A chitosanase was concentrated from the culture broth of Bacillus circulans MH-K 1 and was purified to homogeneity by CM-cellulose and gel permeation chromatography. The enzyme has a molecular weight of about 30,000, its Km is 0.63 mg chitosan/ml and its pus 9.2. The maximum velocity of chitosan degradation by the enzyme was obtained at 50°C when pH was maintained at 6.5. The enzyme was stable within the range of 0-40°C and pH 4.0-9.0. p-Chloromercuribenzoate and the metal ions of Cue +, Hg2 +, Nit +, and Zn2 + inhibited the enzyme activity. The enzyme degraded chitosan, glycolchitosan and CM-chitosan, but f-1,4-glucans such as chitin or its derivatives and CM-cellulose were not susceptible to the enzyme. The degree of deacetylation of chitosan significantly affected its susceptibility to the enzyme action. The most susceptible substrate was 80 deacetylated chitosan, and the substrates with less than 40% deacetylation were not affected by the enzyme. It is suggested that the presence of Nacetylglucosamine residues in the molecule of chitosan play an important role in the recognition of the substrate by the enzyme. The enzyme showed an endo-splitting type of activity, and the end product of chitosan degradation contained a mixture of the dimer and trimer of glucosamine. The smallest of the substrates was a tetramer of glucosamine.Chitosan is a polymer with 18-1,4-linked glucosamine residues. It is usually obtained by artificial deacetylation of chitin, a polymer of N-acetyl;/3-Dglucosamine, with a concentrated NaOH solution. Recently, as the medical uses of chitosan, its derivatives or its partially degraded oligosaccharides have been developed, the demands are growing for chitosanases, needed for mild degradation of chitosan (1).
Chitinase (EC 3.2.1.14) and chitobiase (EC 3.2.1.30) from culture broths of our isolate Aeromonas hydrophila subsp. anaerogenes A52 were concentrated by 80% saturation with ammonium sulfate into a crude enzyme preparation. The crude enzyme preparation was purified by separating the chitinase from the chitobiase by affinity adsorption of the chitinase on chitin. The chitinase was further purified by ion-exchange chromatography with C M-Sephadex C-50. The chitobiase was purified by successive ion-exchange chromatography with CM-cellulose and DEAE-Sephadex A-50. The homogeneity of the purified enzyme preparations was evaluated by polyacrylamide gel disc electrophoresis. The isoelectric points of the chitinase and chitobiase were determined to be 4.60 and 5.35, respectively. The chitinase showed a molecular weight of approximately 110,000, slightly higher than the chitobiase at 105,000. These values are the highest ever reported among chitinolytic enzymes from microbial origins. In addition, both the chitinase and the chitobiase showed a glycoprotein nature on polyacrylamide gel disc electrophoretogram when stained with Schiff's reagent. On examination of the effects of chemical reagents upon the activities of the enzymes, sulfhydryl groups appeared to be involved in the expression of the activities, even though they behaved differently in detail with the different reagents tested. The Km value of the chitinase was 2.8 mg chitin ml -1 and of the chitobiase was 1 x 10 -3 M of p-nitrophenyl-/3-N-acetyl-D-glucosamine. The effect of pH and temperature on the reaction rate or stability of the enzymes are also presented. By following the decrease in turbidity of chitin suspension and the formation of N-acetylglucosamine caused by the action of chitinase, the enzyme was found to degrade the substrate in an endo-splitting manner.25
A rapid induction system for synthesis of alpha-amylase by the funga Aspergillus oryzae M-13 was established. The mycelia were prepared from 20-h cultures grown on a peptone-glycerol medium and starved for 5 h; maltose was the optimum inducer tested. During h 1 of induction, formation of both intra- and extracellular alpha-amylases occurred at an almost identical rate (70 to 80 microgram/g of cells-h) without a detectable lag period. After a 1-h induction period, a remarkable increase in the extracellular concentration of the enzyme occurred, and a maximum rate (330 microgram/g of cells-h) was reached after 1.5 h of induction. During h 2 of induction, no significant change in mycelial weight was observed. Purified samples of intra- and extracellular enzymes formed in the induction system showed identical properties as examined by behavior in diethylaminoethyl-cellulose column chromatography, gel filtration, discontinuous gel electrophoresis, electrofocusing, optimal conditions for the reaction, heat stability, and molecular weight.
To determine the primary structure of chitosanase, which was produced by Bacillus circulans MH-K 1, its amino acid sequence was analyzed. Total 183 amino acids were determined. Two kinds of primer were synthesized according to the obtained amino acid sequence, and were used for PCR amplification of chitosanase gene. A 620 by fragment was amplified, and was used for a probe for Southern hybridization of the genomic DNA which was cut by some restriction enzymes. A 5.6 kb PstI fragment was isolated and introduced into pUC 19 vector. Colonies which harbored chitosanase gene containing pUC 19 were detected by colony hybridization with the probe. HindIII/HindIII fragment (1.2 kb) and HindIII/SacI fragment (0.7 kb) were sub-cloned and sequenced. The chitosanase gene (open reading frame is 900 by containing 259 amino acids and a signal peptide) was coded by the fragments. There was no meaningful homology to other enzymes including chitinase.The difference between chitin and chitosan is the degree of acetylation of D-glucosamine residues. Usually those of chitin are acetylated more than 60% and those of chitosan are acetylated less than 40%. The enzyme which hydrolyzes chitin is chitinase, and for chitosan is chitosanase.
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