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Scalable production, fast purification, and spray drying of native Pycnoporus laccase and circular dichroism characterization
Journal of Cleaner Production xxx (2016) 1e10
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Scalable production, fast purification, and spray drying of native Pycnoporus laccase and circular dichroism characterization Jiayang Liu a, *, Zhonghua Yu a, Xiangru Liao b, Junhe Liu a, Feijun Mao c, Qingguo Huang d, ** a
Fermentation Technology Division, School of Bioengineering, Huanghuai University, Zhumadian 463000, China The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China Wuxi Jinkun Bio-technology Co., Ltd., Wuxi 214131, China d Department of Crop and Soil Sciences, University of Georgia, Griffin, GA30223, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 July 2015 Received in revised form 12 March 2016 Accepted 28 March 2016 Available online xxx
Laccase has been emerged as a valuable green biocatalyst in cleaner production, which requires vast amount of laccase at low production cost and with good properties. The current work evaluated a wild strain Pycnoporus sp. SYBC-L3 as an efficient microbial cell factory for laccase production. High-cell density cultivation (HCDC) of L3 was developed in a 5-L bioreactor by adjusting the ratio of inoculum to medium, achieving over 4-fold of both laccase activity (240 U/mL) and fungal biomass (20 g/L) than those obtained under routine procedures. Laccase productivity was further assessed at pilot-scale in a 500-L and a 5-ton bioreactor, where the highest activity of around 60 U/mL and 80 U/mL were produced, respectively. Cost percentage analysis for the 5-ton reactor demonstrated that labor was the highest portion (50%) in total cost for industrial production in this case. Using a one-step purification scheme, laccase was purified to homogeneity from the culture broth with yield of 44% and 97% purity. Circular dichroism (CD) analyses suggested that the secondary structure of laccase was mainly composed of bsheet that played a vital role in maintaining laccase activity, and also revealed the potential mechanisms for the changes in laccase activities under varying conditions. Powdered laccase was efficiently prepared from cell-free culture broth using spray drying technique with above 74% activity retained. This study provides important information for an efficient and scalable production of laccase. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Scalability Laccase Purification High-cell density cultivation Circular dichroism Powdered enzyme
1. Introduction Many eco-friendly enzymes have been used in industries to promote cleaner production via enzymatic processes (Boruah et al., sz et al., 2015; Skoronski et al., 2016). Laccase (EC 2016; Madara 1.10.3.2) is a very useful green catalyst for industrial, environmental, and medical applications due to its diverse capability in converting a wide range of substrates through catabolic or anabolic reactions (Jeon and Chang, 2013). As a green catalytic reagent, laccase has been increasingly employed in cleaner production, covering biofuel production, textile processing, biodegradation, biografting (Liu et al., 2015; Maryan et al., 2015; Spina et al., 2015; Thakur et al., 2016). As such, a rapid growth in the demand of
* Corresponding author. Tel.: þ86 18839648638. ** Corresponding author. Tel.: þ1 770 229 3302; fax: þ1 770 412 4734. E-mail addresses: [email protected] (J. Liu), [email protected] (Q. Huang).
laccase is expected worldwide, and the gap between production and demand will enlarge. It is thus of practical significance to seek microbes as potent cell factories to produce laccase with high yield at low cost. The major obstacle of broad application is the presumed low productivity by target organisms. Some strategies have been attempted to improve laccase productivity, including medium and process optimization, inducer supplementation, and ultrasonic treatment (Jing and Wang, 2012; Wang et al., 2012). High cell density cultivation (HCDC) has emerged as a research interest among fermentation industries, which can shorten production cycle and thus improve production efficiency (Lee, 1996). By adjusting parameters, such as dissolved oxygen (Matsui et al., 2006), fedbatch (Babaeipour et al., 2007), hyper-production of target products via HCDC can be achieved. There has not been a report on HCDC production of laccase by fungus to the best of our knowledge. In our previous experiments as well as in other reports, fungal biomass was normally limited to a certain level under routine
http://dx.doi.org/10.1016/j.jclepro.2016.03.154 0959-6526/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Liu, J., et al., Scalable production, fast purification, and spray drying of native Pycnoporus laccase and circular dichroism characterization, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.03.154
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J. Liu et al. / Journal of Cleaner Production xxx (2016) 1e10
cultivation procedure, for example 6 g/L in our previous study (Liu et al., 2013), thereby limiting hyper-production of laccase. HCDC was therefore designed for the first time in this study by means of adjusting seed cultivation and inoculation to explore the possibility of robust laccase production. Scalability of laccase production is one major factor that has to be addressed for its industrial applications (Couto and TocaHerrera, 2007). The cost of laccase production in industrial bioreactors can be reduced by several folds in comparison to that at bench scale (Osma et al., 2011). Previous studies reported laccase production with varied efficiency at different scales, most of which were below 50 L (Couto and Toca-Herrera, 2007). Although a multitude of higher fungi have been proven to be able to produce laccase, specific fungus that is propitious to scaling up at desirable level is relatively few. Among various ligninolytic fungi, the genus Pycnoporus attracted research interest for its overproduction of high redox potential laccase and as a strong contender for green biotechnology (Lomascolo et al., 2011). We in our previous study have achieved efficient laccase production by Pycnoporus in a 65 L air-lift reactor (Liu et al., 2013), yet evaluation of laccase production in larger bioreactors, such as in the 500-L or 5-ton reactors in this study, is still of great significance. As far as we know, description of laccase production at such scales has not been reported to date. As an important spectroscopic technology, circular dichroism (CD) analysis has been extensively used in characterizing protein structures (Schneider et al., 1999). The near-UV CD spectrum mainly reflects the tertiary structure while far-UV CD spectrum can provide information on the secondary structure of a protein (Greenfield and Fasman, 1969). The properties of laccase produced by Pycnoporus sp. SYBC-L3 has been previously studied in both crude and purified forms, which exhibited excellent thermostability, acidic pH optima, and metal tolerance (Liu et al., 2012, 2013). It is interesting to explore the underlying mechanisms for these properties in terms of its secondary structures, which have however not been studied before. Therefore a systematic characterization of the Pycnoporus laccase under various conditions, e.g., temperature, pH, and some heavy metal ions, was performed using far-UV CD spectral analysis in this study. The specific goals of this study were: 1) to develop a HCDC strategy for fungal cultivation to enhance laccase production via fermentation in bioreactors at different scales, 2) to purify laccase from the culture broth with an one-step scheme and explain the catalytic properties of laccase by CD spectral analysis, and 3) to prepare powdered laccase using a spray-drying technique. The results will provide experimental data for industrial production and application of laccases.
enzyme sample was measured using a SBA-40E Biosensing analyzer (Shandong Academy of Sciences, Jinan, China). Each experiment was performed in triplicate and the data were used to calculate the mean ± standard deviation (SD). 2.2. Fermentation in 5-L STR under a routine or HCDC operation To perform laccase production under a routine operation, the fungus was firstly cultured on potato dextrose agar (PDA) plate at 30 C for 5 days and subsequently inoculated into growth medium containing 5% glucose, 1% yeast extract, and 0.5% peptone to gain a seed culture under 200 rpm and 30 C. After 48 h of incubation, 600 mL of the inoculum was transferred into a 5-L STR bioreactor (Baoxing Bioengineering Equipment Co., Ltd, Shanghai, China) for laccase production in a 3-L optimized production medium as previously described (Liu et al., 2013). The two major process parameters, pH and dissolved oxygen (DO), were online monitored and recorded by a pH and a DO probe. The culture broth was periodically sampled every 12 h followed by centrifugation to separate mycelia and crude enzyme for subsequent analysis. The fermentation parameters were set as follows: 30 C, agitation rate 200 rpm, constant aeration rate 3 L/min. The cultured fungus in bioreactor for 3 days was taken out for morphological observation using scanning electron microscope (SEM). As for HCDC operation, a minor modification was made on a basis of the routine procedure, shown in Fig. 1. Specifically, a 3 L of seed culture was obtained by growing the fungus in the 5-L STR bioreactor (phase I) and then 1 L of optimized medium was added in the seed culture which was further cultivated for 7 days (phase II), while all other conditions were maintained the same as above mentioned in the routine operation. 2.3. Fermentation at pilot-scale in a 500-L or 5-ton bioreactor In order to produce laccase in 500-L STR, a secondary seed culture was prepared in a 50-L bioreactor (working volume 35 L) for 2 days under conditions: agitation 200 rpm, temperature 30 C, constant aeration rate 10 L/min, constant reactor pressure 0.05 Mpa. The seed culture was then transferred in a 300 L of production medium (glucose was replaced with the same gram of maltose) in a 500-L reactor for laccase production under the following conditions: agitation 200 rpm, temperature 30 C, constant aeration rate 6 m3/h (100 L/min), constant reactor pressure 0.05 Mpa. A volume of 30 mL antifoaming agent was added into the
2. Materials and methods 2.1. Chemicals, fungus, and assays Laccase substrate 2,6-Dimethoxyphenol (DMP) was obtained from Sigma (St. Louis, MO), and other chemicals of analytical-grade were purchased from Sinopharm Chemical Reagent Co., Ltd. Wild fungus, Pycnoporus sp. SYBC-L3, was a stock culture for laccase production in our laboratory (Liu et al., 2013). Laccase activity was determined by monitoring the increase in absorption at 469 nm in 100 mM phosphate-citrate buffer (pH 3.5) with DMP (ε ¼ 49.5 mM1cm1) as substrate, where one unit of laccase activity was defined as the amount of enzyme required to oxidize 1 mol of DMP per min (Litthauer et al., 2007). Protein content was determined using bovine serum albumin (BSA) as standard according to the method of Bradford (1976). The vacuum filtered or centrifuged mycelia were oven dried at 80 C to constant weight for fungal biomass determination. Glucose concentration in crude
Fig. 1. Schematics for routine submerged fermentation (A) and HCDC (B) for laccase production in 5-L STR.
Please cite this article in press as: Liu, J., et al., Scalable production, fast purification, and spray drying of native Pycnoporus laccase and circular dichroism characterization, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.03.154
J. Liu et al. / Journal of Cleaner Production xxx (2016) 1e10
production medium prior to steam sterilization (121 C, 30 min). Fermentation parameters, such as pH, temperature, agitation, aeration, and dissolved oxygen were controlled and/or recorded on-site during seed cultivation, while laccase activity, fungal biomass, and residual sugar was determined by taking 20 mL aliquot of culture medium from the reactor every day. Content of residual sugar was determined using dinitrosalycyclic acid (DNS) method with glucose as standard (Ghose, 1987). After each sampling, the volume of medium was not adjusted to its original volume in each batch run. In order to scale up laccase production in a 5-ton STR, a secondary seed culture was prepared likewise but in a 2-ton bioreactor (working volume 1 ton) for 54 h under conditions: agitation 280 rpm, temperature 30 C, constant aeration rate 0.4 vvm, constant reactor pressure 0.05 Mpa. The seed culture was then transferred in 2.5 ton production medium (glucose was replaced with the same gram of maltose) in a 5-ton reactor for laccase production under the following conditions: agitation 260 rpm, temperature 30 C, constant aeration rate 0.38 vvm, constant reactor pressure 0.05 Mpa. A volume of 300 mL antifoaming agent was added into the production medium prior to steam sterilization (121 C, 30 min). Laccase production cost was analyzed based on the actual consumption in this batch operation, with all five major components included: feedstock, electricity, steam, manpower and equipment depreciation. The cost of each component to carry through the entire operation was calculated. The costs of all components were the actual local prices, and the labor cost was estimated by the total manpower input and the actual salary paid to each worker. 2.4. Purification, gel electrophoresis, and capillary electrophoresis For laccase purification, culture broth was collected after 6 days of cultivation and centrifuged at 8000 g for 10 min to remove fungal biomass and then the supernatant was filtered through a 0.45 mm cellulose nitrate membrane by a vacuum pump. Afterward, the filtrate was loaded on a DEAE 52 anion exchange chromatography column pre-equilibrated with citric-phosphate buffer (pH 6.0, 20 mM) and then eluted with a linear gradient (0e1 M NaCl in 500 mL buffer). The flow rate was set at 2 mL/min and fractions of every 10 mL were pooled in tubes for the subsequent laccase activity assay and electrophoresis analysis. Eluate in each tube was further subjected to native polyacrylamide gel electrophoresis (Native-PAGE) for active staining with 1 mM DMP as substrate or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) for homogeneity verification with 0.2% Coomassie brilliant blue G-250. Purity of laccase was also verified by capillary electrophoresis using a P/ACE 5000 system (Beckman Instruments, Fullerton, CA, USA) equipped with a UV detector (Bornscheuer et al., 1994). 2.5. UVevis and CD spectra analysis of laccase A spectrophotometer (U-3010, HITACHI, Japan) was employed to record UVevis spectra (190e700 nm) of purified laccase. As for CD spectra, the elutions from DEAE 52 with laccase activity in peak A were pooled and powdered with a freeze-dryer (Labconco, USA). Then the powdered laccase was dissolved in 10 mM sodium phosphate buffer (pH 6.0) to prepare concentrated laccase which was further dialyzed against deionized water for 48 h. The dialyzate was used as control sample (correcting the baseline of the spectrum) and the reserved pure laccase (protein content of 0.02 mg/ mL) was scanned for CD spectra using a MOS-450 circular dichroism spectropolarimeter (Bio-Logic, France). The far-UV CD spectra of purified laccase were determined from wavelength
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190e250 nm at 25 C. The scan speed was set at 60 nm/min with a response of 1 s and band width at 1 nm adapted from Salony et al. (2008). The effect of temperature on CD spectra of laccase was measured in a quartz cuvette of 1 cm path length, while the effect of pH and metal ions on CD spectra was measured in a quartz cuvette of 0.1 cm path length. Every experiment was carried out in duplicate and the average value was used for calculation of secondary structure using the software K2d (Salony et al., 2008). 2.6. Powder laccase preparation by spray drying The culture broth that was collected from selected production procedures was firstly filtered through 8-layer gauze to remove fungal biomass and then subjected to spray drier (Uniform Drying Co., Ltd.,Changzhou, China) without further concentration. Two batches were separately carried out with the following conditions: inlet temperature 200 C, outlet temperature 90 C, flow rate of 1 L/ h or 2 L/h. After spray drying, the powdered laccase was collected from the collecting vessel and the inner wall of the pipeline. The activity of liquid or powdered laccase was determined respectively to calculate the activity recovery. 3. Results and discussion 3.1. Laccase production in 5-L bench-scale STR under routine operation As shown in Fig. 2 for laccase production in 5-L STR, pH value of culture media decreased slightly from its initial point (4.5) to the lowest number (3.8) in the first 2 days and then gradually rose up to 6.5 during the following cultivation period. This trend is in accordance with that in our previous report in an air-lift reactor (Liu et al., 2013) and also with that by Stereum ostrea and Phanerochaete chrysosporium for laccase production (Praveen et al., 2011), but slightly different from that by Coriolus versicolor, which continuously dropped to pH 3 from the initial pH 6 during the entire fermentation course (Que et al., 2014). Dissolved oxygen (DO) can greatly impact the growth of free cell and subsequent secretion of extracellular laccase. We in the present study adopted a constant ventilation rate which resulted in variable DO level (Fig. 2A), while some studies chose adjusted ventilation approach to maintain constant DO level (e.g. 30%) throughout the fermentation process (Colao et al., 2006; Que et al., 2014). Fungal biomass and laccase reached the highest value of approximately 3.5 mg/mL on day 2 and 60 U/mL on day 7, respectively (Fig. 2B&C), which were comparable with that obtained (6 mg/mL on day 3 and 72 U/mL on day 6) in an air-lift reactor (Liu et al., 2013). Que et al. (2014) also performed fungal cultivation in a 5-L bioreactor and produced the highest fungal biomass of 30 mg/mL on day 7 and laccase activity of 7.5 U/mL on day 5, respectively. Along with the rapid fungal growth, glucose content dramatically decreased (Fig. 2B). Extracellular laccase accumulated gradually only in the late phase of cultivation presumably because of the release of intracellular laccase caused by cell lysis (Bose et al., 2007). Protein content versus time course kept a similar pace with laccase production, but was produced in low quantities with its maximum of 0.24 mg/mL at day 6 or 7. Recombinant laccase was expressed and produced in Aspergillus niger, bringing about the highest activity of 42 U/mL and a specific activity of 50 U/mg protein (Mekmouche et al., 2014), which were still lower than those in our study (60 U/mL and 240 U/mg, respectively). As seen in the pictures shown in Fig. 3A & B, growth of Pycnoporus sp. SYBC-L3 in bioreactor tended to form trophosome in the form of mycelial pellets (F z 2 mm) during liquid cultivation under agitation, as observed in other fungus such as C. versicolor and
Please cite this article in press as: Liu, J., et al., Scalable production, fast purification, and spray drying of native Pycnoporus laccase and circular dichroism characterization, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.03.154
J. Liu et al. / Journal of Cleaner Production xxx (2016) 1e10
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speed (150 rpm) and inoculum (105 spore/mL) can give rise to the highest enzyme production with pellet size of 2.78 mm, which was similar to our observation (pellet size 1e2 mm)
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Tetraploa aristata (Que et al., 2014; Saparrat et al., 2002). The fungus actually exhibited different morphological characteristics at different stages during its growth and development, i.e., arthrospores were produced by means of fragmentation in later phase (Fig. 3C) under static cultivation on PDA plate, while stirred liquid cultivation hardly generated single spores during the entire cultivation (Fig. 3D). Obviously, the morphological form of hypha can exert significant effect on metabolite accumulation, like laccase and nez-Tobon et al. (1997) exopolysaccharides (Que et al., 2014). Jime studied the effect of inoculum and rotate speed on both pellet size and MnP production and found that higher inoculum can result in smaller pellet but higher enzyme activity, and appropriate rotate
HCDC strategy was designed for Pycnoporus sp. SYBC-L3 (Fig. 1) and the result of laccase production was illustrated in Fig. 4. A 6fold increase in the highest fungal biomass (20 g/L on day 5) was observed under HCDC but requiring 3 more days than the routine operation (Fig. 3B). After a two-phase manipulation, the highest laccase production (240 U/mL) was achieved on day 8, which is about 4-fold of that under routine procedure and comparable to that by Trametes (280 U/mL) within 6 days, the highest that have been reported at flask scale to our knowledge (Ling et al., 2015). To overcome limitations that hamper applicable exploitation due to low enzyme production from native hosts, heterologous expression in some efficient hosts is commonly employed for anticipated hyper-production. Colao et al. (2006) heterologously expressed Trametes trogii laccase gene in Pichia pastoris with a protein yield of 0.017 mg/mL and an activity of 2.52 U/mL after 8-d fermentation, which were far below than those in our result, i.e., 1 mg/mL and 240 U/mL, respectively. It is worth noting that the maximum laccase activity under HCDC could double to over 500 U/mL if the preferable substrate ABTS, instead of DMP, was adopted for activity assay (Liu et al., 2012). 3.3. Laccase production in pilot-scale 500-L STR For fungal fermentation in 500-L STR for laccase, the first-order seed culture from flask was inoculated and amplified in 50-L STR for the second-order seed culture, and the growth behaviors of three separate batches were described in Fig. 5. It can be seen that rapid fungal biomass build-up took place in 2-d cultivation. The pH value of growth medium experienced slight fluctuation but was in
Please cite this article in press as: Liu, J., et al., Scalable production, fast purification, and spray drying of native Pycnoporus laccase and circular dichroism characterization, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.03.154
J. Liu et al. / Journal of Cleaner Production xxx (2016) 1e10
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reached 60% on day 4, which was higher than that (40%) in 5-L reactor (Fig. 1A) because of enhanced ventilation in 500-L STR. The maximum biomass was approximately 10 g/L on day 3, about 3-fold of that (3.5 g/L) in 5-L STR. An interesting phenomenon that the residual sugar content (determined as reducing sugar) increased rather than decreased was observed in the first 3-day (Fig. 6D), possibly owing to increased glucose molecule derived from maltose hydrolysis by fungal extracellular hydrolytic enzymes. Up scaling from 5-L to 500-L STR, the laccase production maintained to the similar level of about 60 U/mL (Fig. 6E). A robust daily output of laccase achieved on day 5e7, close to 12 U/mL/ d (Fig. 6F). During the 8-d cultivation, about 50 L of culture medium was lost from the starting volume of 300 L as a result of evaporation, suggesting evaporation rate of around 8 L per day.
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Cultivation time (h) Fig. 4. Fermentation behavior of Pycnoporus sp. SYBC-L3 in 5-L STR under HCDC: pH and DO (A), biomass and sugar consumption (B), laccase activity and protein content (C). phase I: seed cultivation, phase II: laccase production.
the range of 4.5e6.0 (Fig. 5A). Steep decline in DO from 100% to almost zero indicated the surge in biomass amount (Fig. 5B). The second-order seed culture propagated slowly in the first 10 h and then dramatically increased in the following 10 h and finally leveled off at 8e10 g/L after 20 h cultivation (Fig. 5C). As for up-scaled production in 500-L STR (Fig. 6), the pH value, DO, biomass, laccase activity versus time course followed similar trends to those in 5 L STR (Fig. 2). The pH value of culture broth remained relatively stable (4e5) throughout the cultivation for all data points recorded, while malfunction of the pH and DO electrodes occurred in the first batch (indicated as black square in Fig. 6A and 6B). The lowest DO
Laccase productivity was further evaluated in a 5-ton STR, in which four major process parameters, namely laccase, sugar, pH, and biomass, were investigated. As seen in Fig. 7A, the four variables followed similar trends in general as those in 5-L or 500-L STR (Figs. 2 and 6). A notable difference was the delayed maximal laccase activity (83 U/mL) on day 9, which might be due to the fact that the first-order fungal seed was directly inoculated in the 5-ton reactor production, instead of second-order seeding. Laccase accumulation in the late phase in 5-ton STR appeared to be more rapid relative to that in 5-L STR. Reducing sugar content dropped steadily from the starting point of 25 g/L until day 7, reaching less than 2.5 g/L and then maintained unchanged for the following 4 days. The residual reducing sugar in culture broth was supposed to be partly from the sugars in laccase for glycosylation. The fungus exhibited robust mycelial growth since the transferring of the firstorder seed culture, achieving the highest biomass of near 10 g/L on day 3, and then decreased over the remaining cultivation time. The pH value of culture broth underwent three phases: a dramatic drop from the initial pH 5.5 to pH 3.5 in the first day, maintaining pH 3.5 for the subsequent 4 days, and then rising up to the final value of about pH 7 after day 6. The cost percentage of this batch was roughly calculated on a basis of actual and predicted consumption of five essential constituents, shown in Fig. 7B. The highest percentage (50%) in the total cost was labor in the form of salary which might vary with different working district. Human power was mainly used to manage microorganisms, measuring procedure parameters, as well as operating the fermentation process. Electricity accounted for 36% of the total cost, the second contribution after labor. Depreciation of the equipment was about one-tenth of the total cost. Feedstock was not a major cost factor, accounting for less than 5% of the total cost. The lowest percentage was the consumption of steam, which was only 1%. The actual production cost was not revealed here in order to avoid possible conflict of commercial interest, however our estimated price for laccase by L3 in 65-L bioreactor was far below than those commercially available (Liu et al., 2013). By examining 26 different culture medium and their corresponding laccase production in flask under submerged fermentation, Osma et al. (2011) concluded that the culture medium, in all cases, was the largest component (90%) in the total cost, followed by the cost in operation (10%). In real pilot-scale fermentation, however, the situation may be greatly different depending on the current feedstock, power and labor costs, as discussed above. It is worth noting that the we believe the cost of laccase production can be further reduced by further enhancing laccase productivity using certain strategies at pilot-scale, such as HCDC, stage pH-control, and fed-batch method.
Please cite this article in press as: Liu, J., et al., Scalable production, fast purification, and spray drying of native Pycnoporus laccase and circular dichroism characterization, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.03.154
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J. Liu et al. / Journal of Cleaner Production xxx (2016) 1e10
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Fig. 5. Mycelial growth behavior of Pycnoporus sp. SYBC-L3 in 50-L STR for second order seed culture: batch 1 (black square), batch 2 (red circle), batch 3 (blue triangle). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 6. Fermentation behavior of Pycnoporus sp. SYBC-L3 in 500-L STR for laccase production: batch 1 (black square), batch 2 (red circle), batch 3 (blue triangle). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.5. Fast purification of laccase Purification of laccase from the culture broth using ionexchange chromatography was performed and the profiles are shown in Fig. 8. As the NaCl concentration increased linearly from 0 to 1 mol/L, two absorption peaks (OD 280) of B and C showed up in order, corresponding to tube 3 and 8, respectively. Laccase activity in each tube from 2 to 10 was examined by Native-PAGE (Fig. 8B), which suggested that the elutions in tube 2 and 3 had the highest activity. Elutions in tube 2 and 3 were also applied to SDS-PAGE (Fig. 8C) and a single band was observed for each tube, indicating a purified form of laccase was obtained. Elutions in tube 2 and 3 were then combined to calculate the laccase recovery from crude laccase, which was around 72%.
The purified laccase did not show the blue color by naked eye, the typical color for blue laccase with absorption peak at around 600 nm (Eggert et al., 1996). When subjected to UVevis spectrophotometer, a broad shoulder peak showed up at wavelength 300 nm for purified laccase combined from tube 2 and 3, which is very similar to some previous reports (Lu et al., 2007; Wang et al., 2010), indicating the presence of type III binuclear Cu (II) pair (Eggert et al., 1996). The purified laccase was also confirmed by capillary electrophoresis and the purity of laccase was found to be around 97.83% (major peak at 2.013 min), shown in Fig. 9. Normally, at least three successive steps were required to purify laccase from white-rot fungi: precipitation, ion exchange, and gel filtration or affinity chromatography (Litthauer et al., 2007; Lu et al., 2007; Wang et al., 2010), while only very few studies reported simpler
Please cite this article in press as: Liu, J., et al., Scalable production, fast purification, and spray drying of native Pycnoporus laccase and circular dichroism characterization, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.03.154
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Fig. 7. (A) Fermentation behavior of Pycnoporus sp. SYBC-L3 in 5-ton STR for laccase production: laccase activity (open circle), reducing sugar (solid square), pH value (open square), and fungal biomass (solid circle). (B) Cost percentage analysis of laccase production performed in 5-ton STR. Total cost (100%) of the batch was the sum of five essential parts, namely feedstock (medium, H2O, and antifoam), electricity (bioreactor, compressor, and water pump), steam (sterilization and heating), labor (culture preservation room, inspection center, and production workshop), and depreciation (including equipment purchase fee).
purification procedures, such as by phenyl-Sepharose High Performance column (Garcia et al., 2006). Although recombinant proteins (e.g., laccase) have been thought to require simple purification procedures (Erjavec et al., 2012), published data revealed that several steps were still needed (Colao et al., 2006). Therefore, the fact the laccase produced from Pycnoporus sp. SYBC-L3 can be readily purified in a one-step column procedure is a great advantage, and this is probably because the laccase is usually excreted as a major extracellular enzyme by genus Pycnoporus (Liu et al., 2013; Lomascolo et al., 2011). 3.6. CD spectra of laccase The Far-UV CD spectra of the purified laccase are displayed in Fig. 10. The CD spectra showed one positive peak around 198 nm and one negative trough around 218 nm, indicating the typical signature for a-helix and b-sheet structure, respectively (Salony et al., 2008; Schneider et al., 1999). Similarly, Bukh and Bjerrum (2010) reported that Coprinus cinereus laccase showed CD spectra with maximum absorption at 199 nm and minimum at 218 nm. The content of secondary structure calculated from the experimental CD values in the purified laccase is shown in Table 1. The laccase consisted of approximately 3% a-helix, 50% b-sheet, and 47% random coil, which is similar to that in C. cinereus laccase (6% ahelix, 40% b-sheet, and 54% random coil) (Bukh and Bjerrum, 2010). In contrast, fungal laccase with higher a-helix content (68%), e.g. C. versicolor laccase, has been documented as well (Que et al., 2014). The potential mechanism of thermostability and inhibition of some metal ions on laccase was explored using CD analysis under different conditions. As shown in Table 1, no significant change in the secondary structure was observed with temperature increasing from 5 to 55 C. However, when temperature was above 75 C, the
Fig. 8. (A) Purification of laccase on DEAE-sepharose 52. Abs 280 (line with triangle), laccase activity (line with circle), NaCl concentration (line). (B) Native-PAGE of laccase collected in corresponding tube from DEAE column. Laccase activity was stained with DMP in phosphorus buffer. The lane number above each reddish band was for each tube number in (A). (C) SDS-PAGE of crude laccase and purified laccase. lane 1: molecular mass markers (94kD: TaqDNA Polymerase; 62kD: T4 DNA Ligase; 45kD: Albumin from chicken serum; 33kD: His-Tag fusion protein). lane 2 and lane 3: laccase in the tube number 2 and 3 from DEAE 52. Lane 4: crude laccase in culture broth. (D) Absorbance spectrum of purified laccase by UVevis spectrophotometer, where tube 2 and 3 were combined.
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J. Liu et al. / Journal of Cleaner Production xxx (2016) 1e10 Table 1 Predicted secondary structure of purified laccase under various conditions. Influencing factor
Alpha helix/%
Beta sheet/%
Random coil/%
Control 5 C 25 C 55 C 75 C AgNO3 (1 mM) AgNO3 (5 mM) AgNO3 (50 mM) FeSO4 (1 mM) FeSO4 (5 mM) FeSO4 (50 mM) FeCl3 (1 mM) FeCl3 (5 mM) FeCl3 (50 mM)
3 3 3 3 46 11 13 0 10 9 4 4 3 3
50 50 50 50 23 41 40 4 43 42 48 49 50 49
47 47 47 47 31 48 47 96 47 48 48 47 47 47
3.7. Preparation of powder laccase Fig. 9. Purity verification of laccase by capillary electrophoresis.
Laccase production was successfully achieved by high cell density cultivation at various scales. The highest laccase production was 80 U/mL in a 5-ton bioreactor, where the labor cost was found to be the highest among five cost components. An one-step purification scheme was proven easy and efficient. Circular dichroism revealed the secondary structure of and explained some properties
(B)
20 control FeSO4/1 mmol/L
10
2
pH3.0 pH4.0 pH5.5
2
10
pH2.5 pH3.5 pH4.5 pH6.5
4. Conclusion
-1
20
-1
Molar Ellipticity (deg.cm .dmol )
(A)
After liquid cultivation in bioreactor, supernatant without fungal biomass was subjected to spray drying to produce powdered laccase and the details are listed in Table 2. Laccase powder was successfully produced with high activity in both two batches with recovery as high as 74.6% and 82.2%, respectively. The prepared laccase powder is uniform and smooth, looked yellowish and smelled like peptone. It should be noted that the drying condition was our first trial, and the final yields may be further improved if the drying condition, such as inlet and outlet temperature and the flow rate, are optimized. There has been an argument that production of laccases in their native hosts is often not economically feasible and natural laccases may not withstand the harsh conditions processes (Erjavec et al., 2012). Our results in this study however clearly prove that certain wild fungus, like Pycnoporus sp. SYBC-L3, can serve as an excellent cell factory for laccase production with outstanding properties and economic viability.
Molar Ellipticity (deg.cm .dmol )
absorbance peak around 208 nm and 220 nm decreased (figure not shown), suggesting thermal denaturing (Table 1), resulting in the loss of activity. Likewise, de Wilde et al. (2008) confirmed less thermal stability of Melanocarpus albomyces laccase expressed in rice than in Trichoderma reesei using CD analysis with temperature in the range of 30e90 C. When exposed to various pH values at the same solute concentration (0.1 M), no evident change in the CD spectra was observed for all pH values below 6.5, even though both the purified and crude laccase lost most of its activity toward DMP when pH is above 4.5 (Liu et al., 2012, 2013). The spectra of laccase exposed to Fe2þ and Fe3þ (from 1 to 50 mM) did not show noticeable changes (Fig. 10B), which were also reflected in the relative content of secondary structure (Table 1). However, the presence of Agþ ranging from 5 to 50 mM remarkably altered CD spectra with messy curved lines emerged (figure not shown), suggesting a dramatic decrease of b-sheet content in laccase (Table 1). The analysis of CD spectral changes strongly indicated the different inhibition mechanisms of Fe2þ/Fe3þ and Agþ on laccase. Although CD analytical technique has been utilized for secondary structures of proteins (Allendorf et al., 1985; Salony et al., 2008), studying CD changes in response to varied conditions is relatively few. This study represents the first attempt to collect series of CD data for laccase exposed to different conditions, providing insight into the differences in enzymatic properties.
0
-10
-20 190 200 210 220 230 240 250 Wavelength (nm)
FeSO4/5 mmol/L FeSO4/50 mmol/L
0
-10
-20 190 200 210 220 230 240 250 Wavelength (nm)
Fig. 10. UV-CD spectrum of laccase affected by varying pH (A) and FeSO4 (B) at 25 C. Protein concentration was 0.5 mg/ml. CD spectrum was measured in a quartz cuvette of 0.1 cm path length and the value of ellipticity was converted to molar ellipticity.
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Table 2 Preparation of powdered laccase using spray-dryer. Description
Batch 1
Batch 2
Experiment site Drying condition Total volume of crude liquid laccase Total activity of crude liquid laccase Total weight of powdered laccase Total activity of powdered laccase Specific activity of powdered laccase Activity recovery
Engineering center at School of Food Science, Jiangnan University, Wuxi, China Inlet T 200 C, outlet T 90 C, flow rate 1 L/h 4L 288000 U 3.28 g 215000 U 65548.7 U/g 74.6%
Uniform drying company, Changzhou, China Inlet T 200 C, outlet T 90 C, flow rate 2.0 L/h 36 L 1946000 U 97.3 g 1600000 U 16443.9 U/g 82.2%
of the laccase, like thermostability. A spray-drying technique was employed to prepare powdered laccase with over 74% activity recovered. By investigating productivity, scalability, purification, property, and powder preparation as well as production cost, the laccase by wild Pycnoporus fungus exhibited a great potential for green and clean applications. Acknowledgments This study was supported in part by Scientific Research Foundation for Advanced Talents of Huanghuai University (1000.12.01.1342). We would like to thank Dr. Xiaolian Zhan (Wuxi Jinkun Biotechnology Co., Ltd.) for providing 500 L bioreactor and general manager Guobao Jiao (Henan Yangshao Bioproducts Co., Ltd.) for 5 ton bioreactor and engineer Huaqiang Xi for collecting data in cost analysis. We also thank Changzhou Uniform Drying Co., Ltd for generous help in preparation of powdered laccase. References Allendorf, M.D., Spira, D.J., Solomon, E.I., 1985. Low-temperature magnetic circular dichroism studies of native laccase: spectroscopic evidence for exogenous ligand bridging at a trinuclear copper active site. P. Natl. Acad. Sci. U. S. A. 82, 3063e3067. Babaeipour, V., Shojaosadati, S.A., Robatjazi, S.M., Khalilzadeh, R., Maghsoudi, N., 2007. Over-production of human interferon-g by HCDC of recombinant Escherichia coli. Proc. Biochem. 42, 112e117. Bornscheuer, U., Reif, O.-W., Lausch, R., Freitag, R., Scheper, T., Kolisis, F.N., Menge, U., 1994. Lipase of Pseudomonas cepacia for biotechnological purposes: purification, crystallization and characterization. BBA-Gen. Subjects 1201, 55e60. Boruah, P., Dowarah, P., Hazarika, R., Yadav, A., Barkakati, P., Goswami, T., 2016. Xylanase from Penicillium meleagrinum var. viridiflavum e a potential source for bamboo pulp bleaching. J. Clean. Prod. 116, 259e267. Bose, S., Mazumder, S., Mukherjee, M., 2007. Laccase production by the white-rot fungus Termitomyces clypeatus. J. Basic Microb. 47, 127e131. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248e254. Bukh, C., Bjerrum, M.J., 2010. The reversible depletion and reconstitution of a copper ion in Coprinus cinereus laccase followed by spectroscopic techniques. J. Inorg. Biochem. 104, 1029e1037. Colao, M., Lupino, S., Garzillo, A., Buonocore, V., Ruzzi, M., 2006. Heterologous expression of lcc1 gene from Trametes trogii in Pichia pastoris and characterization of the recombinant enzyme. Microb. Cell Fact. 5, 31. Couto, S.R., Toca-Herrera, J.L., 2007. Laccase production at reactor scale by filamentous fungi. Biotechnol. Adv. 25, 558e569. de Wilde, C., Uzan, E., Zhou, Z., Kruus, K., Andberg, M., Buchert, J., Record, E., Asther, M., Lomascolo, A., 2008. Transgenic rice as a novel production system for Melanocarpus and Pycnoporus laccases. Transgenic Res. 17, 515e527. Eggert, C., Temp, U., Eriksson, K., 1996. The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase. Appl. Environ. Microbiol. 62, 1151e1158. Erjavec, J., Kos, J., Ravnikar, M., Dreo, T., Saboti c, J., 2012. Proteins of higher fungi e from forest to application. Trends. Biotechnol. 30, 259e273. Garcia, T., Santiago, M., Ulhoa, C., 2006. Properties of laccases produced by Pycnoporus sanguineus induced by 2,5-xylidine. Biotechnol. Lett. 28, 633e636. Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257e268. Greenfield, N.J., Fasman, G.D., 1969. Computed circular dichroism spectra for the evaluation of protein conformation. Biochemistry 8, 4108e4116.
Jeon, J.-R., Chang, Y.-S., 2013. Laccase-mediated oxidation of small organics: bifunctional roles for versatile applications. Trends. Biotechnol. 31, 335e341. nez-Tobon, G.A., Penninckx, M.J., Lejeune, R., 1997. The relationship between Jime pellet size and production of Mn(II) peroxidase by Phanerochaete chrysosporium in submerged culture. Enzyme. Microb. Technol. 21, 537e542. Jing, D., Wang, J., 2012. Controlling the simultaneous production of laccase and lignin peroxidase from Streptomyces cinnamomensis by medium formulation. Biotechnol. Biofuels 5, 1e7. Lee, S.Y., 1996. High cell-density culture of Escherichia coli. Trends. Biotechnol. 14, 98e105. Ling, Z.-R., Wang, S.-S., Zhu, M.-J., Ning, Y.-J., Wang, S.-N., Li, B., Yang, A.-Z., Zhang, G.-Q., Zhao, X.-M., 2015. An extracellular laccase with potent dye decolorizing ability from white rot fungus Trametes sp. LAC-01. Int. J. Biol. Macromol. 81, 785e793. Litthauer, D., van Vuuren, M.J., van Tonder, A., Wolfaardt, F.W., 2007. Purification and kinetics of a thermostable laccase from Pycnoporus sanguineus (SCC 108). Enzyme. Microb. Technol. 40, 563e568. Liu, J., Cai, Y., Liao, X., Huang, Q., Hao, Z., Hu, M., Zhang, D., Li, Z., 2013. Efficiency of laccase production in a 65-liter air-lift reactor for potential green industrial and environmental application. J. Clean. Prod. 39, 154e160. Liu, J., Cai, Y., Liao, X., Huang, Q., Hao, Z., Zhang, D., 2012. Purification and Characterization of a novel thermal stable laccase from Pycnoporus sp. SYBC-L3 and its use in dye decolorization. Biol. Environ. 113, 1e13. Liu, J., Sidhu, S.S., Wang, M.L., Tonnis, B., Habteselassie, M., Mao, J., Huang, Q., 2015. Evaluation of various fungal pretreatment of switchgrass for enhanced saccharification and simultaneous enzyme production. J. Clean. Prod. 104, 480e488. €l-Gimbert, I., Sigoillot, J.-C., LesageLomascolo, A., Uzan-Boukhris, E., Herpoe Meessen, L., 2011. Peculiarities of Pycnoporus species for applications in biotechnology. Appl. Microbiol. Biotechnol. 92, 1129e1149. Lu, L., Zhao, M., Zhang, B.B., Yu, S.Y., Bian, X.J., Wang, W., Wang, Y., 2007. Purification and characterization of laccase from Pycnoporus sanguineus and decolorization of an anthraquinone dye by the enzyme. Appl. Microbiol. Biotechnol. 74, 1232e1239. sz, J., Ne meth, D., Bakos, J., Gubicza, L., Bakonyi, P., 2015. Solvent-free Madara enzymatic process for biolubricant production in continuous microfluidic reactor. J. Clean. Prod. 93, 140e144. Maryan, A.S., Montazer, M., Damerchely, R., 2015. Discoloration of denim garment with color free effluent using montmorillonite based nano clay and enzymes: nano bio-treatment on denim garment. J. Clean. Prod. 91, 208e215. Matsui, T., Shinzato, N., Yokota, H., Takahashi, J., Sato, S., 2006. High cell density cultivation of recombinant E. coli with a pressurized culture. Proc. Biochem. 41, 920e924. Mekmouche, Y., Zhou, S., Cusano, A.M., Record, E., Lomascolo, A., Robert, V., Simaan, A.J., Rousselot-Pailley, P., Ullah, S., Chaspoul, F., Tron, T., 2014. Gramscale production of a basidiomycetous laccase in Aspergillus niger. J. Biosci. Bioeng. 117, 25e27. Osma, J.F., Toca-Herrera, J.L., Rodríguez-Couto, S., 2011. Cost analysis in laccase production. J. Environ. Manage. 92, 2907e2912. Praveen, K., Viswanath, B., Usha, K.Y., Pallavi, H., Venkata Subba Reddy, G., Naveen, M., Rajasekhar Reddy, B., 2011. Lignolytic enzymes of a mushroom Stereum ostrea isolated from wood logs. Enzyme Res. 2011, 6. Que, Y., Sun, S., Xu, L., Zhang, Y., Zhu, H., 2014. High-level coproduction, purification and characterisation of laccase and exopolysaccharides by Coriolus versicolor. Food Chem. 159, 208e213. Salony, Garg, N., Baranwal, R., Chhabra, M., Mishra, S., Chaudhuri, T.K., Bisaria, V.S., 2008. Laccase of Cyathus bulleri: structural, catalytic characterization and expression in Escherichia coli. Biochimica Biophysica Acta 1784, 259e268. Saparrat, M.C.N., Cabello, M.N., Arambarri, A.M., 2002. Extracellular laccase activity in Tetraploa aristata. Biotechnol. Lett. 24, 1375e1377. Schneider, P., Caspersen, M.B., Mondorf, K., Halkier, T., Skov, L.K., Østergaard, P.R., Brown, K.M., Brown, S.H., Xu, F., 1999. Characterization of a Coprinus cinereus laccase. Enzyme. Microb. Technol. 25, 502e508. Skoronski, E., de Oliveira, D.C., Fernandes, M., da Silva, G.F., Magalh~ aes, M.d.L.B., Jo~ ao, J.J., 2016. Valorization of agro-industrial by-products: analysis of biodiesel production from porcine fat waste. J. Clean. Prod. 112 (Part 4), 2553e2559.
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Scalable production, fast purification, and spray drying of native Pycnoporus laccase and circular dichroism characterization Jiayang Liu a, *, Zhonghua Yu a, Xiangru Liao b, Junhe Liu a, Feijun Mao c, Qingguo Huang d, ** a
Fermentation Technology Division, School of Bioengineering, Huanghuai University, Zhumadian 463000, China The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China Wuxi Jinkun Bio-technology Co., Ltd., Wuxi 214131, China d Department of Crop and Soil Sciences, University of Georgia, Griffin, GA30223, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 July 2015 Received in revised form 12 March 2016 Accepted 28 March 2016 Available online xxx
Laccase has been emerged as a valuable green biocatalyst in cleaner production, which requires vast amount of laccase at low production cost and with good properties. The current work evaluated a wild strain Pycnoporus sp. SYBC-L3 as an efficient microbial cell factory for laccase production. High-cell density cultivation (HCDC) of L3 was developed in a 5-L bioreactor by adjusting the ratio of inoculum to medium, achieving over 4-fold of both laccase activity (240 U/mL) and fungal biomass (20 g/L) than those obtained under routine procedures. Laccase productivity was further assessed at pilot-scale in a 500-L and a 5-ton bioreactor, where the highest activity of around 60 U/mL and 80 U/mL were produced, respectively. Cost percentage analysis for the 5-ton reactor demonstrated that labor was the highest portion (50%) in total cost for industrial production in this case. Using a one-step purification scheme, laccase was purified to homogeneity from the culture broth with yield of 44% and 97% purity. Circular dichroism (CD) analyses suggested that the secondary structure of laccase was mainly composed of bsheet that played a vital role in maintaining laccase activity, and also revealed the potential mechanisms for the changes in laccase activities under varying conditions. Powdered laccase was efficiently prepared from cell-free culture broth using spray drying technique with above 74% activity retained. This study provides important information for an efficient and scalable production of laccase. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Scalability Laccase Purification High-cell density cultivation Circular dichroism Powdered enzyme
1. Introduction Many eco-friendly enzymes have been used in industries to promote cleaner production via enzymatic processes (Boruah et al., sz et al., 2015; Skoronski et al., 2016). Laccase (EC 2016; Madara 1.10.3.2) is a very useful green catalyst for industrial, environmental, and medical applications due to its diverse capability in converting a wide range of substrates through catabolic or anabolic reactions (Jeon and Chang, 2013). As a green catalytic reagent, laccase has been increasingly employed in cleaner production, covering biofuel production, textile processing, biodegradation, biografting (Liu et al., 2015; Maryan et al., 2015; Spina et al., 2015; Thakur et al., 2016). As such, a rapid growth in the demand of
* Corresponding author. Tel.: þ86 18839648638. ** Corresponding author. Tel.: þ1 770 229 3302; fax: þ1 770 412 4734. E-mail addresses: [email protected] (J. Liu), [email protected] (Q. Huang).
laccase is expected worldwide, and the gap between production and demand will enlarge. It is thus of practical significance to seek microbes as potent cell factories to produce laccase with high yield at low cost. The major obstacle of broad application is the presumed low productivity by target organisms. Some strategies have been attempted to improve laccase productivity, including medium and process optimization, inducer supplementation, and ultrasonic treatment (Jing and Wang, 2012; Wang et al., 2012). High cell density cultivation (HCDC) has emerged as a research interest among fermentation industries, which can shorten production cycle and thus improve production efficiency (Lee, 1996). By adjusting parameters, such as dissolved oxygen (Matsui et al., 2006), fedbatch (Babaeipour et al., 2007), hyper-production of target products via HCDC can be achieved. There has not been a report on HCDC production of laccase by fungus to the best of our knowledge. In our previous experiments as well as in other reports, fungal biomass was normally limited to a certain level under routine
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Please cite this article in press as: Liu, J., et al., Scalable production, fast purification, and spray drying of native Pycnoporus laccase and circular dichroism characterization, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.03.154
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cultivation procedure, for example 6 g/L in our previous study (Liu et al., 2013), thereby limiting hyper-production of laccase. HCDC was therefore designed for the first time in this study by means of adjusting seed cultivation and inoculation to explore the possibility of robust laccase production. Scalability of laccase production is one major factor that has to be addressed for its industrial applications (Couto and TocaHerrera, 2007). The cost of laccase production in industrial bioreactors can be reduced by several folds in comparison to that at bench scale (Osma et al., 2011). Previous studies reported laccase production with varied efficiency at different scales, most of which were below 50 L (Couto and Toca-Herrera, 2007). Although a multitude of higher fungi have been proven to be able to produce laccase, specific fungus that is propitious to scaling up at desirable level is relatively few. Among various ligninolytic fungi, the genus Pycnoporus attracted research interest for its overproduction of high redox potential laccase and as a strong contender for green biotechnology (Lomascolo et al., 2011). We in our previous study have achieved efficient laccase production by Pycnoporus in a 65 L air-lift reactor (Liu et al., 2013), yet evaluation of laccase production in larger bioreactors, such as in the 500-L or 5-ton reactors in this study, is still of great significance. As far as we know, description of laccase production at such scales has not been reported to date. As an important spectroscopic technology, circular dichroism (CD) analysis has been extensively used in characterizing protein structures (Schneider et al., 1999). The near-UV CD spectrum mainly reflects the tertiary structure while far-UV CD spectrum can provide information on the secondary structure of a protein (Greenfield and Fasman, 1969). The properties of laccase produced by Pycnoporus sp. SYBC-L3 has been previously studied in both crude and purified forms, which exhibited excellent thermostability, acidic pH optima, and metal tolerance (Liu et al., 2012, 2013). It is interesting to explore the underlying mechanisms for these properties in terms of its secondary structures, which have however not been studied before. Therefore a systematic characterization of the Pycnoporus laccase under various conditions, e.g., temperature, pH, and some heavy metal ions, was performed using far-UV CD spectral analysis in this study. The specific goals of this study were: 1) to develop a HCDC strategy for fungal cultivation to enhance laccase production via fermentation in bioreactors at different scales, 2) to purify laccase from the culture broth with an one-step scheme and explain the catalytic properties of laccase by CD spectral analysis, and 3) to prepare powdered laccase using a spray-drying technique. The results will provide experimental data for industrial production and application of laccases.
enzyme sample was measured using a SBA-40E Biosensing analyzer (Shandong Academy of Sciences, Jinan, China). Each experiment was performed in triplicate and the data were used to calculate the mean ± standard deviation (SD). 2.2. Fermentation in 5-L STR under a routine or HCDC operation To perform laccase production under a routine operation, the fungus was firstly cultured on potato dextrose agar (PDA) plate at 30 C for 5 days and subsequently inoculated into growth medium containing 5% glucose, 1% yeast extract, and 0.5% peptone to gain a seed culture under 200 rpm and 30 C. After 48 h of incubation, 600 mL of the inoculum was transferred into a 5-L STR bioreactor (Baoxing Bioengineering Equipment Co., Ltd, Shanghai, China) for laccase production in a 3-L optimized production medium as previously described (Liu et al., 2013). The two major process parameters, pH and dissolved oxygen (DO), were online monitored and recorded by a pH and a DO probe. The culture broth was periodically sampled every 12 h followed by centrifugation to separate mycelia and crude enzyme for subsequent analysis. The fermentation parameters were set as follows: 30 C, agitation rate 200 rpm, constant aeration rate 3 L/min. The cultured fungus in bioreactor for 3 days was taken out for morphological observation using scanning electron microscope (SEM). As for HCDC operation, a minor modification was made on a basis of the routine procedure, shown in Fig. 1. Specifically, a 3 L of seed culture was obtained by growing the fungus in the 5-L STR bioreactor (phase I) and then 1 L of optimized medium was added in the seed culture which was further cultivated for 7 days (phase II), while all other conditions were maintained the same as above mentioned in the routine operation. 2.3. Fermentation at pilot-scale in a 500-L or 5-ton bioreactor In order to produce laccase in 500-L STR, a secondary seed culture was prepared in a 50-L bioreactor (working volume 35 L) for 2 days under conditions: agitation 200 rpm, temperature 30 C, constant aeration rate 10 L/min, constant reactor pressure 0.05 Mpa. The seed culture was then transferred in a 300 L of production medium (glucose was replaced with the same gram of maltose) in a 500-L reactor for laccase production under the following conditions: agitation 200 rpm, temperature 30 C, constant aeration rate 6 m3/h (100 L/min), constant reactor pressure 0.05 Mpa. A volume of 30 mL antifoaming agent was added into the
2. Materials and methods 2.1. Chemicals, fungus, and assays Laccase substrate 2,6-Dimethoxyphenol (DMP) was obtained from Sigma (St. Louis, MO), and other chemicals of analytical-grade were purchased from Sinopharm Chemical Reagent Co., Ltd. Wild fungus, Pycnoporus sp. SYBC-L3, was a stock culture for laccase production in our laboratory (Liu et al., 2013). Laccase activity was determined by monitoring the increase in absorption at 469 nm in 100 mM phosphate-citrate buffer (pH 3.5) with DMP (ε ¼ 49.5 mM1cm1) as substrate, where one unit of laccase activity was defined as the amount of enzyme required to oxidize 1 mol of DMP per min (Litthauer et al., 2007). Protein content was determined using bovine serum albumin (BSA) as standard according to the method of Bradford (1976). The vacuum filtered or centrifuged mycelia were oven dried at 80 C to constant weight for fungal biomass determination. Glucose concentration in crude
Fig. 1. Schematics for routine submerged fermentation (A) and HCDC (B) for laccase production in 5-L STR.
Please cite this article in press as: Liu, J., et al., Scalable production, fast purification, and spray drying of native Pycnoporus laccase and circular dichroism characterization, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.03.154
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production medium prior to steam sterilization (121 C, 30 min). Fermentation parameters, such as pH, temperature, agitation, aeration, and dissolved oxygen were controlled and/or recorded on-site during seed cultivation, while laccase activity, fungal biomass, and residual sugar was determined by taking 20 mL aliquot of culture medium from the reactor every day. Content of residual sugar was determined using dinitrosalycyclic acid (DNS) method with glucose as standard (Ghose, 1987). After each sampling, the volume of medium was not adjusted to its original volume in each batch run. In order to scale up laccase production in a 5-ton STR, a secondary seed culture was prepared likewise but in a 2-ton bioreactor (working volume 1 ton) for 54 h under conditions: agitation 280 rpm, temperature 30 C, constant aeration rate 0.4 vvm, constant reactor pressure 0.05 Mpa. The seed culture was then transferred in 2.5 ton production medium (glucose was replaced with the same gram of maltose) in a 5-ton reactor for laccase production under the following conditions: agitation 260 rpm, temperature 30 C, constant aeration rate 0.38 vvm, constant reactor pressure 0.05 Mpa. A volume of 300 mL antifoaming agent was added into the production medium prior to steam sterilization (121 C, 30 min). Laccase production cost was analyzed based on the actual consumption in this batch operation, with all five major components included: feedstock, electricity, steam, manpower and equipment depreciation. The cost of each component to carry through the entire operation was calculated. The costs of all components were the actual local prices, and the labor cost was estimated by the total manpower input and the actual salary paid to each worker. 2.4. Purification, gel electrophoresis, and capillary electrophoresis For laccase purification, culture broth was collected after 6 days of cultivation and centrifuged at 8000 g for 10 min to remove fungal biomass and then the supernatant was filtered through a 0.45 mm cellulose nitrate membrane by a vacuum pump. Afterward, the filtrate was loaded on a DEAE 52 anion exchange chromatography column pre-equilibrated with citric-phosphate buffer (pH 6.0, 20 mM) and then eluted with a linear gradient (0e1 M NaCl in 500 mL buffer). The flow rate was set at 2 mL/min and fractions of every 10 mL were pooled in tubes for the subsequent laccase activity assay and electrophoresis analysis. Eluate in each tube was further subjected to native polyacrylamide gel electrophoresis (Native-PAGE) for active staining with 1 mM DMP as substrate or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) for homogeneity verification with 0.2% Coomassie brilliant blue G-250. Purity of laccase was also verified by capillary electrophoresis using a P/ACE 5000 system (Beckman Instruments, Fullerton, CA, USA) equipped with a UV detector (Bornscheuer et al., 1994). 2.5. UVevis and CD spectra analysis of laccase A spectrophotometer (U-3010, HITACHI, Japan) was employed to record UVevis spectra (190e700 nm) of purified laccase. As for CD spectra, the elutions from DEAE 52 with laccase activity in peak A were pooled and powdered with a freeze-dryer (Labconco, USA). Then the powdered laccase was dissolved in 10 mM sodium phosphate buffer (pH 6.0) to prepare concentrated laccase which was further dialyzed against deionized water for 48 h. The dialyzate was used as control sample (correcting the baseline of the spectrum) and the reserved pure laccase (protein content of 0.02 mg/ mL) was scanned for CD spectra using a MOS-450 circular dichroism spectropolarimeter (Bio-Logic, France). The far-UV CD spectra of purified laccase were determined from wavelength
3
190e250 nm at 25 C. The scan speed was set at 60 nm/min with a response of 1 s and band width at 1 nm adapted from Salony et al. (2008). The effect of temperature on CD spectra of laccase was measured in a quartz cuvette of 1 cm path length, while the effect of pH and metal ions on CD spectra was measured in a quartz cuvette of 0.1 cm path length. Every experiment was carried out in duplicate and the average value was used for calculation of secondary structure using the software K2d (Salony et al., 2008). 2.6. Powder laccase preparation by spray drying The culture broth that was collected from selected production procedures was firstly filtered through 8-layer gauze to remove fungal biomass and then subjected to spray drier (Uniform Drying Co., Ltd.,Changzhou, China) without further concentration. Two batches were separately carried out with the following conditions: inlet temperature 200 C, outlet temperature 90 C, flow rate of 1 L/ h or 2 L/h. After spray drying, the powdered laccase was collected from the collecting vessel and the inner wall of the pipeline. The activity of liquid or powdered laccase was determined respectively to calculate the activity recovery. 3. Results and discussion 3.1. Laccase production in 5-L bench-scale STR under routine operation As shown in Fig. 2 for laccase production in 5-L STR, pH value of culture media decreased slightly from its initial point (4.5) to the lowest number (3.8) in the first 2 days and then gradually rose up to 6.5 during the following cultivation period. This trend is in accordance with that in our previous report in an air-lift reactor (Liu et al., 2013) and also with that by Stereum ostrea and Phanerochaete chrysosporium for laccase production (Praveen et al., 2011), but slightly different from that by Coriolus versicolor, which continuously dropped to pH 3 from the initial pH 6 during the entire fermentation course (Que et al., 2014). Dissolved oxygen (DO) can greatly impact the growth of free cell and subsequent secretion of extracellular laccase. We in the present study adopted a constant ventilation rate which resulted in variable DO level (Fig. 2A), while some studies chose adjusted ventilation approach to maintain constant DO level (e.g. 30%) throughout the fermentation process (Colao et al., 2006; Que et al., 2014). Fungal biomass and laccase reached the highest value of approximately 3.5 mg/mL on day 2 and 60 U/mL on day 7, respectively (Fig. 2B&C), which were comparable with that obtained (6 mg/mL on day 3 and 72 U/mL on day 6) in an air-lift reactor (Liu et al., 2013). Que et al. (2014) also performed fungal cultivation in a 5-L bioreactor and produced the highest fungal biomass of 30 mg/mL on day 7 and laccase activity of 7.5 U/mL on day 5, respectively. Along with the rapid fungal growth, glucose content dramatically decreased (Fig. 2B). Extracellular laccase accumulated gradually only in the late phase of cultivation presumably because of the release of intracellular laccase caused by cell lysis (Bose et al., 2007). Protein content versus time course kept a similar pace with laccase production, but was produced in low quantities with its maximum of 0.24 mg/mL at day 6 or 7. Recombinant laccase was expressed and produced in Aspergillus niger, bringing about the highest activity of 42 U/mL and a specific activity of 50 U/mg protein (Mekmouche et al., 2014), which were still lower than those in our study (60 U/mL and 240 U/mg, respectively). As seen in the pictures shown in Fig. 3A & B, growth of Pycnoporus sp. SYBC-L3 in bioreactor tended to form trophosome in the form of mycelial pellets (F z 2 mm) during liquid cultivation under agitation, as observed in other fungus such as C. versicolor and
Please cite this article in press as: Liu, J., et al., Scalable production, fast purification, and spray drying of native Pycnoporus laccase and circular dichroism characterization, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.03.154
J. Liu et al. / Journal of Cleaner Production xxx (2016) 1e10
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speed (150 rpm) and inoculum (105 spore/mL) can give rise to the highest enzyme production with pellet size of 2.78 mm, which was similar to our observation (pellet size 1e2 mm)
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Tetraploa aristata (Que et al., 2014; Saparrat et al., 2002). The fungus actually exhibited different morphological characteristics at different stages during its growth and development, i.e., arthrospores were produced by means of fragmentation in later phase (Fig. 3C) under static cultivation on PDA plate, while stirred liquid cultivation hardly generated single spores during the entire cultivation (Fig. 3D). Obviously, the morphological form of hypha can exert significant effect on metabolite accumulation, like laccase and nez-Tobon et al. (1997) exopolysaccharides (Que et al., 2014). Jime studied the effect of inoculum and rotate speed on both pellet size and MnP production and found that higher inoculum can result in smaller pellet but higher enzyme activity, and appropriate rotate
HCDC strategy was designed for Pycnoporus sp. SYBC-L3 (Fig. 1) and the result of laccase production was illustrated in Fig. 4. A 6fold increase in the highest fungal biomass (20 g/L on day 5) was observed under HCDC but requiring 3 more days than the routine operation (Fig. 3B). After a two-phase manipulation, the highest laccase production (240 U/mL) was achieved on day 8, which is about 4-fold of that under routine procedure and comparable to that by Trametes (280 U/mL) within 6 days, the highest that have been reported at flask scale to our knowledge (Ling et al., 2015). To overcome limitations that hamper applicable exploitation due to low enzyme production from native hosts, heterologous expression in some efficient hosts is commonly employed for anticipated hyper-production. Colao et al. (2006) heterologously expressed Trametes trogii laccase gene in Pichia pastoris with a protein yield of 0.017 mg/mL and an activity of 2.52 U/mL after 8-d fermentation, which were far below than those in our result, i.e., 1 mg/mL and 240 U/mL, respectively. It is worth noting that the maximum laccase activity under HCDC could double to over 500 U/mL if the preferable substrate ABTS, instead of DMP, was adopted for activity assay (Liu et al., 2012). 3.3. Laccase production in pilot-scale 500-L STR For fungal fermentation in 500-L STR for laccase, the first-order seed culture from flask was inoculated and amplified in 50-L STR for the second-order seed culture, and the growth behaviors of three separate batches were described in Fig. 5. It can be seen that rapid fungal biomass build-up took place in 2-d cultivation. The pH value of growth medium experienced slight fluctuation but was in
Please cite this article in press as: Liu, J., et al., Scalable production, fast purification, and spray drying of native Pycnoporus laccase and circular dichroism characterization, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.03.154
J. Liu et al. / Journal of Cleaner Production xxx (2016) 1e10
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reached 60% on day 4, which was higher than that (40%) in 5-L reactor (Fig. 1A) because of enhanced ventilation in 500-L STR. The maximum biomass was approximately 10 g/L on day 3, about 3-fold of that (3.5 g/L) in 5-L STR. An interesting phenomenon that the residual sugar content (determined as reducing sugar) increased rather than decreased was observed in the first 3-day (Fig. 6D), possibly owing to increased glucose molecule derived from maltose hydrolysis by fungal extracellular hydrolytic enzymes. Up scaling from 5-L to 500-L STR, the laccase production maintained to the similar level of about 60 U/mL (Fig. 6E). A robust daily output of laccase achieved on day 5e7, close to 12 U/mL/ d (Fig. 6F). During the 8-d cultivation, about 50 L of culture medium was lost from the starting volume of 300 L as a result of evaporation, suggesting evaporation rate of around 8 L per day.
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the range of 4.5e6.0 (Fig. 5A). Steep decline in DO from 100% to almost zero indicated the surge in biomass amount (Fig. 5B). The second-order seed culture propagated slowly in the first 10 h and then dramatically increased in the following 10 h and finally leveled off at 8e10 g/L after 20 h cultivation (Fig. 5C). As for up-scaled production in 500-L STR (Fig. 6), the pH value, DO, biomass, laccase activity versus time course followed similar trends to those in 5 L STR (Fig. 2). The pH value of culture broth remained relatively stable (4e5) throughout the cultivation for all data points recorded, while malfunction of the pH and DO electrodes occurred in the first batch (indicated as black square in Fig. 6A and 6B). The lowest DO
Laccase productivity was further evaluated in a 5-ton STR, in which four major process parameters, namely laccase, sugar, pH, and biomass, were investigated. As seen in Fig. 7A, the four variables followed similar trends in general as those in 5-L or 500-L STR (Figs. 2 and 6). A notable difference was the delayed maximal laccase activity (83 U/mL) on day 9, which might be due to the fact that the first-order fungal seed was directly inoculated in the 5-ton reactor production, instead of second-order seeding. Laccase accumulation in the late phase in 5-ton STR appeared to be more rapid relative to that in 5-L STR. Reducing sugar content dropped steadily from the starting point of 25 g/L until day 7, reaching less than 2.5 g/L and then maintained unchanged for the following 4 days. The residual reducing sugar in culture broth was supposed to be partly from the sugars in laccase for glycosylation. The fungus exhibited robust mycelial growth since the transferring of the firstorder seed culture, achieving the highest biomass of near 10 g/L on day 3, and then decreased over the remaining cultivation time. The pH value of culture broth underwent three phases: a dramatic drop from the initial pH 5.5 to pH 3.5 in the first day, maintaining pH 3.5 for the subsequent 4 days, and then rising up to the final value of about pH 7 after day 6. The cost percentage of this batch was roughly calculated on a basis of actual and predicted consumption of five essential constituents, shown in Fig. 7B. The highest percentage (50%) in the total cost was labor in the form of salary which might vary with different working district. Human power was mainly used to manage microorganisms, measuring procedure parameters, as well as operating the fermentation process. Electricity accounted for 36% of the total cost, the second contribution after labor. Depreciation of the equipment was about one-tenth of the total cost. Feedstock was not a major cost factor, accounting for less than 5% of the total cost. The lowest percentage was the consumption of steam, which was only 1%. The actual production cost was not revealed here in order to avoid possible conflict of commercial interest, however our estimated price for laccase by L3 in 65-L bioreactor was far below than those commercially available (Liu et al., 2013). By examining 26 different culture medium and their corresponding laccase production in flask under submerged fermentation, Osma et al. (2011) concluded that the culture medium, in all cases, was the largest component (90%) in the total cost, followed by the cost in operation (10%). In real pilot-scale fermentation, however, the situation may be greatly different depending on the current feedstock, power and labor costs, as discussed above. It is worth noting that the we believe the cost of laccase production can be further reduced by further enhancing laccase productivity using certain strategies at pilot-scale, such as HCDC, stage pH-control, and fed-batch method.
Please cite this article in press as: Liu, J., et al., Scalable production, fast purification, and spray drying of native Pycnoporus laccase and circular dichroism characterization, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.03.154
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J. Liu et al. / Journal of Cleaner Production xxx (2016) 1e10
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Fig. 5. Mycelial growth behavior of Pycnoporus sp. SYBC-L3 in 50-L STR for second order seed culture: batch 1 (black square), batch 2 (red circle), batch 3 (blue triangle). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 6. Fermentation behavior of Pycnoporus sp. SYBC-L3 in 500-L STR for laccase production: batch 1 (black square), batch 2 (red circle), batch 3 (blue triangle). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.5. Fast purification of laccase Purification of laccase from the culture broth using ionexchange chromatography was performed and the profiles are shown in Fig. 8. As the NaCl concentration increased linearly from 0 to 1 mol/L, two absorption peaks (OD 280) of B and C showed up in order, corresponding to tube 3 and 8, respectively. Laccase activity in each tube from 2 to 10 was examined by Native-PAGE (Fig. 8B), which suggested that the elutions in tube 2 and 3 had the highest activity. Elutions in tube 2 and 3 were also applied to SDS-PAGE (Fig. 8C) and a single band was observed for each tube, indicating a purified form of laccase was obtained. Elutions in tube 2 and 3 were then combined to calculate the laccase recovery from crude laccase, which was around 72%.
The purified laccase did not show the blue color by naked eye, the typical color for blue laccase with absorption peak at around 600 nm (Eggert et al., 1996). When subjected to UVevis spectrophotometer, a broad shoulder peak showed up at wavelength 300 nm for purified laccase combined from tube 2 and 3, which is very similar to some previous reports (Lu et al., 2007; Wang et al., 2010), indicating the presence of type III binuclear Cu (II) pair (Eggert et al., 1996). The purified laccase was also confirmed by capillary electrophoresis and the purity of laccase was found to be around 97.83% (major peak at 2.013 min), shown in Fig. 9. Normally, at least three successive steps were required to purify laccase from white-rot fungi: precipitation, ion exchange, and gel filtration or affinity chromatography (Litthauer et al., 2007; Lu et al., 2007; Wang et al., 2010), while only very few studies reported simpler
Please cite this article in press as: Liu, J., et al., Scalable production, fast purification, and spray drying of native Pycnoporus laccase and circular dichroism characterization, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.03.154
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Fig. 7. (A) Fermentation behavior of Pycnoporus sp. SYBC-L3 in 5-ton STR for laccase production: laccase activity (open circle), reducing sugar (solid square), pH value (open square), and fungal biomass (solid circle). (B) Cost percentage analysis of laccase production performed in 5-ton STR. Total cost (100%) of the batch was the sum of five essential parts, namely feedstock (medium, H2O, and antifoam), electricity (bioreactor, compressor, and water pump), steam (sterilization and heating), labor (culture preservation room, inspection center, and production workshop), and depreciation (including equipment purchase fee).
purification procedures, such as by phenyl-Sepharose High Performance column (Garcia et al., 2006). Although recombinant proteins (e.g., laccase) have been thought to require simple purification procedures (Erjavec et al., 2012), published data revealed that several steps were still needed (Colao et al., 2006). Therefore, the fact the laccase produced from Pycnoporus sp. SYBC-L3 can be readily purified in a one-step column procedure is a great advantage, and this is probably because the laccase is usually excreted as a major extracellular enzyme by genus Pycnoporus (Liu et al., 2013; Lomascolo et al., 2011). 3.6. CD spectra of laccase The Far-UV CD spectra of the purified laccase are displayed in Fig. 10. The CD spectra showed one positive peak around 198 nm and one negative trough around 218 nm, indicating the typical signature for a-helix and b-sheet structure, respectively (Salony et al., 2008; Schneider et al., 1999). Similarly, Bukh and Bjerrum (2010) reported that Coprinus cinereus laccase showed CD spectra with maximum absorption at 199 nm and minimum at 218 nm. The content of secondary structure calculated from the experimental CD values in the purified laccase is shown in Table 1. The laccase consisted of approximately 3% a-helix, 50% b-sheet, and 47% random coil, which is similar to that in C. cinereus laccase (6% ahelix, 40% b-sheet, and 54% random coil) (Bukh and Bjerrum, 2010). In contrast, fungal laccase with higher a-helix content (68%), e.g. C. versicolor laccase, has been documented as well (Que et al., 2014). The potential mechanism of thermostability and inhibition of some metal ions on laccase was explored using CD analysis under different conditions. As shown in Table 1, no significant change in the secondary structure was observed with temperature increasing from 5 to 55 C. However, when temperature was above 75 C, the
Fig. 8. (A) Purification of laccase on DEAE-sepharose 52. Abs 280 (line with triangle), laccase activity (line with circle), NaCl concentration (line). (B) Native-PAGE of laccase collected in corresponding tube from DEAE column. Laccase activity was stained with DMP in phosphorus buffer. The lane number above each reddish band was for each tube number in (A). (C) SDS-PAGE of crude laccase and purified laccase. lane 1: molecular mass markers (94kD: TaqDNA Polymerase; 62kD: T4 DNA Ligase; 45kD: Albumin from chicken serum; 33kD: His-Tag fusion protein). lane 2 and lane 3: laccase in the tube number 2 and 3 from DEAE 52. Lane 4: crude laccase in culture broth. (D) Absorbance spectrum of purified laccase by UVevis spectrophotometer, where tube 2 and 3 were combined.
Please cite this article in press as: Liu, J., et al., Scalable production, fast purification, and spray drying of native Pycnoporus laccase and circular dichroism characterization, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.03.154
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J. Liu et al. / Journal of Cleaner Production xxx (2016) 1e10 Table 1 Predicted secondary structure of purified laccase under various conditions. Influencing factor
Alpha helix/%
Beta sheet/%
Random coil/%
Control 5 C 25 C 55 C 75 C AgNO3 (1 mM) AgNO3 (5 mM) AgNO3 (50 mM) FeSO4 (1 mM) FeSO4 (5 mM) FeSO4 (50 mM) FeCl3 (1 mM) FeCl3 (5 mM) FeCl3 (50 mM)
3 3 3 3 46 11 13 0 10 9 4 4 3 3
50 50 50 50 23 41 40 4 43 42 48 49 50 49
47 47 47 47 31 48 47 96 47 48 48 47 47 47
3.7. Preparation of powder laccase Fig. 9. Purity verification of laccase by capillary electrophoresis.
Laccase production was successfully achieved by high cell density cultivation at various scales. The highest laccase production was 80 U/mL in a 5-ton bioreactor, where the labor cost was found to be the highest among five cost components. An one-step purification scheme was proven easy and efficient. Circular dichroism revealed the secondary structure of and explained some properties
(B)
20 control FeSO4/1 mmol/L
10
2
pH3.0 pH4.0 pH5.5
2
10
pH2.5 pH3.5 pH4.5 pH6.5
4. Conclusion
-1
20
-1
Molar Ellipticity (deg.cm .dmol )
(A)
After liquid cultivation in bioreactor, supernatant without fungal biomass was subjected to spray drying to produce powdered laccase and the details are listed in Table 2. Laccase powder was successfully produced with high activity in both two batches with recovery as high as 74.6% and 82.2%, respectively. The prepared laccase powder is uniform and smooth, looked yellowish and smelled like peptone. It should be noted that the drying condition was our first trial, and the final yields may be further improved if the drying condition, such as inlet and outlet temperature and the flow rate, are optimized. There has been an argument that production of laccases in their native hosts is often not economically feasible and natural laccases may not withstand the harsh conditions processes (Erjavec et al., 2012). Our results in this study however clearly prove that certain wild fungus, like Pycnoporus sp. SYBC-L3, can serve as an excellent cell factory for laccase production with outstanding properties and economic viability.
Molar Ellipticity (deg.cm .dmol )
absorbance peak around 208 nm and 220 nm decreased (figure not shown), suggesting thermal denaturing (Table 1), resulting in the loss of activity. Likewise, de Wilde et al. (2008) confirmed less thermal stability of Melanocarpus albomyces laccase expressed in rice than in Trichoderma reesei using CD analysis with temperature in the range of 30e90 C. When exposed to various pH values at the same solute concentration (0.1 M), no evident change in the CD spectra was observed for all pH values below 6.5, even though both the purified and crude laccase lost most of its activity toward DMP when pH is above 4.5 (Liu et al., 2012, 2013). The spectra of laccase exposed to Fe2þ and Fe3þ (from 1 to 50 mM) did not show noticeable changes (Fig. 10B), which were also reflected in the relative content of secondary structure (Table 1). However, the presence of Agþ ranging from 5 to 50 mM remarkably altered CD spectra with messy curved lines emerged (figure not shown), suggesting a dramatic decrease of b-sheet content in laccase (Table 1). The analysis of CD spectral changes strongly indicated the different inhibition mechanisms of Fe2þ/Fe3þ and Agþ on laccase. Although CD analytical technique has been utilized for secondary structures of proteins (Allendorf et al., 1985; Salony et al., 2008), studying CD changes in response to varied conditions is relatively few. This study represents the first attempt to collect series of CD data for laccase exposed to different conditions, providing insight into the differences in enzymatic properties.
0
-10
-20 190 200 210 220 230 240 250 Wavelength (nm)
FeSO4/5 mmol/L FeSO4/50 mmol/L
0
-10
-20 190 200 210 220 230 240 250 Wavelength (nm)
Fig. 10. UV-CD spectrum of laccase affected by varying pH (A) and FeSO4 (B) at 25 C. Protein concentration was 0.5 mg/ml. CD spectrum was measured in a quartz cuvette of 0.1 cm path length and the value of ellipticity was converted to molar ellipticity.
Please cite this article in press as: Liu, J., et al., Scalable production, fast purification, and spray drying of native Pycnoporus laccase and circular dichroism characterization, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.03.154
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Table 2 Preparation of powdered laccase using spray-dryer. Description
Batch 1
Batch 2
Experiment site Drying condition Total volume of crude liquid laccase Total activity of crude liquid laccase Total weight of powdered laccase Total activity of powdered laccase Specific activity of powdered laccase Activity recovery
Engineering center at School of Food Science, Jiangnan University, Wuxi, China Inlet T 200 C, outlet T 90 C, flow rate 1 L/h 4L 288000 U 3.28 g 215000 U 65548.7 U/g 74.6%
Uniform drying company, Changzhou, China Inlet T 200 C, outlet T 90 C, flow rate 2.0 L/h 36 L 1946000 U 97.3 g 1600000 U 16443.9 U/g 82.2%
of the laccase, like thermostability. A spray-drying technique was employed to prepare powdered laccase with over 74% activity recovered. By investigating productivity, scalability, purification, property, and powder preparation as well as production cost, the laccase by wild Pycnoporus fungus exhibited a great potential for green and clean applications. Acknowledgments This study was supported in part by Scientific Research Foundation for Advanced Talents of Huanghuai University (1000.12.01.1342). We would like to thank Dr. Xiaolian Zhan (Wuxi Jinkun Biotechnology Co., Ltd.) for providing 500 L bioreactor and general manager Guobao Jiao (Henan Yangshao Bioproducts Co., Ltd.) for 5 ton bioreactor and engineer Huaqiang Xi for collecting data in cost analysis. We also thank Changzhou Uniform Drying Co., Ltd for generous help in preparation of powdered laccase. References Allendorf, M.D., Spira, D.J., Solomon, E.I., 1985. Low-temperature magnetic circular dichroism studies of native laccase: spectroscopic evidence for exogenous ligand bridging at a trinuclear copper active site. P. Natl. Acad. Sci. U. S. A. 82, 3063e3067. Babaeipour, V., Shojaosadati, S.A., Robatjazi, S.M., Khalilzadeh, R., Maghsoudi, N., 2007. Over-production of human interferon-g by HCDC of recombinant Escherichia coli. Proc. Biochem. 42, 112e117. Bornscheuer, U., Reif, O.-W., Lausch, R., Freitag, R., Scheper, T., Kolisis, F.N., Menge, U., 1994. Lipase of Pseudomonas cepacia for biotechnological purposes: purification, crystallization and characterization. BBA-Gen. Subjects 1201, 55e60. Boruah, P., Dowarah, P., Hazarika, R., Yadav, A., Barkakati, P., Goswami, T., 2016. Xylanase from Penicillium meleagrinum var. viridiflavum e a potential source for bamboo pulp bleaching. J. Clean. Prod. 116, 259e267. Bose, S., Mazumder, S., Mukherjee, M., 2007. Laccase production by the white-rot fungus Termitomyces clypeatus. J. Basic Microb. 47, 127e131. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248e254. Bukh, C., Bjerrum, M.J., 2010. The reversible depletion and reconstitution of a copper ion in Coprinus cinereus laccase followed by spectroscopic techniques. J. Inorg. Biochem. 104, 1029e1037. Colao, M., Lupino, S., Garzillo, A., Buonocore, V., Ruzzi, M., 2006. Heterologous expression of lcc1 gene from Trametes trogii in Pichia pastoris and characterization of the recombinant enzyme. Microb. Cell Fact. 5, 31. Couto, S.R., Toca-Herrera, J.L., 2007. Laccase production at reactor scale by filamentous fungi. Biotechnol. Adv. 25, 558e569. de Wilde, C., Uzan, E., Zhou, Z., Kruus, K., Andberg, M., Buchert, J., Record, E., Asther, M., Lomascolo, A., 2008. Transgenic rice as a novel production system for Melanocarpus and Pycnoporus laccases. Transgenic Res. 17, 515e527. Eggert, C., Temp, U., Eriksson, K., 1996. The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase. Appl. Environ. Microbiol. 62, 1151e1158. Erjavec, J., Kos, J., Ravnikar, M., Dreo, T., Saboti c, J., 2012. Proteins of higher fungi e from forest to application. Trends. Biotechnol. 30, 259e273. Garcia, T., Santiago, M., Ulhoa, C., 2006. Properties of laccases produced by Pycnoporus sanguineus induced by 2,5-xylidine. Biotechnol. Lett. 28, 633e636. Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257e268. Greenfield, N.J., Fasman, G.D., 1969. Computed circular dichroism spectra for the evaluation of protein conformation. Biochemistry 8, 4108e4116.
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Please cite this article in press as: Liu, J., et al., Scalable production, fast purification, and spray drying of native Pycnoporus laccase and circular dichroism characterization, Journal of Cleaner Production (2016), http://dx.doi.org/10.1016/j.jclepro.2016.03.154