普鲁兰酶在解淀粉芽孢杆菌中表达方法的探索
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摘要
普鲁兰酶水解支链淀粉中α-1,6-糖苷键,在淀粉糖生产中具有重要用途。目前已发现的来源于芽孢杆菌及其近缘种属的普鲁兰酶分子量都在100 kDa左右,由于分子量大其分泌表达有很大的困难。本文探索以解淀粉芽孢杆菌为宿主表达普鲁兰酶的方法。
解淀粉芽孢杆菌BF7658菌株(Bacillus amyloliquefaciens BF7658)是我国自主开发的中温α-淀粉酶工业生产菌。由于解淀粉芽孢杆菌BF7658具有高产淀粉酶的特点,因此其淀粉酶基因启动子是构建表达载体的理想材料。本文以解淀粉芽孢杆菌BF7658染色体DNA为模板扩增获得其启动子和信号肽编码区基因片段Pamy,与大肠杆菌质粒pBR322的复制原件pMB1 ori、穿梭质粒pHY300PLK的芽孢杆菌复制元件ori-pama以及四环素抗性基因tet进行DNA重组,构建了适用于解淀粉芽孢杆菌的分泌表达载体pCHA03。
解淀粉芽孢杆菌BF7658菌株在发酵后期有芽孢形成并停止产酶的特点,破坏其芽孢形成能力有可能延长发酵产酶时间。以红霉素抗性为标记构建了针对解淀粉芽孢杆菌BF7658菌株芽孢形成关键基因spoIIAC(psp)的基因敲除载体pHY-psp-pem。利用载体pHY-psp-pem转化解淀粉芽孢杆菌BF7658,通过传代筛选最终获得一株spoIIAC基因缺失的菌株,命名为解淀粉芽孢杆菌sj08。解淀粉芽孢杆菌sj08与表达载体pCHA03共同组成一套新型芽孢杆菌分泌表达系统。
将普鲁兰酶编码基因BdP插入pCHA03以及枯草芽孢杆菌表达载体pUC980多克隆位点,构建重组质粒pUC980-BdP和pCHA03-BdP。将上述重组质粒分别转化枯草芽孢杆菌1A717和解淀粉芽孢杆菌BF7658。摇瓶发酵实验显示两种质粒转化枯草芽孢杆菌得到的转化子发酵液中均只有微弱的普鲁兰酶酶活。pUC980-BdP和pCHA03-BdP转化解淀粉芽孢杆菌BF7658得到的重组菌摇瓶发酵最高酶活分别达到1.1 ASPU/mL和3.1 ASPU/mL。以解淀粉芽孢杆菌BF7658为宿主的重组菌发酵水平明显高于以枯草芽孢杆菌为宿主的重组菌。以解淀粉芽孢杆菌BF7658为宿主时,利用其自身淀粉酶基因启动子和信号肽能更有效的实现普鲁兰酶的分泌表达。将pCHA03-BdP转入解淀粉芽孢杆菌sj08,获得重组菌解淀粉芽孢杆菌sj08/pCHA03-BdP。15 L发酵罐发酵试验表明解淀粉芽孢杆菌sj08/pCHA03-BdP的发酵产酶时间比解淀粉芽孢杆菌BF7658/pCHA03-BdP延长8 h,发酵液酶活提高23%以上。对解淀粉芽孢杆菌sj08/pCHA03-BdP发酵条件进行优化,在优化条件下重组菌普鲁兰酶发酵水平达到6.9 ASPU/mL。纯化了解淀粉芽孢杆菌sj08/pCHA03-BdP表达的普鲁兰酶,研究了纯酶的酶学性质。通过纯化,从发酵液中获得了电泳纯的普鲁兰酶。酶回收率为20.1%,纯化倍数为49.9。该酶分子量大约为100 kDa。重组普鲁兰酶最适作用温度为60℃,最适pH为5.0,60℃保温3 h后酶活仍保存50%左右。
Pullulanase is a debranching enzyme for the hydrolysis ofα-1,6-linkages in starch. It is wildly used in the starch processing industry. The molecular weight of the pullulanases produced by Bacillus sp. or its relatives is always approximately 100 kDa; hence, its secretory expression is difficult. In the current study, the secretory expression of pullulanase is investigated using Bacillus amyloliquefaciens as expression host.
Bacillus amyloliquefaciens BF7658 was used for the commercial production of mesophilicα-amylase in China. Considering that B. amyloliquefaciens BF7658 secretes a large amount ofα-amylase, the promoter ofα-amylase could be used to drive the gene expression in an expression vector. The DNA fragment Pamy, which contains the promoter and the signal peptide-encoding region ofα-amylase was amplified using PCR, with the genomic DNA of B. amyloliquefaciens 7658 used as a template. A B. amyloliquefaciens espression vector was constructed with Pamy as promoter, the pMB1 ori from pBR322 as the original replication region in E. coli, with the ori-pama and the tetracycline-resistance (tet) gene from pHY300PLK as the replication original region in Bacillus and the selection marker, respectively. The constructed vector was designated as pCHA03.
In B. amyloliquefaciens BF7658, the production ofα-amylase always ends because of spore germination; therefore, disruption of the gene responsible for sporulation may delay spore germination,which would increase the time for enzyme production. In B. amyloliquefaciens, spoIIAC is the key gene responsible for initiation of sporulation. A vector used for the deletion of spoIIAC, designated as pHY-psp-pem, was constructed with erythromycin resistance as the selection marker. B. amyloliquefaciens BF7658 was transformed with pHY-psp-pem, a mutant strain with spoIIAC, which was disrupted, selected, and designated as B. amyloliquefaciens sj08. Together with pCHA03, a new expression system was formed.
The BdP gene that encodes pullulanase was inserted into the MCS of secretary expression vector pUC980 and pCHA03, resulting in recombinant plasmids pUC980-BdP and pCHA03-BdP. The two recombinant plasmids were transformed into B. subtilis 1A717 and B. amyloliquefaciens BF7658, respectively. The pullulanase activities produced by B. subtilis 1A717/pUC980-BdP and B. subtilis 1A717/pCHA03-BdP were rather low. The pullulanase activities produced by B. amyloliquefaciens BF7658/pUC980-BdP and B. amyloliquefaciens BF7658/pCHA03-BdP were at 1.1 ASPU/mL, and 3.1 ASPU/mL, respectively. The results indicate that the pullulanase activity expressed in B. amyloliquefaciens were higher than that in B. subtilis. The gene expression, driven by Pamy is more efficient than that by the P43 promoter of pUC980. Plasmid pCHA03-BdP was transformed into B. amyloliquefaciens sj08. The fermentation experiment in a 15 L fermentor showed that compared with B. amyloliquefaciens BF7658/pCHA03-BdP, pullulanase production with B. amyloliquefaciens sj08/pCHA03-BdP was prolonged to 8 h, and the pullulanase activity increased by 23%. Culture condition of B. amyloliquefaciens sj08/pCHA03-BdP in flask scale was optimized for pullulanase production. The maximum enzyme activity under the optimized conditions reached 6.9 ASPU/mL. The recombinant pullulanase produced by B. amyloliquefaciens sj08/pCHA03-BdP was purified to homogeneity and the enzyme properties were investigated. The recover rate of the enzyme was 20.1% and fold of purification was 49.9. The molecular weight of the enzyme was about 100 kDa. It showed optimum enzymatic activity at pH5.0 and has an optimal temperature of about 60℃; more than 50% of its initial enzyme activity was still detectable after incubation at 60℃for 3 h.
解淀粉芽孢杆菌BF7658菌株(Bacillus amyloliquefaciens BF7658)是我国自主开发的中温α-淀粉酶工业生产菌。由于解淀粉芽孢杆菌BF7658具有高产淀粉酶的特点,因此其淀粉酶基因启动子是构建表达载体的理想材料。本文以解淀粉芽孢杆菌BF7658染色体DNA为模板扩增获得其启动子和信号肽编码区基因片段Pamy,与大肠杆菌质粒pBR322的复制原件pMB1 ori、穿梭质粒pHY300PLK的芽孢杆菌复制元件ori-pama以及四环素抗性基因tet进行DNA重组,构建了适用于解淀粉芽孢杆菌的分泌表达载体pCHA03。
解淀粉芽孢杆菌BF7658菌株在发酵后期有芽孢形成并停止产酶的特点,破坏其芽孢形成能力有可能延长发酵产酶时间。以红霉素抗性为标记构建了针对解淀粉芽孢杆菌BF7658菌株芽孢形成关键基因spoIIAC(psp)的基因敲除载体pHY-psp-pem。利用载体pHY-psp-pem转化解淀粉芽孢杆菌BF7658,通过传代筛选最终获得一株spoIIAC基因缺失的菌株,命名为解淀粉芽孢杆菌sj08。解淀粉芽孢杆菌sj08与表达载体pCHA03共同组成一套新型芽孢杆菌分泌表达系统。
将普鲁兰酶编码基因BdP插入pCHA03以及枯草芽孢杆菌表达载体pUC980多克隆位点,构建重组质粒pUC980-BdP和pCHA03-BdP。将上述重组质粒分别转化枯草芽孢杆菌1A717和解淀粉芽孢杆菌BF7658。摇瓶发酵实验显示两种质粒转化枯草芽孢杆菌得到的转化子发酵液中均只有微弱的普鲁兰酶酶活。pUC980-BdP和pCHA03-BdP转化解淀粉芽孢杆菌BF7658得到的重组菌摇瓶发酵最高酶活分别达到1.1 ASPU/mL和3.1 ASPU/mL。以解淀粉芽孢杆菌BF7658为宿主的重组菌发酵水平明显高于以枯草芽孢杆菌为宿主的重组菌。以解淀粉芽孢杆菌BF7658为宿主时,利用其自身淀粉酶基因启动子和信号肽能更有效的实现普鲁兰酶的分泌表达。将pCHA03-BdP转入解淀粉芽孢杆菌sj08,获得重组菌解淀粉芽孢杆菌sj08/pCHA03-BdP。15 L发酵罐发酵试验表明解淀粉芽孢杆菌sj08/pCHA03-BdP的发酵产酶时间比解淀粉芽孢杆菌BF7658/pCHA03-BdP延长8 h,发酵液酶活提高23%以上。对解淀粉芽孢杆菌sj08/pCHA03-BdP发酵条件进行优化,在优化条件下重组菌普鲁兰酶发酵水平达到6.9 ASPU/mL。纯化了解淀粉芽孢杆菌sj08/pCHA03-BdP表达的普鲁兰酶,研究了纯酶的酶学性质。通过纯化,从发酵液中获得了电泳纯的普鲁兰酶。酶回收率为20.1%,纯化倍数为49.9。该酶分子量大约为100 kDa。重组普鲁兰酶最适作用温度为60℃,最适pH为5.0,60℃保温3 h后酶活仍保存50%左右。
Pullulanase is a debranching enzyme for the hydrolysis ofα-1,6-linkages in starch. It is wildly used in the starch processing industry. The molecular weight of the pullulanases produced by Bacillus sp. or its relatives is always approximately 100 kDa; hence, its secretory expression is difficult. In the current study, the secretory expression of pullulanase is investigated using Bacillus amyloliquefaciens as expression host.
Bacillus amyloliquefaciens BF7658 was used for the commercial production of mesophilicα-amylase in China. Considering that B. amyloliquefaciens BF7658 secretes a large amount ofα-amylase, the promoter ofα-amylase could be used to drive the gene expression in an expression vector. The DNA fragment Pamy, which contains the promoter and the signal peptide-encoding region ofα-amylase was amplified using PCR, with the genomic DNA of B. amyloliquefaciens 7658 used as a template. A B. amyloliquefaciens espression vector was constructed with Pamy as promoter, the pMB1 ori from pBR322 as the original replication region in E. coli, with the ori-pama and the tetracycline-resistance (tet) gene from pHY300PLK as the replication original region in Bacillus and the selection marker, respectively. The constructed vector was designated as pCHA03.
In B. amyloliquefaciens BF7658, the production ofα-amylase always ends because of spore germination; therefore, disruption of the gene responsible for sporulation may delay spore germination,which would increase the time for enzyme production. In B. amyloliquefaciens, spoIIAC is the key gene responsible for initiation of sporulation. A vector used for the deletion of spoIIAC, designated as pHY-psp-pem, was constructed with erythromycin resistance as the selection marker. B. amyloliquefaciens BF7658 was transformed with pHY-psp-pem, a mutant strain with spoIIAC, which was disrupted, selected, and designated as B. amyloliquefaciens sj08. Together with pCHA03, a new expression system was formed.
The BdP gene that encodes pullulanase was inserted into the MCS of secretary expression vector pUC980 and pCHA03, resulting in recombinant plasmids pUC980-BdP and pCHA03-BdP. The two recombinant plasmids were transformed into B. subtilis 1A717 and B. amyloliquefaciens BF7658, respectively. The pullulanase activities produced by B. subtilis 1A717/pUC980-BdP and B. subtilis 1A717/pCHA03-BdP were rather low. The pullulanase activities produced by B. amyloliquefaciens BF7658/pUC980-BdP and B. amyloliquefaciens BF7658/pCHA03-BdP were at 1.1 ASPU/mL, and 3.1 ASPU/mL, respectively. The results indicate that the pullulanase activity expressed in B. amyloliquefaciens were higher than that in B. subtilis. The gene expression, driven by Pamy is more efficient than that by the P43 promoter of pUC980. Plasmid pCHA03-BdP was transformed into B. amyloliquefaciens sj08. The fermentation experiment in a 15 L fermentor showed that compared with B. amyloliquefaciens BF7658/pCHA03-BdP, pullulanase production with B. amyloliquefaciens sj08/pCHA03-BdP was prolonged to 8 h, and the pullulanase activity increased by 23%. Culture condition of B. amyloliquefaciens sj08/pCHA03-BdP in flask scale was optimized for pullulanase production. The maximum enzyme activity under the optimized conditions reached 6.9 ASPU/mL. The recombinant pullulanase produced by B. amyloliquefaciens sj08/pCHA03-BdP was purified to homogeneity and the enzyme properties were investigated. The recover rate of the enzyme was 20.1% and fold of purification was 49.9. The molecular weight of the enzyme was about 100 kDa. It showed optimum enzymatic activity at pH5.0 and has an optimal temperature of about 60℃; more than 50% of its initial enzyme activity was still detectable after incubation at 60℃for 3 h.
引文
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