芽枝霉菌Lcxs9产两种耐热β-葡萄糖苷酶的快速纯化及酶学特性研究

Rapid purification and enzymatic properties of two thermostableβ-glucosidase from Cladosporium cladosporioides Lcxs9

以高产β-葡萄糖苷酶的芽枝霉菌(Cladosporium cladosporioides)Lcxs9为研究对象,对其进行固态发酵,采用硫酸铵沉淀及强阴离子交换柱对其所产β-葡萄糖苷酶进行分离纯化,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测其分子质量,并研究其酶学性质。结果表明,快速纯化获得两种β-葡萄糖苷酶,分别命名为BG CC1和BG CC2,分子质量分别为41 kDa、53 kDa,比酶活力分别为8.6 U/mg、12.2 U/mg。两种酶都具有水解活性,可以水解纤维二糖得到葡萄糖;同时具有转苷活性,可以利用低分子质量的单糖合成纤维三糖和纤维四糖。两种酶的最适作用pH及温度均分别为6.0、60℃,均在pH 4~10和20~80℃条件下较稳定,Ag^(2+)、Co^(2+)、Cu^(2+)、Hg^(2+)对两种酶均有抑制作用,而Mn^(2+)、Ca^(2+)和Mg^(2+)均有明显的激活作用,Zn^(2+)和Ni^(+)无明显影响。利用4-硝基苯基-β-D-吡喃葡萄糖苷(pNPG)为底物,两种酶的动力学常数Km和最大反应速率(V_(max))均分别为2.76 mg/mL、20.6 U/mg。

Taking Cladosporium cladosporioides Lcxs9 with highβ-glucosidase production as the research object,it was subjected to solid-state fermentation,theβ-glucosidase was separated and purified by ammonium sulfate precipitation and strong anion exchange column,the molecular mass was detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis(SDS-PAGE),and its enzymatic properties were studied.The results showed that two kinds ofβ-glucosidases were obtained by rapid purification,named as BG CC1 and BG CC2,respectively.The molecular mass were 41 kDa and 53 kDa,respectively,and the specific enzyme activities were 8.6 U/mg and 12.2 U/mg,respectively.Two kinds of enzymes both had hydrolytic activity and could hydrolyze cellobiose to obtain glucose.Meanwhile,they had transglycosidation activity and could synthesize cellotriose and cellotetraose with monosaccharides of low molecular mass.The optimum pH and temperature of the two kinds of enzymes were 6.0 and 60℃,respectively,and they were both stable at pH 4-10 and 20-80℃.Ag^(2+),Co^(2+),Cu^(2+)and Hg^(2+)had inhibition effect on the two kinds of enzymes,while Mn^(2+),Ca^(2+)and Mg^(2+)all had obvious activation effect,Zn^(2+)and Ni^(+)hadn't obvious effect.Using 4-nitrophenyl-β-D-glucopyranoside(pNPG)as the substrate,the kinetic constant Km and maximum reaction rate(V_(max))of the two kinds of enzymes were 2.76 mg/ml and 20.6 U/mg,respectively.