两种组分的纤维素酶基因随机突变及紫外诱变
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摘要
为了进一步提高基因重组菌的β-葡萄糖苷酶和内切葡聚糖酶的分泌表达能力,以及为这两种酶的深入研究和开发利用提供技术指导,论文探索了β-葡萄糖苷酶和内切葡聚糖酶基因的定向进化及其基因工程菌诱变育种的可行性和方法,主要结果如下:
(1)采用易错PCR技术对本实验室已克隆出的β-葡萄糖苷酶基因进行随机突变,将突变后构建的重组质粒pTRC99A-bgl1导入E.coli BL21 CodonPlus(DE3)-RIL中进行表达,结果表明,β-葡萄糖苷酶基因未能实现分泌性表达,而是形成了包涵体。
(2)对酵母基因工程菌GS115-bgl1-38进行紫外诱变育种。筛选出的酵母突变株GS115-bgl1-9和GS115-bgl1-37的酶活分别为出发菌株GS115-bgl1-38产β-葡萄糖苷酶酶活的2倍和1.5倍。通过十次传代实验证明其遗传性能相对稳定。通过对GS115-bgl1-38、GS115-bgl1-9和GS115-bgl1-37的酶学性质比较可知,突变株的最适温度与出发菌株变化不大,但最适pH范围呈扩大趋势,受金属离子的影响与出发菌株差异也较大。
(3)采用单因素和正交实验对酵母突变株GS115-bgl1-9分泌表达β-葡萄糖苷酶的条件进行了初步研究,适宜的条件为:接种量为4%(v/v),每24h添加0.5%(v/v)甲醇,Tween80添加量为0.2%(v/v),L-组氨酸添加量为0.5%(v/v),发酵液初始pH为5.0。
(4)以内切葡聚糖酶基因为模板进行易错PCR,向内切葡聚糖酶引入突变位点,然后将PCR产物连接到载体pTRC99A上构建突变后的表达质粒pTRC99A-Cel5A,导入E.coli BL21 CodonPlus(DE3)-RIL中表达。获得透明圈明显小于未突变菌株的4个菌株pTRC99A-Cel5A-1、pTRC99A-Cel5A-2、pTRC99A-Cel5A-3和pTRC99A-Cel5A-4。4个菌株的诱导产酶实验结果表明,它们的总酶活分别为0.050 IU/mL、0.050 IU/ mL、0.048 IU/ mL、0.043 IU/ mL,均低于未突变株的表达量。但4个突变菌株的酶最适pH和最适温度有一定程度的改变。测序结果表明,突变后的Cel5A基因中少数碱基发生了改变,突变株酶学性质的改变可能就是由于这些碱基的改变所致。
In order to further enhance the expression ability ofβ-glucosidase and endoglucanase from the strain of genetic engineering and provide technical guidance for further research and application on two kinds of enzymes,we explored the feasibility and methods on directed evolution of two kinds of enzymes and mutation breeding of the strain of genetic engineering,the main results are as follows:
(1)β-glucosidase was directedly evolved in vitro by error-prone PCR,then it was ligated with pTRC99A vector and transferred into E.coli BL21 CodonPlus(DE3)-RIL.The results show that the gene ofβ-glucosidase was unable to express in E.coli BL21 CodonPlus(DE3)-RIL, the protein had formed into inclusion body.
(2)The strain of genetic engineering GS115-bgl1-38 was mutated by ultraviolet light,two high production strain was obtained:GS115-bgl1-9 mutation and GS115-bgl1-37 mutation,their enzyme production capacities increased by 2 times and 1.5 times separately,two mutants kept stable after 10 times subculture.We also have studied on characters of two mutants,compared with the contrast,mutations’s optimal reaction temperature had little change,but the optimal pH had expanding tendency, metal ions had most impact on ability.
(3)By single and orthogonal test,we studied the culture medium for the expression ofβ-glucosidase from GS115-bgl1-9 mutation. The results indicated that the appropriate conditions were initial pH5.0 of BMGY,adding 0.5% histidine, inoculumed 4% with additive amount of methanol 0.5% every 24h. In addition, adding 0.2% Tween80 can enhanceβ-glucosidase activity.
(4)Endoglucanase gene from B.thermoliquefaciens-NL was directedly evolved in vitro by error-prone PCR,then it was ligated with pTRC99A vector and transferred into E.coli BL21 CodonPlus(DE3)-RIL,the mutant library had been construsted.Four lower production strains were obtained ,they were named: pTRC99A-Cel5A-1,pTRC99A-Cel5A-2,pTRC99A-Cel5A-3 and pTRC99A-Cel5A-4,the ability of 4 mutative strains were: 0.050 IU/ml,0.050 IU/ml,0.048 IU/ml,0.043 IU/ml. Compared with the contrast, characters of 4 mutations were changed a little, and through sequencing we knew the bases of genes of 4 mutations were changed a little , so we thought that the change of bases from gene may lead to the change of enzymes'character.
(1)采用易错PCR技术对本实验室已克隆出的β-葡萄糖苷酶基因进行随机突变,将突变后构建的重组质粒pTRC99A-bgl1导入E.coli BL21 CodonPlus(DE3)-RIL中进行表达,结果表明,β-葡萄糖苷酶基因未能实现分泌性表达,而是形成了包涵体。
(2)对酵母基因工程菌GS115-bgl1-38进行紫外诱变育种。筛选出的酵母突变株GS115-bgl1-9和GS115-bgl1-37的酶活分别为出发菌株GS115-bgl1-38产β-葡萄糖苷酶酶活的2倍和1.5倍。通过十次传代实验证明其遗传性能相对稳定。通过对GS115-bgl1-38、GS115-bgl1-9和GS115-bgl1-37的酶学性质比较可知,突变株的最适温度与出发菌株变化不大,但最适pH范围呈扩大趋势,受金属离子的影响与出发菌株差异也较大。
(3)采用单因素和正交实验对酵母突变株GS115-bgl1-9分泌表达β-葡萄糖苷酶的条件进行了初步研究,适宜的条件为:接种量为4%(v/v),每24h添加0.5%(v/v)甲醇,Tween80添加量为0.2%(v/v),L-组氨酸添加量为0.5%(v/v),发酵液初始pH为5.0。
(4)以内切葡聚糖酶基因为模板进行易错PCR,向内切葡聚糖酶引入突变位点,然后将PCR产物连接到载体pTRC99A上构建突变后的表达质粒pTRC99A-Cel5A,导入E.coli BL21 CodonPlus(DE3)-RIL中表达。获得透明圈明显小于未突变菌株的4个菌株pTRC99A-Cel5A-1、pTRC99A-Cel5A-2、pTRC99A-Cel5A-3和pTRC99A-Cel5A-4。4个菌株的诱导产酶实验结果表明,它们的总酶活分别为0.050 IU/mL、0.050 IU/ mL、0.048 IU/ mL、0.043 IU/ mL,均低于未突变株的表达量。但4个突变菌株的酶最适pH和最适温度有一定程度的改变。测序结果表明,突变后的Cel5A基因中少数碱基发生了改变,突变株酶学性质的改变可能就是由于这些碱基的改变所致。
In order to further enhance the expression ability ofβ-glucosidase and endoglucanase from the strain of genetic engineering and provide technical guidance for further research and application on two kinds of enzymes,we explored the feasibility and methods on directed evolution of two kinds of enzymes and mutation breeding of the strain of genetic engineering,the main results are as follows:
(1)β-glucosidase was directedly evolved in vitro by error-prone PCR,then it was ligated with pTRC99A vector and transferred into E.coli BL21 CodonPlus(DE3)-RIL.The results show that the gene ofβ-glucosidase was unable to express in E.coli BL21 CodonPlus(DE3)-RIL, the protein had formed into inclusion body.
(2)The strain of genetic engineering GS115-bgl1-38 was mutated by ultraviolet light,two high production strain was obtained:GS115-bgl1-9 mutation and GS115-bgl1-37 mutation,their enzyme production capacities increased by 2 times and 1.5 times separately,two mutants kept stable after 10 times subculture.We also have studied on characters of two mutants,compared with the contrast,mutations’s optimal reaction temperature had little change,but the optimal pH had expanding tendency, metal ions had most impact on ability.
(3)By single and orthogonal test,we studied the culture medium for the expression ofβ-glucosidase from GS115-bgl1-9 mutation. The results indicated that the appropriate conditions were initial pH5.0 of BMGY,adding 0.5% histidine, inoculumed 4% with additive amount of methanol 0.5% every 24h. In addition, adding 0.2% Tween80 can enhanceβ-glucosidase activity.
(4)Endoglucanase gene from B.thermoliquefaciens-NL was directedly evolved in vitro by error-prone PCR,then it was ligated with pTRC99A vector and transferred into E.coli BL21 CodonPlus(DE3)-RIL,the mutant library had been construsted.Four lower production strains were obtained ,they were named: pTRC99A-Cel5A-1,pTRC99A-Cel5A-2,pTRC99A-Cel5A-3 and pTRC99A-Cel5A-4,the ability of 4 mutative strains were: 0.050 IU/ml,0.050 IU/ml,0.048 IU/ml,0.043 IU/ml. Compared with the contrast, characters of 4 mutations were changed a little, and through sequencing we knew the bases of genes of 4 mutations were changed a little , so we thought that the change of bases from gene may lead to the change of enzymes'character.
引文
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