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CN116218801B - 一种耐热葡萄糖氧化酶突变体及其应用 - Google Patents

一种耐热葡萄糖氧化酶突变体及其应用 Download PDF

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CN116218801B
CN116218801B CN202211588610.9A CN202211588610A CN116218801B CN 116218801 B CN116218801 B CN 116218801B CN 202211588610 A CN202211588610 A CN 202211588610A CN 116218801 B CN116218801 B CN 116218801B
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游帅
卢宇
张伊欣
张温馨
张颖
徐远志
赵卫国
王俊
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Nanning Harworld Biotechnology Co ltd
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Abstract

一种耐热葡萄糖氧化酶突变体及其应用，其特征在于，包括以葡萄糖氧化酶PaGOD‑1GPE为母本对A263和K424两个位点突变后得到的突变体，命名为PaGOD‑1GPE_A263P和PaGOD‑1GPE_K424F；所述PaGOD‑1GPE_A263P的氨基酸序列如SEQ ID NO.1所示；所述PaGOD‑1GPE_K424F的氨基酸序列如SEQ ID NO.2所示。相对于盲目筛菌或人工（自然）诱变等手段，理性设计缩短了酶学性质改造时间。因此，将本发明的耐热葡萄糖氧化酶突变体应用于饲料添加产业中，具有广阔的应用前景。

Description

一种耐热葡萄糖氧化酶突变体及其应用
技术领域
本发明涉及基因工程和蛋白质工程领域，涉及一种来源于真菌P.amagasakiense的葡萄糖氧化酶PaGOD-1GPE突变体及其应用。
背景技术
葡萄糖氧化酶(glucose oxidase，GOD)是一种需氧脱氢酶，能专一地氧化β-D-葡萄糖成为葡萄糖酸和过氧化氢。葡萄糖氧化酶广泛分布于动植物和微生物体内，微生物是其主要来源，主要生产菌株为黑曲霉和青霉。葡萄糖氧化酶作为替代抗生素的新型饲料酶制剂，具有保护动物肠道，促进消化吸收，提高机体免疫力等多种功能。产自特异青霉和黑曲霉的葡萄糖氧化酶已被列入农业部《饲料添加剂品种目录(2013)》第4大类酶制剂。
Pen-GOD以同型二聚体的形式存在，每个亚基可非共价结合一个FAD。对β-D-葡萄糖专一性较强，通常来源于青霉属比来源于曲霉属的GOD对底物的催化效率更高，大多数GOD在30 50℃和pH 6.0的条件下表现出最大的酶活性，但是稳定性较差。目前，大多数GOD在pH 4.0 7.0的缓冲条件下可以维持稳定，当pH值小于3.0或高于10.0时，基本没有酶活。青霉属来源的GOD一般在50℃以下维持稳定。
葡萄糖氧化酶的应用十分广泛，主要包括饲料、食品、医药、纺织和生物燃料电池。在工业应用中，大多数应用环境都属于高温环境，因此获得热稳定性优良的葡萄糖氧化酶对工业生产至关重要。
目前，蛋白质工程在酶分子改良方面应用广泛，即通过对基因或者蛋白本身进行修改或修饰以改变蛋白结构从而实现酶功能的改造。蛋白质工程主要用于酶的热稳定性、催化效率、底物特异性和极端环境耐受性等酶学性质的设计和改造。主要涉及的方法有定向进化、理性设计和半理性设计。其中理性设计是一种快速且有效的改造手段，如通过该方法，将A.niger和P.amagasakiense来源的GOD基因进行相互替换，成功获得催化效率和稳定性均提高的GOD突变体。
发明内容
解决的技术问题：本发明提供一种葡萄糖氧化酶突变体及其应用，通过对Penicillium amagasakiense来源的PaGOD-1GPE关键氨基酸位点A263和K424突变后筛选得到的突变体，具体得到2种热稳定性提高的葡萄糖氧化酶突变体，对于应用到饲料防霉和替代抗生素产业中具有广阔的前景。
技术方案：一种耐高温葡萄糖氧化酶突变体，包括以葡萄糖氧化酶PaGOD-1GPE为母本对A263和K424两个位点突变后得到的突变体，命名为PaGOD-1GPE_A263P和PaGOD-1GPE_K424F；所述PaGOD-1GPE_A263P的氨基酸序列如SEQ ID NO.1所示；所述PaGOD-1GPE_K424F的氨基酸序列如SEQ ID NO.2所示。
翻译上述葡萄糖氧化酶突变体的核苷酸，所述PaGOD-1GPE_A263P的核苷酸序列如SEQ ID NO.3所示；所述PaGOD-1GPE_K424F的核苷酸序列如SEQ ID NO.4所示
一种重组载体，所述重组载体包括上述的核苷酸序列。
一种重组菌株，含有上述重组载体。
上述重组菌株在制备饲料添加剂中的应用。
上述重组菌株在抑制病原微生物中的应用。
有益效果：本发明通过以来源于Penicillium amagasakiense的葡萄糖氧化酶PaGOD-1GPE为母本对A263和K424位点突变后得到的两种突变体，具体通过构建含有该突变体的重组菌株，诱导培养后筛选出在热稳定性提高的两种葡萄糖氧化酶突变体PaGOD-1GPE_A263P和PaGOD-1GPE_K424F。在热稳定性方面，突变体PaGOD-1GPE_A263P和PaGOD-1GPE_K424F在60℃下的半衰期(t1/2)分别为9min和10min，分别是野生酶PaGOD-1GPE(5min)的1.8倍和2倍；在催化活力方面，突变体PaGOD-1GPE_A263P和PaGOD-1GPE_K424F的比活分别为78.32U/mg和75.94U/mg，与野生型PaGOD-1GPE(74.93U/mg)相比差距不大；最适pH值和最适温度与野生型基本一致。相对于盲目筛菌或人工(自然)诱变等手段，理性设计缩短了酶学性质改造时间。因此，将本发明的耐热葡萄糖氧化酶突变体应用于饲料添加产业中，具有广阔的应用前景。
附图说明
图1为野生型葡萄糖氧化酶和两个葡萄糖氧化酶突变体PaGOD-1GPE_A263P和PaGOD-1GPE_K424F的最适pH测定结果；
图2为野生型葡萄糖氧化酶和两个葡萄糖氧化酶突变体PaGOD-1GPE_A263P和PaGOD-1GPE_K424F的最适温度测定结果；
图3为野生型葡萄糖氧化酶和两个葡萄糖氧化酶突变体PaGOD-1GPE_A263P和PaGOD-1GPE_K424F的pH稳定性测定结果。
图4为野生型葡萄糖氧化酶和两个葡萄糖氧化酶突变体PaGOD-1GPE_A263P和PaGOD-1GPE_K424F在60℃下的半衰期t1/2的测定结果。
图5为野生型葡萄糖氧化酶和两个葡萄糖氧化酶突变体PaGOD-1GPE_A263P和PaGOD-1GPE_K424F的抑菌圈直径测定结果。
具体实施方式
以下实施例所用的试验材料：
1.菌株及载体：表达宿主Pichia pastoris GS115，表达质粒载体pPIC9r实验室自备；
2.酶类及其它生化试剂：Taq酶购自全式金公司，内切酶购自全式金公司，邻联茴香胺购自Sigma公司，过氧化物酶购自源叶公司；其它都为国产分析纯试剂(均从国药集团购买)；
3.培养基：
(1)LB培养基：0.5％酵母提取物，1％蛋白胨，1％ NaCl，pH 7.0；
(2)YPD培养基：1％酵母提取物，2％蛋白胨，2％葡萄糖；
(3)MD固体培养基：2％葡萄糖，1.5％琼脂糖，1.34％ YNB，0.00004％ Biotin；
(4)MM固体培养基：1.5％琼脂糖，1.34％ YNB，0.00004％ Biotin，0.5％甲醇；
(5)BMGY培养基：1％酵母提取物，2％蛋白胨，1％甘油(V/V)，1.34％ YNB，0.00004％Biotin；
(6)BMMY培养基：1％酵母提取物，2％蛋白胨，1.34％YNB，0.00004％ Biotin，0.5％甲醇(V/V)。
实施例1耐热葡萄糖氧化酶突变体编码基因的获得
以来源于Penicillium amagasakiense的葡萄糖氧化酶基因PaGOD-1GPE(核苷酸序列如SEQ ID NO.5所示)的重组表达载体pic9r-PaGOD-1GPE为模板，采用定点突变的方法对A263和K424位点进行定点突变，引物设计如表1所示，突变方法以及克隆方法参考文献(Exploiting the activity-stability trade-off of glucose oxidase fromAspergillus niger using a simple approach to calculate thermostability ofmutants.Jiang,et al.,2021)。
表1葡萄糖氧化酶PaGOD-1GPE中定点突变引物
实施例2耐热葡萄糖氧化酶突变体的制备
将经实施例1PCR获得的线性重组表达载体直接转化DMT感受态，菌落PCR验证，获得目标位点突变体的核酸序列，将重组质粒线性化后转化毕赤酵母GS115，获得重组酵母菌株GS115/PaGOD-1GPE_A263P和GS115/PaGOD-1GPE_K424F。
将含有重组质粒的GS115菌株，接种于2mL BMGY培养基的10mL试管中，置于30℃，220rpm摇床培养48h后将培养液3000g离心5min，弃上清，沉淀用2mL含有0.5％甲醇的BMMY培养基重悬，并再次置于30℃，220rpm条件下诱导培养48h。取上清用于酶活性检测，筛选到热稳定性较野生酶提高的突变体PaGOD-1GPE_A263P(氨基酸序列如SEQ ID NO.1所示，核苷酸序列如SEQ ID NO.3所示)和PaGOD-1GPE_K424F(氨基酸序列如SEQ ID NO.2所示，核苷酸序列如SEQ ID NO.4所示)。
将野生型GS115/PaGOD-1GPE和两个突变体GS115/PaGOD-1GPE_A263P和GS115/PaGOD-1GPE_K424F放大发酵体系，首先接种于YPD培养基中获得种子培养液，按1％接种量接种于300mL BMGY培养基的1L三角瓶中，置于30℃，220rpm摇床培养48h；后将培养液3000g离心5min，弃上清，沉淀用100mL含有0.5％甲醇的BMMY培养基重悬，并再次置于30℃，220rpm条件下诱导培养。每隔12h补加0.5mL甲醇，使菌液中的甲醇浓度保持在0.5％，同时取上清用于酶活性检测。最后将上清液浓缩至20mL，用于酶学性质测定和比较。
实施例3重组耐热葡萄糖氧化酶突变体和野生型的酶学性质比较分析
一、邻联茴香胺法测定
具体方法如下：在标准条件(pH 6.0，30℃)下，3mL的反应体系包括2.5mL邻联茴香胺缓冲液，300μL底物，100μL过氧化物酶(90U/mL)，100μL稀释酶液，反应3min，加入2mL 2MH2SO4终止反应。在540nm测定OD值。1个酶活单位(U)定义为在标准条件下，每分钟分解1μmoL底物生成过氧化氢所需的酶量。
二、重组耐热葡萄糖氧化酶突变体和野生型的性质测定
1、重组耐热葡萄糖氧化酶突变体和野生型的最适pH测定方法
将实施例2的葡萄糖氧化酶突变体和野生型葡萄糖氧化酶在不同的pH(1.0-11.0)下进行酶促反应，以测定其最适pH。底物β-D-葡萄糖用不同pH(1.0、2.0、3.0、4.0、5.0、6.0、7.0、8.0、9.0、10.0、11.0)的0.1mol/L柠檬酸-磷酸氢二钠缓冲液在30℃下进行葡萄糖氧化酶活力测定。
结果如图1所示，野生型葡萄糖氧化酶和葡萄糖氧化酶突变体的最适反应pH在6.0。
2、重组耐热葡萄糖氧化酶突变体和野生型的最适温度测定方法
重组耐热葡萄糖氧化酶突变体和野生型葡萄糖氧化酶的最适温度的测定为：在0.1mol/L柠檬酸-磷酸氢二钠缓冲液(pH 6)缓冲液体系及不同温度(25℃、30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃)下进行酶促反应。
结果如图2所示，表明重组野生型葡萄糖氧化酶的最适温度为45℃，而两个耐热葡萄糖氧化酶突变体的最适温度为40℃，且在高温(65℃)下的相对酶活较野生酶明显提高。
3、野生型葡萄糖氧化酶和突变体pH稳定性测定方法
将葡萄糖氧化酶突变体和野生型葡萄糖氧化酶用不同pH(1.0、2.0、3.0、4.0、5.0、6.0、7.0、8.0、9.0、10.0、11.0)的0.1mol/L柠檬酸-磷酸氢二钠缓冲液稀释后置于37℃恒温水浴锅中处理1h，然后在pH 6.0、30℃条件下测定其相对剩余酶活，未进行处理酶活为100％对照。
结果如图3所示，表明在酸性环境下葡萄糖氧化酶突变体PaGOD-1GPE_A263P的pH稳定性优于重组野生型葡萄糖氧化酶。
4、野生型葡萄糖氧化酶和突变体热稳定性测定方法
60℃条件下半衰期(t1/2)：突变体与野生型在60℃处理不同时间，最多处理30min，检测各自的剩余酶活。
60℃的半衰期测定结果如图4所示，表明葡萄糖氧化酶突变体PaGOD-1GPE_A263P和PaGOD-1GPE_K424F的t1/2分别为9min和10min，分别较野生型葡萄糖氧化酶(5min)延长1.8倍和2倍，即突变体PaGOD-1GPE_K424F热稳定性最好。
5、重组耐热葡萄糖氧化酶突变体和野生型的动力学参数测定方法
检测方法参照文献(Improving the thermostability and catalyticefficiency of glucose oxidase from Aspergillus niger by molecularevolution.Tu,et al.,2019)。
用pH 6.0，0.1mol/L柠檬酸-磷酸氢二钠缓冲液配制不同浓度(3.125mM、6.25mM、12.5mM、25mM、50mM、62.5mM、100mM、125mM、200mM、250mM、500mM，1000mM)的葡萄糖溶液作为底物，在30℃、pH 6.0标准条件下测定酶活性。将测定的酶活性数据利用GraphPad Prism5.01软件分析，获得重组耐热葡萄糖氧化酶野生型及各突变体的Km值及Vmax。
在标准条件下以葡萄糖为底物时，重组耐热葡萄糖氧化酶突变体PaGOD-1GPE_A263P和PaGOD-1GPE_K424F的催化效率(kcat/Km)分别为13.07mM-1min-1和38.01mM-1min-1，突变体PaGOD-1GPE_K424F的催化效率(kcat/Km)是野生型(25.74mM-1min-1)的1.47倍；重组耐热葡萄糖氧化酶突变体的比活分别为78.32U/mg和75.94U/mg，较野生型(74.93U/mg)无明显变化(见表2)。
表2重组耐热葡萄糖氧化酶突变体与野生型的比活及动力学参数比较表
实施例4重组耐热葡萄糖氧化酶突变体和野生型的体外抑菌效果分析
将37℃培养12h的铜绿假单胞菌菌液浓度稀释至1x107 CFU·mL-1，吸取100μL涂布于LB固体平板，使用打孔器(6mm)等距离打孔。
将实施例2的葡萄糖氧化酶突变体和野生型葡萄糖氧化酶分别与10％β-D-葡萄糖底物等体积混合，并取200μL混合物样液加入提前打好的孔内。在37℃培养24h，以测定其抑菌圈直径。
结果如图5所示，表明重组野生型葡萄糖氧化酶的抑菌圈直径为12.5mm，重组耐热葡萄糖氧化酶突变体PaGOD-1GPE_A263P和PaGOD-1GPE_K424F的抑菌圈直径分别为13mm和16.25mm。葡萄糖氧化酶突变体PaGOD-1GPE_A263P和PaGOD-1GPE_K424F的抑菌圈直径分别是重组野生型葡萄糖氧化酶的1.04倍和1.3倍，即突变体PaGOD-1GPE_K424F对铜绿假单胞菌的抑制效果最好。

Claims (5)

1.一种耐高温葡萄糖氧化酶突变体，其特征在于，包括以葡萄糖氧化酶PaGOD-1GPE为母本对A263和K424两个位点突变后得到的突变体，命名为PaGOD-1GPE_A263P和PaGOD-1GPE_K424F；所述PaGOD-1GPE_A263P的氨基酸序列如SEQ ID NO.1所示；所述PaGOD-1GPE_K424F的氨基酸序列如SEQ ID NO.2所示。
2.编码权利要求1所述葡萄糖氧化酶突变体的核苷酸分子，其特征在于，所述PaGOD-1GPE_A263P的核苷酸序列如SEQ ID NO.3所示；所述PaGOD-1GPE_K424F的核苷酸序列如SEQID NO.4所示。
3.一种重组载体，其特征在于，所述重组载体包括权利要求2所述的核苷酸序列。
4.一种重组菌株，其特征在于，含有权利要求3所述的重组载体。
5.权利要求4所述重组菌株在制备饲料添加剂中的应用。
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