经紫外-甲基磺酸乙酯复合诱变选育得到的匍枝根霉诱变株TZ-03,其产β-葡萄糖苷酶(β-glycosidase,BG)能力显著提高,酶活峰值高达10.53 IU/mL,较原菌TP-02提高1.52倍。为研究该诱变株产酶差异内因,比较诱变前后bg基因转录水平,并克隆得到BG编码基因bgl4,利用Swiss model和Discovery Studio 3.0等生物信息学软件进行结构建模及分析。结果表明,诱变后bgl4基因转录水平上调90.19%,其可导致蛋白表达量的提高,从而可能引起BG酶活的提高。且诱变后BGL IV活性中心构象变化可能更有利于酶与底物的结合,对酶活的提高也有一定的促进作用。为进一步提高TZ-03的BG产量,经摇瓶优化确定最佳培养基为:微晶纤维素20 g/L、鱼粉蛋白胨10 g/L、谷氨酸1 g/L、CaCl2 2 g/L、KH2PO4 3 g/L、MgSO4·7H2O 4 g/L、PEG-4000 0.25 g/L、麸皮浸出汁2.5%、Tween 80 200 μL/L、微量元素液1 mL/L,TZ-03可在培养108 h达到BG酶活峰值22.15 IU/mL。利用10 L发酵罐进行放大培养TZ-03,使BG酶活在84 h达到最大值41.62 IU/mL。
The β-glycosidase (BG) of Rhizopus stolonifer TZ-03, which obtained by UV mutagenesis combined with ethyl methanesulfonate (EMS) mutagenesis, was 10.53 IU/mL (152% higher than that of wild strain TP-02). In order to investigate the difference of β-glycosidase production of TZ-03, the transcription level of bgl and bgl4 was compared, the gene bgl4 encoding β-glycosidase was cloned and structural modeling and analysis was conducted by an array of bioinformatics software, Swiss model and Discovery Studio 3.0. Results showed that improved effect of β-glycosidase might cause by the increased expressing quantity probably due to the higher translation level of bgl4 (90.19% higher than that of bgl) after induction. And the conformational change of active sites might facilitate the binding of BG-cellobiose, which also had a certain promotion effect on BG. In order to improve the yield of β-glycosidase, the optimal medium in shake flask was confirmed as follows: Avicel 20 g/L, peptone 10 g/L, glutamate 1 g/L, CaCl2 2 g/L,KH2PO4 3 g/L, MgSO4·7H2O 4 g/L, PEG-4000 0.25 g/L, wheat bran extract 2.5%, Tween 80 200 μL/L, trace element solution 1 mL/L. Under the condition, the BG was peaked at 22.15 IU/mL in 108 h. The amplification experiment was carried out in 10 L fermentor with the control of fermentation process, and the maximum of β-glycosidase was 41.62 IU/mL at 84 h. In this paper, the possibility of structural and function change of β-glycosidase after mutagenesis was analyzed and increased production of β-glycosidase was achieved by the optimization of fermentation conditions. All of these results provided certain support for modification of β-glucosidase gene and industrial application of β-glucosidase.
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