α-淀粉酶AmyP融合三个外源淀粉结合结构（SBD）后的酶学性质研究
发布时间：2018-10-21 16:30
【摘要】：α-淀粉酶(α-amylase)是一个重要的工业用酶,广泛应用在食品、化工、酿造等行业。具有生淀粉降解能力的α-淀粉酶AmyP来自海洋宏基因文库,它不仅能快速的降解多种生淀粉,而且对生大米淀粉具有偏好性。AmyP有一个淀粉结合结构域SBD,该SBD的缺失不会急剧降低AmyP的生淀粉降解能力。为了提高AmyP的降解能力,我们对 AmyP 的 SBD 进行了替换,选择来自 Cryptococcus sp.S-2,Thermobifidafusca NTU22和Clostrid u butyricum T--7 的α淀粉酶酶的SBBD 与 AyPP△BBD(缺失SBD)进行融合,在大肠杆菌中重组表达,获得三个融合酶AmyP-Cr、AmyP-Th和AmyP-Cl。融合酶AmyP-Cr是AmyP△SBD与Cryptococcussp.S-2的α-淀粉酶的SBD的融合,在三个融合酶中酶学性质最好。AmyP-Cr对生大米淀粉的比活达到327.9±31.8 U/mg;是野生型AmyP的1.7倍。4h内,AmyP-Cr对大米生淀粉的最高降解率为46.6%,是野生型AmyP的1.8倍。AmyP-Cr的对生大米淀粉的最高结合率为0.113 uM/g,是同条件下AmyP的5.1倍。而且AmyP-Cr的热稳定性也获得了提高,40°℃的半衰期超过12 h,是野生型AmyP的5.1倍。这些数据表明融合酶AmyP-Cr对生大米淀粉降解能力的提高,是由于来自Crpytococcussp.S-2的SBD能同时提高酶对生大米底物的吸附力和热稳定性。AmyP-Cr是一个新的性质比较优良的生大米淀粉降解酶。融合酶 AmyP-Th 是 AmyP△SBD与Thermobifda fusca NTU22 的α-淀粉酶的 SBD 融合。AmyP-Th对生大米淀粉的比活为254.2±26.5 U/mg,为野生型AmyP的1.3倍。4h内,AmyP-Th对大米生淀粉的最高降解率为39.6%,虽然比野生型AmyP的提高了1.5倍,但是略低于AmyP-Cr的降解率。AmyP-Th的热稳定性没有明显改善。融合酶 AmyP-C l是 AmyPASBD 与 Clostridium butyricum T-7 的 α-淀粉酶的 SBD 融合。AmyP-Cl对生大米淀粉的比活为468.6±43.5 U/mg,为野生型AmyP的2.4倍。虽然AmyP-Cl的比活在三个融合蛋白中最高,但是AmyP-Cl在4h内对大米生淀粉的最高降解率为41.6%,略低于AmyP-Cr。此外,AmyP-Cl的热稳定性也没有明显改善。
[Abstract]:伪-amylase (伪-amylase) is an important industrial enzyme, widely used in food, chemical, brewing and other industries. The 伪 -amylase AmyP, which has the ability to degrade raw starch, comes from the marine macro gene library. AmyP has a starch binding domain SBD, the absence of SBD does not dramatically reduce the ability of AmyP to degrade raw starch. In order to improve the degradation ability of AmyP, the SBD of AmyP was replaced by SBBD from Cryptococcus sp.S-2,Thermobifidafusca NTU22 and Clostrid u butyricum T- 7 伪 -amylase, which was fused with AyPP BBD (missing SBD) and expressed in Escherichia coli. Three fusion enzymes AmyP-Cr,AmyP-Th and AmyP-Cl. were obtained The fusion enzyme AmyP-Cr is the fusion of AmyP SBD and SBD of 伪 -amylase of Cryptococcussp.S-2. The specific activity of AmyP-Cr to raw rice starch was 327.9 卤31.8 U / mg, which was 1.7 times of that of wild type AmyP. Within 4 hours, the highest degradation rate of AmyP-Cr to rice starch was 46.6 and 1.8 times that of wild type AmyP, and the highest of AmyP-Cr to raw rice starch. The high binding rate of 0.113 uM/g, was 5.1 times higher than that of AmyP under the same conditions. The thermal stability of AmyP-Cr was also improved, and the half-life of 40 掳C was over 12 h, which was 5.1 times of that of wild-type AmyP. These data indicated that the ability of the fusion enzyme AmyP-Cr to degrade raw rice starch was improved because the SBD from Crpytococcussp.S-2 could improve the adsorption ability and thermal stability of the enzyme to the raw rice substrate simultaneously. AmyP-Cr is a new rice starch degrading enzyme with better properties. AmyP-Th was the SBD fusion of 伪 -amylase of AmyP SBD and Thermobifda fusca NTU22. The specific activity of AmyP-Th to raw rice starch was 254.2 卤26.5U / mg, which was 1.3 times of that of wild-type AmyP. Within 4 hours, the highest degradation rate of AmyP-Th to rice starch was 39.6%, although it was 1.5-fold higher than that of wild type AmyP. However, the degradation rate of AmyP-Th was slightly lower than that of AmyP-Cr. The thermal stability of AmyP-Th did not improve significantly. The specific activity of AmyP-Cl to raw rice starch was 468.6 卤43.5 U / mg, which was 2.4-fold of that of wild type AmyP. The fusion enzyme AmyP-C l was the SBD fusion of 伪 -amylase of AmyPASBD and Clostridium butyricum T-7. The specific activity of AmyP-Cl to raw rice starch was 468.6 卤43.5Umg. Although the specific activity of AmyP-Cl was the highest among the three fusion proteins, the highest degradation rate of rice raw starch by AmyP-Cl within 4 hours was 41.6, slightly lower than that of AmyP-Cr.. In addition, the thermal stability of AmyP-Cl has not been significantly improved.
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q55
本文编号：2285694
[Abstract]:伪-amylase (伪-amylase) is an important industrial enzyme, widely used in food, chemical, brewing and other industries. The 伪 -amylase AmyP, which has the ability to degrade raw starch, comes from the marine macro gene library. AmyP has a starch binding domain SBD, the absence of SBD does not dramatically reduce the ability of AmyP to degrade raw starch. In order to improve the degradation ability of AmyP, the SBD of AmyP was replaced by SBBD from Cryptococcus sp.S-2,Thermobifidafusca NTU22 and Clostrid u butyricum T- 7 伪 -amylase, which was fused with AyPP BBD (missing SBD) and expressed in Escherichia coli. Three fusion enzymes AmyP-Cr,AmyP-Th and AmyP-Cl. were obtained The fusion enzyme AmyP-Cr is the fusion of AmyP SBD and SBD of 伪 -amylase of Cryptococcussp.S-2. The specific activity of AmyP-Cr to raw rice starch was 327.9 卤31.8 U / mg, which was 1.7 times of that of wild type AmyP. Within 4 hours, the highest degradation rate of AmyP-Cr to rice starch was 46.6 and 1.8 times that of wild type AmyP, and the highest of AmyP-Cr to raw rice starch. The high binding rate of 0.113 uM/g, was 5.1 times higher than that of AmyP under the same conditions. The thermal stability of AmyP-Cr was also improved, and the half-life of 40 掳C was over 12 h, which was 5.1 times of that of wild-type AmyP. These data indicated that the ability of the fusion enzyme AmyP-Cr to degrade raw rice starch was improved because the SBD from Crpytococcussp.S-2 could improve the adsorption ability and thermal stability of the enzyme to the raw rice substrate simultaneously. AmyP-Cr is a new rice starch degrading enzyme with better properties. AmyP-Th was the SBD fusion of 伪 -amylase of AmyP SBD and Thermobifda fusca NTU22. The specific activity of AmyP-Th to raw rice starch was 254.2 卤26.5U / mg, which was 1.3 times of that of wild-type AmyP. Within 4 hours, the highest degradation rate of AmyP-Th to rice starch was 39.6%, although it was 1.5-fold higher than that of wild type AmyP. However, the degradation rate of AmyP-Th was slightly lower than that of AmyP-Cr. The thermal stability of AmyP-Th did not improve significantly. The specific activity of AmyP-Cl to raw rice starch was 468.6 卤43.5 U / mg, which was 2.4-fold of that of wild type AmyP. The fusion enzyme AmyP-C l was the SBD fusion of 伪 -amylase of AmyPASBD and Clostridium butyricum T-7. The specific activity of AmyP-Cl to raw rice starch was 468.6 卤43.5Umg. Although the specific activity of AmyP-Cl was the highest among the three fusion proteins, the highest degradation rate of rice raw starch by AmyP-Cl within 4 hours was 41.6, slightly lower than that of AmyP-Cr.. In addition, the thermal stability of AmyP-Cl has not been significantly improved.
【学位授予单位】：安徽大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q55
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