构建了枯草芽孢杆菌CH2菌株(Bacillus subtilis CH2)壳聚糖酶基因的真核表达载体pPIC9K-CH2-Cns,在毕赤酵母GS115(Pichia pastoris)中进行重组表达,获得了有生物学活性的重组壳聚糖酶,对重组壳聚糖酶的酶学特性及酶解产物进行了分析.结果显示,重组壳聚糖酶的分子量为29 kD,粗酶的比酶活达到133.60 U/mg,纯化后重组蛋白的比酶活为338.08 U/mg;该酶的最适作用温度为50℃,最适pH为4.5,酶动力学常数Vmax=24.39(μmol/mg)·min-1,Km=5.48 mg/mL;Fe2+、K+等对其酶活力有一定的激活作用,其中Mn2+能使酶活力提升2.4倍,Ag+、Mg2+、Hg2+、EDTA、EGTA和SDS等存在时则对其酶活力有强烈抑制作用.利用重组壳聚糖酶酶解壳聚糖,酶解产物主要为聚合度2~10的壳寡糖,且分布均匀.以上结果表明,枯草芽孢杆菌CH2菌株壳聚糖酶基因在毕赤酵母GS115中成功表达,获得了一种内切型的高酶活力重组壳聚糖酶,该重组酶具有反应条件温和、酶解产物均一等特点,可被应用于海洋甲壳质加工应用中.%Chitosanase is important in carbon and nitrogen recycles that occur extensively in nature, and is useful in the preparation of biofunctional chitooligosaccharides. Chitosanase occurs in a variety of microorganisms, in-cluding bacteria and fungi. Transformation by genetic engineering has increased the enzyme activity and enzyme content of recombinant chitosanase. To obtain abundant chitosanases possessing high chitosanolytic activity for large-scale production of chitosan-oligosaccharide, the chitosanase of Bacillus subtilis CH2 was best induced by fructose and not induced with chitosan, unlike other chitosanases. This study investigated recombinant expression of chitosanase from B. subtilis CH2, which was cloned and expressed in Pichia pastoris, and characterized the application of the recombinant enzyme for chitosan hydrolysis. Chitosanase may have important industrial appli-cations in the utilization of the enormous chitosan substrates. Mass spectrometry (MS) and thin-layer chromatog-raphy (TLC) were used to analyze the enzymatic products. The molecular weight of the recombinant chitosanase was estimated to be 29 kD using SDS-PAGE. The specific activity of crude enzymes was 133.60 U/mg. The spe-cific activity of the purified chitosanase was up to 338.08 U/mg. The optimal pH and temperature of the purified chitosanase was 50℃ and 4.5, respectively. The Km and Vmax values with soluble chitosan as a substrate were 5.48 mg/mL and 24.39 (μmol/mg)·min-1, respectively. Fe2+, K+, Na+, Li+, Ca2+, and especially Mn2+ enhanced the en-zyme activity of the recombinant chitosanase. Whereas, Ag+, Mg2+, Hg2+, EDTA, EGTA, and SDS inhibited the enzyme activity, and the other metal ion tested had no effect on enzyme activity. This characteristic of the recom-binant chitosanase was better than the chitosanase of B. subtilis CH2. Furthermore, the enzymatic production of chitooligosaccharides from chitosans of various deacetylation degrees ranged mainly from chitobiose to chitopen-tamer, and the enzymic products contained 2-10 degrees of polymerization of chito-oligosaccharide after the re-combinant chitosanase hydrolysis, but no monose. These results indicated that the enzyme was an endo-type chi-tosanase and a typical acidic metalloproteinase, which might be a good candidate for biotechnological application in producing chitooligosaccharides.
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