Molecular cloning, expression and biochemical characterisation of a cold-adapted novel recombinant chitinase from Glaciozyma antarctica PI12

BackgroundCold-adapted enzymes are proteins produced by psychrophilic organisms that display a high catalytic efficiency at extremely low temperatures. Chitin consists of the insoluble homopolysaccharide β-(1, 4)-linked N-acetylglucosamine, which is the second most abundant biopolymer found in nature. Chitinases (EC 3.2.1.14) play an important role in chitin recycling in nature. Biodegradation of chitin by the action of cold-adapted chitinases offers significant advantages in industrial applications such as the treatment of chitin-rich waste at low temperatures, the biocontrol of phytopathogens in cold environments and the biocontrol of microbial spoilage of refrigerated food.ResultsA gene encoding a cold-adapted chitinase (CHI II) from Glaciozyma antarctica PI12 was isolated using Rapid Amplification of cDNA Ends (RACE) and RT-PCR techniques. The isolated gene was successfully expressed in the Pichia pastoris expression system. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 1,215 bp, which encodes a 404 amino acid protein. The recombinant chitinase was secreted into the medium when induced with 1% methanol in BMMY medium at 25°C. The purified recombinant chitinase exhibited two bands, corresponding to the non-glycosylated and glycosylated proteins, by SDS-PAGE with molecular masses of approximately 39 and 50 kDa, respectively. The enzyme displayed an acidic pH characteristic with an optimum pH at 4.0 and an optimum temperature at 15°C. The enzyme was stable between pH 3.0-4.5 and was able to retain its activity from 5 to 25°C. The presence of K+, Mn2+ and Co2+ ions increased the enzyme activity up to 20%. Analysis of the insoluble substrates showed that the purified recombinant chitinase had a strong affinity towards colloidal chitin and little effect on glycol chitosan. CHI II recombinant chitinase exhibited higher Vmax and Kcat values toward colloidal chitin than other substrates at low temperatures.ConclusionBy taking advantage of its high activity at low temperatures and its acidic pH optimum, this recombinant chitinase will be valuable in various biotechnological applications under low temperature and acidic pH conditions.

[1]  J. Yim,et al.  Expression of recombinant endochitinase from the Antarctic bacterium, Sanguibacter antarcticus KOPRI 21702 in Pichia pastoris by codon optimization. , 2010, Protein expression and purification.

[2]  R. Kaldenhoff,et al.  Fast and reliable mini-prep RNA extraction from Neurospora crassa , 1990 .

[3]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[4]  K. Mavromatis,et al.  Cloning, Sequences, and Characterization of Two Chitinase Genes from the Antarctic Arthrobacter sp. Strain TAD20: Isolation and Partial Characterization of the Enzymes , 2001, Journal of bacteriology.

[5]  B. Bhushan,et al.  Isolation, purification and properties of a thermostable chitinase from an alkalophilic Bacillus sp. BG-11 , 1998, Biotechnology Letters.

[6]  Sven-Olof Enfors,et al.  Temperature limited fed-batch technique for control of proteolysis in Pichia pastoris bioreactor cultures , 2003, Microbial cell factories.

[7]  Søren Brunak,et al.  Prediction of Glycosylation Across the Human Proteome and the Correlation to Protein Function , 2001, Pacific Symposium on Biocomputing.

[8]  G. Feller.  Molecular adaptations to cold in psychrophilic enzymes , 2003, Cellular and Molecular Life Sciences CMLS.

[9]  L. Selbmann,et al.  Chitinolytic activity at low temperature of an Antarctic strain (A3) of Verticillium lecanii. , 1998, Research in microbiology.

[10]  M. Wakayama,et al.  A Chitinase Indispensable for Formation of Protoplast of Schizophyllum commune in Basidiomycete-lytic Enzyme Preparation Produced by Bacillus circulans KA-304 , 2004, Bioscience, biotechnology, and biochemistry.

[11]  N. Tsuji,et al.  Identification and Molecular Characterization of a Chitinase from the Hard Tick Haemaphysalis longicornis * , 2003, The Journal of Biological Chemistry.

[12]  R. Cavicchioli,et al.  Low-temperature extremophiles and their applications. , 2002, Current opinion in biotechnology.

[13]  J. Gatehouse,et al.  Cloning, expression and functional characterisation of chitinase from larvae of tomato moth (Lacanobia oleracea): a demonstration of the insecticidal activity of insect chitinase. , 2004, Insect biochemistry and molecular biology.

[14]  K. Robra,et al.  Characterization of a chitinase and an endo-β-1,3-glucanase from Trichoderma harzianum Rifai T24 involved in control of the phytopathogen Sclerotium rolfsii , 2001, Applied Microbiology and Biotechnology.

[15]  A. Rincón,et al.  Increased antifungal and chitinase specific activities of Trichoderma harzianum CECT 2413 by addition of a cellulose binding domain , 2004, Applied Microbiology and Biotechnology.

[16]  J. Zhang,et al.  Cloning and characterization of a novel chitinase gene (chi46) from Chaetomium globosum and identification of its biological activity , 2008, Applied Microbiology and Biotechnology.

[17]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[18]  W. Pearson,et al.  Current Protocols in Bioinformatics , 2002 .

[19]  M. Morange,et al.  Microbial Cell Factories , 2006 .

[20]  N. Meinander,et al.  Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris. , 2002, Biotechnology and bioengineering.

[21]  G. Somero,et al.  Hot spots in cold adaptation: localized increases in conformational flexibility in lactate dehydrogenase A4 orthologs of Antarctic notothenioid fishes. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[22]  Xiang Xiao,et al.  Chitinase Genes in Lake Sediments of Ardley Island, Antarctica , 2005, Applied and Environmental Microbiology.

[23]  Stefano Pascarella,et al.  Structural adaptation to low temperatures − analysis of the subunit interface of oligomeric psychrophilic enzymes , 2007, The FEBS journal.

[24]  R. Tewari,et al.  Chitinase from Enterobacter sp. NRG4: Its purification, characterization and reaction pattern , 2005 .

[25]  G. Feller,et al.  Cloning and expression in Escherichia coli of three lipase-encoding genes from the psychrotrophic antarctic strain Moraxella TA144. , 1991, Gene.

[26]  J. V. Van Beeumen,et al.  Purification, characterization, and nucleotide sequence of the thermolabile alpha-amylase from the antarctic psychrotroph Alteromonas haloplanctis A23. , 1992, The Journal of biological chemistry.

[27]  P. Robbins,et al.  Molecular cloning and characterization of chitinase genes from Candida albicans. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[28]  G. Feller,et al.  Structural determinants of cold adaptation and stability in a psychrophilic enzyme , 2001 .

[29]  Julie D Thompson,et al.  Multiple Sequence Alignment Using ClustalW and ClustalX , 2003, Current protocols in bioinformatics.

[30]  R. Illias,et al.  Expression and characterization of Trichoderma virens UKM-1 endochitinase in Escherichia coli , 2009 .

[31]  Ryo Kanai,et al.  Distribution and Phylogenetic Analysis of Family 19 Chitinases in Actinobacteria , 2004, Applied and Environmental Microbiology.

[32]  S. Brunak,et al.  Improved prediction of signal peptides: SignalP 3.0. , 2004, Journal of molecular biology.

[33]  Gennaro Marino,et al.  Recombinant protein expression system in cold loving microorganisms , 2006 .

[34]  Qian Yang,et al.  Cloning and Expression of a Novel Chitinase chi58 from Chaetomium cupreum in Pichia pastoris , 2009, Biochemical Genetics.

[35]  G. Feller,et al.  Activity, stability and flexibility in glycosidases adapted to extreme thermal environments. , 2003, Journal of molecular biology.

[36]  Jinkui Yang,et al.  Cloning and expression analysis of a chitinase gene Crchi1 from the mycoparasitic fungus Clonostachys rosea (syn. Gliocladium roseum). , 2007, Journal of microbiology.

[37]  Shann-Tzong Jiang,et al.  Cloning, Expression and Purification of Bacillus cereus Endochitinase in the Escherichia coli AD494(DE3)pLysS Expression System , 2009, Bioscience, biotechnology, and biochemistry.

[38]  Patil,et al.  Chitinolytic enzymes: an exploration. , 2000, Enzyme and microbial technology.

[39]  Heui-Dong Park,et al.  An extracellular Bacillus sp. chitinase for the production of chitotriose as a major chitinolytic product , 2003, Biotechnology Letters.

[40]  Seung-moon Park,et al.  Heterologous expression and characterization of class III chitinases from rice (Oryza sativa L.) , 2002 .

[41]  M. Yasuda,et al.  Molecular Analysis of the Gene Encoding a Novel Cold-Adapted Chitinase (ChiB) from a Marine Bacterium, Alteromonas sp. Strain O-7 , 2003, Journal of bacteriology.

[42]  Rupinder Tewari,et al.  Biotechnological aspects of chitinolytic enzymes: a review , 2006, Applied Microbiology and Biotechnology.

[43]  S. Brunak,et al.  Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites. , 2005, Glycobiology.

[44]  N. Willassen,et al.  Cold adapted enzymes. , 2000, Biotechnology annual review.

[45]  G. Thomas,et al.  Cold active microbial lipases: some hot issues and recent developments. , 2008, Biotechnology advances.

[46]  G. Thottappilly,et al.  Molecular cloning and structural analysis of the gene encoding Bacillus cereus exochitinase Chi36. , 2001, Journal of bioscience and bioengineering.

[47]  T. Schweder,et al.  Cloning, expression, and characterization of a chitinase gene from the Antarctic psychrotolerant bacterium Vibrio sp. strain Fi:7 , 2001, Extremophiles.

[48]  G. Gooday.  Physiology of microbial degradation of chitin and chitosan , 1990, Biodegradation.

[49]  G. L. Miller.  Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar , 1959 .

[50]  K. Nampoothiri,et al.  Extracellular chitinase production by Trichoderma harzianum in submerged fermentation , 2004, Journal of basic microbiology.

[51]  C. Vorgias,et al.  Molecular analysis of the gene encoding a new chitinase from the marine psychrophilic bacterium Moritella marina and biochemical characterization of the recombinant enzyme , 2008, Extremophiles.

[52]  Toshihide Nakamura,et al.  Characterization of Cold-Responsive Extracellular Chitinase in Bromegrass Cell Cultures and Its Relationship to Antifreeze Activity1[OA] , 2008, Plant Physiology.

[53]  A Bairoch,et al.  New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. , 1993, The Biochemical journal.

[54]  Michael Sauer,et al.  Stress in recombinant protein producing yeasts. , 2004, Journal of biotechnology.

[55]  Robert D. Finn,et al.  InterPro: the integrative protein signature database , 2008, Nucleic Acids Res..

[56]  George N. Phillips,et al.  Structures and Analysis of Highly Homologous Psychrophilic, Mesophilic, and Thermophilic Adenylate Kinases* , 2004, Journal of Biological Chemistry.

[57]  Jinkui Yang,et al.  Cloning of the gene Lecanicillium psalliotae chitinase Lpchi1 and identification of its potential role in the biocontrol of root-knot nematode Meloidogyne incognita , 2007, Applied Microbiology and Biotechnology.

[58]  P. Robbins,et al.  Chitinase is required for cell separation during growth of Saccharomyces cerevisiae. , 1991, The Journal of biological chemistry.

[59]  T. Watanabe,et al.  Structure of the gene encoding chitinase D of Bacillus circulans WL-12 and possible homology of the enzyme to other prokaryotic chitinases and class III plant chitinases , 1992, Journal of bacteriology.