Characterization and Secretive Expression in Bacillus subtilis of Endoglucanase from Bacillus safensis Isolated from Freshwater Swamp Forest

Pimpikar KANCHANADUMKERNG; Makiko SAKKA; Kazuo SAKKA; Chanpen WIWAT

Authors

Pimpikar KANCHANADUMKERNG Department of Microbiology, Faculty of Pharmacy, Mahidol University, Bangkok 10400
Makiko SAKKA Applied Microbiology Laboratory, Graduate School of Bioresources, Mie University, Mie
Kazuo SAKKA Applied Microbiology Laboratory, Graduate School of Bioresources, Mie University, Mie
Chanpen WIWAT Department of Microbiology, Faculty of Pharmacy, Mahidol University, Bangkok 10400

Keywords:

Bacillus safensis, cellulases, endoglucanase, recombinant expression, freshwater swamp forest

Abstract

Bacillus safensis M3 was newly isolated from freshwater swamp forest soil in western Thailand. The endoglucanase gene of B. safensis M3, cel9A, had an open reading frame of 1,848 bp encoding a 616 amino acid protein. Initial expression in Escherichia coli yielded a low amount of soluble protein in the cytosolic and secreted fractions. Cel9A was successfully expressed by recombinant B. subtilis with a 4-fold greater total enzyme activity than from recombinant E. coli. By SDS-PAGE analysis, the molecular weight of Cel9A was estimated to be 70 kDa. The optimal temperature of Cel9A was 55 °C and the optimal pH was 5 - 8. Cel9A had the highest activity in the pH range from 5 - 8, and the highest stability in pH range 4 to 10, which is useful for industrial applications. Notably, Cel9A was able to hydrolyze both mixed linkage glucan (lichenan) and hemicellulose (konjac glucomannan and oat spelt xylan) better than carboxymethylcellulose. Cel9A also showed a tolerance to metal ions and surfactants. In addition, recombinant B. subtilis with endoglucanase activity has potential for biotechnological applications and benefits in the optimization of large scale enzyme production with minimal medium using agriculture wastes and other inexpensive feedstock materials.

Downloads

Download data is not yet available.

Metrics

Metrics Loading ...

References

V Juturu and JC Wu. Microbial cellulose: Engineering, production and applications. Renew. Sustain. Energ. Rev. 2014; 33, 188-203.

S Sadhu, P Saha, SK Sen, S Mayilraj and TK Maiti. Production, purification and characterization of a novel thermotolerant endoglucanase (CMCase) from Bacillus strain isolated from cow dung. SpringerPlus 2013; 2, 10.

SL Liu and K Du. Enhanced expression of an endoglucanase in Bacillus subtilis by using the sucrose-inducible sacB promoter and improved properties of the recombinant enzyme. Protein Expr. Purif. 2012; 83, 164-8.

S Pandy, J Kushwah, R Tiwari, R Kumar, VS Somvanshi, L Nain and AKS Saxena. Cloning and expression of β-1,4-endoglucanase gene from Bacillus subtilis isolated from soil long and term irrigated with effluent of paper and pulp mill. Microbial. Res. 2014; 169, 693-8.

N Balasubramanian and N Simoes. Bacillus pumilus S124A carboxynethyl cellulose; A thermostable enzyme with a wide substrate spectrum utility. Int. J. Biol. Macromol. 2014; 67, 132-9.

S Ruangsanka. Identification of phosphate-solubilizing bacteria from the bamboo rhizosphere. Science Asia 2014; 40, 204-11.

XZ Zhang and YHP Zhang. One-step production of biocommodities from lignocellulosic biomass by recombinant cellulytic Bacillus subtilis: Opportunities and challenges. Eng. Life Sci. 2010; 10, 398-406.

K Ozaki, Y Hayashi, N Sumitomo, S Kawai and S Ito. Construction, purification, and properties of a truncated alkaline endoglucanase from Bacillus sp. KSM-635. Biosci. Biotech. Biochem. 1995; 59, 1613-8.

JM Liu, XJ Xin, CX Li, JH Xu and J Bao. Cloning of thermostable cellulose genes of Clostridium thermocellum and their secretive expression in Bacillus subtilis. Appl. Biochem. Biotech. 2012; 166, 652-62.

M Lertcanawanichakul and C Wiwat. Improved shuttle vector for expression of chitinase gene in Bacillus thuringiensis. Lett. Appl. Microbiol. 2000; 31, 123-8.

RM Teather and PJ Wood. Use of Congo red-polysaccharide interaction in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl. Environ. Microbiol. 1982; 43, 777-80.

Z Xioa, R Stroms and A Tsang. Microplate-based carboxymethylcellulose assay for endoglucanase activity. Anal. Biochem. 2005; 342, 176-8.

JS Van Dyk, M Sakka, K Sakka and BI Pletschke. The cellulolytic and hemicellulolytic system of Bacillus lichemniformis SVD and the evidence for production of a large multi-enzyme complex. Enzyme Microb. Tech. 2009; 45, 372-8.

S Pandey, S Singh, AN Yadav, L Nain and AN Saxena. Phylogenetic diversity and characterization of novel and efficient cellulose producing bacterial isolates from various extreme environments. Biosci. Biotech. Biochem. 2013; 77, 1474-80.

A Lateef, IA Adelere and EB Gueguim-Kana. Bacillus safensis LAU 13: A new source of keratinase and its multi-functional biocatalytic application. Biotech. Equip. 2015; 29, 54-63.

A Nath, S Datta, R Chowdhurry and C Bhattacharjee. Fermentative production of intracellular β-galactosidase by Bacillus safensis (JUCHE 1) growing on lactose and glucose-Modeling and experimental. Biocatal. Agr. Biotech. 2014; 3, 246-58.

RS Singh and RP Singh. Response surface optimization of endoinulinase production from a cost effective substrate by Bacillus safensis AS-08 for hydrolysis of inulin. Biocatal. Agr. Biotech. 2014; 3, 365-72.

P Vos, G Garrity, D Jones, NR Krieg, W Ludwig, FA Rainey, KH Schleifer and W Whitman. Bergey’s Manual of Systematic Bacteriology. Vol III. The Firmicutes. 2nd ed. Springer, New York, 2009.

M Satomi, T Myron, L Duc and K Venkateswaran. Bacillus safensis sp. nov., isolated from spacecraft and assembly-facility surfaces. Int. J. Syst. Evol. Microbiol. 2006; 56, 1735-40.

AA Telke, SS Ghatge, SH Kang, S Thangapandian, KW Lee, HD Shin and SW Kim. Construction and characterization of chimeric cellulases with enhanced catalytic activity towards insoluble cellulosic substrates. Bioresour. Technol. 2012; 112, 10-7.

S Jindou, Q Xu, R Kenig, M Shulman, Y Shoham, EA Bayer and R Lamed. Novel architecture of family-9 glycoside hydrolases identified in cellulosomal enzymes of Acetivibrio cellulolyticus and Clostridium thermocellum. FEMS Microbiol. Lett. 2006; 254, 308-16.

J Hitomi, Y Hatada, S Kawaminami, S Kawai and S Ito. Amino acid sequence and stereoselective hydrolytic reaction of an endo-1, 4-β-glucanase from a Bacillus strain. Biosci. Biotech. Biochem. 1997; 61, 2004-9.

AOS Lima, MC Quecine, MHP Fungara, FD Andreote, W Maccherono Jr, WL Araujo, MC Siliva-Filho, AA Pizzirami-Kleiner and JL Azevedo. Molecular characterization of a β-1,4-endoglucanse from an endophytic Bacillus pumilus strain. Appl. Microbiol. Biotech. 2005; 68, 57-65.

Y Liu, J Zhang, Q Liu, C Zhang and Q Ma. Molecular cloning of novel cellulase genes cel9A and cel12A from Bacillus licheniformis GXN151 and synergism of their encoded polypeptides. Curr. Microbiol. 2004; 49, 234-8.

S Zhang, QY Yin, YH Li, M Ding, GJ Xu and FK Zhao. Molecular and biochemical characterization of Ba-EGA, a cellulase secreted by Bacillus sp. AC-1 from Ampullaria crosseans. Appl. Microbiol. Biotech. 2007; 75, 1327-34.

A Davison and M Blaxter. Ancient origin of glycosyl hydrolase family 9 cellulase genes. Mol. Biol. Evol. 2005; 22, 1273-84.

YH Li, M Ding, J Wang, GJ Xu and F Zhao. A novel thermoacidophilic endoglucanse, Ba-EGA, from a new cellulose-degrading bacterium, Bacillus sp. AC-1. Appl. Microbiol. Biotech. 2006; 70, 430-6.

XZ Zhang, N Sathitsuksanoh, Z Zhu and YHP Zhang. One-step production of lactate from cellulose as the sole carbon source without any other organic nutrient by recombinant cellulolytic Bacillus subtilis. Metab. Eng. 2011; 13, 364-72.

P Christakopoulos, DG Hatzinikolaou, G Fountoukidis, D Kekos, M Claeyssens and BJ Macris. Purification and mode of action of an alkali-resistant endo-1,4,-β-glucanase from Bacillus pumilus. Arch. Biochem. Biophys. 1999; 364, 61-6.

M Zafar, S Ahmed, MIM Khan and A Jamil. Recombinant expression and characterization of a novel endoglucanase from Bacillus subtilis in Escherichia coli. Mol. Biol. Rep. 2014; 41, 3295-302.

E Vlasenko, M Schulein, J Cherry and F Xu. Substrate specificity of family 5, 6, 7, 9, 12, and 45 endoglucanases. Bioresour. Technol. 2010; 101, 2405-11.