Original Research

Enhancing the soluble expression of recombinant chitobiase using osmolytes in Escherichia coli

Arun Kumar Dangi1, Praveen Rishi 2, Rupinder Tewari1*

1 Department of Microbial Biotechnology, Panjab University, Chandigarh, India
2Department of Microbiology, Panjab University, Chandigarh, India

J Innov Biol (2014) Volume 2, Issue 4: Pages: 250-253

Abstract: Chitobiase (CHB) is an important enzyme for the production of N-acetyl-D-glucosamine from the chitin polysaccharide in the series of chitinolytic enzymes. Majority of over-expressed CHB (58%) in Escherichia coli expression system led to formation of inclusion bodies even after optimization of inducer concentrations and culture conditions. The production and solubility of active CHB was enhanced by supplementation of osmolytes in the culture medium and optimizing culture conditions in the presence of osmolytes. The presence of sorbitol (0.25 M) in the culture medium after optimization of conditions significantly enhanced the yield of active CHB at 37oC. Approximately 1.7 fold increase in the activity of over-expressed CHB was observed which is step forward in replacing hazardous chemical technology by biotechnological process for the production of NAG from chitinous waste.

Received: 05 December 2015
Accepted: 19 December 2015
Published Online: 27 Decenber 2015


Corresponding Author:
Tiwari R.,
Department of Microbial Biotechnology, Panjab University, Chandigarh, India
email: rupinder@pu.ac.in

Keywords: chitobiase, chitin, osmotic stress, osmolytes

Introduction

IntroductionReferences
The over-expression of industrially important proteins in Escherichia coli has become widely used method to get the higher yield due to its simple approach, easy to culture, fast growth rate, availability of genetic information (Rosano and Ceccarelli 2014). However, the soluble yield of the expressed proteins is often reduced by mis-folding and aggregation of the newly formed heterologous proteins also known as inclusion bodies (Thomas and Baneyx 1996). Many factors are responsible for the formation of inclusion bodies such as protein expression rate, intrinsic nature of recombinant protein or intermediates formed during protein folding, viscosity and temperature of the cellular environment (Baneyx and Mujacic 2004). Protein aggregation may also cause some neurodegenerative diseases like Alzheimer’s disease, cystic fibrosis, and Huntington’s disease (Irvine et al. 2008; Ross and Poirier 2004).
Generally, bacteria have two important mechanisms to combat the inclusion bodies formation by using molecular chaperones and chemical chaperones also known as osmolytes (Saunders et al. 2000). Several reports have been shown that osmolytes (small organic molecules) significantly enhances the yield of soluble recombinant protein by reducing the inclusion bodies formation (de Marco et al. 2005; Oganesyana et al. 2007; Thapliyal and Chattopadhyay 2015). The solvophobic thermodynamics is mainly responsible for the in vivo folding the proteins into their native states. These small organic molecules enter into the cytoplasm of the cell by crossing the cell membrane results formation of the crowded environment. The high concentration of osmolytes stabilizes the structure of newly formed proteins by involving strong exclusion of the osmolytes from the surface of proteins resulting increase the free energy of the unfolded peptide by unfavorable interaction with the peptide backbone, thus push the equilibrium of protein folding toward the native state (Wu and Bolen 2006).
Chitobiase (CHB; EC 3.2.1.52 ) also known as β-N-acetylglucosaminidase is one of the important enzyme used for the chitin degradation which is second most biopolymer present in nature after cellulose(Dahiya et al. 2005). This enzyme typical not acts directly on chitin polymer instead degrade the chito-oligosaccharides, especially chitobiose produced by chitinase (EC 3.2.1.14) which hydrolyze the internal β-1,4linkages in chitin polymers. CHB acts on terminal non-reducing β-1,4linkages of chitobiose, a dimeric product results production of medically and industrially important monomer called as n-acetyl glucosamine (NAG) (Chen et al. 2010). The gene (nagZ) from E. coli K12 strain was isolated and cloned into E. coli M15 by corresponding author of this manuscript (Kumar et al. 2011). Although, large amount of recombinant CHB was produced but major fraction was in the form of inclusion bodies. In the present study, we have investigated the effects of various osmolytes to enhance the soluble expression of CHB in E. coli M15 cells. These osmolytes were supplemented in the growth media.

MATERIALS AND METHODS

Materials, plasmid and bacterial strain
E. coli M15 cells harboring a plasmid, pQE-30 containing nagZ gene, under the control of T5 promoter which corresponds to expression of CHB was taken from the laboratory of corresponding author. Isopropyl β-D-1-thiogalactopyranoside (IPTG), p-nitrophenyl-β-N-acetyl-D-glucosaminide (pNP-NAG) and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma-Aldrich while rest of the reagents and chemicals used in this study were purchased from Hi Media Laboratories (India).

Effect of osmolytes on solubility of CHB
The recombinant E. coli M15 cells were grown overnight in 20 ml Luria Broth (LB) having ampicillin (100 µg/ml) and kanamycin (25 µg/ml) at 37oC, 200 rpm in orbital shaker. To perform the expression, 1% (v/v) of overnight grown culture was inoculated into 50 ml LB broth media containing necessary antibiotics in 250 ml flask and incubated at 37oC, 200 rpm. Effect of different osmolytes as chemical chaperones on the in vivo solubility of CHB was investigated by adding various osmolytes (Table 1) in the culture medium before 30 min of IPTG induction when OD600 was reached at 0.4-0.5. The CHB was induced by 0.5 mM IPTG (already optimized) at an OD600 of 0.6-0.7. The cells were further grown at 37oC, 200 rpm till final OD600 2.0, post IPTG induction and cells were harvested by centrifugation (8000×g, 4oC for10 min). The pellets were resuspended in 10 ml lysis buffer (50 mM Tris-HCl, 300 mM NaCl with 1mM PMSF, pH 7.5), lysed by sonication (10 cycles, 30 sec on and 30 sec off, 24%) and centrifuged (12000×g, 4oC for 10 min). The cell free supernatant was preserved for chitobiase activity assay.

Optimization of conditions
Various conditions such as temperatures (25oC, 30oC and 37oC), sorbitol concentrations (0.1 – 1.5 M) and sequence of sorbitol supplementation (in the medium, before 30 min, simultaneous, after 30 min of IPTG induction) were also optimized in 50 ml LB broth media for the higher expression of soluble recombinant CHB enzymes.

Chitobiase activity assay
The activity of CHB was determined by measuring the release of p-Nitrophenol (pNP) form the pNP-NAG(Cheng et al. 2000), with slight modifications. The reaction mixture containing 0.5 ml of pNP-NAG (1 mM, prepared in 50 mM Tris-HCl, pH 7.5) and 0.4 ml of diluted enzyme followed by incubation at 37°C for 15 min. The reaction was stopped by adding 0.2 ml of K2CO3 (1.25 M). The amount of pNP liberated was estimated by measuring the absorbance at 420 nm with reference to the pNP standard curve. Enzyme activity was expressed as the amount of enzyme required to liberate 1 µmol of pNP per min at 37°C.

SDS-PAGE analysis
The expression of protein was determined using SDS-PAGE in 12% polyacrylamide gel according the method described by Laemmli et al. (1970). The samples were mixed with SDS loading dye (5 X), boiled for 5 min and loaded in equal volume (10 µL) on 12% SDS-PAGE gel. The gel was stained with Coomassie brilliant blue R-250 and destained with destaining solution.

RESULTS AND DISCUSSION
Chitobiase is one of the important enzymes which produce the NAG (a globally acclaimed pain killer in osteoarthritis) from hydrolysis of chitobiose (a dimeric product of chitin hydrolysis). Previously, we successfully cloned and over expressed the CHB in E. coli M15. But very low amount of CHB (~42%) was obtained in soluble and active form while major fraction of expressed CHB (~58%) was in the form of inclusion bodies (Fig. 1) even culture grown at lower temperature(Kumar et al. 2011). In the present study, we further extended the work and tried to enhance the yield of soluble CHB by providing the osmotic stress to the cell by supplementing the various osmolytes in culture medium. The osmolytes used in this study is described in Table 1.

Effect of osmolytes
E. coli M15 cells harboring the recombinant plasmid (pSTR1) under T5 promoter, corresponded to expression of CHB enzyme with molecular weight ~38.5 kDa was used to enhance the solubility of CHB in the presence of small organic molecules which can act as chemical chaperones or osmolytes caused osmotic stress to cell (Thapliyal and Chattopadhyay 2015). These osmolytes protect the native structure of proteins and compatible with the structure even at higher concentration(Ma et al. 2010). From all the osmolytes used in this study it was found that addition of 0.5 M sorbitol in culture media before 30 min of IPTG induction (0.5 mM) showed maximum increase in the activity of CHB (470.50 IU/ml) than the control without osmolytes (320.12 IU/ml). This increase in the activity was 1.56 fold higher at 37oC (Fig. 2). This enhanced activity in the presence of sorbitol might be due the stabilizing nature of sorbitol. It has the ability to inhibit the unfolding of native confirmation to unfolded state in intracellular space by entering into E. coli via sorbitol-specific phosphoenolpyruvate phosphotransferase system (Prasad et al. 2011).

Optimization of temperature conditions
The effect of above osmolytes on the soluble expression of CHB was also determined at 30oC and 25oC (Table 2) and it was observed that at both the temperatures only 10 mM benzyl-alcohol (BA) showed the significant increase in activities which were 332 IU/ml and 238 IU/ml corresponding to 1.39 and 1.32 fold increase at 30oC and 25oC respectively, but these values were less than the obtained values with 0.5 M sorbitol at 37oC. In addition, reducing the cultivation temperature not only hampers cell growth but also cooling instruments to maintain the constant incubation temperature. Thus, further studies were carried out at 37oC in the presence of sorbitol.

Optimization of sorbitol concentrations
The soluble expression of CHB in E. coli M15 was optimized by change in the activities in the presence of variable concentrations of sorbitol (0.1-1.5 M) supplemented into the culture medium before 30 min of IPTG (0.5 mM) induction and results were depicted in (Fig. 3). It was found that the maximum activity (516.6 IU/ml) was obtained in the presence 0.25 M sorbitol corresponding to increase of 1.54 fold. Further increase in concentration of sorbitol results gradual decrease in the activity of CHB was observed. Thus, further experiments were performed at 0.25 M of sorbitol at 37oC.

Sequence of sorbitol addition
The sequence of supplementation of sorbitol into the culture media was carried out into four sets. First, 1% (v/v) of overnight culture was added in to LB media already supplemented with previously optimized sorbitol concentration (0.25 M). In second set, the sorbitol was added before 30 min of IPTG induction, in third set of experiment, sorbitol was added simultaneously and in the last set, sorbitol was added after 30 min of IPTG induction and results were summarized into (Fig. 4). The maximum CHB activity (545.45 IU/ml) was observed when sorbitol was added at the time of inoculation into LB media which was 1.7 fold higher than the control without sorbitol (320.72 IU/ml). Sorbitol has been very well documented as chemical chaperone for the in vivo folding of many proteins such as dihydrofolate reductase (1.6 fold), green florescent protein (1.5 fold), human Hep27scFv fragment (1.5 fold) and Chandipura virus P-protein showing 80% enhanced soluble expression in E. coli(Majumder et al. 2001; Prasad et al. 2011; Sandee et al. 2005; Thapliyal and Chattopadhyay 2015).

Conclusions
Using optimized culture conditions in the presence of osmolytes especially sorbitol, nearly 1.7 fold increases in the activity of recombinant CHB was obtained which is directly correlated to the increase in the yields of soluble and functionally active enzyme. These results suggested that the in vivo supplementation of osmolytes in optimized concentration could improve the soluble expression of recombinant proteins in E. coli. The potential of this process to be scaled up for large scale production of CHB and a step forward in designing a biotechnological route for NAG production that can replace existing chemical method.

Acknowledgements
This work was financially supported by CSIR-UGC, Government of India for the fellowship.
The authors declare that they have no conflict of interest.

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