豆豉纤溶酶定向进化及突变酶基因的表达研究
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摘要
豆豉纤溶酶(Douchi fibrinolytic enzyme,DFE)是从中国传统发酵食品豆豉中发现的新型纤溶酶,具有开发成新一代溶栓药物和相关功能食品的巨大潜力。本文从产纤溶酶的枯草芽孢杆菌DC12中克隆豆豉纤溶酶基因并对其进行了验证,在此基础上通过定向进化得到催化效率提高的突变酶,将突变酶基因在枯草芽孢杆菌蛋白酶缺陷株WB800中进行了诱导型分泌表达及发酵优化。主要内容与结果如下:
利用PCR技术从产纤溶酶的枯草芽孢杆菌DC-12中扩增豆豉纤溶酶基因,为验证豆豉纤溶酶基因正确与否以及方便在后续研究中对随机突变文库的筛选,将豆豉纤溶酶基因连接到表达载体pET-32a中,构建豆豉纤溶酶重组表达质粒然后转化到E.coli BL21(DE3)中,重组菌株于37℃培养至OD600为0.6左右,加入终浓度为0.4 mmol/L的IPTG于24℃进行诱导,SDS-PAGE显示表达产物同时存在于菌体破碎后的上清液和沉淀中,菌体破碎后上清液的纤溶酶活力为200 IU/mL。通过Ni-NTA亲和柱层析和Sephadex G-75分子筛层析对重组豆豉纤溶酶进行纯化,得到分子量约为28 kDa的目的蛋白,重组蛋白纯化后N-端氨基酸序列测定结果与表达质粒构建时的DNA序列推导的氨基酸序列一致。通过表达产物的纤溶酶活性、纯化后目的蛋白的分子量、纯化后目的蛋白N末端氨基酸序列测定证实所克隆的基因为豆豉纤溶酶基因。
通过易错PCR技术对豆豉纤溶酶基因进行体外随机突变,并构建突变体文库,利用血纤溶酶最适底物H-D-Val-Leu-Lys-pNA对突变体文库进行筛选,经过三轮易错PCR,筛选到对H-D-Val-Leu-Lys-pNA底物特异性提高,催化效率为未突变豆豉纤溶酶2.57倍的突变酶mDFE3。序列分析表明突变酶基因发生六处碱基突变(C119G /T236C /A308T/G397A/A633T/T705C),其中四处突变发生氨基酸取代(P40R/ V79A/ Q103L/ A133T),另两处为同义突变。
为了提高突变酶的产量并缩短发酵周期,利用PCR方法从质粒pBE3中扩增卡那霉素抗性基因并将其插入到载体pHT43中,构建了携带卡那霉素抗性基因的重组载体pHK11。PCR扩增突变酶基因并插入到pHK11中,构建了突变酶基因表达质粒并将其转入枯草芽孢杆菌蛋白酶缺陷株WB800中进行表达。将重组菌株培养至对数中期,加入终浓度为1 mmol/L IPTG进行诱导,经诱导5 h,发酵上清液纤溶酶活力达1164 IU/mL。发酵液经SDS-PAGE分析可见约28 kDa目标蛋白条带。质粒稳定性试验表明突变酶重组表达质粒在无卡那霉素的LB培养基中具有一定的分裂不稳定性,而在30μg/mL卡那霉素的选择压力下重组质粒具有良好的结构稳定性,传代50代仍具有良好的产酶能力。
对突变酶基因重组菌株进行了摇瓶条件下的产酶发酵优化研究,优化后发酵培养基:碳源为葡萄糖,浓度为2 %;氮源为大豆蛋白胨,浓度为2 %;无机盐组合为:0.6 % K_2HPO_4、0.2 % KH2PO4、0.04 % CaCl2、0.075 % MgSO_4;培养基初始pH为7.0。优化后诱导产酶条件为:250 mL三角瓶中装液量40 mL;以2 %接种量接入种子培养液;在37℃振荡培养至对数生长中期,加入终浓度为1 mmol/L的IPTG后转至30℃进行诱导。在此条件下,重组菌pHK-mDFE3/WB800发酵上清液纤溶酶活性为1948 IU/mL。
DFE(Douchi fibrinolytic enzyme) is a new fibrinolytic enzyme which was discovered in Douchi. DFE is worthy developing as either natural thrombolytic agent or health food to prevent thrombotic diseases.The gene encoding pro-DFE from DC-12 was cloned and expressed in E. col.i The catalytic efficiency of DFE was improved by directed evolution. The mutant DFE was expressed in Bacillus subtiilis WB800 by induction with IPTG and the fermentation conditions were optimized. The main results of the thesis are as follows:
A functional expression is favorable for high throughput screening(HTS) which is applied to screen libraries consisting of a large number of variants produced by random mutation experiments such as directed evolution. The pro-DFE gene was amplified from DNA of Bacillus subtiilis DC-12 isolated from Douchi-a traditional Chinese fermented-soybean food, The fragment was cloned into PET-32a, and then transformed into E.coli BL21(DE3).The recombinant strain was grown at 37℃. At an OD600 of 0.6,cells were induced by additional IPTG(0.4 mmol/L final concentration). Growth was continued at 24℃. The expressed products were analyzed through SDS-PAGE, the recombinant protein exist in the supernatants and pellets. The strong fibrinolytic activity was detected in the supernatants and reached 200 IU/mL. The recombinant protein with a molecular weight of 28 kDa was purified by Ni2+-NTA column and Sephadex G-75 column.N-terminal sequence of the recombinant protein is in according with the result deduced by DNA sequence.DFE gene was confirmed by the functional expression in E.coli, the molecular weight and N-terminal sequence of the recombinant protein.
Mutagenesis on Douchi fibrinolytic enzyme gene was performed by using error-prone PCR strategy. After three cycles of error prone PCR and screening by substrate H-D-Val-Leu-Lys-pNA, the mutant enzyme with improved substrate specificity and catalytic efficiency was obtained. Gene analysis of the mutant enzyme gene showed that the mutant had six nucleotide substitutions(C119G/T236C/A308T/G397A/A633T/T705C)and four of them caused amino acid changes(P40R/ V79A/ Q103L/ A133T).
In order to highly express of mDFE3 gene in B.subtilis WB800 and shorten the fermentation period,the full Kanr gene including its promoter seuqence was cloned from pBE3 and inserted into a shuttle vector pHT43 to construct the expression vector pHK11. The mDFE3 encoding sequence was cloned into pHK11 and expressed in B. subtilis WB800. The recombinant strain was grown in LB to the mid-log phase,cells were induced by additional IPTG (1 mmol/L final concentration). Growth was continued at 37℃for up to 5 h. Its maximum fibrinolytic activity of the supernatant in LB medium was 1164 IU/mL. The results of SDS-PAGE analysis showed that there were indeed recombinant proteins with a molecular mass of 28 kDa in supernants. Plasmid stability was also measured. The recombinant plasmid didn't appear obvious gene deletion, showed structural stability after 50 generations. However, the plasmid showed some segregational instability, the plasmid-free cells appeared under no Kanamycin selection pressure.This result indicates that cells carrying certain recombinant pHK-mDFE3 plasmids should be grown in the presence of Kanamycin to avoid loss of the plasmids.
In order to further enhance production of recombinant Douchi fibrinolytic enzyme,the optimal fermentation conditions of pHK-mDFE3/WB800 were determined. The compositions of fermentation medium are 2% glucose;2% soybean peptone;0.6% K2HPO4,0.2% KH_2PO_4, 0.04% CaCl2,0.075% MgSO_4;pH7.0; Initial growth of the cells at 37℃to the mid-log phase and subsequent induction with IPTG(1mmol/L final concentration) at 30℃;40 mL of media are in 250 mL flask. Under these conditions the fibrinolytic activity of the supernatant can reach 1948 IU/mL.
利用PCR技术从产纤溶酶的枯草芽孢杆菌DC-12中扩增豆豉纤溶酶基因,为验证豆豉纤溶酶基因正确与否以及方便在后续研究中对随机突变文库的筛选,将豆豉纤溶酶基因连接到表达载体pET-32a中,构建豆豉纤溶酶重组表达质粒然后转化到E.coli BL21(DE3)中,重组菌株于37℃培养至OD600为0.6左右,加入终浓度为0.4 mmol/L的IPTG于24℃进行诱导,SDS-PAGE显示表达产物同时存在于菌体破碎后的上清液和沉淀中,菌体破碎后上清液的纤溶酶活力为200 IU/mL。通过Ni-NTA亲和柱层析和Sephadex G-75分子筛层析对重组豆豉纤溶酶进行纯化,得到分子量约为28 kDa的目的蛋白,重组蛋白纯化后N-端氨基酸序列测定结果与表达质粒构建时的DNA序列推导的氨基酸序列一致。通过表达产物的纤溶酶活性、纯化后目的蛋白的分子量、纯化后目的蛋白N末端氨基酸序列测定证实所克隆的基因为豆豉纤溶酶基因。
通过易错PCR技术对豆豉纤溶酶基因进行体外随机突变,并构建突变体文库,利用血纤溶酶最适底物H-D-Val-Leu-Lys-pNA对突变体文库进行筛选,经过三轮易错PCR,筛选到对H-D-Val-Leu-Lys-pNA底物特异性提高,催化效率为未突变豆豉纤溶酶2.57倍的突变酶mDFE3。序列分析表明突变酶基因发生六处碱基突变(C119G /T236C /A308T/G397A/A633T/T705C),其中四处突变发生氨基酸取代(P40R/ V79A/ Q103L/ A133T),另两处为同义突变。
为了提高突变酶的产量并缩短发酵周期,利用PCR方法从质粒pBE3中扩增卡那霉素抗性基因并将其插入到载体pHT43中,构建了携带卡那霉素抗性基因的重组载体pHK11。PCR扩增突变酶基因并插入到pHK11中,构建了突变酶基因表达质粒并将其转入枯草芽孢杆菌蛋白酶缺陷株WB800中进行表达。将重组菌株培养至对数中期,加入终浓度为1 mmol/L IPTG进行诱导,经诱导5 h,发酵上清液纤溶酶活力达1164 IU/mL。发酵液经SDS-PAGE分析可见约28 kDa目标蛋白条带。质粒稳定性试验表明突变酶重组表达质粒在无卡那霉素的LB培养基中具有一定的分裂不稳定性,而在30μg/mL卡那霉素的选择压力下重组质粒具有良好的结构稳定性,传代50代仍具有良好的产酶能力。
对突变酶基因重组菌株进行了摇瓶条件下的产酶发酵优化研究,优化后发酵培养基:碳源为葡萄糖,浓度为2 %;氮源为大豆蛋白胨,浓度为2 %;无机盐组合为:0.6 % K_2HPO_4、0.2 % KH2PO4、0.04 % CaCl2、0.075 % MgSO_4;培养基初始pH为7.0。优化后诱导产酶条件为:250 mL三角瓶中装液量40 mL;以2 %接种量接入种子培养液;在37℃振荡培养至对数生长中期,加入终浓度为1 mmol/L的IPTG后转至30℃进行诱导。在此条件下,重组菌pHK-mDFE3/WB800发酵上清液纤溶酶活性为1948 IU/mL。
DFE(Douchi fibrinolytic enzyme) is a new fibrinolytic enzyme which was discovered in Douchi. DFE is worthy developing as either natural thrombolytic agent or health food to prevent thrombotic diseases.The gene encoding pro-DFE from DC-12 was cloned and expressed in E. col.i The catalytic efficiency of DFE was improved by directed evolution. The mutant DFE was expressed in Bacillus subtiilis WB800 by induction with IPTG and the fermentation conditions were optimized. The main results of the thesis are as follows:
A functional expression is favorable for high throughput screening(HTS) which is applied to screen libraries consisting of a large number of variants produced by random mutation experiments such as directed evolution. The pro-DFE gene was amplified from DNA of Bacillus subtiilis DC-12 isolated from Douchi-a traditional Chinese fermented-soybean food, The fragment was cloned into PET-32a, and then transformed into E.coli BL21(DE3).The recombinant strain was grown at 37℃. At an OD600 of 0.6,cells were induced by additional IPTG(0.4 mmol/L final concentration). Growth was continued at 24℃. The expressed products were analyzed through SDS-PAGE, the recombinant protein exist in the supernatants and pellets. The strong fibrinolytic activity was detected in the supernatants and reached 200 IU/mL. The recombinant protein with a molecular weight of 28 kDa was purified by Ni2+-NTA column and Sephadex G-75 column.N-terminal sequence of the recombinant protein is in according with the result deduced by DNA sequence.DFE gene was confirmed by the functional expression in E.coli, the molecular weight and N-terminal sequence of the recombinant protein.
Mutagenesis on Douchi fibrinolytic enzyme gene was performed by using error-prone PCR strategy. After three cycles of error prone PCR and screening by substrate H-D-Val-Leu-Lys-pNA, the mutant enzyme with improved substrate specificity and catalytic efficiency was obtained. Gene analysis of the mutant enzyme gene showed that the mutant had six nucleotide substitutions(C119G/T236C/A308T/G397A/A633T/T705C)and four of them caused amino acid changes(P40R/ V79A/ Q103L/ A133T).
In order to highly express of mDFE3 gene in B.subtilis WB800 and shorten the fermentation period,the full Kanr gene including its promoter seuqence was cloned from pBE3 and inserted into a shuttle vector pHT43 to construct the expression vector pHK11. The mDFE3 encoding sequence was cloned into pHK11 and expressed in B. subtilis WB800. The recombinant strain was grown in LB to the mid-log phase,cells were induced by additional IPTG (1 mmol/L final concentration). Growth was continued at 37℃for up to 5 h. Its maximum fibrinolytic activity of the supernatant in LB medium was 1164 IU/mL. The results of SDS-PAGE analysis showed that there were indeed recombinant proteins with a molecular mass of 28 kDa in supernants. Plasmid stability was also measured. The recombinant plasmid didn't appear obvious gene deletion, showed structural stability after 50 generations. However, the plasmid showed some segregational instability, the plasmid-free cells appeared under no Kanamycin selection pressure.This result indicates that cells carrying certain recombinant pHK-mDFE3 plasmids should be grown in the presence of Kanamycin to avoid loss of the plasmids.
In order to further enhance production of recombinant Douchi fibrinolytic enzyme,the optimal fermentation conditions of pHK-mDFE3/WB800 were determined. The compositions of fermentation medium are 2% glucose;2% soybean peptone;0.6% K2HPO4,0.2% KH_2PO_4, 0.04% CaCl2,0.075% MgSO_4;pH7.0; Initial growth of the cells at 37℃to the mid-log phase and subsequent induction with IPTG(1mmol/L final concentration) at 30℃;40 mL of media are in 250 mL flask. Under these conditions the fibrinolytic activity of the supernatant can reach 1948 IU/mL.
引文
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