Improving the Chitinolytic Activity of Bacillus pumilus SG2 by Random Mutagenesis

Majid  Vahed; Ebrahim  Motalebi; Garshasb  Rigi; Kambiz  Akbari Noghabi; Mohammad Reza  Soudi; Mehdi  Sadeghi; Gholamreza  Ahmadian

doi:10.4014/jmb.1301.01048

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Research article

Microbial Cell Biology

Improving the Chitinolytic Activity of Bacillus pumilus SG2 by Random Mutagenesis

Majid Vahed ^{1, 2}, Ebrahim Motalebi ¹, Garshasb Rigi ¹, Kambiz Akbari Noghabi ¹, Mohammad Reza Soudi ³, Mehdi Sadeghi ¹ and Gholamreza Ahmadian ^1*

¹Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran, ²Islamic Azad University, Karaj, Iran, ³National Laboratory of Industrial Microbiology (NLIM), Department of Biology, Faculty of Sciences, Alzahra University, Tehran, Iran

Received: January 18, 2013; Accepted: July 18, 2013

J. Microbiol. Biotechnol. 2013; 23(11): 1519-1528

Published November 28, 2013 https://doi.org/10.4014/jmb.1301.01048

Abstract

Bacillus pumilus SG2, a halotolerant strain, expresses two major chitinases designated ChiS and ChiL that were induced by chitin and secreted into the supernatant. The present work aimed to obtain a mutant with higher chitinolytic activity through mutagenesis of Bacillus pumilus SG2 using a combination of UV irradiation and nitrous acid treatment. Following mutagenesis and screening on chitin agar and subsequent formation of halos, the mutated strains were examined for degradation of chitin under different conditions. A mutant designated AV2-9 was selected owing to its higher chitinase activity. To search for possible mutations in the whole operon including ChiS and ChiL, the entire chitinase operon, including the intergenic region, promoter, and two areas corresponding to the ChiS and ChiL ORF, was suquenced. Nucleotide sequence analysis of the complete chitinase operon from the SG2 and AV2-9 strains showed the presence of a mutation in the catalytic domain (GH18) of chitinase (ChiL). The results demonstrated that a single base change had occurred in the ChiL sequence in AV2- 9. The wild-type chitinase, ChiL, and the mutant (designated ChiLm) were cloned, expressed, and purified in E. coli. Both enzymes showed similar profiles of activity at different ranges of pH, NaCl concentration, and temperature, but the mutant enzyme showed approximately 30% higher catalytic activity under all the conditions tested. The results obtained in this study showed that the thermal stability of chitinase increased in the mutant strain. Bioinformatics analysis was performed to predict changes in the stability of proteins caused by mutation.

Keywords

Bacillus pumilus, chitinase, mutagenesis, AV2-9

References

Ahmadian G, Degrassi G, Venturi V, Zeigler D, Soudi MR, Zanguinejad P. 2007. Bacillus pumilus SG2 isolated from saline conditions produces and secretes two chitinases. J. Appl. Microbiol. 103: 1081-1089.
Araya CL, Fowler DM, Chenc W, Munieza I, Kelly JW, Fields S. 2012. A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function. Proc. Natl. Acad. Sci. USA 109: 16858-16863.
Arnold K, Bordoli L, Kopp J, Schwede T. 2006. The SWISSMODEL Workspace: A Web-based environment for protein structure homology modeling. Bioinformatics 22: 195-201.
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.
Chen HM, Chan SC, Leung KW, Wu JM, Fang HJ, Tsong TY. 2005. Local stability identification and the role of key acidic amino acid residues in staphylococcal nuclease unfolding. FEBS J. 272: 3967-3974.
Courcelle J, Khodursky A, Peter B, Brown PO, Hanawalt PC. 2001. Comparative gene expression profiles following UV exposure in wild type and SOS-deficient Escherichia coli. Genetics 158: 41-64.
Cozzi R, Nuccitelli A, D’Onofrio M, Necchi F, Rosini R, Zerbini F, et al. 2012. New insights into the role of the glutamic acid of the E-box motif in group B Streptococcus pilus 2a assembly. FASEB J. 26: 1-11.
Dehestani A, Kazemitabar K, Ahmadian G, Jelodar BN, Salmanian AH, Seyedi M, et al. 2010. Chitinolytic and antifungal activity of a Bacillus pumilus chitinase expressed in Arabidopsis. Biotechnol. Lett. 32: 539-546.
Dehouck Y, Kwasigroch JM, Gilis D, Rooman M. 2011. PoPMuSiC 2.1: a Web server for the estimation of protein stability changes upon mutation and sequence optimality. BMC Bioinformatics 12: 151. 10.Ghasemi SH, Ahmadian G, Sadeghi M, Zeigler DR. 2011. First report of a bifunctional chitinase/lysozyme produced by Bacillus pumilus SG2. Enzyme Microb. Technol. 48: 225–231.
Ghribi D, Zouari N, Jaoua S. 2004. Improvement of bioinsecticides production through mutagenesis of Bacillus thuringiensis by U.V. and nitrous acid affecting metabolic pathways and/or delta-endotoxin synthesis. J. Appl. Microbiol. 97: 338-346.
Gomes RC, Semedo LT, Soares RM, Soares RMA, Linhares LF, Ulhoa CJ, et al. 2001. Purification of a thermostable endochitinase from Streptomyces RC1071 isolated from a cerrado soil and its antagonism against phytopathogenic fungi. J. Appl. Microbiol. 90: 653-661.
Henrissat B. 1991. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 280:309–316.
Henrissat B, Bairoch A. 1993. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 29: 781–788.
Keyhani NO, Roseman S. 1999. Physiological aspects of chitin catabolism in marine bacteria. Biochim. Biophys. Acta 1473: 108-122.
Kitamura E, Kamei Y. 2003. Molecular cloning, sequencing, and expression of the gene encoding a novel chitinase A from a marine bacterium, Pseudomonas sp. PE2, and its domain structure. Appl. Microbiol. Biotechnol. 61: 140-149.
Koschorreck K, Schmid RD, Urlacher VB. 2009. Improving the functional expression of a Bacillus licheniformis laccase by random and site-directed mutagenesis. BMC Biotechnol. 9: 12.
Kutchuk ian PS, Yang JS, Verdine GL, Shakhnovich EI. 2009. All-atom model for stabilization of alpha-helical structure in peptides by hydrocarbon staples. J. Am. Chem. Soc. 131:4622-4627.
Lee YS, Park IH, Yoo JS, Chung SY, Lee YC, YS Cho, et al. 2007. Cloning, purification, and characterization of chitinase from Bacillus sp. DAU101. Bioresour. Technol. 98: 2734-2741.
Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426-428
Ordentlich A, Elad Y, Chet L. 1988. The role of chitinase of Serratia marcescens in biocontrol of Sclerotium rolfsii. Phytopathology 78: 84-88.
Parker BM, Taylor IN, Woodley JM, Ward JM, Dalby PA. 2011. Directed evolution of a thermostable l-aminoacylase biocatalyst. J. Biotechnol. 155: 396-405.
Perrakis A, Tews I, Dauter Z, Oppenheim AB, Chet I, Wilson KS, et al. 1994. Crystal structure of a bacterial chitinase at 2.3 Å resolution. Structure 2: 1169-1180.
Roberts WK, Selitrennikoff CP. 1986. Isolation and partial characterization of two antifungal proteins from barley. Biochim. Biophys. Acta 880: 161-70.
Sanchez-Salas JL, Santiago-Lara ML, Setlow B, Sussman MD, Setlow P. 1992. Properties of Bacillus megaterium and Bacillus subtilis mutants which lack the protease that degrades small, acid-soluble proteins during germination. J. Bacteriol. 174: 807-814.
Sambrook J, Russell DW. 2000. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY.
Shali A, Ghasemi SH, Ahmadian G, Ranjbar G, Dehestani A, Khalesi N, et al. 2010. Bacillus pumilus SG2 chitinases induced and regulated by chitin, show inhibitory activity against Fusarium graminearum and Bipolaris sorokiniana. Phytoparasitica 38: 141-147.
van der Sloot AM, Mullally MM, Fernandez-Ballester G, Serrano L, Quax WJ. 2004. Stabilization of TRAIL, an allbetasheet multimeric protein, using computational redesign. Protein Eng. Des. Sel. 17: 673-680.
Watanabe T, Kobori K, Miyashita K, Fujii T, Sakai H, Uchida M, et al. 1993. Identification of glutamic acid 204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity. J. Biol. Chem. 268: 18567-18572.
Worth CL, Preissner R, Blundell TL. 2011. A server for predicting effects of mutations on protein stability and malfunction. Nucleic Acids Res. 39: 215-222.

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Article

Research article

J. Microbiol. Biotechnol. 2013; 23(11): 1519-1528

Published online November 28, 2013 https://doi.org/10.4014/jmb.1301.01048

Improving the Chitinolytic Activity of Bacillus pumilus SG2 by Random Mutagenesis

Majid Vahed ^{1, 2}, Ebrahim Motalebi ¹, Garshasb Rigi ¹, Kambiz Akbari Noghabi ¹, Mohammad Reza Soudi ³, Mehdi Sadeghi ¹ and Gholamreza Ahmadian ^1*

¹Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran, ²Islamic Azad University, Karaj, Iran, ³National Laboratory of Industrial Microbiology (NLIM), Department of Biology, Faculty of Sciences, Alzahra University, Tehran, Iran

Received: January 18, 2013; Accepted: July 18, 2013

Abstract

Bacillus pumilus SG2, a halotolerant strain, expresses two major chitinases designated ChiS and
ChiL that were induced by chitin and secreted into the supernatant. The present work aimed
to obtain a mutant with higher chitinolytic activity through mutagenesis of Bacillus pumilus
SG2 using a combination of UV irradiation and nitrous acid treatment. Following mutagenesis
and screening on chitin agar and subsequent formation of halos, the mutated strains were
examined for degradation of chitin under different conditions. A mutant designated AV2-9
was selected owing to its higher chitinase activity. To search for possible mutations in the
whole operon including ChiS and ChiL, the entire chitinase operon, including the intergenic
region, promoter, and two areas corresponding to the ChiS and ChiL ORF, was suquenced.
Nucleotide sequence analysis of the complete chitinase operon from the SG2 and AV2-9
strains showed the presence of a mutation in the catalytic domain (GH18) of chitinase (ChiL).
The results demonstrated that a single base change had occurred in the ChiL sequence in AV2-
9. The wild-type chitinase, ChiL, and the mutant (designated ChiLm) were cloned, expressed,
and purified in E. coli. Both enzymes showed similar profiles of activity at different ranges of
pH, NaCl concentration, and temperature, but the mutant enzyme showed approximately 30%
higher catalytic activity under all the conditions tested. The results obtained in this study
showed that the thermal stability of chitinase increased in the mutant strain. Bioinformatics
analysis was performed to predict changes in the stability of proteins caused by mutation.

Keywords: Bacillus pumilus, chitinase, mutagenesis, AV2-9

Abstract

References

Ahmadian G, Degrassi G, Venturi V, Zeigler D, Soudi MR, Zanguinejad P. 2007. Bacillus pumilus SG2 isolated from saline conditions produces and secretes two chitinases. J. Appl. Microbiol. 103: 1081-1089.
Araya CL, Fowler DM, Chenc W, Munieza I, Kelly JW, Fields S. 2012. A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function. Proc. Natl. Acad. Sci. USA 109: 16858-16863.
Arnold K, Bordoli L, Kopp J, Schwede T. 2006. The SWISSMODEL Workspace: A Web-based environment for protein structure homology modeling. Bioinformatics 22: 195-201.
Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.
Chen HM, Chan SC, Leung KW, Wu JM, Fang HJ, Tsong TY. 2005. Local stability identification and the role of key acidic amino acid residues in staphylococcal nuclease unfolding. FEBS J. 272: 3967-3974.
Courcelle J, Khodursky A, Peter B, Brown PO, Hanawalt PC. 2001. Comparative gene expression profiles following UV exposure in wild type and SOS-deficient Escherichia coli. Genetics 158: 41-64.
Cozzi R, Nuccitelli A, D’Onofrio M, Necchi F, Rosini R, Zerbini F, et al. 2012. New insights into the role of the glutamic acid of the E-box motif in group B Streptococcus pilus 2a assembly. FASEB J. 26: 1-11.
Dehestani A, Kazemitabar K, Ahmadian G, Jelodar BN, Salmanian AH, Seyedi M, et al. 2010. Chitinolytic and antifungal activity of a Bacillus pumilus chitinase expressed in Arabidopsis. Biotechnol. Lett. 32: 539-546.
Dehouck Y, Kwasigroch JM, Gilis D, Rooman M. 2011. PoPMuSiC 2.1: a Web server for the estimation of protein stability changes upon mutation and sequence optimality. BMC Bioinformatics 12: 151. 10.Ghasemi SH, Ahmadian G, Sadeghi M, Zeigler DR. 2011. First report of a bifunctional chitinase/lysozyme produced by Bacillus pumilus SG2. Enzyme Microb. Technol. 48: 225–231.
Ghribi D, Zouari N, Jaoua S. 2004. Improvement of bioinsecticides production through mutagenesis of Bacillus thuringiensis by U.V. and nitrous acid affecting metabolic pathways and/or delta-endotoxin synthesis. J. Appl. Microbiol. 97: 338-346.
Gomes RC, Semedo LT, Soares RM, Soares RMA, Linhares LF, Ulhoa CJ, et al. 2001. Purification of a thermostable endochitinase from Streptomyces RC1071 isolated from a cerrado soil and its antagonism against phytopathogenic fungi. J. Appl. Microbiol. 90: 653-661.
Henrissat B. 1991. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 280:309–316.
Henrissat B, Bairoch A. 1993. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 29: 781–788.
Keyhani NO, Roseman S. 1999. Physiological aspects of chitin catabolism in marine bacteria. Biochim. Biophys. Acta 1473: 108-122.
Kitamura E, Kamei Y. 2003. Molecular cloning, sequencing, and expression of the gene encoding a novel chitinase A from a marine bacterium, Pseudomonas sp. PE2, and its domain structure. Appl. Microbiol. Biotechnol. 61: 140-149.
Koschorreck K, Schmid RD, Urlacher VB. 2009. Improving the functional expression of a Bacillus licheniformis laccase by random and site-directed mutagenesis. BMC Biotechnol. 9: 12.
Kutchuk ian PS, Yang JS, Verdine GL, Shakhnovich EI. 2009. All-atom model for stabilization of alpha-helical structure in peptides by hydrocarbon staples. J. Am. Chem. Soc. 131:4622-4627.
Lee YS, Park IH, Yoo JS, Chung SY, Lee YC, YS Cho, et al. 2007. Cloning, purification, and characterization of chitinase from Bacillus sp. DAU101. Bioresour. Technol. 98: 2734-2741.
Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31: 426-428
Ordentlich A, Elad Y, Chet L. 1988. The role of chitinase of Serratia marcescens in biocontrol of Sclerotium rolfsii. Phytopathology 78: 84-88.
Parker BM, Taylor IN, Woodley JM, Ward JM, Dalby PA. 2011. Directed evolution of a thermostable l-aminoacylase biocatalyst. J. Biotechnol. 155: 396-405.
Perrakis A, Tews I, Dauter Z, Oppenheim AB, Chet I, Wilson KS, et al. 1994. Crystal structure of a bacterial chitinase at 2.3 Å resolution. Structure 2: 1169-1180.
Roberts WK, Selitrennikoff CP. 1986. Isolation and partial characterization of two antifungal proteins from barley. Biochim. Biophys. Acta 880: 161-70.
Sanchez-Salas JL, Santiago-Lara ML, Setlow B, Sussman MD, Setlow P. 1992. Properties of Bacillus megaterium and Bacillus subtilis mutants which lack the protease that degrades small, acid-soluble proteins during germination. J. Bacteriol. 174: 807-814.
Sambrook J, Russell DW. 2000. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY.
Shali A, Ghasemi SH, Ahmadian G, Ranjbar G, Dehestani A, Khalesi N, et al. 2010. Bacillus pumilus SG2 chitinases induced and regulated by chitin, show inhibitory activity against Fusarium graminearum and Bipolaris sorokiniana. Phytoparasitica 38: 141-147.
van der Sloot AM, Mullally MM, Fernandez-Ballester G, Serrano L, Quax WJ. 2004. Stabilization of TRAIL, an allbetasheet multimeric protein, using computational redesign. Protein Eng. Des. Sel. 17: 673-680.
Watanabe T, Kobori K, Miyashita K, Fujii T, Sakai H, Uchida M, et al. 1993. Identification of glutamic acid 204 and aspartic acid 200 in chitinase A1 of Bacillus circulans WL-12 as essential residues for chitinase activity. J. Biol. Chem. 268: 18567-18572.
Worth CL, Preissner R, Blundell TL. 2011. A server for predicting effects of mutations on protein stability and malfunction. Nucleic Acids Res. 39: 215-222.