Molecular cloning and purification of an endochitinase from Serratia marcescens (Nima)

An endochitinase gene from the Serratia marcescens Nima strain (chiA Nima) was cloned, sequenced, and expressed in Escherichia coli DH5αF′, and the recombinant protein (ChiA Nima) was purified by hydrophobic interaction chromatography. chiA Nima contains an open reading frame (ORF) that encodes an endochitinase with a deduced molecular weight and an isoelectric point of 61 kDa and 6.84, respectively. A sequence at the 5′-end was identified as a signal peptide, recognized by Gram-negative bacteria transport mechanism. Comparison of ChiA Nima with other chitinases revealed a modular structure formed by the catalytic domain and a putative chitin-binding domain. The purified chitinase was able to hydrolyze both trimeric and tetrameric fluorogenic substrates, but not a chitobiose analog substrate. ChiA Nima showed high enzymatic activity within a broad pH range (pH 4.0–10.0), with a peak activity at pH 5.5. The optimal temperature for enzymatic activity was detected at 55°C.

[1]  J. E. Barboza-Corona,et al.  Chitinases from Serratia marcescens Nima , 2005, Biotechnology Letters.

[2]  M. Chang,et al.  Biochemical characterization and site-directed mutational analysis of the double chitin-binding domain from chitinase 92 of Aeromonas hydrophila JP101. , 2004, FEMS microbiology letters.

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

[4]  T. Suslow,et al.  Isolation and characterization of genes encoding two chitinase enzymes from Serratia marcescens , 1986, The EMBO journal.

[5]  J. Contreras,et al.  Selection of chitinolytic strains of Bacillus thuringiensis , 1999, Biotechnology Letters.

[6]  B. Jiménez,et al.  Cloning, Sequencing, and Expression of the Chitinase Gene chiA74 from Bacillus thuringiensis , 2003, Applied and Environmental Microbiology.

[7]  K S Wilson,et al.  Crystal structure of a bacterial chitinase at 2.3 A resolution. , 1994, Structure.

[8]  W. Panbangred,et al.  Chitinase from Bacillusthuringiensis subsp. pakistani , 2001, Applied Microbiology and Biotechnology.

[9]  T Watanabe,et al.  Genetic analysis of the chitinase system of Serratia marcescens 2170 , 1997, Journal of bacteriology.

[10]  D. Lereclus,et al.  Transformation and expression of a cloned δ-endotoxin gene in bacillus thuringiensis , 1989 .

[11]  J. Ibarra,et al.  Antagonism between Cry1Ac1 and Cyt1A1 Toxins ofBacillus thuringiensis , 1999, Applied and Environmental Microbiology.

[12]  S. Dauenhauer,et al.  Cloning and expression in Escherichia coli of Serratia marcescens genes encoding prodigiosin biosynthesis , 1984, Journal of bacteriology.

[13]  M. Cho,et al.  Cloning of the 52-kDa chitinase gene from Serratia marcescens KCTC2172 and its proteolytic cleavage into an active 35-kDa enzyme. , 1998, FEMS microbiology letters.

[14]  V. Eijsink,et al.  Characterization of a chitinase gene (chiA) from Serratia marcescens BJL200 and one-step purification of the gene product. , 1994, FEMS microbiology letters.

[15]  Ming-Qun Xu,et al.  A single surface tryptophan in the chitin-binding domain from Bacillus circulans chitinase A1 plays a pivotal role in binding chitin and can be modified to create an elutable affinity tag. , 2003, Biochimica et biophysica acta.

[16]  C. Koncz,et al.  Synergistic activity of a Bacillus thuringiensis delta-endotoxin and a bacterial endochitinase against Spodoptera littoralis larvae , 1996, Applied and environmental microbiology.

[17]  Y. Chuang,et al.  Identification and Characterization of the Three Chitin-Binding Domains within the Multidomain Chitinase Chi92 fromAeromonas hydrophila JP101 , 2001, Applied and Environmental Microbiology.

[18]  T. Uchiyama,et al.  Roles of the Exposed Aromatic Residues in Crystalline Chitin Hydrolysis by Chitinase A from Serratia marcescens2170* , 2001, The Journal of Biological Chemistry.

[19]  R. Laine,et al.  Mutation of active site residues in the chitin-binding domain ChBDChiA1 from chitinase A1 of Bacillus circulans alters substrate specificity: use of a green fluorescent protein binding assay. , 2004, Archives of biochemistry and biophysics.

[20]  T. Uchiyama,et al.  Chitinases A, B, and C1 of Serratia marcescens 2170 Produced by Recombinant Escherichia coli: Enzymatic Properties and Synergism on Chitin Degradation , 2002, Bioscience, biotechnology, and biochemistry.

[21]  J. E. Barboza-Corona,et al.  Antifungal Activity of Bacillus thuringiensis Chitinase and Its Potential for the Biocontrol of Phytopathogenic Fungi in Soybean Seeds , 2006 .

[22]  L. Chernin,et al.  Chitinolytic Enterobacter agglomerans Antagonistic to Fungal Plant Pathogens , 1995, Applied and environmental microbiology.

[23]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[24]  J. Dow,et al.  pH GRADIENTS IN LEPIDOPTERAN MIDGUT. , 1992, The Journal of experimental biology.

[25]  R. Rodríguez-Vázquez,et al.  Selection and characterization of a proteo-chitinolytic strain of Bacillus thuringiensis, able to grow in shrimp waste media , 1999 .

[26]  T. Ikegami,et al.  Expression and Characterization of the Chitin-Binding Domain of Chitinase A1 from Bacillus circulans WL-12 , 2000, Journal of bacteriology.