棘孢曲霉β-葡萄糖苷酶Ⅰ在毕赤酵母中的表达及烷基糖苷的催化合成

Expression of Aspergillus aculeatus β-Glucosidase Ⅰ Gene in Pichia pastoris and Its Application on the Synthsis of Alkyl Glucoside

将来自棘孢曲霉(Aspergillus aculeatus NO.F-50)的β-葡萄糖苷酶Ⅰ在毕赤酵母中分泌表达.初步研究表明,目的蛋白得到较好表达,以对硝基酚-β-D-葡萄糖苷(4-Nitrophenyl-β-D-glucopyranoside,pNPG)为底物,重组β-葡萄糖苷酶Ⅰ酶促反应的最适温度为65℃,最适pH为5.0,50℃下反应发酵上清液中的酶活力可达33.8 U/mL,蛋白表达量最高可达0.388 mg/mL.该重组酶可通过逆水解或转糖苷反应催化合成烷基糖苷.在有机-水双相反应体系中,初步优化了pH值、含水量、葡萄糖浓度及酶量等条件.结果表明,在优化的反应条件下,丁基、己基、辛基和癸基葡萄糖苷最大产率分别为51.4%,28.8%,6.9%和3.0%.

β-Glucosidases,as a specific group of glycosyl hydrolase could catalyze the hydrolysis of β-1,4-glycosidic bond presented in short-chain oligosaccharides（containing 2—6 monosaccharides）,are widely used in the degradation of cellulose,the improvement of food flavor and so on.The gene of Aspergillus aculeatus NO.F-50 β-glucosidase Ⅰ was cloned and integrated into Pichia pastoris GS115,in which β-glucosidase Ⅰ can be expressed and secreted in an active form.The recombinant β-glucosidase Ⅰ had an optimum pH of 5.0 and an optimum temperature of 65 ℃ using 4-nitrophenyl-β-D-glucopyranoside（pNPG） as substrate.The highest hydrolytic activity and protein expression level at 50 ℃ in the culture supernatant of recombinant strain were up to 33.8 U/mL and 0.388 mg/mL,respectively.The recombinant β-glucosidase Ⅰ was found to be able to catalyze the synthesis of alkyl glucoside through reverse-hydrolysis or trans-glycosylation reaction.Some major influential factors in water/organic two-phase system such as pH value,water content,overall concentration of glucose and enzyme concentration were optimized.The yield of butyl glucoside,hexyl glucoside,octyl glucoside and decyl glucoside was 51.4%,28.8%,6.9% and 3.0%,respectivly.