古菌Sulfolobus sofaltaricus P2嗜热嗜酸淀粉酶基因的克隆和表达
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摘要
耐热淀粉酶是现在工业生产中一个很重要的酶制剂，因其热稳定好、水解淀粉效率高，而广泛地应用在食品、制药等行业。目前工业上大规模使用的耐热淀粉酶大部分来自芽孢杆菌，主要是地衣芽孢杆菌。但地衣芽孢杆菌耐高温α-淀粉酶其最适pH为6.0左右，液化完成后进行糖化时所用的糖化酶最适pH为4.5，需要进行pH值的调整。这样不但增加了生产成本，而且造成后续处理设备的增加，排出的废水由于盐浓度的增加，还会造成处理困难和处理成本的上升。因此寻找和糖化酶最适pH相一致的耐高温α-淀粉酶对改进淀粉转化工艺有重要意义。
Sulfolobus sofaltaricus P2属于极端嗜热古菌，最适生长温度为80℃，最适pH为3.0-4.5。它的基因组全序列的测定已于2001年完成，其中有一编码α-淀粉酶的基因。通过PCR扩增，获得了该菌的α-淀粉酶基因sspa。将该基因插入表达载体pET29a中，构建重组表达载体pET29a-sspa，经IPTG诱导在E.coli BL21(DE3)得到了表达，但是活性较低。通过软件SMART分析，sspa基因的功能域为7-447位氨基酸。所以将sspa基因进行了部分切除，使酶活有了提高。
另外选择大肠杆菌和枯草芽孢杆菌的穿梭表达载体pP43NMK，构建重组表达载体pP43NMK-sspa，实现α-淀粉酶基因sspa在在枯草芽孢杆菌中的分泌表达，但表达量非常低。分析表明可能是该基因中含有大量枯草芽孢杆菌的稀有密码子所致，因此使用重叠延伸PCR的方法，对该基因中的4个稀有密码子进行定点突变，最终提高了在枯草芽孢杆菌中的表达量。
Thermostableα-amylase is one of the most important enzyme in industry, which iswidely used in food and pharmacy for its heat-resistant and efficient starch hydrolyzing. Atpresent mostα-amylase used in industry in large scale is from Bacillus licheniformis. Butthe optimal pH ofα-amylase from Bacillus licheniformis is 6.0, which was used inindustry in large scale. But the optimal pH of glucosidase is 4.5, which was used in thefollowing saccharification process. So before the following technics, product of starchhydrolyzation must be adjusted pH, which enhanced the producing cost and producedwaste water not easily to be treated. Accordingly finding hyperthermophilic and acidophilicα-amylase is very important.
Sulfolobus solfataricus P2 is a hyperthermophilic and acidophilic archaeon living optimallyat 80℃and pH3.0-4.5. The its genomic DNAwas sequenced in 2001, which contains aα-amylase gene.α-amylase gene of Sulfolobus solfataricus P2(sspa) was got by PCR andcloned into expression vector pET29a. The recombinant plasmid pET29a- sspa wastransformed into E.coli BL21(DE3). Induced by IPTG, theα-amylase gene was expressed.But the expressedα-amylase had low activity. By analysing the domain of theα-amylasewith SMART software. It found that the functional domain of sspa is from 7 to 447 aminoacid. After exciseing part sequences, the rest sequences of sspa was cloned and expressedonce again in E.coli BL21(DE3). Theα-amylase activity was largely enhanced.
The shuttle vector pP43NMK was chosen to constructed the recombinant secretingexpression plasmid pP43NMK-sspa, and expressed in B. subtilis1A771. But expressionlevel was very low, which could be due to rare codons present in sspa. After site-directedmutagenesis expression level was largely enhanced.
Sulfolobus sofaltaricus P2属于极端嗜热古菌，最适生长温度为80℃，最适pH为3.0-4.5。它的基因组全序列的测定已于2001年完成，其中有一编码α-淀粉酶的基因。通过PCR扩增，获得了该菌的α-淀粉酶基因sspa。将该基因插入表达载体pET29a中，构建重组表达载体pET29a-sspa，经IPTG诱导在E.coli BL21(DE3)得到了表达，但是活性较低。通过软件SMART分析，sspa基因的功能域为7-447位氨基酸。所以将sspa基因进行了部分切除，使酶活有了提高。
另外选择大肠杆菌和枯草芽孢杆菌的穿梭表达载体pP43NMK，构建重组表达载体pP43NMK-sspa，实现α-淀粉酶基因sspa在在枯草芽孢杆菌中的分泌表达，但表达量非常低。分析表明可能是该基因中含有大量枯草芽孢杆菌的稀有密码子所致，因此使用重叠延伸PCR的方法，对该基因中的4个稀有密码子进行定点突变，最终提高了在枯草芽孢杆菌中的表达量。
Thermostableα-amylase is one of the most important enzyme in industry, which iswidely used in food and pharmacy for its heat-resistant and efficient starch hydrolyzing. Atpresent mostα-amylase used in industry in large scale is from Bacillus licheniformis. Butthe optimal pH ofα-amylase from Bacillus licheniformis is 6.0, which was used inindustry in large scale. But the optimal pH of glucosidase is 4.5, which was used in thefollowing saccharification process. So before the following technics, product of starchhydrolyzation must be adjusted pH, which enhanced the producing cost and producedwaste water not easily to be treated. Accordingly finding hyperthermophilic and acidophilicα-amylase is very important.
Sulfolobus solfataricus P2 is a hyperthermophilic and acidophilic archaeon living optimallyat 80℃and pH3.0-4.5. The its genomic DNAwas sequenced in 2001, which contains aα-amylase gene.α-amylase gene of Sulfolobus solfataricus P2(sspa) was got by PCR andcloned into expression vector pET29a. The recombinant plasmid pET29a- sspa wastransformed into E.coli BL21(DE3). Induced by IPTG, theα-amylase gene was expressed.But the expressedα-amylase had low activity. By analysing the domain of theα-amylasewith SMART software. It found that the functional domain of sspa is from 7 to 447 aminoacid. After exciseing part sequences, the rest sequences of sspa was cloned and expressedonce again in E.coli BL21(DE3). Theα-amylase activity was largely enhanced.
The shuttle vector pP43NMK was chosen to constructed the recombinant secretingexpression plasmid pP43NMK-sspa, and expressed in B. subtilis1A771. But expressionlevel was very low, which could be due to rare codons present in sspa. After site-directedmutagenesis expression level was largely enhanced.
引文
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陈陶声．酶制剂的生产技术[M]．上海科学技术出版社．1994
华南理工学院 等．酒精与白酒工艺学[M]．北京：轻工业出版社，1980，44-45
胡学智，凌晨，候琴芳等．高温α—淀粉酶生产菌种选育的研究[J]．微生物报．1991，31(4)：267-273
姜锡瑞等．酶制剂应用手册[M]．北京：中国轻工出版社，1999
蒋如璋，耿运琪，倪津等．地衣芽孢杆菌热稳定α—淀粉酶基因的分子克隆及其在枯草芽孢杆菌中的表达[J]．遗传学报．1987，14(5)：323-331
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孔显良，王俊英，江洪涛等．地衣芽孢杆菌胞外耐高温α—淀粉酶的研究[J]．微生物学报．1993，33(4)：274-279
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林耀建，林影，吴晓英．极端微生物的研究概况[J]．工业微生物．2000，30(3)：53-55
刘莉，陈炜，金城．芝田硫化叶菌新型α—淀粉酶基因在大肠杆菌的克隆和表达[J]．微生物学报．2000，40(3)：323-326
刘仲敏，曾辉，何新民等．耐高温α—淀粉酶的研制开发及酶学特性的研究[J]．食品工业科技．1999，20(4)：18-20
罗贵民等．酶工程[M]．北京：化学工业出版社，2002，337
马向东，康海霞，黄春华等．枯草芽孢杆菌α—淀粉酶基因的克隆及表达[J]．河南农业大学学报．2000，34(4)：223-226
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