产壳聚糖酶菌株的筛选、鉴定及酶学特性分析

Screening, identification and enzymatic properties of chitosanase-producing strain

【目的】利用筛选培养基,从福建沿海潮间带泥样中分离筛选产壳聚糖酶的菌株,并研究菌株的产酶特性。【方法】通过形态学观察,结合26S rDNA序列进行分类鉴定,采用DNS法测定酶活力。【结果】筛选得到产壳聚糖酶的菌株KQ-1002与草酸青霉(Penicillium oxalicum)的同源性为99%,并初步鉴定为青霉属的一种。发酵培养的最适温度为30°C,最适碳源为1.0%水溶性壳聚糖,最适氮源为1.87%(NH4)2SO4,最适pH为6.0。该菌株液体发酵培养72 h产壳聚糖酶活性最高,经优化后最高产酶量为18 U/mL。纯化后的壳聚糖酶经SDS-PAGE分析其分子量约40 kD。酶促反应最适pH为5.0,最适反应温度为55°C,Km值为1.293 g/L。在离子浓度为1.0×10 3mol/L时,金属离子Cu2+、Hg2+、Ag+对酶的活性均有强烈的抑制作用。壳聚糖酶对不同底物及脱乙酰度的壳聚糖具有不同的降解作用。【结论】筛选获得产壳聚糖酶的真菌菌株KQ-1002的壳聚糖酶活力经优化后提高了约7倍,是一株具有研究和应用潜力的产壳聚糖酶菌株。

[Objective] By screening of chitosanase strains in mud samples from the Fujian coastal intertidal with screening medium, studied the characteristics of the strains for enzyme production. [Methods] Through morphological observation, combined with 26S rDNA sequences were classified and identified, DNS method was use for the determination of enzyme activity. [Results] The conclusion showed a 99% similarity between the chitosanase strain KQ-1002 and Penicillium oxalicum. The strain was tentatively identified as a species of Penicillium fungus. The optimal fermentation temperature was 30 °C, the optimum carbon source was the 1.0% soluble chitosan, the optimal nitrogen source was 1.87% （NH4）2SO4, and the optimum pH was 6.0. Cultured for 72 h under liquid fermentation on the strains, the chitosanase-producing activity reached the maximum, and the maximum of enzyme production had been optimized to 18 U/mL. The molecular masses of purified enzymes was 40 kD which analyzed by SDS-PAGE. Enzymatic reactions’ optimum pH was 5.0, the optimum temperature was 55 °C, and Km was 1.293 g/L. When ion concentration reached 1.0×10^-3 mol/L, metal ions of Cu^2＋, Hg^2＋, Ag^＋ would strongly inhibit the enzyme’ activity. There was a different degradation of chitosanase on various substrates and deacetylation of chitosan. [Conclusion] Through screening, the chitosanase activity of chitosanase-producing fungal strains KQ-1002 which had been optimized boosted by approximately 7 times, and it is a chitosanase-producing strains of research and potential applications.