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Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli
Bioresource Technology xxx (2015) xxx–xxx
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Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli Sujata Vijay Sohoni a,b, Dhanaraj Nelapati a, Sneha Sathe a, Vaishali Javadekar-Subhedar c, Raghavendra P. Gaikaiwari c, Pramod P. Wangikar a,b,d,⇑ a
Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India DBT-Pan IIT Center for Bioenergy, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India Hi Tech Biosciences India Ltd., C-2, 102/103, Saudamini Complex, Right Bhusari Colony, Paud Road, Kothrud, Pune 411038, India d Wadhwani Research Center for Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India b c
h i g h l i g h t s Optimized batch process resulted in 5-folds increase in recombinant nitrilase. Fed-batch in shake flasks yielded fourfolds increase than optimized batch process. Glycerol–yeast extract feed was developed for improving nitrilase production. Scale up of fed-batch process to bioreactor resulted in further eightfolds increase.
a r t i c l e
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Article history: Received 29 November 2014 Received in revised form 9 February 2015 Accepted 10 February 2015 Available online xxxx Keywords: Nitrilase High cell density fermentation E. coli BL21 (DE3)
a b s t r a c t Nitrilases constitute an important class of biocatalysts for chiral synthesis. This work was undertaken with the aim to optimize nitrilase production in a host that is well-studied for protein production. Process parameters were optimized for high cell density fermentation, in batch and fed-batch modes, of Escherichia coli BL21 (DE3) expressing Pseudomonas fluorescens nitrilase with a T7 promoter based expression system. Effects of different substrates, temperature and isopropyl b-D-1-thiogalactopyranoside (IPTG) induction on nitrilase production were studied. Super optimal broth containing glycerol but without an inducer gave best results in batch mode with 32 °C as the optimal temperature. Use of IPTG led to insoluble protein and lower enzyme activity. Optimized fed-batch strategy resulted in significant improvement in specific activity as well as volumetric productivity of the enzyme. On a volumetric basis, the activity improved 40-fold compared to the unoptimized batch process. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Nitrilases (EC 3.5.5.1) have potential as commercial biocatalysts for chiral synthesis of various drug intermediates. Nitrilases belong to nitrile hydrolase family and are involved in conversion of a variety of aromatic as well as aliphatic nitriles (R-CN) into carboxylic acids (R-COOH) and ammonia without producing intermediate amides (Pace and Brenner, 2001). Chiral carboxylic acid products of nitrilase reaction e.g., R-mandelic acid or 2-aryl propionic acid are precursors for synthesis of antibiotics and other pharmaceuticals (Brady et al., 2004; MartÍnková and Krˇen, 2002). Several nitri⇑ Corresponding author at: Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India. Tel.: +91 22 2576 72 32; fax: +91 22 2572 68 95. E-mail address: [email protected] (P.P. Wangikar).
lase enzymes are reported that show high enantioselectivity toward compounds of commercial interest (Naik et al., 2008). Enzymes are typically produced in smaller amounts in the native host. Once the enzyme is characterized for desired properties, large scale production is essential. Hence cloning and high level expression in heterologous hosts is generally helpful. Escherichia coli is greatly exploited as a host for production of recombinant proteins and enzymes. E. coli grows faster, has simple nutrient requirements and can be easily grown to high cell densities (Gopal and Kumar, 2013; Terpe, 2006). E. coli BL21 strain that is deficient in lon and ompT proteases is routinely used for larger scale protein production. A derivative of this strain E. coli BL21 (DE3) harbors T7 DNA polymerase copy under the control of lac UV5 promoter on the genome and is used for T7 driven protein expression (Studier and Moffatt, 1986; Zerbs et al., 2009). Synthetic, semi-synthetic as well as complex media and various feeding
http://dx.doi.org/10.1016/j.biortech.2015.02.038 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Sohoni, S.V., et al. Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.02.038
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strategies based on glucose have been used for production of recombinant proteins (Fass et al., 1991; Bae et al., 1997; Shin et al., 1997). Several reports available on high cell density fermentations (HCDF) for production of recombinant proteins focus on E. coli BL21 (DE3) as a host cell. Most of these reports demonstrate use of glucose feeding for protein production (Shin et al., 1997; Son et al., 2010). Grossman et al. (1998) reported that addition of glucose reduces basal level expression of the target gene. Hence glucose based feeding strategies are not ideal. There are a few reports that discuss optimization of culture conditions for Pseudomonas putida nitrilase expressed in E. coli (Banerjee et al., 2009; Naik et al., 2008; Nigam et al., 2012). However, these do not report enzyme activity on volumetric basis and hence it is difficult to estimate the production cost of biocatalyst. Liu et al. (2011) report nitrilase enzyme activity of 19 units/ml with glycerol feeding in E. coli JM109. This study was initiated with the motivation of developing a feeding strategy based on favorable carbon and nitrogen substrate to achieve enzyme activity of greater than 400 units/ml of fermentation broth. Codon optimized and previously characterized nitrilase from Pseudomonas fluorescens (Layh et al., 1992; Kiziak et al., 2005) was cloned in pET21a plasmid and expressed in E. coli BL21 (DE3). Effect of glucose on plasmid stability and thereby nitrilase production was also studied. Further, the effect of growth temperature and induction with Isopropyl b-D-1-thiogalactopyranoside (IPTG) was examined. Feeding strategy with combination of glycerol and yeast extract was developed for HCDF and maximizing nitrilase production. 2. Methods 2.1. Chemicals All the chemicals used in this study were analytical grade unless otherwise stated. All solvents were HPLC grade. All the chemicals, solvents and racemic mixture of mandelonitrile were purchased from Merck (Massachusetts, USA). Antibiotics were purchased from Sigma Aldrich (Missouri, USA). Water (MQ) was purified with a Milli-Q-system (Millipore, Bedford, MA). 2.2. Bacterial strain, media and culture conditions E. coli BL21 (DE3) is an all-purpose strain for high-level protein expression and easy induction. Synthesis of codon optimized nitrilase gene from P. fluorescens (PF) was outsourced to Biomatik Corporation (Ontario, Canada). It was cloned in pET21a plasmid at NdeI and BamHI sites and expressed in E. coli BL21 (DE3). The resulting strain E. coli BL21 (DE3)_PF nit was used to study recombinant nitrilase production in this study. LB broth (HiMedia Laboratories) was used in initial studies. Super Optimal broth (SOB) medium (Tryptone 20 g/l, Yeast extract 5 g/l, NaCl 0.5 g/l, KCl 0.186 g/l) was used subsequently in the shake flask and reactor cultivations in this study. MgCl2 (final concentration 10 mM) and carbenicillin (150 lg/ml) were added to all the media as well as feed after autoclaving. Feed used in fed-batch consisted of 50% yeast extract solution or 2.2 M glycerol solution. Feed used in the reactor for continuous feeding consisted of 200 g/l yeast extract and 67 g/l glycerol. Carbon substrates wherever applicable were also added to SOB medium after autoclaving at 1% final concentration for sugars or glycerol 30 mM. All the shake flask cultivations were carried out at 37 °C or 32 °C and shaking at 180 rpm. 2XYT agar (Tryptone 15 g/l, Yeast extract 10 g/l, NaCl 5 g/l, agar 15 g/l) with and without carbenicillin (150 lg/ml) was used for determining plasmid stability.
2.3. Seed conditions Two seed stages namely, pre-seed and seed preceded the production. In the Pre-seed stage, 1 ml of glycerol stock culture was inoculated in 5 ml SOB broth. Incubation was at 37 °C or 32 °C for 9 h under static conditions. In the seed stage, 0.6 ml of Pre-seed culture was inoculated in 20 ml SOB in 100 ml shake flask. Incubation was at 37 °C or 32 °C, 180 rpm and for 12 h. 2.4. Protein production in shake flasks and bioreactor 3 ml of seed culture was inoculated in 100 ml SOB broth in 500 ml shake flask. Incubation was at 37 °C or 32 °C, 180 rpm and for 12–24 h. The nitrilase production was induced by the addition of IPTG to the final concentration of 1 mM in the production medium after 3 h of inoculation wherever applicable. Samples taken at different time points were analyzed for dry cell weight (DCW), Optical density (OD600nm) and nitrilase activity. Fed-batch cultivations in bioreactor were performed at 32 °C, pH 7.0, at the agitation rate of 800 rpm and aeration rate of 1 vvm (volume per volume per minute). Sartorius BIOSTAT bioreactors with 2 l working volume were used in this study (Sartorius AG, Gottingen, Germany). One l SOB containing 30 mM glycerol was inoculated with 30 ml of the seed. Solutions of 2 M NaOH and 2 M HCl were used to maintain the pH. Feed was initiated after 4 h of growth and at the constant flow of 22 ml/min. A BlueInOne Cell exit gas analyzer (Bluesens, Herten, Germany) was used to monitor the concentrations of CO2 and O2 in the exit gas. Samples taken from bioreactors at different time points were analyzed for DCW, OD600nm and nitrilase activity. 2.5. Analytical methods Samples taken from shake flasks and bioreactor were centrifuged at 8000 rpm for 10 min. Pellets were washed with 0.1 M Na-phosphate buffer (pH 7.5) and resuspended in the same buffer. Optical density was measured at 600 nm using a UV–vis spectrophotometer (V-540 Jasco, Tokyo, Japan). Supernatants from centrifugation of the fermentation broth were saved for estimation of glycerol wherever applicable. Glycerol was estimated using HPLC with RI detector (HitachiMerck, Darmstadt, Germany) and HP-Aminex 87-H column (BioRad, Hercules, CA). Samples were analyzed using 0.01 N H2SO4 as a mobile phase, 0.6 ml/min flow rate and column temperature of 50 °C. Free ammonia in the supernatants was analyzed using phenol-hypochlorite assay described by Weatherburn (1967), after suitable modifications. 2.6. Determination of nitrilase activity The nitrilase activity in resuspended cell pellets was determined. The reaction mix (1 ml each) contained 50 mM Tris–HCl buffer (pH 7.5), 10 mM mandelonitrile and 50 ll cells. Mandelonitrile stock solution (100 mM) was prepared in methanol. The reaction mixtures with appropriate blanks were incubated at 37 °C for 30 min. The reactions were stopped by the addition of 500 ll methanol. The samples were centrifuged at 12,000 rpm for 5 min. The resulting supernatants were analyzed for ammonia released during the reaction using phenol-hypochlorite assay. 2.7. Testing plasmid stability Plasmid stability testing was carried out at different stages of cultivation i.e. pre-seed (at 0 and 9 h), seed (at 12 h) and production medium (at 12 h) as described by Zhang et al. (2003). Two combinations were used at each stage, one containing only SOB
Please cite this article in press as: Sohoni, S.V., et al. Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.02.038
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and the other containing SOB with 1% glucose. 2X YT agar plates with and without 150 lg/ml carbenicillin were used for testing plasmid stability. Samples taken for plasmid stability were diluted and plated on non selective 2X YT agar plates. The plates were incubated at 32 °C for 16–20 h. 100 colonies were randomly picked and spotted on 2X YT plates with and without 150 lg/ml carbenicillin. The plates were incubated at 32 °C for 9–12 h. The colonies were counted and the ratio of number of colonies on plates with and without carbenicillin was used to calculate percentage stability. 3. Results and discussion 3.1. Batch fermentation Initial batch fermentations were carried out in LB medium. SOB medium is rich in nutrients compared to LB and it additionally contains MgCl2 that is essential for growth of E coli. Hence batch fermentation was also carried out in SOB medium. Induction with IPTG was performed after 3 h of growth. Table 1 summarizes nitrilase production in (u/l) and (u/g DCW) in both media. Nitrilase activity per unit biomass in LB medium at 5 h was 0.14 103 u/g DCW. Nitrilase activity per unit biomass in SOB medium was 3.4 103 u/g DCW. SOB medium demonstrated better activity due to its higher nitrogen content and growth. Hence SOB was used as a basal medium in all the experiments in this study. 3.2. Effect of different carbon substrates Carbon substrates have been successfully used in complex media for HCDF of E. coli to improve biomass and thereby the amount of protein produced (Panda et al., 2000; Tabandeh et al.,
2004). The vector pET21a used in this study contains T7 promoter controlled by lac operator. T7 RNA polymerase required to transcribe T7 promoter is expressed in E. coli BL21 (DE3) strain under lacUV5 promoter. Lac promoter is repressed by glucose (Grossman et al., 1998). However, presence of glucose in the medium is reported to enhance plasmid stability (Zhang et al., 2003) and hence effect of different substrates was studied to improve nitrilase production in SOB medium. Different sugars such as glucose, sucrose, maltose and lactose were added at 1% concentration in SOB medium. Glycerol was added at 30 mM final concentration. The cultures were induced with 1 mM IPTG after 3 h of growth. Fig. 1A represents OD profile at 600 nm and Fig. 1B represents nitrilase production profile with different substrates. Maltose demonstrated highest growth of all followed by lactose and glycerol. Glucose and sucrose exhibited poor growth compared to other substrates. Maltose, glucose and sucrose did not result in higher nitrilase production compared to control. Lactose on the other hand even if acts as an inducer of lac promoter, did not support highest nitrilase production. Maltose, lactose and sucrose yield glucose upon hydrolysis and hence it is possible that nitrilase production was repressed in presence of these sugars. Glycerol demonstrated highest nitrilase production amongst all the substrates (2.7 103 u/l). Hence glycerol was chosen to be used as a substrate for further experiments.
3.3. Effect of IPTG IPTG is used as an inducer of lac operon but is also shown to repress growth (Marbach and Bettenbrock, 2012). P. fluorescens nitrilase was expressed under the control of T7 promoter and lac operator while, T7 polymerase is expressed in E. coli BL21 (DE3) under the control of lac UV5 promoter which could be induced
Table 1 Nitrilase activity obtained in different media combinations of batch/fed-batch cultivations in shake flasks and bioreactor. Initial medium
Dose given
Nitrilase production (u/l)
Nitrilase activity (u/g) of DCW
LB SOB SOB + 30 mM SOB + 30 mM SOB + 30 mM SOB + 30 mM
Batch run Batch run Batch run 1% (final concentration) yeast extract dose at 6 h 30 mM (final concentration) glycerol dose at 9 h 1% (final concentration) yeast extract dose at 6 h & 30 mM (final concentration) glycerol dose at 9 h 1% (final concentration) yeast extract dose at 6 h, 30 mM (final concentration) glycerol dose at 9 h and 1% (final concentration) yeast extract 30 mM (final concentration) glycerol dose Continuous feeding of combination of yeast extract (200 g/l) and glycerol (67 g/l) at 23 ml/h
2.06 102 10.8 103 53.5 103 64.5 103 57.7 103 106.8 103
0.14 103 3.4 103 8.8 103 10 103 9 103 16 103
210.7 103
23.9 103
419.2 103
21 103
glycerol + 10 mM glycerol + 10 mM glycerol + 10 mM glycerol + 10 mM
MgCl2 MgCl2 MgCl2 MgCl2
SOB + 30 mM glycerol + 10 mM MgCl2
SOB + 30 mM glycerol + 10 mM MgCl2 in 2 l bioreactor
Fig. 1. Effect of different substrates on nitrilase production: E. coli BL21 (DE3)_PF nit strain was grown in the shake flasks with SOB medium containing different substrates. The culture were induced with 1 mM of IPTG at 3 h (A) represents OD profile at 600 nm and (B) nitrilase production profile represented in u/l. SOB control (-d-), SOB with 1% glucose (-j-), SOB with 1% sucrose (--), SOB with 1% lactose (-.-), SOB with 1% maltose(-N-) and SOB with 30 mM glycerol (-X-).
Please cite this article in press as: Sohoni, S.V., et al. Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.02.038
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Fig. 2. Effect of IPTG induction on nitrilase production: E. coli BL21 (DE3)_PF nit strain was grown in the shake flasks with SOB medium. The culture was induced with different concentrations of IPTG (A) represents OD profile at 600 nm and (B) nitrilase production profile represented in u/l. No induction (-d-), induction with 0.1 mM IPTG (j-), 0.25 mM IPTG (--), 0.5 mM IPTG (-.-), 1 mM IPTG (-N-).
by IPTG or lactose. E. coli BL21 (DE3)_pET21a_PF nit strain was inoculated in SOB containing 30 mM glycerol. In order to find out the optimal concentration of IPTG required for induction, cultures were induced at 3 h with different concentrations of IPTG (0, 0.1 mM, 0.25 mM, 0.5 mM and 1 mM). Fig. 2A represents OD profile at 600 nm and Fig. 2B represents nitrilase production profile at different concentrations of IPTG. IPTG induction did not affect growth (Fig. 2A) but seemed to reduce nitrilase production (Fig. 2B). Maximum nitrilase production was observed in the flask without IPTG induction with nitrilase activity per unit biomass of 8.8 103 u/g DCW. T7 promoter is known to yield steady basal level expression, even in absence of an inducer (Pan and Malcolm, 2000). Induction with IPTG leads to high levels of protein production, sometimes improperly folded into inclusion bodies, making it inactive (Makrides, 1996). The results from SDS PAGE gel in this study support these observations. During the course of this study protein production in each flask was assessed in soluble and insoluble fraction. The flasks with IPTG yielded greater protein in the insoluble fraction (i.e., in the form of inclusion bodies) compared to the soluble fraction (data not shown). Hence reduced nitrilase activity in presence of IPTG can be attributed to formation of inclusion bodies. Clearly, the promoter showed leaky expression. Since the nitrilase produced is not toxic to E. coli cells and resulted in higher soluble nitrilase production than in cultures induced with IPTG, all the remaining experiments were performed without IPTG induction. 3.4. Determination of plasmid stability Plasmid stability at various stages of nitrilase production was determined for improving nitrilase production. Pre-seed and seed stages of cultures were grown in SOB medium with and without glucose. Production medium on the other hand contained SOB with 30 mM glycerol with and without glucose. Plasmid stability was assessed in the culture for Pre-seed at 0 and 9 h, seed at 12 h and production medium at 12 h. Glucose is reported to improve plasmid stability by repressing lac UV5 and T7 promoter (Zhang et al., 2003). Hence two combinations were set up at each stage of cultivation for testing plasmid stability. One of them contained only SOB while, the other combination contained SOB with 1% glucose. Plasmid stability was comparable in both the combinations at pre-seed and seed levels (Table 2). In production medium however only 47% of the cells contained plasmid in only SOB while, 70% cells contained plasmid in SOB with 1% glucose (Table 2). Excessive protein synthesis in the production medium may have led to the selection of cells without plasmids and hence reduction in plasmid stability at production stage compared to seed. Medium combination with 1% glucose demonstrated better plasmid stability than
one without glucose and hence effect of glucose on nitrilase production was further studied. 3.5. Effect of glucose on nitrilase production Glucose in SOB medium led to improved plasmid stability. Hence effect of glucose on nitrilase production was studied. Three combinations namely, SOB with 30 mM glycerol as control and SOB with 30 mM glycerol containing either 0.5% or 1% glucose were used. Fig. 3A represents OD profile at 600 nm and Fig. 3B represents nitrilase production profile at different glucose concentrations. Less growth was observed in combinations containing glucose than those in without glucose (Fig. 3B). This result was in disagreement with Fig. 1B (studying effect of carbon substrates). In Fig. 1B experiment the culture was induced with IPTG after 3 h of growth. It is reported that LacUV5 promoter shows limited decreased dependency on cAMP and can be efficiently induced in presence of glucose (Hirschel et al., 1980; Studier, 1991). The activity obtained in Fig. 1B could be attributed to IPTG induction. The current experiment did not involve IPTG induction step and could be the possible reason for disagreement. No nitrilase production was observed in the combination with 1% glucose without IPTG induction. Nitrilase production was repressed for 8 h in combination with 0.5% glucose. A combination without glucose demonstrated highest nitrilase production. Even though glucose improved plasmid stability, it did not enhance the resulting protein production. Hence glucose was not incorporated in the medium in further studies. 3.6. Effect of temperature Temperature plays an important role in protein production. Soluble protein expression increases when the temperature is lowered (Weickert et al., 1996). E. coli BL21 (DE3)_pET21a_PF nit
Table 2 Effect of glucose on plasmid stability – Plasmid stability is reported as the ratio of number of colony forming units on plates with and without carbenicillin was used to calculate percentage (%) of plasmid stability in medium with and without glucose. Stage
+Glucose (%)
Pre-seed Plasmid stability Pre-seed Plasmid stability Seed Plasmid stability Production Plasmid stability
98
96
Glucose (%)
95
95
93
92
71
40
(at 0 h) (at 9 h) (at 12 h) (at 12 h)
Please cite this article in press as: Sohoni, S.V., et al. Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.02.038
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Fig. 3. Effect glucose on nitrilase production: E. coli BL21 (DE3)_PF nit strain was grown in the shake flasks with SOB medium containing 30 mM glycerol with 0.5 and 1.0% glucose. SOB with 30 mM glycerol was used as a control (A) represents OD profile at 600 nm and (B) nitrilase production profile represented in u/l. 0.5% glucose (-d-), 1% glucose (-j-) and (-.-) without glucose.
Fig. 4. Effect of temperature on nitrilase production: E. coli BL21 (DE3)_PF nit strain was grown in the shake flasks with SOB medium containing 30 mM glycerol. (A) Represents OD profile at 600 nm and (B) nitrilase activity profile represented in u/l. 37 °C (-d-), 32 °C (-j-), 27 °C (--).
strain was inoculated in SOB containing 30 mM glycerol at three different temperatures (27 °C, 32 °C and 37 °C) to deduce optimal temperature for soluble nitrilase production. These cultures were not induced with IPTG. Fig. 4A represents OD profile at 600 nm and Fig. 4B represents nitrilase production profile at different temperatures. Maximum nitrilase production was observed at 32 °C. However, the expression of soluble protein was not found to be higher at 32 °C when compared with that at 37 °C or 27 °C (data not shown). 3.7. Optimization of fed-batch fermentation Fed-batch experiments were set up to improve nitrilase production further. PF nitrilase expressed in E. coli BL21 (DE3) is growth associated and hence the aim was to maximize the biomass. Feeding of a desired carbon and nitrogen substrate in an optimal ratio is crucial. In order to get an idea of feeding strategy, glycerol and yeast extract were fed to the culture growing in SOB containing 30 mM glycerol at different time points. Initial fed-batch experiments were carried out in shake flasks. All the experiments were monitored for 24 h. Doses of glycerol and yeast extract were given at different time points during cultivations. Table 1 represents various combinations of doses tried and the resulting nitrilase production (u/l) as well as nitrilase activity per unit biomass (u/g DCW). Batch fermentation in SOB with 30 mM glycerol resulted in 5.3 103 u/l nitrilase production and nitrilase activity per unit biomass of 8.8 103 u/g DCW. SOB containing 30 mM glycerol was used as a basal medium when developing fed-batch fermentations. Individual addition of yeast extract (6 h) or glycerol (9 h) did not lead to significant increase in nitrilase activity per unit biomass (Table 1). However, sequential dosing of glycerol and yeast extract (yeast extract
dose at 6 h, glycerol dose at 9 h and yeast extract- glycerol mixed dose at 12 h) resulted in fourfolds increase in the production of nitrilase and 2.8-folds increase in nitrilase activity per unit biomass (Table 1).
3.8. Fed-batch fermentation in bioreactor According to the results from fed-batch experiments in shake flasks, bioreactor was set up with initial medium SOB containing 30 mM glycerol. Continuous feeding strategy was used here in which yeast extract (200 g/l) and glycerol (67 g/l) was fed at the flow rate of 23 ml/h after 4 h of growth. Fig. 5A represents growth and nitrilase production profile and Fig. 5B represents data from exit gas analyzer. Exit CO2 (%) demonstrated a sharp peak at 5 h representing highly active state of culture. CO2 (%) reduced thereafter and remained constant at 0.5% throughout the cultivation. Continuous feeding resulted in 19.5 g/l DCW and 419 103 u/l nitrilase production. Liu et al. (2011) optimized glycerol fed-batch strategy and reported 19.3 103 u/l nitrilase production. Continuous feeding strategy improved nitrilase production by eightfolds and nitrilase activity per unit biomass by 2.4-folds compared to optimized batch fermentations in this study (Table 1). The nitrilase activity per unit biomass obtained in this study was 21.4 103 u/g DCW, while Liu et al. (2011) report nitrilase activity of 1.8 103 u/g DCW. Liu et al. (2011) report feeding of glycerol in the fed-batch mode, while in this study a mixture of yeast extract and glycerol was fed. Here twelvefolds increase in nitrilase activity per unit biomass was reported compared to the report from Liu et al. (2011).
Please cite this article in press as: Sohoni, S.V., et al. Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.02.038
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Fig. 5. Scale up in 2 l bioreactor: E. coli BL21 (DE3)_PF nit strain was grown in 2 l bioreactor with SOB medium with 30 mM glycerol. Continuous feeding of mix of yeast extract and glycerol was achieved at 23 ml/h after 4 h of growth for 24 h. (A) Represents OD profile at 600 nm (-j-), dry cell weight g/l (-d-), nitrilase production profile u/l (.-) and glycerol consumption (-N-) (B) exit gas profile – solid line representing % O2 and dotted line representing %CO2.
4. Conclusion HCDF was optimized for production of P. fluorescens nitrilase cloned in pET21a and expressed in E. coli BL21 (DE3). Nitrilase production was improved by fivefold in optimized batch fermentations in shake flasks compared to initial batches. Optimized fedbatch strategy further improved nitrilase production by fortyfold compared to initial batches in SOB and eightfolds against optimized batch. The strategy developed here can be readily applied to optimize production of other proteins and enzymes in the E. coli expression host. Acknowledgement Authors would like to acknowledge Department of Biotechnology, India (grant no. BT/SBIRI/846/54/B16/2011) for providing financial support for this work. References Bae, C.-S., Hong, M.-S., Chang, S.-G., Kim, D.-Y., Shin, H.-C., 1997. Optimization of fusion proinsulin production by high cell-density fermentation of recombinant E. coli. Biotechnol. Bioprocess Eng. 2, 27–32. Banerjee, A., Dubey, S., Kaul, P., Barse, B., Piotrowski, M., Banerjee, U.C., 2009. Enantioselective nitrilase from Pseudomonas putida: cloning, heterologous expression, and bioreactor studies. Mol. Biotechnol. 41, 35–41.
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Studier, F.W., 1991. Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J. Mol. Biol. 219, 37–44. Tabandeh, F., Shojaosadati, S.A., Zomorodipour, A., Khodabandeh, M., Sanati, M.H., Yakhchali, B., 2004. Heat-induced production of human growth hormone by high cell density cultivation of recombinant Escherichia coli. Biotechnol. Lett. 26, 245–250. Terpe, K., 2006. Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 72, 211–222. Weatherburn, M.W., 1967. Phenol-hypochlorite reaction for determination of ammonia. Anal. Chem. 39, 971–974. Weickert, M.J., Doherty, D.H., Best, E.A., Olins, P.O., 1996. Optimization of heterologous protein production in Escherichia coli. Curr. Opin. Biotechnol. 7, 494–499. Zerbs, S., Frank, A.M., Collart, F.R., 2009. Bacterial systems for production of heterologous proteins. Methods Enzymol. 463, 149–168. Zhang, Y., Taiming, L., Liu, J., 2003. Low temperature and glucose enhanced T7 RNA polymerase-based plasmid stability for increasing expression of glucagon-like peptide-2 in Escherichia coli. Protein Expr. Purif. 29, 132–139.
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Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli Sujata Vijay Sohoni a,b, Dhanaraj Nelapati a, Sneha Sathe a, Vaishali Javadekar-Subhedar c, Raghavendra P. Gaikaiwari c, Pramod P. Wangikar a,b,d,⇑ a
Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India DBT-Pan IIT Center for Bioenergy, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India Hi Tech Biosciences India Ltd., C-2, 102/103, Saudamini Complex, Right Bhusari Colony, Paud Road, Kothrud, Pune 411038, India d Wadhwani Research Center for Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India b c
h i g h l i g h t s Optimized batch process resulted in 5-folds increase in recombinant nitrilase. Fed-batch in shake flasks yielded fourfolds increase than optimized batch process. Glycerol–yeast extract feed was developed for improving nitrilase production. Scale up of fed-batch process to bioreactor resulted in further eightfolds increase.
a r t i c l e
i n f o
Article history: Received 29 November 2014 Received in revised form 9 February 2015 Accepted 10 February 2015 Available online xxxx Keywords: Nitrilase High cell density fermentation E. coli BL21 (DE3)
a b s t r a c t Nitrilases constitute an important class of biocatalysts for chiral synthesis. This work was undertaken with the aim to optimize nitrilase production in a host that is well-studied for protein production. Process parameters were optimized for high cell density fermentation, in batch and fed-batch modes, of Escherichia coli BL21 (DE3) expressing Pseudomonas fluorescens nitrilase with a T7 promoter based expression system. Effects of different substrates, temperature and isopropyl b-D-1-thiogalactopyranoside (IPTG) induction on nitrilase production were studied. Super optimal broth containing glycerol but without an inducer gave best results in batch mode with 32 °C as the optimal temperature. Use of IPTG led to insoluble protein and lower enzyme activity. Optimized fed-batch strategy resulted in significant improvement in specific activity as well as volumetric productivity of the enzyme. On a volumetric basis, the activity improved 40-fold compared to the unoptimized batch process. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Nitrilases (EC 3.5.5.1) have potential as commercial biocatalysts for chiral synthesis of various drug intermediates. Nitrilases belong to nitrile hydrolase family and are involved in conversion of a variety of aromatic as well as aliphatic nitriles (R-CN) into carboxylic acids (R-COOH) and ammonia without producing intermediate amides (Pace and Brenner, 2001). Chiral carboxylic acid products of nitrilase reaction e.g., R-mandelic acid or 2-aryl propionic acid are precursors for synthesis of antibiotics and other pharmaceuticals (Brady et al., 2004; MartÍnková and Krˇen, 2002). Several nitri⇑ Corresponding author at: Department of Chemical Engineering, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India. Tel.: +91 22 2576 72 32; fax: +91 22 2572 68 95. E-mail address: [email protected] (P.P. Wangikar).
lase enzymes are reported that show high enantioselectivity toward compounds of commercial interest (Naik et al., 2008). Enzymes are typically produced in smaller amounts in the native host. Once the enzyme is characterized for desired properties, large scale production is essential. Hence cloning and high level expression in heterologous hosts is generally helpful. Escherichia coli is greatly exploited as a host for production of recombinant proteins and enzymes. E. coli grows faster, has simple nutrient requirements and can be easily grown to high cell densities (Gopal and Kumar, 2013; Terpe, 2006). E. coli BL21 strain that is deficient in lon and ompT proteases is routinely used for larger scale protein production. A derivative of this strain E. coli BL21 (DE3) harbors T7 DNA polymerase copy under the control of lac UV5 promoter on the genome and is used for T7 driven protein expression (Studier and Moffatt, 1986; Zerbs et al., 2009). Synthetic, semi-synthetic as well as complex media and various feeding
http://dx.doi.org/10.1016/j.biortech.2015.02.038 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Sohoni, S.V., et al. Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.02.038
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strategies based on glucose have been used for production of recombinant proteins (Fass et al., 1991; Bae et al., 1997; Shin et al., 1997). Several reports available on high cell density fermentations (HCDF) for production of recombinant proteins focus on E. coli BL21 (DE3) as a host cell. Most of these reports demonstrate use of glucose feeding for protein production (Shin et al., 1997; Son et al., 2010). Grossman et al. (1998) reported that addition of glucose reduces basal level expression of the target gene. Hence glucose based feeding strategies are not ideal. There are a few reports that discuss optimization of culture conditions for Pseudomonas putida nitrilase expressed in E. coli (Banerjee et al., 2009; Naik et al., 2008; Nigam et al., 2012). However, these do not report enzyme activity on volumetric basis and hence it is difficult to estimate the production cost of biocatalyst. Liu et al. (2011) report nitrilase enzyme activity of 19 units/ml with glycerol feeding in E. coli JM109. This study was initiated with the motivation of developing a feeding strategy based on favorable carbon and nitrogen substrate to achieve enzyme activity of greater than 400 units/ml of fermentation broth. Codon optimized and previously characterized nitrilase from Pseudomonas fluorescens (Layh et al., 1992; Kiziak et al., 2005) was cloned in pET21a plasmid and expressed in E. coli BL21 (DE3). Effect of glucose on plasmid stability and thereby nitrilase production was also studied. Further, the effect of growth temperature and induction with Isopropyl b-D-1-thiogalactopyranoside (IPTG) was examined. Feeding strategy with combination of glycerol and yeast extract was developed for HCDF and maximizing nitrilase production. 2. Methods 2.1. Chemicals All the chemicals used in this study were analytical grade unless otherwise stated. All solvents were HPLC grade. All the chemicals, solvents and racemic mixture of mandelonitrile were purchased from Merck (Massachusetts, USA). Antibiotics were purchased from Sigma Aldrich (Missouri, USA). Water (MQ) was purified with a Milli-Q-system (Millipore, Bedford, MA). 2.2. Bacterial strain, media and culture conditions E. coli BL21 (DE3) is an all-purpose strain for high-level protein expression and easy induction. Synthesis of codon optimized nitrilase gene from P. fluorescens (PF) was outsourced to Biomatik Corporation (Ontario, Canada). It was cloned in pET21a plasmid at NdeI and BamHI sites and expressed in E. coli BL21 (DE3). The resulting strain E. coli BL21 (DE3)_PF nit was used to study recombinant nitrilase production in this study. LB broth (HiMedia Laboratories) was used in initial studies. Super Optimal broth (SOB) medium (Tryptone 20 g/l, Yeast extract 5 g/l, NaCl 0.5 g/l, KCl 0.186 g/l) was used subsequently in the shake flask and reactor cultivations in this study. MgCl2 (final concentration 10 mM) and carbenicillin (150 lg/ml) were added to all the media as well as feed after autoclaving. Feed used in fed-batch consisted of 50% yeast extract solution or 2.2 M glycerol solution. Feed used in the reactor for continuous feeding consisted of 200 g/l yeast extract and 67 g/l glycerol. Carbon substrates wherever applicable were also added to SOB medium after autoclaving at 1% final concentration for sugars or glycerol 30 mM. All the shake flask cultivations were carried out at 37 °C or 32 °C and shaking at 180 rpm. 2XYT agar (Tryptone 15 g/l, Yeast extract 10 g/l, NaCl 5 g/l, agar 15 g/l) with and without carbenicillin (150 lg/ml) was used for determining plasmid stability.
2.3. Seed conditions Two seed stages namely, pre-seed and seed preceded the production. In the Pre-seed stage, 1 ml of glycerol stock culture was inoculated in 5 ml SOB broth. Incubation was at 37 °C or 32 °C for 9 h under static conditions. In the seed stage, 0.6 ml of Pre-seed culture was inoculated in 20 ml SOB in 100 ml shake flask. Incubation was at 37 °C or 32 °C, 180 rpm and for 12 h. 2.4. Protein production in shake flasks and bioreactor 3 ml of seed culture was inoculated in 100 ml SOB broth in 500 ml shake flask. Incubation was at 37 °C or 32 °C, 180 rpm and for 12–24 h. The nitrilase production was induced by the addition of IPTG to the final concentration of 1 mM in the production medium after 3 h of inoculation wherever applicable. Samples taken at different time points were analyzed for dry cell weight (DCW), Optical density (OD600nm) and nitrilase activity. Fed-batch cultivations in bioreactor were performed at 32 °C, pH 7.0, at the agitation rate of 800 rpm and aeration rate of 1 vvm (volume per volume per minute). Sartorius BIOSTAT bioreactors with 2 l working volume were used in this study (Sartorius AG, Gottingen, Germany). One l SOB containing 30 mM glycerol was inoculated with 30 ml of the seed. Solutions of 2 M NaOH and 2 M HCl were used to maintain the pH. Feed was initiated after 4 h of growth and at the constant flow of 22 ml/min. A BlueInOne Cell exit gas analyzer (Bluesens, Herten, Germany) was used to monitor the concentrations of CO2 and O2 in the exit gas. Samples taken from bioreactors at different time points were analyzed for DCW, OD600nm and nitrilase activity. 2.5. Analytical methods Samples taken from shake flasks and bioreactor were centrifuged at 8000 rpm for 10 min. Pellets were washed with 0.1 M Na-phosphate buffer (pH 7.5) and resuspended in the same buffer. Optical density was measured at 600 nm using a UV–vis spectrophotometer (V-540 Jasco, Tokyo, Japan). Supernatants from centrifugation of the fermentation broth were saved for estimation of glycerol wherever applicable. Glycerol was estimated using HPLC with RI detector (HitachiMerck, Darmstadt, Germany) and HP-Aminex 87-H column (BioRad, Hercules, CA). Samples were analyzed using 0.01 N H2SO4 as a mobile phase, 0.6 ml/min flow rate and column temperature of 50 °C. Free ammonia in the supernatants was analyzed using phenol-hypochlorite assay described by Weatherburn (1967), after suitable modifications. 2.6. Determination of nitrilase activity The nitrilase activity in resuspended cell pellets was determined. The reaction mix (1 ml each) contained 50 mM Tris–HCl buffer (pH 7.5), 10 mM mandelonitrile and 50 ll cells. Mandelonitrile stock solution (100 mM) was prepared in methanol. The reaction mixtures with appropriate blanks were incubated at 37 °C for 30 min. The reactions were stopped by the addition of 500 ll methanol. The samples were centrifuged at 12,000 rpm for 5 min. The resulting supernatants were analyzed for ammonia released during the reaction using phenol-hypochlorite assay. 2.7. Testing plasmid stability Plasmid stability testing was carried out at different stages of cultivation i.e. pre-seed (at 0 and 9 h), seed (at 12 h) and production medium (at 12 h) as described by Zhang et al. (2003). Two combinations were used at each stage, one containing only SOB
Please cite this article in press as: Sohoni, S.V., et al. Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.02.038
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and the other containing SOB with 1% glucose. 2X YT agar plates with and without 150 lg/ml carbenicillin were used for testing plasmid stability. Samples taken for plasmid stability were diluted and plated on non selective 2X YT agar plates. The plates were incubated at 32 °C for 16–20 h. 100 colonies were randomly picked and spotted on 2X YT plates with and without 150 lg/ml carbenicillin. The plates were incubated at 32 °C for 9–12 h. The colonies were counted and the ratio of number of colonies on plates with and without carbenicillin was used to calculate percentage stability. 3. Results and discussion 3.1. Batch fermentation Initial batch fermentations were carried out in LB medium. SOB medium is rich in nutrients compared to LB and it additionally contains MgCl2 that is essential for growth of E coli. Hence batch fermentation was also carried out in SOB medium. Induction with IPTG was performed after 3 h of growth. Table 1 summarizes nitrilase production in (u/l) and (u/g DCW) in both media. Nitrilase activity per unit biomass in LB medium at 5 h was 0.14 103 u/g DCW. Nitrilase activity per unit biomass in SOB medium was 3.4 103 u/g DCW. SOB medium demonstrated better activity due to its higher nitrogen content and growth. Hence SOB was used as a basal medium in all the experiments in this study. 3.2. Effect of different carbon substrates Carbon substrates have been successfully used in complex media for HCDF of E. coli to improve biomass and thereby the amount of protein produced (Panda et al., 2000; Tabandeh et al.,
2004). The vector pET21a used in this study contains T7 promoter controlled by lac operator. T7 RNA polymerase required to transcribe T7 promoter is expressed in E. coli BL21 (DE3) strain under lacUV5 promoter. Lac promoter is repressed by glucose (Grossman et al., 1998). However, presence of glucose in the medium is reported to enhance plasmid stability (Zhang et al., 2003) and hence effect of different substrates was studied to improve nitrilase production in SOB medium. Different sugars such as glucose, sucrose, maltose and lactose were added at 1% concentration in SOB medium. Glycerol was added at 30 mM final concentration. The cultures were induced with 1 mM IPTG after 3 h of growth. Fig. 1A represents OD profile at 600 nm and Fig. 1B represents nitrilase production profile with different substrates. Maltose demonstrated highest growth of all followed by lactose and glycerol. Glucose and sucrose exhibited poor growth compared to other substrates. Maltose, glucose and sucrose did not result in higher nitrilase production compared to control. Lactose on the other hand even if acts as an inducer of lac promoter, did not support highest nitrilase production. Maltose, lactose and sucrose yield glucose upon hydrolysis and hence it is possible that nitrilase production was repressed in presence of these sugars. Glycerol demonstrated highest nitrilase production amongst all the substrates (2.7 103 u/l). Hence glycerol was chosen to be used as a substrate for further experiments.
3.3. Effect of IPTG IPTG is used as an inducer of lac operon but is also shown to repress growth (Marbach and Bettenbrock, 2012). P. fluorescens nitrilase was expressed under the control of T7 promoter and lac operator while, T7 polymerase is expressed in E. coli BL21 (DE3) under the control of lac UV5 promoter which could be induced
Table 1 Nitrilase activity obtained in different media combinations of batch/fed-batch cultivations in shake flasks and bioreactor. Initial medium
Dose given
Nitrilase production (u/l)
Nitrilase activity (u/g) of DCW
LB SOB SOB + 30 mM SOB + 30 mM SOB + 30 mM SOB + 30 mM
Batch run Batch run Batch run 1% (final concentration) yeast extract dose at 6 h 30 mM (final concentration) glycerol dose at 9 h 1% (final concentration) yeast extract dose at 6 h & 30 mM (final concentration) glycerol dose at 9 h 1% (final concentration) yeast extract dose at 6 h, 30 mM (final concentration) glycerol dose at 9 h and 1% (final concentration) yeast extract 30 mM (final concentration) glycerol dose Continuous feeding of combination of yeast extract (200 g/l) and glycerol (67 g/l) at 23 ml/h
2.06 102 10.8 103 53.5 103 64.5 103 57.7 103 106.8 103
0.14 103 3.4 103 8.8 103 10 103 9 103 16 103
210.7 103
23.9 103
419.2 103
21 103
glycerol + 10 mM glycerol + 10 mM glycerol + 10 mM glycerol + 10 mM
MgCl2 MgCl2 MgCl2 MgCl2
SOB + 30 mM glycerol + 10 mM MgCl2
SOB + 30 mM glycerol + 10 mM MgCl2 in 2 l bioreactor
Fig. 1. Effect of different substrates on nitrilase production: E. coli BL21 (DE3)_PF nit strain was grown in the shake flasks with SOB medium containing different substrates. The culture were induced with 1 mM of IPTG at 3 h (A) represents OD profile at 600 nm and (B) nitrilase production profile represented in u/l. SOB control (-d-), SOB with 1% glucose (-j-), SOB with 1% sucrose (--), SOB with 1% lactose (-.-), SOB with 1% maltose(-N-) and SOB with 30 mM glycerol (-X-).
Please cite this article in press as: Sohoni, S.V., et al. Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.02.038
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Fig. 2. Effect of IPTG induction on nitrilase production: E. coli BL21 (DE3)_PF nit strain was grown in the shake flasks with SOB medium. The culture was induced with different concentrations of IPTG (A) represents OD profile at 600 nm and (B) nitrilase production profile represented in u/l. No induction (-d-), induction with 0.1 mM IPTG (j-), 0.25 mM IPTG (--), 0.5 mM IPTG (-.-), 1 mM IPTG (-N-).
by IPTG or lactose. E. coli BL21 (DE3)_pET21a_PF nit strain was inoculated in SOB containing 30 mM glycerol. In order to find out the optimal concentration of IPTG required for induction, cultures were induced at 3 h with different concentrations of IPTG (0, 0.1 mM, 0.25 mM, 0.5 mM and 1 mM). Fig. 2A represents OD profile at 600 nm and Fig. 2B represents nitrilase production profile at different concentrations of IPTG. IPTG induction did not affect growth (Fig. 2A) but seemed to reduce nitrilase production (Fig. 2B). Maximum nitrilase production was observed in the flask without IPTG induction with nitrilase activity per unit biomass of 8.8 103 u/g DCW. T7 promoter is known to yield steady basal level expression, even in absence of an inducer (Pan and Malcolm, 2000). Induction with IPTG leads to high levels of protein production, sometimes improperly folded into inclusion bodies, making it inactive (Makrides, 1996). The results from SDS PAGE gel in this study support these observations. During the course of this study protein production in each flask was assessed in soluble and insoluble fraction. The flasks with IPTG yielded greater protein in the insoluble fraction (i.e., in the form of inclusion bodies) compared to the soluble fraction (data not shown). Hence reduced nitrilase activity in presence of IPTG can be attributed to formation of inclusion bodies. Clearly, the promoter showed leaky expression. Since the nitrilase produced is not toxic to E. coli cells and resulted in higher soluble nitrilase production than in cultures induced with IPTG, all the remaining experiments were performed without IPTG induction. 3.4. Determination of plasmid stability Plasmid stability at various stages of nitrilase production was determined for improving nitrilase production. Pre-seed and seed stages of cultures were grown in SOB medium with and without glucose. Production medium on the other hand contained SOB with 30 mM glycerol with and without glucose. Plasmid stability was assessed in the culture for Pre-seed at 0 and 9 h, seed at 12 h and production medium at 12 h. Glucose is reported to improve plasmid stability by repressing lac UV5 and T7 promoter (Zhang et al., 2003). Hence two combinations were set up at each stage of cultivation for testing plasmid stability. One of them contained only SOB while, the other combination contained SOB with 1% glucose. Plasmid stability was comparable in both the combinations at pre-seed and seed levels (Table 2). In production medium however only 47% of the cells contained plasmid in only SOB while, 70% cells contained plasmid in SOB with 1% glucose (Table 2). Excessive protein synthesis in the production medium may have led to the selection of cells without plasmids and hence reduction in plasmid stability at production stage compared to seed. Medium combination with 1% glucose demonstrated better plasmid stability than
one without glucose and hence effect of glucose on nitrilase production was further studied. 3.5. Effect of glucose on nitrilase production Glucose in SOB medium led to improved plasmid stability. Hence effect of glucose on nitrilase production was studied. Three combinations namely, SOB with 30 mM glycerol as control and SOB with 30 mM glycerol containing either 0.5% or 1% glucose were used. Fig. 3A represents OD profile at 600 nm and Fig. 3B represents nitrilase production profile at different glucose concentrations. Less growth was observed in combinations containing glucose than those in without glucose (Fig. 3B). This result was in disagreement with Fig. 1B (studying effect of carbon substrates). In Fig. 1B experiment the culture was induced with IPTG after 3 h of growth. It is reported that LacUV5 promoter shows limited decreased dependency on cAMP and can be efficiently induced in presence of glucose (Hirschel et al., 1980; Studier, 1991). The activity obtained in Fig. 1B could be attributed to IPTG induction. The current experiment did not involve IPTG induction step and could be the possible reason for disagreement. No nitrilase production was observed in the combination with 1% glucose without IPTG induction. Nitrilase production was repressed for 8 h in combination with 0.5% glucose. A combination without glucose demonstrated highest nitrilase production. Even though glucose improved plasmid stability, it did not enhance the resulting protein production. Hence glucose was not incorporated in the medium in further studies. 3.6. Effect of temperature Temperature plays an important role in protein production. Soluble protein expression increases when the temperature is lowered (Weickert et al., 1996). E. coli BL21 (DE3)_pET21a_PF nit
Table 2 Effect of glucose on plasmid stability – Plasmid stability is reported as the ratio of number of colony forming units on plates with and without carbenicillin was used to calculate percentage (%) of plasmid stability in medium with and without glucose. Stage
+Glucose (%)
Pre-seed Plasmid stability Pre-seed Plasmid stability Seed Plasmid stability Production Plasmid stability
98
96
Glucose (%)
95
95
93
92
71
40
(at 0 h) (at 9 h) (at 12 h) (at 12 h)
Please cite this article in press as: Sohoni, S.V., et al. Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.02.038
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Fig. 3. Effect glucose on nitrilase production: E. coli BL21 (DE3)_PF nit strain was grown in the shake flasks with SOB medium containing 30 mM glycerol with 0.5 and 1.0% glucose. SOB with 30 mM glycerol was used as a control (A) represents OD profile at 600 nm and (B) nitrilase production profile represented in u/l. 0.5% glucose (-d-), 1% glucose (-j-) and (-.-) without glucose.
Fig. 4. Effect of temperature on nitrilase production: E. coli BL21 (DE3)_PF nit strain was grown in the shake flasks with SOB medium containing 30 mM glycerol. (A) Represents OD profile at 600 nm and (B) nitrilase activity profile represented in u/l. 37 °C (-d-), 32 °C (-j-), 27 °C (--).
strain was inoculated in SOB containing 30 mM glycerol at three different temperatures (27 °C, 32 °C and 37 °C) to deduce optimal temperature for soluble nitrilase production. These cultures were not induced with IPTG. Fig. 4A represents OD profile at 600 nm and Fig. 4B represents nitrilase production profile at different temperatures. Maximum nitrilase production was observed at 32 °C. However, the expression of soluble protein was not found to be higher at 32 °C when compared with that at 37 °C or 27 °C (data not shown). 3.7. Optimization of fed-batch fermentation Fed-batch experiments were set up to improve nitrilase production further. PF nitrilase expressed in E. coli BL21 (DE3) is growth associated and hence the aim was to maximize the biomass. Feeding of a desired carbon and nitrogen substrate in an optimal ratio is crucial. In order to get an idea of feeding strategy, glycerol and yeast extract were fed to the culture growing in SOB containing 30 mM glycerol at different time points. Initial fed-batch experiments were carried out in shake flasks. All the experiments were monitored for 24 h. Doses of glycerol and yeast extract were given at different time points during cultivations. Table 1 represents various combinations of doses tried and the resulting nitrilase production (u/l) as well as nitrilase activity per unit biomass (u/g DCW). Batch fermentation in SOB with 30 mM glycerol resulted in 5.3 103 u/l nitrilase production and nitrilase activity per unit biomass of 8.8 103 u/g DCW. SOB containing 30 mM glycerol was used as a basal medium when developing fed-batch fermentations. Individual addition of yeast extract (6 h) or glycerol (9 h) did not lead to significant increase in nitrilase activity per unit biomass (Table 1). However, sequential dosing of glycerol and yeast extract (yeast extract
dose at 6 h, glycerol dose at 9 h and yeast extract- glycerol mixed dose at 12 h) resulted in fourfolds increase in the production of nitrilase and 2.8-folds increase in nitrilase activity per unit biomass (Table 1).
3.8. Fed-batch fermentation in bioreactor According to the results from fed-batch experiments in shake flasks, bioreactor was set up with initial medium SOB containing 30 mM glycerol. Continuous feeding strategy was used here in which yeast extract (200 g/l) and glycerol (67 g/l) was fed at the flow rate of 23 ml/h after 4 h of growth. Fig. 5A represents growth and nitrilase production profile and Fig. 5B represents data from exit gas analyzer. Exit CO2 (%) demonstrated a sharp peak at 5 h representing highly active state of culture. CO2 (%) reduced thereafter and remained constant at 0.5% throughout the cultivation. Continuous feeding resulted in 19.5 g/l DCW and 419 103 u/l nitrilase production. Liu et al. (2011) optimized glycerol fed-batch strategy and reported 19.3 103 u/l nitrilase production. Continuous feeding strategy improved nitrilase production by eightfolds and nitrilase activity per unit biomass by 2.4-folds compared to optimized batch fermentations in this study (Table 1). The nitrilase activity per unit biomass obtained in this study was 21.4 103 u/g DCW, while Liu et al. (2011) report nitrilase activity of 1.8 103 u/g DCW. Liu et al. (2011) report feeding of glycerol in the fed-batch mode, while in this study a mixture of yeast extract and glycerol was fed. Here twelvefolds increase in nitrilase activity per unit biomass was reported compared to the report from Liu et al. (2011).
Please cite this article in press as: Sohoni, S.V., et al. Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.02.038
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S.V. Sohoni et al. / Bioresource Technology xxx (2015) xxx–xxx
Fig. 5. Scale up in 2 l bioreactor: E. coli BL21 (DE3)_PF nit strain was grown in 2 l bioreactor with SOB medium with 30 mM glycerol. Continuous feeding of mix of yeast extract and glycerol was achieved at 23 ml/h after 4 h of growth for 24 h. (A) Represents OD profile at 600 nm (-j-), dry cell weight g/l (-d-), nitrilase production profile u/l (.-) and glycerol consumption (-N-) (B) exit gas profile – solid line representing % O2 and dotted line representing %CO2.
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Please cite this article in press as: Sohoni, S.V., et al. Optimization of high cell density fermentation process for recombinant nitrilase production in E. coli. Bioresour. Technol. (2015), http://dx.doi.org/10.1016/j.biortech.2015.02.038