High-level Expression and Characterization of Thermostable Esterase from Thermoanaerobacter Tengcongensis in Escherichia coli

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1 High-level Expression and Characterization of Thermostable Esterase from Thermoanaerobacter Tengcongensis in Escherichia coli Ruobing Deng, Xun Li, Hong Gao, Fei Wang China College of Chemical Engineering, Nanjing Forestry University, Nanjing , China Jiangsu Key Lab of Biomass-Based Green Fuels and Chemicals, Nanjing , China Abstract A novel thermostable esterase from Thermoanaerobacter tengcongensis MB4T was successfully over-expressed in Escherichia coli and characterized. Two plasmids pet28a (T7 strong promoter) and ptrc99a (trc strong promoter) were used as the expression vectors. The results indicated that plasmid ptrc99a was more suitable for this esterase expression and the efficiency of the trc promoter for our analysis is much higher than T7 promoter. The recombinant esterase, having a molecular mass of 43 kda determined with SDS-PAGE, was purified to homogeneity through heat treatment and DEAE-SepharoseCL-6B. The ptrc99a-est esterase showed maximum activities towards short-chain p-np esters (C4). The esterase was optimally active at 70 0 C (over 15 min) and at ph 7.5. It is highly thermostable, with a residual activity greater than 80% after incubation at 70 0 C for more than 2 h. Key words: Esterase, over-expression, characterization, Thermoanaerobacter tengcongensis, E.coli Paper PS-10 1 of 8

2 Introduction Lipolytic enzymes (EC x), represent a hydrolases group, which specifically works over carboxylic ester, and hence has a potentially broad spectrum of biotechnological uses. Lipolytic enzymes can be classified into three main groups: esterase/carboxylesterase (EC , carboxyl ester hydrolases), which prefer water-soluble substrates and catalyze the hydrolysis of glycerol esters with short acyl chain ( 10 carbon atoms) to partial gly cerides and fatty acids; true lipases (EC , triacylglycerol hydrolases), which display maximal activity towards water- insoluble long-chain triglycerides( 10 carbon atoms); and various types of phospholipase (Arpigny et al. 1999, Pleiss, J. et al. 1998). The three-dimensional (3D) structures of both enzymes show the characteristic -hydrolase fold (David L. et al. 1992). Recently, esterase has been identified containing a Gly-x-x-Leu motif (Wei Y. et al. 1995) as well as enzymes showing high homology to class α/β-lactamases (Petersen et al. 2001). Moreover, Esterase plays a major role in degradation of synthetic materials and the synthesis of optically pure compounds, perfumes, and antioxidants (Panda, T. et al. 2005). As applications for esterase are found in various fields, there are growing interest in this enzyme. Furthermore, the esterase in this report is not a true lipase but a novel thermostable esterase (Zhang, J. et al. 2003), which have been isolated from thermophilic organism, Thermoanaerobacter tengcongensis (Xue, Y. et al. 2001), and this kind of enzymes have been found a number of commercial applications because of their overall inherent stability ( Demirijan, D. et al. 2001), such as the field of the petroleum, chemical and pulp and paper industries, for example, thermostable enzymes have been used for the elimination of sulphur containing pollutants through the biodegradation of compounds like dibenzothiophene ( Bahrami, A. et al. 2001). Besides, the gene of this enzyme has been cloned and expressed in E.coli by using the plasmid pet23b as the vector (Zhang, J. et al. 2003), E. coli remains the most widely used host for recombinant protein expression. It is easy to transform, grows quickly in simple media, and requires inexpensive equipment for growth and storage. Different strategies like promoter strength (Boer, H. et al. 1983), Combination of plasmids (Khushoo, A. et al. 2005) were used to overcome the limitations. In this work, we compared the expression of the thermostable esterase from Thermoanaerobacter tengcongensis cloned in two different plasmids pet28a (T7 strong promoter) and ptrc99a (trc strong promoter) in order to find a suitable plasmid for high-level expression of this esterase. As a result we found that ptrc99a (trc strong promoter) was more suitable for this esterase expression. Then we purified and characterized the esterase cloned in plasmid ptrc99a. This knowledge may be applied in the strategy for the selection of suitable plasmid for this esterase expression. Paper PS-10 2 of 8

3 Results Cloning and Overexpression of Est from T. tengcongensis in E. coli Genomic DNA obtained from the T. tengcongensis was used as a PCR template. A DNA fragment of about 1100 bp length was obtained with PCR amplification with primers described above, and was confirmed by the sequence analysis. The expression plasmid ptrc99a-est and pet28a-est were successfully constructed and were transformed into E. coli TOP10 and E. coli BL21DE3 respectively for protein expression. For strain Top10/pTrc-99A-est, the expression of the esterase was confirmed by SDS-PAGE analysis (Fig.1, lane 1). And the molecular weight of this protein (43 kda) on SDS-PAGE (Fig.1, lane 2) was found consistent with the expected molecular weight size. As a negative control, no T. tengcongensis esterase protein or activity was detected in the vector-transformed strain (Fig.1, lane 1). However, the result of the enzyme expression in strain BL21DE3/pET-28a-est (Fig.2), shows that the esterase was not successfully over-expressed with pet-28a in E. coli. Figure 1. Overexpression and purification of T. tengcongensis lipase (est) in E. coli. Lane M1, Lane M2, protein molecular weight markers (in kda); lane 1, total proteins from strain Top10/pTrc-99A; lane 2 total proteins from strain Top10/pTrc-99A::est; lane 3, supernatant of concentrated proteins fromtop10/ ptrc-99a::est. lane 4, purified lipase. Figure 2. Overexpression and purification of T. tengcongensis lipase (est) in E. coli. Lane M, protein molecular weight markers; lane 1, total proteins from strain BL21DE3 (condon plus)/pet-28a; lane 2 total proteins from strain BL21DE3 (condon plus)/pet-28a::est Paper PS-10 3 of 8

4 Enzyme purification Purfication was achieved by heat treatment and DEAE Sepharose Fast Flow. Table 1 depicts specific activity and the purification fold after these steps. The purity of the esterase after the purification increased approximately 12-fold over the crude extract. The purified lipase showed a single band in 12% SDS PAGE gel (Fig.1, lane 4). Total protein (mg) Total acctivity (U) Specific activity (U/ mg) yield (%) Purification (fold) Crude extract Heat treatment DEAE-SepharoseCL-6B Table 1 Activity and yield of the recombinant esterase in purification procedure Substrates specificity The esterase expressed with ptrc99a showed maximum activities towards short-chain p-np esters (C2 and C4), middle activities towards middle-chain p-np esters (C6 to C10) and little activity towards long-chain p-np ester (C16) (Fig. 3). However the esterase expressed with pet-28a showed little activity towards all of these substrates. Figure 3. Determination of the substrate specificity of the esterase (Est). The p-np-acetate (C2), p-np-butyrate (C4), p-np-caproate (C6), p-np-caprate (C10), and p-np-palmitate (C16) were selected as the substrates. The reactions were conducted using the standard assay conditions (see Materials and methods section), and the highest esterase activity towards p-np-butyrate (C4) was defined as the 100% level (about 99 U mg 1). Effects of ph and temperature on the esterase activity The ph profiles showed that the esterase was active over a broad ph range (ph ) and the optimum ph was 7.5 (Fig. 4B). The temperature activity curve for the recombinant lipase Paper PS-10 4 of 8

5 was shown in Fig. 4A. The temperature for the optimum of the esterase was 70 0 C. The thermal stability of the esterase was studied by measuring the residual activity after incubation for different periods at temperatures ranging between 65 and 80 0 C. The half life of the thermostability at 80 0 C was more than 60 min. (Fig. 4C). Figure 4. Effects of temperature and ph on esterase activity and stability. (A) Determination of esterase activity at various temperatures at ph 7.5 for 15 min, using p-np- butyrate as a substrate. The highest activity at 70 0 C was defined as 100% level (5.014 U ml 1 ). (B) Determination of esterase activity at various phs at 65 0 C for 15 min, using p-np- butyrate as a substrate. The highest activity at ph 7.5 was defined as 100% level (3.51 U ml 1 ). (C) Determination of the effects of temperature on esterase stability by incubating the purified esterase at various temperatures for up to 2 h and measuring the remaining activity. The original enzyme activity before treatment was defined as 100% (4.01 U ml 1 ). (D) Determination of the effects of ph on esterase stability by incubating the purified esterase at various phs for up to 2 h and measuring the remaining activity. The original enzyme activity before treatment was defined as 100% (3.008 U ml 1 ). Discussion Our results showed that the recombinant esterase of T. tengcongensis MB4T was successfully over-expressed in E. coli. The expression level of the esterase in ptrc99a was about 100 times higher than that in pet-28a. A number of central elements are essential in the design of Paper PS-10 5 of 8

6 recombinant expression systems (Baneyx, F. 1999, Jonasson, P. et al. 2002). Promoter strength affects the amount of transcripts produced and thereby increasing the protein expression. So the plasmids we selected in this study (ptrc99a and pet-28a) were both controlled by strong promoter (trc and T7). However the result shows that the efficiency of the trc promoter for our analysis is much higher than T7 promoter. Briefly many different types of promoters can affect the level of gene expression in E. coli and suitability of promoters is important for hith-level expression (Hannig, G. and Makrides, S.C. 1998). Furthermore, the copy number of the expression vector is an important factor for high-level expression (Kobayashi, M. et al. 1991). Plasmid ptrc99a is a high-copy-number of about 100 (Amann, E. et al. 1988), while plasmid pet-28a is a medium-copy-number of copies/cell (Kleefeld, A. et al. 2009). It is consistent with the result of our study. In addition, a highly repressible promoter is particularly important for case in which the protein of interest is toxic or detrimental to the growth of the host cell (Hannig, G. and Makrides, S.C. 1998, Amann, E. et al. 1988, Yike, I. et al. ). So the reason why pet-28a can not be suitable for this esterase expression maybe the target protein expressed with pet28a is toxic or detrimental to the growth of to the host cell E. coli BL21DE3. The recombinant enzyme was purified by the heat treatment and DEAE Sepharose Fast Flow presented as a single protein band on SDS-PAGE with a molecular weight of 43 kda. In addition, the result shows that this esterase is indeed highly thermostable. The studies on substrate specificity demonstrated that the recombinant esterase had the strong catalytic ability to the substrates with short carbon chain. These properties would be important for the lipase to be applied in industry. However, modern methods of enzyme engineering, especially directed evolution will certainly provide suitable esterase variants with increased use in organic synthesis and other areas of application in the near future. Acknowledgments This work was supported by grants from the programs of State Forestry Administration, China (No , ), and a project funded by the priority academic program development of Jiangsu Higher Education Institutions (PAPD). Reference Amann, E. Ochs, B. and Abel, K.J Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene. 69: Kleefeld, A. Ackermann, B. Bauer, J. et al The fumarate/succinate antiporter DcuB of Escherichia coli is a bifunctional protein with sites for regulation of DcuSdependent gene expression. Journal of Biological Chemistry. 284: Paper PS-10 6 of 8

7 Arpigny, J.L. Jaeger, K.E Bacterial lipolytic enzymes: classification and properties. Biochem. J. 343: Bahrami, A. Shojaosadati, S. Mahbeli, G Biodegradation of dibenzothiophene by thermophilic bacteria. Biotechnol. Lett. 23: Baneyx, F Recombinant protein expression in Escherichia coli. Curr. Opin. Biotechnol. 10: Boer, H. Comstock, L.J. Vasser, M The tac Promoter: A Functional Hybrid Derived from the trp and lac Promoters. Proc. Natl. Acad. Sci. 80: David, L. Ollis, Cheah, E. et al The α/β hydrolase fold. Protein Engneering. 5: Demirijan, D. Moris-Varas, F. Cassidy, C Enzymes from extremophiles. Curr. Opin. Chem. Boil. 5: Hannig, G. Makrides, S.C Strategies for optimizing heterol- ogous protein expression in Escherichia coli. Trends Biotechnol. 16: Zhang, J. Liu J.f. Zhou, J. et al Thermostable esterase from Thermoanaerobacter tengcongensis: high-level expression, purification and characterization. Biotechnology Letters. 25: Jonasson, P. Liljeqvist, S. Nygren, P.A. et al Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli. Biotechnol Appl Biochem. 35: Khushoo, A. Pal, Y. Mukherjee, K.J Optimization of extracellular production of recombinant asparaginase in Escherichia coli in shake-flask and bioreactor. Appl Microbiol Biotechnol. 68: Kobayashi, M. Kurusu, Y. Yukawa, H High-Expression of a Target Gene and High-Stability of the Plasmid, Applied Biochemistry and Biotechnology. 27: Petersen, E.I. Valinger, G. Solkner, B. et al A novel esterase from Burkholderia gladioli shows high deacetylation activity on cephalosporins is related to -lactamases and DD-peptidases. J. Biotechnol. 89: Pleiss, J. Fischer, M. Schmid, R.D Anatomy of lipase binding sites: the scissile fatty acid binding site. Chem Phys Lipids. 93: Paper PS-10 7 of 8

8 T. Panda, B.S Gowrishankar, Production and applications of esterases, Appl Microbiol Biotechnol. 67, Wei, Y. Schottel, J.L. Derewenda, U. et al A novel variant of the catalytic triad in the Streptomyces scabies esterase. Nat. Struct. Biol. 2: Xue, Y. Xu, Y. Liu, Y. et al Thermoanaerobacter tengcongensis sp. nov., a novel anaerobic, saccharolytic, thermophilic bacterium isolated from a hot spring in Tengcong, China. Int. J. Syst. Evol. Microbiol. 51: Yike, I. Zhang, Y. Ye, J. et al Protein Expression, Purif. 7: Paper PS-10 8 of 8