两种酸性耐热α-淀粉酶的表达研究
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摘要
α-淀粉酶(1,4-α-D-葡萄糖苷葡萄糖苷水解酶,EC3.2.1.1)能够以淀粉为底物,从淀粉内部水解1,4-α-D-葡萄糖苷键,广泛应用在淀粉糖生产、发酵、纺织、造纸等许多行业,具有巨大的经济价值。本研究尝试利用巴斯德毕赤酵母Pichia pastoris作为生物反应器表达两种极具应用潜力的极端α-淀粉酶,并研究了重组α-淀粉酶的酶学性质。
从嗜酸耐热的酸热脂环酸杆菌Alicyclobacillus acidocaldarius中克隆到一种α-淀粉酶的基因amy,基因amy全长3903bp,编码1301个氨基酸,理论分子量约140kD。基因amy被分别克隆到质粒表达载体pET-22b(+)和pPIC9α,并分别在大肠杆菌Escherichia coli BL21(DE3)利巴斯德毕赤酵母P. pastoris GS115中得到表达,表达产物具有淀粉酶的活性。
纯化酵母中表达的重组α-淀粉酶rAMY,并研究了它的酶学性质。以可溶性淀粉为底物,rAMY作用最适pH3.2,在pH2.5-4.6范围内,酶活性保留50%以上,它的最适温度65℃,在70℃下处理30分钟,酶活性维持50%以上。重组α-淀粉酶rAMY基本保留了天然酶蛋白的耐热性和嗜酸性特点。
根据对α-淀粉酶AMY的氨基酸序列分析和同源建模,去除基因amy的+1～+1174bp和+3288～3903bp的核酸序列,获得全长2115bp的基因片断amy',amy'编码705个氨基酸,理论分子量约79kD。amy'被克隆到质粒表达载体pET-22b(+),并在大肠杆菌E. coli BL21(DE3)获得表达,表达产物具有淀粉酶的活性,证明该截断肽链包含能折叠形成α-淀粉酶催化结构域所需的氨基酸序列。
来源于嗜热球菌Thermococcus sp.的酸性高温α-淀粉酶BD5063理论分子量约49kD,不存在潜在N-连接糖基化位点,编码基因BD5063全长1299bp。根据巴斯德毕赤酵母高效表达基因的密码子使用偏好性,对BD5063进行密码子优化并全基因合成获得新基因BD5063r。巴斯德毕赤酵母能高效表达基因BD5063r,表达量达剑60mg/L,表达产物rBD5063水解可溶性淀粉主要产物是单糖和低聚寡糖,结合活性染色结果证明rBD5063具有α-淀粉酶的活性。
纯化rBD5063,获得纯度大于90%的样品,并研究了rBD5063酶学性质。以可溶性淀粉为底物,rBD5063作用最适pH5.0,在pH3.5～10.0范围内处理30分钟,酶活性保留50%以上;作用最适温度100-110℃,在90℃下酶活性半衰期约30分钟。低浓度Ca~(2+)对激活rBD5063活性和维持稳定性是必需的,而且在一定浓度范围内Ca~(2+)浓度对活性影响不显著,Mg~(2+)、SDS利Trition X-100都能够部分抑制rBD5063活性,Cu~(2+)和EDTA严重抑制rBD5063活性。
研究了rBD5063的多组分情况,表达产物包括三种分子量分别是64、57、44kD的活性组分,在通常情况下主要以大分子量活性多聚体的形式存在。rBD5063不存在N-连接的糖基化,但是存在O-连接的糖基化现象,O-连接对rBD5063的热稳定性和最适温度没有显著影响。
本研究利用巴斯德毕赤酵母成功表达两种不同来源和性质的酸性耐热α-淀粉酶,通过对这两种α-淀粉酶异源表达情况分析,证实了巴斯德毕赤酵母作为表达极端α-淀粉酶的生物反应器的可行性。
Alpha-amylases (1,4-alpha-D-glucan-glucanohydrolase, EC 3.2.1.1) hydrolyze alpha-1,4-linkages in starch. The action of alpha-amylases on starch leads to a rapid decrease of the starch solution viscosity. Alpha-amylases are widely applied in numerous areas, such as in starch saccharification, fermentation, spin, paper. Thus any research on alpha-amylase is particularly significant in both industorial production and scientific investigation. In this project, in order to explore the way of using P. pastoris as bioreactor to express extremophilic alpha-amylases, two non-coincident acidothermophilic alpha-amylases were expressed in Escherichia coli and Pichia pastoris, respectively, and then characterization of the recombinant alpha-amylases were described.Alpha-amylase AMY from the Gram-positive Alicyclobacillus acidocaldarius is one kind of acidothermophilic enzyme, with the optimal temperature and pH of 75℃ and 3. The nucleotide sequence of the gene amy was cloned by PCR. The gene amy was 3903bp long, comprising one open reading frame encoding a polypeptide of 1301amino acids. The calculated molecular weight of AMY was about 140kD. The gene amy was expressed in E. coli BL21(DE3) and P. pastoris GS115 respectively, and both of the recombinant proteins had bioactivity.rAMY expressed in P. pastoris was further purified and characterized. The apparent molecular weight of rAMY was about 160kD according to SDS-PAGE. When rAMY hydrolyzed soluble starch, the optimal pH value was 3.2, which is the same as that of the native enzyme. However, the optimum of temperature was 65℃, a little lower than that of the native enzyme. Above 50% of relative activity remained after incubation for 30 minutes at 70 ℃. Anyway rAMY expressed in P. pastoris is acidothermophilic.Based on analysis of AMY amino acid sequence and homology model of its catalytic domain, the middle segment of the gene amy, which ranged from +1174 bp to +3288 bp, was cloned by PCR. The truncated gene amy' encoded 705 amino acids with the calculated molecular weight of 79kD. amy' was expressed in E. coli BL21(DE3) induced by 1mmol/L IPTG, and the expressed enzyme also retained α-amylase activity. So the truncated peptide AMY' should include essential amino acid sequences which can fold catalytic domain.Acidohermophlic alpha-amylase BD5063 is found in Thermococcus sp.. The encoding gene BD5063 was reconstructed for the optimal expression in P. pastoris. Modification was based on unequal usage of synonymous codon of high-level expressed genes in P. pastoris. The gene BD5063r consisted of 1299bp, encoding 433 amino acids with the calculated molecular mass 49kD. The recombinant a-amylase rBD5063 was secreted by P. pastoris up to a yield of 60mg/L. TLC results for the various hydrolysis products of soluble starch showed that rBD5063 cleaved alpha-1,4-glucosidic linkages, and most products were glucose, maltose, and other oligosaccharides.rBD5063 was purified and characterized. When rBD5063 hydrolyzed soluble starch, the optimal
pH was 5.0 and more than 50% of its maximal activity remained after treatment in the buffers ranged from pH 3.5-10.0 for 30 minutes. The optimum temperature was 100-110°C, and the half-life at 90°C, pH 5.0 was 30 minutes. Low content of Ca + is enough to activate and stabilize its activity, and even up to 20mmol/L, Ca2+ only affected its activity slightly. Mg2+, SDS and Trition X-100 partially inhibited its activity, while Cu2+ and EDTA thoroughly inhibited its activity.Most rBD5063 was inclined to aggregate into bioactive polymers form, which would be disaggregated into three bioactive proteins with molecular weight of 64, 57, 44kD, respectively. rBD5063 is not modified by N-linked glycosyiation but by O-linked glycosyiation. The O-linked glycosyiation had little influence on thermostability and the optimal temperature of rBD5063.In conclusion, two acidothermophilic alpha-amylases were high-level expressed in P. pastoris. Those recombinant alpha-amylases expressed in P. pastoris held essential characterization of native alpha-amylases. As a result, P. pastoris has been of great value as the bioreactor to produce extremophilic alpha-amylases.
从嗜酸耐热的酸热脂环酸杆菌Alicyclobacillus acidocaldarius中克隆到一种α-淀粉酶的基因amy,基因amy全长3903bp,编码1301个氨基酸,理论分子量约140kD。基因amy被分别克隆到质粒表达载体pET-22b(+)和pPIC9α,并分别在大肠杆菌Escherichia coli BL21(DE3)利巴斯德毕赤酵母P. pastoris GS115中得到表达,表达产物具有淀粉酶的活性。
纯化酵母中表达的重组α-淀粉酶rAMY,并研究了它的酶学性质。以可溶性淀粉为底物,rAMY作用最适pH3.2,在pH2.5-4.6范围内,酶活性保留50%以上,它的最适温度65℃,在70℃下处理30分钟,酶活性维持50%以上。重组α-淀粉酶rAMY基本保留了天然酶蛋白的耐热性和嗜酸性特点。
根据对α-淀粉酶AMY的氨基酸序列分析和同源建模,去除基因amy的+1～+1174bp和+3288～3903bp的核酸序列,获得全长2115bp的基因片断amy',amy'编码705个氨基酸,理论分子量约79kD。amy'被克隆到质粒表达载体pET-22b(+),并在大肠杆菌E. coli BL21(DE3)获得表达,表达产物具有淀粉酶的活性,证明该截断肽链包含能折叠形成α-淀粉酶催化结构域所需的氨基酸序列。
来源于嗜热球菌Thermococcus sp.的酸性高温α-淀粉酶BD5063理论分子量约49kD,不存在潜在N-连接糖基化位点,编码基因BD5063全长1299bp。根据巴斯德毕赤酵母高效表达基因的密码子使用偏好性,对BD5063进行密码子优化并全基因合成获得新基因BD5063r。巴斯德毕赤酵母能高效表达基因BD5063r,表达量达剑60mg/L,表达产物rBD5063水解可溶性淀粉主要产物是单糖和低聚寡糖,结合活性染色结果证明rBD5063具有α-淀粉酶的活性。
纯化rBD5063,获得纯度大于90%的样品,并研究了rBD5063酶学性质。以可溶性淀粉为底物,rBD5063作用最适pH5.0,在pH3.5～10.0范围内处理30分钟,酶活性保留50%以上;作用最适温度100-110℃,在90℃下酶活性半衰期约30分钟。低浓度Ca~(2+)对激活rBD5063活性和维持稳定性是必需的,而且在一定浓度范围内Ca~(2+)浓度对活性影响不显著,Mg~(2+)、SDS利Trition X-100都能够部分抑制rBD5063活性,Cu~(2+)和EDTA严重抑制rBD5063活性。
研究了rBD5063的多组分情况,表达产物包括三种分子量分别是64、57、44kD的活性组分,在通常情况下主要以大分子量活性多聚体的形式存在。rBD5063不存在N-连接的糖基化,但是存在O-连接的糖基化现象,O-连接对rBD5063的热稳定性和最适温度没有显著影响。
本研究利用巴斯德毕赤酵母成功表达两种不同来源和性质的酸性耐热α-淀粉酶,通过对这两种α-淀粉酶异源表达情况分析,证实了巴斯德毕赤酵母作为表达极端α-淀粉酶的生物反应器的可行性。
Alpha-amylases (1,4-alpha-D-glucan-glucanohydrolase, EC 3.2.1.1) hydrolyze alpha-1,4-linkages in starch. The action of alpha-amylases on starch leads to a rapid decrease of the starch solution viscosity. Alpha-amylases are widely applied in numerous areas, such as in starch saccharification, fermentation, spin, paper. Thus any research on alpha-amylase is particularly significant in both industorial production and scientific investigation. In this project, in order to explore the way of using P. pastoris as bioreactor to express extremophilic alpha-amylases, two non-coincident acidothermophilic alpha-amylases were expressed in Escherichia coli and Pichia pastoris, respectively, and then characterization of the recombinant alpha-amylases were described.Alpha-amylase AMY from the Gram-positive Alicyclobacillus acidocaldarius is one kind of acidothermophilic enzyme, with the optimal temperature and pH of 75℃ and 3. The nucleotide sequence of the gene amy was cloned by PCR. The gene amy was 3903bp long, comprising one open reading frame encoding a polypeptide of 1301amino acids. The calculated molecular weight of AMY was about 140kD. The gene amy was expressed in E. coli BL21(DE3) and P. pastoris GS115 respectively, and both of the recombinant proteins had bioactivity.rAMY expressed in P. pastoris was further purified and characterized. The apparent molecular weight of rAMY was about 160kD according to SDS-PAGE. When rAMY hydrolyzed soluble starch, the optimal pH value was 3.2, which is the same as that of the native enzyme. However, the optimum of temperature was 65℃, a little lower than that of the native enzyme. Above 50% of relative activity remained after incubation for 30 minutes at 70 ℃. Anyway rAMY expressed in P. pastoris is acidothermophilic.Based on analysis of AMY amino acid sequence and homology model of its catalytic domain, the middle segment of the gene amy, which ranged from +1174 bp to +3288 bp, was cloned by PCR. The truncated gene amy' encoded 705 amino acids with the calculated molecular weight of 79kD. amy' was expressed in E. coli BL21(DE3) induced by 1mmol/L IPTG, and the expressed enzyme also retained α-amylase activity. So the truncated peptide AMY' should include essential amino acid sequences which can fold catalytic domain.Acidohermophlic alpha-amylase BD5063 is found in Thermococcus sp.. The encoding gene BD5063 was reconstructed for the optimal expression in P. pastoris. Modification was based on unequal usage of synonymous codon of high-level expressed genes in P. pastoris. The gene BD5063r consisted of 1299bp, encoding 433 amino acids with the calculated molecular mass 49kD. The recombinant a-amylase rBD5063 was secreted by P. pastoris up to a yield of 60mg/L. TLC results for the various hydrolysis products of soluble starch showed that rBD5063 cleaved alpha-1,4-glucosidic linkages, and most products were glucose, maltose, and other oligosaccharides.rBD5063 was purified and characterized. When rBD5063 hydrolyzed soluble starch, the optimal
pH was 5.0 and more than 50% of its maximal activity remained after treatment in the buffers ranged from pH 3.5-10.0 for 30 minutes. The optimum temperature was 100-110°C, and the half-life at 90°C, pH 5.0 was 30 minutes. Low content of Ca + is enough to activate and stabilize its activity, and even up to 20mmol/L, Ca2+ only affected its activity slightly. Mg2+, SDS and Trition X-100 partially inhibited its activity, while Cu2+ and EDTA thoroughly inhibited its activity.Most rBD5063 was inclined to aggregate into bioactive polymers form, which would be disaggregated into three bioactive proteins with molecular weight of 64, 57, 44kD, respectively. rBD5063 is not modified by N-linked glycosyiation but by O-linked glycosyiation. The O-linked glycosyiation had little influence on thermostability and the optimal temperature of rBD5063.In conclusion, two acidothermophilic alpha-amylases were high-level expressed in P. pastoris. Those recombinant alpha-amylases expressed in P. pastoris held essential characterization of native alpha-amylases. As a result, P. pastoris has been of great value as the bioreactor to produce extremophilic alpha-amylases.
引文
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