Purification and characterization of a novel chitinase from the entomopathogenic fungus, Metarhizium anisopliae.

A novel chitinase was detected in extracellular culture fluids of the entomopathogenic fungus Metarhizium anisopliae (ATCC 20500) grown in liquid medium containing chitin as a sole carbon source. A chitinase was purified to near homogeneity from culture broth of M. anisopliae by DEAE-Sephacel, CM-Sepharose CL-6B ion-exchange chromatography, and gel filtration with Superose 12HR. The molecular mass of the enzyme determined by SDS-polyacrylamide gel electrophoresis was approximately 60 kDa and the optimum pH of the enzyme was 5.0. This molecular mass is different from values of 33, 43.5, and 45 kDa for endochitinases and 110 kDa for an exochitinase (N-acetylglucosaminidase) from M. anisopliae ME-1 published previously. In addition, N-terminal sequences of 60-kDa chitinase are different from those of 43.4- and 45-kDa endochitinases. The purified enzyme showed high chitinolytic activity against colloidal, crystalline chitin of crab shells as well as against p-nitrophenyl-beta-d-N-acetylglucosamide, p-nitrophenyl-beta-d-N, N'-diacetylchitobiose, and p-nitrophenyl-N, N'-N"-triacetylchitotriose, indicating that this enzyme has both endo- and exochitinase activity.

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

[2]  J. Lockwood,et al.  Powdered chitin agar as a selective medium for enumeration of actinomycetes in water and soil. , 1975, Applied microbiology.

[3]  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.

[4]  D. Young,et al.  Purification and characterization of chitinase enzymes from healthy and Verticillium albo-atrum-infected tomato plants, and from V. albo-atrum , 1982 .

[5]  C. Ignoffo,et al.  Levels of chitinolytic activity during development of three entomopathogenic fungi , 1984 .

[6]  R. Leger,et al.  Cuticle-degrading enzymes of entomopathogenic fungi: cuticle degradation in vitro by enzymes from entomopathogens , 1986 .

[7]  R. Leger,et al.  Cuticle-degrading enzymes of entomopathogenic fungi: Synthesis in culture on cuticle , 1986 .

[8]  R. Leger,et al.  Cuticle-degrading Enzymes of Entomopathogenic Fungi: Regulation of Production of Chitinolytic Enzymes , 1986 .

[9]  R. Cooper,et al.  Production of Cuticle-degrading Enzymes by the Entomopathogen Metarhizium Anisopliae during Infection of Cuticles from Calliphora Vomitoria and Manduca Sexta , 1987 .

[10]  J. Latgé,et al.  Atlas of Entomopathogenic Fungi , 1988, Springer Berlin Heidelberg.

[11]  G. Riba,et al.  Chitinolytic activity and virulence associated with native and mutant isolates of an entomopathogenic fungus , 1989 .

[12]  Y. Fujii,et al.  Purification and characterization of chitinase produced by Streptomyces erythraeus. , 1989, Journal of biochemistry.

[13]  M. Llewellyn,et al.  Characteristics relating to the pathogenicity of Metarhizium anisopliae toward Nilaparvata lugens , 1989 .

[14]  G. Charnley,et al.  Characterization of chitinase and chitobiase produced by the entomopathogenic fungus , 1991 .

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

[16]  A. Ohta,et al.  Purification of two chitinases from Rhizopus oligosporus and isolation and sequencing of the encoding genes , 1992, Journal of bacteriology.

[17]  Y. Nishizawa,et al.  Induction and Purification of Beauveria bassiana Chitinolytic Enzymes , 1993 .

[18]  R. Staples,et al.  Entomopathogenic Isolates of Metarhizium anisopliae, Beauveria bassiana, and Aspergillus flavus Produce Multiple Extracellular Chitinase Isozymes , 1993 .

[19]  S. O. Andersen,et al.  Insect cuticular proteins. , 1995, Insect biochemistry and molecular biology.

[20]  R. S. St. Leger,et al.  Construction of an improved mycoinsecticide overexpressing a toxic protease. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[21]  N. Rizzo,et al.  Characterization and Ultrastructural Localization of Chitinases from Metarhizium anisopliae, M. flavoviride, and Beauveria bassiana during Fungal Invasion of Host (Manduca sexta) Cuticle , 1996, Applied and environmental microbiology.

[22]  R. Leger,et al.  Chapter VIII-3 – The application of molecular techniques to insect pathology with emphasis on entomopathogenic fungi , 1997 .

[23]  L. Lacey.  Manual of techniques in insect pathology , 1997 .

[24]  R. S. St. Leger,et al.  Ambient pH Is a Major Determinant in the Expression of Cuticle-Degrading Enzymes and Hydrophobin by Metarhizium anisopliae , 1998, Applied and Environmental Microbiology.

[25]  S. Kang,et al.  Isolation and characterization of a chitinase cDNA from the entomopathogenic fungus, Metarhizium anisopliae. , 1998, FEMS microbiology letters.