Reaction mechanisms of chitinase and chitosanase produced by bacteria, and application of the enzymes to protoplast fusion of fungi

嗜水气单胞菌亚种发酵液中产生的几丁质酶。厌氧菌A52经几丁质亲和吸附和CM-Sephadex C-50离子交换层析得到纯品。测得该酶的等电点为4.60。该酶的相对分子质量约为105,000。通过CM-纤维素柱层析和凝胶渗透层析，从环状芽孢杆菌MH-K1的发酵液中分离纯化出一种壳聚糖酶。该酶的相对分子质量约为3万，其Km值为0.63 mg壳聚糖/ml，pI为9.2。该酶具有内切裂型活性，最终产物含有氨基葡萄糖二聚体和三聚体的混合物。最小的底物是氨基葡萄糖四聚体。该酶的N-端氨基酸序列为Ala-Ser-Pro-Asp-Asp-Asn-Phe-Ser-Pro-Glu-Thr-Leu-Gln-Phe-Leu-Arg-Asn-Asn-Cys-Gly-Leu-Asp-.酶中未发现蛋氨酸残基。用该几丁质酶处理细胞，可有效地产生乌萨米曲霉和泡盛曲霉原生质体。该壳聚糖酶可用于制备爪哇毛霉和杭州根霉的原生质体。

A chitinase produced in the culture broth by Aeromonas hydrophila subsp. anaerogenes A52 was purified to homogeneity by affinity abso-rption with chitin and ion-exchange chromatography with CM-Sephadex C-50. The isoelectric point of the enzyme was determined to be 4.60. The enzyme showed a molecular weight of about 105,000.A chitosanase was purified from the culture broth of Bacillus circulans MH-Kl 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 pi is 9.2. The enzyme showed an endo-splitting type of activity, and the end product of chitosan deg-radation contained a mixture of the dimer and trimer of glucosamine. The smallest of the substrate was a tetramer of glucosamine. The N-terminal amino acid sequence of the enzyme was determined to be Ala-Ser-Pro-Asp-Asp-Asn-Phe-Ser-Pro-Glu-Thr-Leu-Gln-Phe-Leu-Arg-Asn-Asn-Cys-Gly-Leu-Asp-. A methionine residue was not found in the enzyme.Protoplasts of aspergillus usami and Aspergillus awamori were efficiently produced by treatment of cells with the above chitinase. The chitosanase was available for preparetion of protoplasts from Mucor javanicus and Rhizopus hangchaoo.