α-淀粉酶AmyP融合三個外源淀粉結合結構（SBD）后的酶學性質研究
發(fā)布時間：2018-10-21 16:30
【摘要】：α-淀粉酶(α-amylase)是一個重要的工業(yè)用酶,廣泛應用在食品、化工、釀造等行業(yè)。具有生淀粉降解能力的α-淀粉酶AmyP來自海洋宏基因文庫,它不僅能快速的降解多種生淀粉,而且對生大米淀粉具有偏好性。AmyP有一個淀粉結合結構域SBD,該SBD的缺失不會急劇降低AmyP的生淀粉降解能力。為了提高AmyP的降解能力,我們對 AmyP 的 SBD 進行了替換,選擇來自 Cryptococcus sp.S-2,Thermobifidafusca NTU22和Clostrid u butyricum T--7 的α淀粉酶酶的SBBD 與 AyPP△BBD(缺失SBD)進行融合,在大腸桿菌中重組表達,獲得三個融合酶AmyP-Cr、AmyP-Th和AmyP-Cl。融合酶AmyP-Cr是AmyP△SBD與Cryptococcussp.S-2的α-淀粉酶的SBD的融合,在三個融合酶中酶學性質最好。AmyP-Cr對生大米淀粉的比活達到327.9±31.8 U/mg;是野生型AmyP的1.7倍。4h內,AmyP-Cr對大米生淀粉的最高降解率為46.6%,是野生型AmyP的1.8倍。AmyP-Cr的對生大米淀粉的最高結合率為0.113 uM/g,是同條件下AmyP的5.1倍。而且AmyP-Cr的熱穩(wěn)定性也獲得了提高,40°℃的半衰期超過12 h,是野生型AmyP的5.1倍。這些數據表明融合酶AmyP-Cr對生大米淀粉降解能力的提高,是由于來自Crpytococcussp.S-2的SBD能同時提高酶對生大米底物的吸附力和熱穩(wěn)定性。AmyP-Cr是一個新的性質比較優(yōu)良的生大米淀粉降解酶。融合酶 AmyP-Th 是 AmyP△SBD與Thermobifda fusca NTU22 的α-淀粉酶的 SBD 融合。AmyP-Th對生大米淀粉的比活為254.2±26.5 U/mg,為野生型AmyP的1.3倍。4h內,AmyP-Th對大米生淀粉的最高降解率為39.6%,雖然比野生型AmyP的提高了1.5倍,但是略低于AmyP-Cr的降解率。AmyP-Th的熱穩(wěn)定性沒有明顯改善。融合酶 AmyP-C l是 AmyPASBD 與 Clostridium butyricum T-7 的 α-淀粉酶的 SBD 融合。AmyP-Cl對生大米淀粉的比活為468.6±43.5 U/mg,為野生型AmyP的2.4倍。雖然AmyP-Cl的比活在三個融合蛋白中最高,但是AmyP-Cl在4h內對大米生淀粉的最高降解率為41.6%,略低于AmyP-Cr。此外,AmyP-Cl的熱穩(wěn)定性也沒有明顯改善。
[Abstract]:偽-amylase (偽-amylase) is an important industrial enzyme, widely used in food, chemical, brewing and other industries. The 偽 -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 偽 -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 偽 -amylase of Cryptococcussp.S-2. The specific activity of AmyP-Cr to raw rice starch was 327.9 鹵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 擄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 偽 -amylase of AmyP SBD and Thermobifda fusca NTU22. The specific activity of AmyP-Th to raw rice starch was 254.2 鹵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 鹵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 偽 -amylase of AmyPASBD and Clostridium butyricum T-7. The specific activity of AmyP-Cl to raw rice starch was 468.6 鹵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.
【學位授予單位】：安徽大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q55
本文編號：2285694
[Abstract]:偽-amylase (偽-amylase) is an important industrial enzyme, widely used in food, chemical, brewing and other industries. The 偽 -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 偽 -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 偽 -amylase of Cryptococcussp.S-2. The specific activity of AmyP-Cr to raw rice starch was 327.9 鹵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 擄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 偽 -amylase of AmyP SBD and Thermobifda fusca NTU22. The specific activity of AmyP-Th to raw rice starch was 254.2 鹵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 鹵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 偽 -amylase of AmyPASBD and Clostridium butyricum T-7. The specific activity of AmyP-Cl to raw rice starch was 468.6 鹵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.
【學位授予單位】：安徽大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q55
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