanch, H.W., Simmons, B.A., Sapra, R., 2010. Ionic liquid tolerant hyperthermophilic cellulases for biomass pretreatment and hydrolysis. Green Chem. 12, 338–345. Dotsenko, G.S., Sinitsyna, O.A., Hinz, S.W.A., Wery, J., Sinitsyn, A.P., 2012. Characterization of a GH family 3 b-glycoside hydrolase from Chrysosporium lucknowense and its application to the hydrolysis of b-glucan and xylan. Bioresour. Technol. 112, 345–349. Engel, P., Mladenov, R., Wulfhorst, H., Jäger, Spiess, A.C., 2010. Point by point analysis: how ionic liquid affects the enzymatic hydrolysis of native and modified cellulose. Green Chem. 12, 1959–1966. Freire, M.G., Teles, A.R.R., Rocha, M.M.A., Schroder, B., Neves, C.M.S.S., Carvalho, P.J., Evtuguin, D.V., Santos, L.M.N.B.F., Coutinho, J.A.P., 2011. Thermophysical characterization of ionic liquids able to dissolve biomass. J. Chem. Eng. Data 56, 4813–4822. Fukuchi, S., Yoshimune, K., Wakayama, M., Moriguchi, M., Nishikawa, K., 2003. Unique amino acid composition of proteins in halophilic bacteria. J. Mol. Biol. 327, 347–357. Ilmberger, N., Meske, D., Juergensen, J., Schulte, Barthen, P., Rabausch, U., Angelov, A., Mientus, M., Liebl, W., Schmitz, R.A., Streit, W.R., 2012. Metagenomic cellulases highly tolerant towards the presence of ionic liquids—linking thermostability and halotolerance. Appl. Microbiol. Biotechnol. 95, 135–146. Kasana, R.C., Salwan, R., Dhar, H., Dutt, S., Gulati, A., 2008. A rapid and easy method for the detection of microbial cellulase on agar plate using Gram’s iodine. Curr. Microbiol. 57, 503–507. Klemm, D., Heublein, B., Fink, H.P., Bohn, A., 2005. Cellulose: fascinating biopolymer and sustainable raw material. Angew. Chem. Int. Ed. 44, 3358–3393. Li, X., Yu, H.Y., 2012. Purification and characterization of an organic-solvent-tolerant cellulase from a halotolerant isolate, Bacillus sp. L1. J. Ind. Microbiol. Biotechnol. 39, 1117–1124. Liang, C., Xue, Y., Fioroni, M., Rodríguez-Ropero, F., Zhou, C., Schwaneberg, U., Ma, Y., 2011. Cloning and characterization of a thermostable and halo-tolerant endoglucanase from Thermoanaerobacter tengcongensis MB4. Appl. Microbiol. Biotechnol. 89, 315–326. Lin, L., Kan, X., Yan, H., Wang, D., 2012. Characterization of extracellular cellulosedegrading enzymes from Bacillus thuringiensis strains. Electron. J. Biotechnol.. http://dx.doi.org/10.2225/vol15-issue3-fulltext-1. Liu, D., Zhang, R., Yang, X., Zhang, Z., Song, S., Miao, Y., Shen, Q., 2012. Characterization of a thermostable b-glucosidase from Aspergillus fumigates Z5 and its functional expression in Pichia pastoris X33. Microb. Cell Factor 11, 1–15. Liu, Z., Wang, H., Li, Z., Lu, X., Zhang, X., Zhang, S., Zhou, K., 2011. Characterization of the regenerated cellulose films in ionic liquids and rheological properties of the solutions. Mater. Chem. Phys. 128, 220–227. Lozano, P., Bernal, B., Bernal, B.M., Pucheault, M., Vaultier, M., 2011. Stabilizing immobilized cellulase by ionic liquids for saccharification of cellulose solutions in 1-butyl-3-methylimidazolium chloride. Green Chem. 13, 1406–1410.

N. Trivedi et al. / Bioresource Technology 132 (2013) 313–319 Mcclendon, S.D., Baath, T., Petzold, C.J., Adams, P.D., Simmons, B.A., Singer, S.W., 2012. Thermoascus aurantiacus is a promising source of enzymes for biomass deconstruction under thermophilic conditions. Biotechnol. Biofuels 5, 1–9. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426–428. Moniruzzaman, M., Kamiya, N., Goto, M., 2010. Activation and stabilization of enzymes in ionic liquids. Org. Biomol. Chem. 8, 2887–2899. Paul, S., Bag, S.K., Das, S., Harvill, E.T., Dutta, C., 2008. Molecular signature of hypersaline adaptation: insights from genome and proteome composition of halophilic prokaryotes. Genom. Biol. 9, R70. Pottkamper, J., Barthen, P., Ilmberger, N., Schwaneberg, U., Schenk, A., Schulte, M., Ignatiev, N., Streit, W.R., 2009. Applying metagenomics for the identification of bacterial cellulases that are stable in ionic liquids. Green Chem. 11, 957–965. Sasaki, M., Kabyemela, B., Malaluan, R., Hirose, S., Takeda, N., Adschiri, T., 1998. Cellulose hydrolysis in subcritical and supercritical water. J. Supercrit. Fluids 13, 261–268. Singer, S.W., Reddy, A.P., Gladden, J.M., Guo, H., Hazen, T.C., Simmons, B.A., Vanderqhenyst, J.S., 2011. Enrichment, isolation and characterization of fungi tolerant to 1-ethyl-3-methylimidazolium acetate. J. Appl. Microbiol. 110, 1023– 1031.

319

Tadeo, X., Mendez, B.L., Castano, D., Trigueros, T., Millet, O., 2009. Protein stabilization and the Hofmeister effect: the role of hydrophobic solvation. Biophys. J. 97, 2595–2603. Trivedi, N., Gupta, V., Kumar, M., Kumari, P., Reddy, C.R.K., Jha, B., 2011. An alkalihalo tolerant cellulase from Bacillus flexus isolated from green seaweed Ulva lactuca. Carbohydr. Polym. 83, 891–897. Turner, M.B., Spear, S.K., Huddleston, J.G., Holbrey, J.D., Rogers, R.D., 2003. Ionic liquid salt-induced inactivation and unfolding of cellulase from Trichoderma reesei. Green Chem. 5, 443–444. Voget, S., Steele, H., Streit, W.R., 2006. Characterization of a metagenome-derived halotolerant cellulase. J. Biotechnol. 126, 26–36. Wang, Y., Radosevich, M., Hayes, D., Labbe, N., 2011. Compatible ionic liquidcellulases system for hydrolysis of lignocellulosic biomass. Biotechnol. Bioeng. 108, 1042–1048. Zhang, T., Datta, S., Eichler, J., Lvanova, N., Axen, S.D., Kerfeld, C.A., Chen, F., Kyrpides, N., Hugenholtz, P., Cheng, J.F., Sale, K.L., Simmons, B., Rubin, E., 2011. Identification of a haloalkaliphilic and thermostable cellulase with improved ionic liquid tolerance. Green Chem. 13, 2083–2090.