来源于脂环酸芽孢杆菌的GH1家族β-葡萄糖苷酶的葡萄糖耐受性分子改造

豆粕中大豆异黄酮主要以无活性的结合型存在。在玉米豆粕型日粮为主的饲料领域,β-葡萄糖苷酶可以将结合型大豆异黄酮转化为游离型活性苷元,但转化过程受肠道中较高浓度葡萄糖的抑制。和GH3家族相比,GH1家族β-葡萄糖苷酶不仅具有较高的葡萄糖耐受性,而且一定浓度范围内的葡萄糖对其具有酶活促进效应,因此在饲料领域显示出巨大的应用潜力。从脂环酸芽孢杆菌Alicyclobacillus sp.A4中克隆表达了一个具有较高葡萄糖耐受性(IC50800 mmol/L)的GH1家族β-葡萄糖苷酶As BG1。为了进一步提高As BG1的葡萄糖耐受性,通过同源建模、序列比对、分子对接技术等生物信息学手段,聚焦于催化通道的非催化葡萄糖结合位点,对As BG1进行了理性设计的分子突变研究。对不同突变体的葡萄糖耐受性测定表明:以5 mmol/L p NPG为底物,100 mmol/L葡萄糖对WT(野生型)、H315R和M325K酶活促进作用分别为125%、163%和162%,其中H315R的IC50提高至1 200 mmol/L;以1 mmol/L大豆苷为底物,10 mmol/L的葡萄糖对WT、H315R和M325K分别具有163%、212%和226%的酶活促进作用,结果表明突变体具有更好的在动物胃肠道水解大豆异黄酮能力。上述结果丰富了对GH1家族葡萄糖耐受性机制的认识,为日后进行耐受性相关的分子改造提供了重要的指导意义。

Glucose-Tolerance Molecular Modification of GH1 β-glucosidase from Alicyclobacillus sp. A4

Soybean isoflavones has the effect of promoting growth and accelerating protein synthesis, but it mainly exists in the soybean meal with inactive and bounded form. In the feed field based on corn and soybean meal, β-glucosidase could convert the bounded soybean isoflavones into the free and active aglycone, but the transformation process was inhibited by the relatively high concentration of glucose in the intestinal tract. Compared with the GH3 family, β-glucosidase from GH1 family has not only higher glucose tolerance, but also can be stimulated by glucose with a certain range of concentration. Hence, it shows great potential for application in the field of feed. This study cloned and expressed a GH1 family β-glucosidase AsBG1 from Alicyclobacillus sp. A4 with high glucose tolerance (IC50 800 mmol/L). In order to further improve the glucose tolerance of AsBG1, the molecular mutation of AsBG1 was conducted focusing on the non-active binding sites of glucose in catalytic channel and based on homology modeling, sequence alignment and molecular docking. The glucose tolerance test showed that with 5 mmol/L pNPG as substrate, the specific activity of WT, H315R and M325K was stimulated to 125%, 163% and 162% by 100 mmol/L glucose, respectively. And the IC50 of H315R increased to 1 200 mmol/L. With 1 mmol/L daidzin as substrate, the activity of WT, H315R and M325K was stimulated to 163%, 212% and 226% by 10 mmol/L glucose, respectively. This study enriched the understanding of glucose tolerance mechanism of GH1 family and had important guiding significance for the relevant molecular modification of tolerance in the future.