CN114806988A - 一种枯草芽孢杆菌及其在谷氨酰胺酶生产中的应用 - Google Patents
一种枯草芽孢杆菌及其在谷氨酰胺酶生产中的应用 Download PDFInfo
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- CN114806988A CN114806988A CN202210458275.4A CN202210458275A CN114806988A CN 114806988 A CN114806988 A CN 114806988A CN 202210458275 A CN202210458275 A CN 202210458275A CN 114806988 A CN114806988 A CN 114806988A
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- glutaminase
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- bacillus subtilis
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Abstract
本发明涉及基因工程技术领域，具体提供了一种高产谷氨酰胺酶的枯草芽孢杆菌突变菌株及其应用。申请人通过紫外诱变筛选获得一株能大幅提高谷氨酰胺酶表达量的突变菌，其保藏编号为CCTCC NO：M20211415。该突变菌在15L罐发酵30h后，发酵上清液中谷氨酰胺酶酶活高达25.3U/ml，比出发菌提高了148%，可以广泛应用于谷氨酰胺酶的生产，有利于降低该酶的生产成本。
Description
技术领域
本发明涉及基因工程技术领域，具体涉及一种枯草芽孢杆菌及其在谷氨酰胺酶生产中的应用。
背景技术
谷氨酰胺酶（Glutaminase，EC3.5.1.2）具有谷氨酰胺水解活力，能够将L-谷氨酰胺的Y-酰胺残基进行加水分解，生成L-谷氨酸和氨，在细菌、酵母菌、真菌等微生物中谷氨酰胺酶分布广泛。目前，米曲霉（Aspergillus oryzae）、假单胞菌（Pseudomonas.spp）、藤黄微球菌（Micrococcus luteus）K-3、芽孢杆菌（Bacillus spp）来源的谷氨酰胺酶已被广泛研究，特别是Kazuaki Yoshimune团队对M. luteus K-3的谷氨酰胺酶的研究较为系统，解析了该酶的晶体结构、催化机制、催化位点及耐盐性等。
酱油中的鲜味成分主要来源于大豆蛋白经微生物酶解之后形成的氨基酸、多肽类物质，而呈鲜味的氨基酸主要是谷氨酸。大豆蛋白中含有约16％谷氨酸，其中约46％谷氨酸以谷氨酰胺的形式存在。在酱油酿造业中，通常利用谷氨酰胺酶水解酱油发酵原料中的谷氨酰胺生成谷氨酸，因此谷氨酰胺酶的酶活力是影响酱油发酵中谷氨酸得率的关键因素。酱油发酵过程中，在谷氨酰胺酶活力不高的情况下，部分谷氨酰胺就会转变成一种无鲜味的物质——焦谷氨酸，此转化过程不可逆且影响酱油的口感和品质；当谷氨酰胺酶活力较高时，谷氨酰胺会尽可能多的转化成谷氨酸，提高酱油鲜味。由于谷氨酰胺酶的酶学性质、菌种制曲环境、微生物菌相、安全性等方面存在差异，故不同的谷氨酰胺酶对酱油谷氨酸含量的提高效果有显著性差距。叶茂等人研究发现来源于解淀粉芽孢杆菌（Bacillus amyloliquefaciens Y-9）的谷氨酰胺酶在酱油高盐稀态发酵过程中，能有效提高谷氨酸的含量、降低焦谷氨酸含量，提高酱油鲜味和口感。
枯草芽孢杆菌作为一种重要的原核表达宿主，具有强大的蛋白分泌表达系统和相对完善的载体/宿主系统，是异源表达蛋白酶的理想宿主。研究发现，通过优化枯草芽孢杆菌表达调控元件可使蛋白酶的表达量获得较大提高，从而降低成本，使其更能适应大生产的需要。在基因工程操作过程中，筛选标记是重组DNA载体的重要组成部分，通常用来验证转化子是否成功。目前，芽孢杆菌表达系统中，常用的筛选标记是抗生素抗性基因。但抗生素抗性基因这一新型污染物不仅能在宿主细菌中伴随细菌增殖而增加丰度，还会通过基因突变和基因水平转移增加多样性、宿主范围及丰度。2016年11月，来自英国牛津大学等机构的研究人员，用新的实验模型证实质粒DNA是扩散抗生素耐药性的全球健康重大威胁的元凶之一。他们发现质粒DNA能够作为一种进化促进剂，加快新的耐药性类型进化。此外，质粒自动地扩增这些新的功能获得改善的耐药性基因的拷贝数。这些发现表明抗生素质粒对公共环境和卫生带来巨大的的威胁。
因此，目前急需开发一种无抗生素筛选标记、稳定高产谷氨酰胺酶的枯草芽孢杆菌。
发明内容
本发明为解决现有技术问题，提供了一种高产谷氨酰胺酶的枯草芽孢杆菌突变菌株及其应用。申请人将来源于解淀粉芽孢杆菌（Bacillusamyloliquefaciens）的谷氨酰胺酶基因，在枯草芽孢杆菌（Bacillus subtilis）宿主中过表达，构建得到重组整合表达菌株；进一步通过敲除谷氨酰胺酶表达的抗性筛选标记基因，得到无抗生素筛选标记的重组菌株；然后以该菌株为出发菌进行紫外诱变，筛选获得一株能大幅提高谷氨酰胺酶表达量的突变菌。该突变菌可以广泛应用于谷氨酰胺酶的生产，有利于降低该酶的生产成本。
本发明一方面提供了一种枯草芽孢杆菌工程菌株，其携带有表达谷氨酰胺酶的重组质粒。
所述谷氨酰胺酶的核苷酸序列为SEQ ID NO：1，其编码的氨基酸序列为SEQ IDNO：2。
本发明一方面提供了一种突变菌株，命名为枯草芽孢杆菌ZXP02（Bacillus subtilis ZXP02），是以上述枯草芽孢杆菌工程菌为出发菌，通过紫外诱变方法获得的，已于2021年11月15日保藏于中国武汉 武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC NO：M20211415。
本发明还提供了一种蛋白质谷氨酰胺酶的生产方法，是以上述突变菌为发酵菌株。
本发明首先提供了整合表达谷氨酰胺酶基因的无抗生素筛选标记的枯草芽孢杆菌工程菌ZXP01，其摇瓶发酵上清液中谷氨酰胺酶酶活达到2.55 U/ml。为了提高谷氨酰胺酶的产量，本发明以枯草芽孢杆菌ZXP01为出发菌株，通过紫外诱变筛选获得的突变菌枯草芽孢杆菌ZXP02，摇瓶发酵上清液中谷氨酰胺酶酶活高达6.04 U/ml，比出发菌提高了136%；15L罐发酵30h后，发酵上清液中谷氨酰胺酶酶活高达25.3U/ml，比出发菌提高了148%，取得了意料不到的技术效果。
本发明所述突变菌株重组表达的谷氨酰胺酶的最适作用温度为60℃，在50℃-65℃范围内能保持60%以上的相对酶活；最适作用pH为9.0，在pH超过8.5的碱性环境中能保持60%以上的相对酶活。
本发明所述突变菌株可广泛应用于谷氨酰胺酶的生产，有利于降低该酶的生产成本，促进其推广和使用。
附图说明
图1为谷氨酰胺酶GP的温度-相对酶活曲线；
图2为谷氨酰胺酶GP的pH-相对酶活曲线；
图3为枯草芽孢杆菌ZXP01、ZXP02的15L罐发酵曲线图。
具体实施方式
下面结合实例对本发明的方法做进一步说明，实施例中未注明具体条件的实验方法，可按常规条件，如J.萨姆布鲁克（Sambrook）等编写的《分子克隆实验指南》中所述的条件，或按照制造厂商所建议的条件运行。本领域相关技术人员可以借助实施例更好地理解和掌握本发明。但是，实现本发明的方法不应限于本发明实施例所记载的具体方法步骤。
本发明实施例中所涉及的培养基的配方如下：
LB液体培养基：胰蛋白胨1%，酵母粉0.5%，NaCl 0.5%；
LB平板：胰蛋白胨1%，酵母粉0.5%，NaCl 0.5%，琼脂2%；
GM I配制方法为：1*最低盐溶液95.6ml，20%葡萄糖2.5 ml，5%水解酪蛋白0.4 ml，10%酵母粉汁1ml；其中1*最低盐溶液的配制方法为：K2HPO4 14 g/L，KH2PO4 6 g/L，（NH4）2SO4 2 g/L，柠檬酸三钠1 g/L，MgSO4•7H2O 0.2 g/L，在蒸馏水中依次溶解；
GM II配制方法为：1*最低盐溶液96.98 ml，20%葡萄糖2.5 ml，5%水解酪蛋白0.08ml，10%酵母粉汁0.04 ml，1 M MgCl2 0.25 ml，1 M CaCl2 0.05 ml；
种子培养基：酵母浸粉0.5%，胰蛋白胨0.5%，甘油1%，K2HPO4 1.8%；
发酵培养基：酵母粉1～2%，豆饼粉2～5%，麦芽糊精5～10%，柠檬酸钠0.1～0.5%，CaCl2 0.1～0.5%，MgSO4 0.1～0.5%，K2HPO40.5～2%。
实施例1谷氨酰胺酶基因的克隆
申请人将来源于解淀粉芽孢杆菌（Bacillusamyloliquefaciens）的谷氨酰胺酶基因命名为GP，其核苷酸序列为SEQ ID NO:1，其编码的氨基酸序列为SEQ ID NO:2。GP基因的表达盒序列，包括启动子、GP基因和终止子序列，由北京六合华大基因科技有限公司合成。然后以合成的基因片段为模板，利用引物GP-F和引物GP-Rv扩增出GP基因表达盒片段。
PCR引物和反应条件如下：
GP-F：ctactctgaatttttttaaaaggagagggtaaagaatgaaaaagaaaaagtttatgaacc；
GP-Rv：gttatctatgaccatgattacgccaagctgggcccttaagcttaaaaata。
PCR条件为：98℃ 2min；98℃ 10s；58℃ 20s，72℃ 60s，30个循环；72℃ 5min。利用凝胶回收试剂盒回收PCR扩增产物。
实施例2重组表达谷氨酰胺酶的枯草芽孢杆菌工程菌的构建与筛选
将来源于变形菌的四环素抗性基因命名为tet，其核苷酸序列为SEQ ID NO:3，其编码的氨基酸序列为SEQ ID NO:4。tet基因的表达盒序列由北京六合华大基因科技有限公司合成。然后以合成的基因片段为模板，利用引物tet-F和引物tet-Rv扩增出四环素抗性基因tet表达盒片段，该表达盒序列的5’端和3’端有loxp位点。
PCR引物和反应条件如下：
tet-F：cttaagggcccagcttggcgtaatcatggtcatagataac；
tet-Rv： ctagagcggataacaatttcacacaggaaacagctatgaccatg。
PCR条件为：98℃ 2min；98℃ 10s；58℃ 20s，72℃ 45s，30个循环；72℃ 5min。利用凝胶回收试剂盒回收PCR扩增产物。
以质粒p302为模板，扩增整合位点上游同源臂片段f1：upstream-1kb，1080bp，其核苷酸序列为SEQ ID NO:5；以质粒p302为模板，扩增四环素抗性基因tet片段和整合位点下游同源臂片段f2：tet- downstream-2kb，下游同源臂的核苷酸序列为SEQ ID NO:6，1048bp。
利用融合PCR，以上游同源臂片段f1、抗性基因和下游同源臂片段f2和谷氨酰胺酶基因表达盒片段GP为模板，扩增整合型表达盒GP cassete。引物为assGP-F和assGP-Rv，PCR条件为：98℃ 2min；98℃ 10s；58℃ 20s，72℃ 2min，30个循环；72℃ 5min。利用凝胶回收试剂盒回收PCR扩增产物，命名为GP cassete。
将上述整合型表达盒GP cassete的PCR产物通过感受态方法转化宿主细胞枯草芽孢杆菌168，具体转化过程如下：将新鲜活化的枯草芽孢杆菌168由LB平板接种到5 ml GMI溶液，在30℃、125 rpm振荡培养过夜；第二天取1 ml转接到9 ml GMI中，37℃、220 rpm培养3.5 h；再取1 ml上一步骤的培养液转接到9 ml GMⅡ溶液中，37℃、125 rpm培养90 min后，5000 g、10 min离心收集菌体；用1 ml GMⅡ溶液轻轻悬浮菌体，悬浮后的菌体即为感受态细胞。然后取0.2 ml感受态细胞，加入10uL GP cassete pcr产物，于37℃、200 rpm振荡培养60 min后，涂布含10 μg/ml四环素的LB平板，37℃培养过夜培养，次日检查转化子。
挑取转化平板上的菌落，在含10 μg/ml四环素的LB平板上划线纯化，分别接种于20 ml种子培养基中，37℃、220 rpm振荡培养约6 h；然后分别取2.5 ml种子液接种到50 ml发酵培养基中，37℃、220 rpm振荡培养48h；离心菌体获得上清液，分别进行谷氨酰胺酶酶活力检测。
结果显示，上述获得的阳性转化子在摇瓶发酵条件下，其发酵上清液中蛋白质谷氨酰胺酶活力最高达到2.52 U/ml。将该酶活水平最高的阳性转化子命名为枯草芽孢杆菌ZXGP（Bacillus subtilis ZXGP）。
assGP-F：cacatggactgaaacatcatcggcatttttttctgcctgc；
assGP-Rv：ggatcgaagcggccgtgccttttcggtttttgcagaccg。
酶活检测方法
（1）谷氨酰胺酶酶活力定义
在37℃、pH6.0条件下每分钟水解底物谷氨酰胺生成1 μmol谷氨酸所需要的酶量，定义为1个谷氨酰胺酶活力单位，以U表示。
（2）酶活测定方法
标准曲线：取1 ml 50 μmol/ml 氯化铵溶液稀释至10 ml，配制 5 μmol/ml氯化铵溶液。用蒸馏水分别稀释 80、 40、 20、 15、 10、 8 倍至浓度0.0625、0.125、0.25、0.333、0.5、0.625 μmol/ml。分别取0.2ml上述溶液及蒸馏水空白，加入 0.8ml 蒸馏水混匀，加入显色液 A 1ml、显色液 B 0.5ml 和显色液 C 1ml，混匀于 37℃下显色 20min 后，在630nm下测吸光值，以氯化铵浓度为横坐标、吸光值为纵坐标，绘制准曲线 y=kx+b。
显色液A：称取2.023g苯酚和 0.0075g亚硝基铁氰化钠，用水溶解定容至50ml，4℃避光保存。
显色液B：称取2.5g氢氧化钾，用蒸馏水溶解后定容至50ml。
显色液C：称取10.5g无水碳酸钾，用蒸馏水溶解后加入0.5ml次氯酸钠，加水定容至 50ml，现配现用。
样品反应：取1ml底物加入到 10*100mm 试管中，37℃预5min，加入 0.1ml 酶液，37℃反应 30min，然后加入1ml 终止液，混合均匀并从水浴锅中取出。
空白反应：取1ml底物加入到10*100mm试管中，37℃预热5min，加入1ml 终止液，37℃反应 30min,补加0.1ml酶液，混合均匀并从水浴锅中取出。取0.2ml终止后溶液至5ml扣盖离心管，加入0.8ml蒸馏水混匀，然后分别加入显色液A 1ml、显色液B 0.5ml和显色液C1ml，混匀后于37℃下显色20min后，在630nm下测样品吸光值AS和空白吸光值Ab。
（3）酶活计算
酶活力 X=（((AS-Ab)-b)/k）× n/T。
式中： X —样品的酶活力，U/g（U/ml）；
AS—样品吸光值；
Ab —空白吸光值；
b —标准曲线截距；
k —标准曲线斜率；
n—样品稀释倍数；
T—反应时间，30min。
实施例3 无抗生素标记的枯草芽孢杆菌工程菌的构建
将Cre蛋白的表达质粒pZXcre转化工程菌ZXGP的感受态细胞，感受态细胞制备的具体过程见实施例2。取0.2 ml感受态细胞，加入10uL 质粒pZXcre，于30℃、200 rpm振荡培养60 min后，涂布含30 μg/ml卡那霉素的LB平板，30℃培养过夜培养，次日检查转化子。
收集转化平板上所有的转化子，涡旋振荡混匀，取100uL菌液转接到20 ml含30 μg/ml卡那霉素的LB液体培养基中，30℃摇床培养至OD600为1.5，取100uL培养菌液转接到含30μg/ml卡那霉素的LB液体培养基中，30℃摇床培养至OD600为1.5时，稀释涂布30μg/ml卡那霉素的LB平板，30℃过夜培养。挑取稀释平板上的转化子，分别点种于含30μg/ml卡那霉素和10μg/ml四环素的LB平板，30℃培养18h，筛选出有卡那霉素抗性、同时对四环素敏感（KanRTetS）的转化子。
收集所有KanRTetS的转化子，涡旋振荡混匀，取100uL菌液转接到20ml LB液体培养基中，37℃摇床培养至OD600为1.5，取100uL培养菌液转接到LB液体培养基中，37℃摇床培养至OD600为1.5时，稀释涂布LB平板，37℃过夜培养。挑取稀释平板上的转化子，分别点种于含30 μg/ml卡那霉素的LB平板和无抗生素的LB平板，筛选出对卡那霉素敏感的转化子（KanS），即为重组表达谷氨酰胺酶的无抗生素标记的枯草芽孢杆菌工程菌。
挑取不同的KanS转化子，在LB平板上划线纯化，分别接种于20 ml种子培养基中，37℃、220 rpm振荡培养约6 h；然后分别取2.5 ml种子液接种到50 ml发酵培养基中，37℃、220 rpm振荡培养48h；离心菌体获得上清液，分别进行谷氨酰胺酶酶活力检测。
结果显示，上述获得的无抗生素标记的阳性转化子在摇瓶发酵条件下，其发酵上清液中谷氨酰胺酶酶活最高达到2.52 U/ml。将该酶活水平最高的阳性转化子命名为枯草芽孢杆菌ZXP01（Bacillus subtilis ZXP01）。
实施例4 谷氨酰胺酶高产菌株的筛选
紫外诱变导致的突变随机性很强，突变产生的效果也是随机的，难以预测。因此，为了获得有效的正突变，技术人员通常需要进行多轮紫外诱变，筛选的工作量较大，而且存在无法获得有效正突变的可能性。但因为紫外诱变所需设备简单、费用少，并且可在短时间内获得大量突变体，因此，它现在仍是一种常用的诱变选育方法。
申请人以枯草芽孢杆菌ZXP01为出发菌株，通过紫外诱变方法对其进行遗传学改造，进一步提高其谷氨酰胺酶的产量。
5.1 紫外诱变处理及诱变剂量确定
取培养至9h的枯草芽孢杆菌ZXP01菌液离心去上清，菌体用无菌生理盐水洗涤两次后，将打散的细胞悬浮于生理盐水中，最后调整细胞浓度至108个/ml。打开9 W紫外灯开关，预热约30 min；取直径9 cm无菌平皿，加入上述细胞浓度为108个/ml的菌悬液10 ml，加入一根无菌磁力搅拌转子，打开磁力搅拌器，打开皿盖，在垂直距离为15 cm处，进行不同时间（0s～300s）的紫外照射，每隔20s取样一次。盖上平皿盖，关闭紫外灯，黑暗中孵育30min。将照射后的菌悬液用0.85%生理盐水梯度稀释至10-1～10-6；取10-4、10-5、10-6三个稀释度的菌悬液各100 μL，涂布LB平板，每个稀释度涂布三个平板；以同样的操作，取未经紫外线照射处理的菌液稀释涂平板作对照。将上述涂布均匀的平板，用黑布或报纸包好后，置于37℃过夜培养。统计不同照射时间下每个稀释度下平板上长出的单菌落数，若在某个稀释度下长出的单菌落数在30～300个之间，则认为该稀释度合适。将该稀释度下三个平板上长出的单菌落数求平均值，按下列公式计算菌悬液浓度：
菌悬液浓度（CFU/ml）=某个稀释度下的菌落平均数×稀释倍数×10。
按下列公式计算某个紫外处理剂量下的致死率：
致死率（%）=（1－某剂量处理后的菌悬液浓度/处理前菌悬液浓度）×100%。
以致死率约80%-90%的诱变剂量处理菌悬液。当照射时间为145s时，致死率达87.62%。因此，申请人最终确定的诱变时间为145s。
5.2 高产蛋白质谷氨酰胺酶的突变菌株筛选
从致死率为87.62%的LB平板上挑取菌落，划线培养获得单菌落后点种于LB平板，每组设3个平行，同时点种出发菌株作为对照，37℃培养18 h，共筛选到1038株突变菌株。将各个单菌落分别接种至装有200ul LB液体培养基的96孔板，37℃、500rpm振荡培养6h后，取30uL转接至装有200uL 发酵培养基（葡萄糖1%、磷酸氢二钠0.2%，蛋白胨1%、氯化钠1%，酵母粉0.5%）的96孔板，37℃ 500rpm振荡培养3 d，离心去除菌体，得到发酵上清液，测定上清液中谷氨酰胺酶的酶活，以出发菌作为对照，筛选出发酵酶活力得到显著提高的突变菌株。
结果显示，第一轮紫外诱变筛选获得的1038株突变菌中，没有一株突变菌发酵上清液中蛋白质谷氨酰胺酶酶活高于出发菌。
申请人按照上述方法继续进行了21轮诱变筛选，最终获得一株蛋白质谷氨酰胺酶产量显著高于出发菌的突变菌株，命名为枯草芽孢杆菌ZXP02（Bacillus subtilisZXP02）。该突变菌株摇瓶发酵上清液中谷氨酰胺酶活力达到6.04 U/ml，比出发菌提高了136%，取得了意料不到的技术效果。
实施例7 谷氨酰胺酶GP的酶学特性检测
1、最适作用温度
利用磷酸缓冲液对上述枯草芽孢杆菌ZXP02发酵上清液进行稀释，分别在 30℃、40℃、50℃、60℃、65℃、70℃，pH6.0条件下测定其谷氨酰胺酶酶活，以最高酶活为100%，计算相对酶活，做温度-相对酶活曲线。
结果如图1所示，本发明所述谷氨酰胺酶GP的最适作用温度为60℃，在50-65℃范围内能保持60%以上的相对酶活。
2、最适作用pH
分别用pH4.0、5.0、6.0、7.0、8.0、9.0、10.0的0.1M磷酸氢二钠-0.05M柠檬酸、硼酸-硼砂、氢氧化钠-硼砂缓冲液对上述枯草芽孢杆菌ZXP02发酵上清液进行稀释，在37℃条件下测定其谷氨酰胺酶酶活，以最高酶活为100%，计算相对酶活，做pH-相对酶活曲线。
结果如图2所示，本发明所述谷氨酰胺酶GP的最适作用pH为9.0，在pH超过8.5的碱性环境中能保持60%以上的相对酶活。
实施例8 15L罐发酵
分别将出发菌枯草芽孢杆菌ZXP01和突变菌枯草芽孢杆菌ZX02在15L发酵罐上发酵，发酵使用的培养基配方为：葡萄糖10 g/L、豆饼粉15 g/L、NaCl 5 g/L、K2HPO4 0. 3 g/L、氯化钙5 g/L、七水合硫酸镁1 g/L，pH7.1。
发酵生产工艺：pH 7.1，温度37℃，搅拌速率800rpm，通风量2.16m3/h，接种量为3%，溶氧控制在25%以上。
通过测定不同时间发酵上清液中谷氨酰胺酶酶活力，可得发酵酶活曲线（图3）。
结果显示：发酵30h后，出发菌枯草芽孢杆菌ZXP01发酵液中蛋白质谷氨酰胺酶酶活为10.2 U/ml，而突变菌枯草芽孢杆菌ZXP02的发酵酶活为25.3U/ml，比出发菌提高了148%，取得了意料不到的技术效果。
综上，本发明提供的突变菌枯草芽孢杆菌ZX02能显著提高谷氨酰胺酶的产量，有利于降低该酶的生产成本，提高其应用效果，从而有利于其在食品工业领域中的广泛应用。
申请人已于2021年11月15日将上述突变菌枯草芽孢杆菌ZXP02（Bacillus subtilis ZXP02）保藏于中国武汉 武汉大学的中国典型培养物保藏中心，保藏编号为CCTCC M 20211415。
序列表
<110> 青岛蔚蓝生物集团有限公司
青岛蔚蓝生物股份有限公司
<120> 一种枯草芽孢杆菌及其在谷氨酰胺酶生产中的应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1779
<212> DNA
<213> 解淀粉芽孢杆菌(Bacillus amyloliquefaciens)
<400> 1
atgaaaaaga aaaagtttat gaacctgtgc tttatcgtcc tgctgtcaac actgctggca 60
gcaggctcaa ttccgtatca tgcacaagca aagaaacatc cgtttagcta tgacgactat 120
aaacaagttg atgtcggcaa agatggcatg gttgcaacag cacatccgct ggcatcacaa 180
attggcgcag atgttctgaa gaaaggcgga aatgcagttg atgcagcagt tgcaattcaa 240
tttgcactga atgttacaga accgatgatg tcaggcattg gcggaggcgg atttatgatg 300
gtttatgatg caaaaacgaa ggacacgaca attatcgatt caagagaaag agcaccggca 360
ggcgcaacac cggatatgtt tctggatgaa aatggcaaag caatcccgtt ttcagaaaga 420
gttacaaaag gcacagcagt tggcgttccg ggaacactga aaggcctgga aaaagcactg 480
gataaatggg gcacaagatc aatgaaacaa ctgattacac cgagcattaa actggcatca 540
aaaggctttc cgattgattc agttctggca gatgcaatca gcgactacaa agataaactg 600
tcacatacag cagcgaaaga tgtttttctg ccggatggcg aaccgctgaa agaaggcgat 660
acactgattc aaaaagatct ggcgaaaaca tttacggcga tcaaatacaa aggcacgaaa 720
gcattttacg atggcgcatt ttcaaagaaa ctggcagaaa cagttcaaga atttggcgga 780
tcaatgacgg aaaaagacat caaaaacttc aacgtcacaa tcgatgaacc gatctgggga 840
gattatcaag gctatcacat tgcgacagca ccgcctccgt catcaggcgg agtttttctt 900
ctgcaaatgc tgaatctgct ggacgatttt aaactgagcc aatatgatat tcgcagctgg 960
caaaaatatc aactgcttgc agaaacaatg catctggcat atgcggatag agcagcgttt 1020
gcaggcgatc cggaatttgt taatgttccg cttaaaggcc tgctgaaccc ggattatatc 1080
aatgcaagac gccagctgat tgatatcgat aaggttaaca aaaagccgaa agctggcgat 1140
ccgtgggcat atcaagaagg ctcagcaaat tacaaacaag tcaaacagcc gacggataaa 1200
caagaaggcc aaacaacaca ttttacagtg acagatagat ttggcaacgt cgtcagctat 1260
acaacaacaa tcgaacaact gtttggcagc ggaattatgg ttcctggcta tggcgttgtt 1320
ctgaataatg aactgacaga ttttgatgca gtccctggcg gagcaaatga agttcaaccg 1380
aataaaagac cgctgtcatc aatgacaccg acaatcctgt ttaaaaacaa tgaaccggtc 1440
ctgacagttg gctcaccagg cggagctaca attatttcat cagttctgca aacgatcctg 1500
aacaaagttg aatatggcat ggatctgaaa gcagcagtcg aagaaccgag aatttataca 1560
aatagcatga cgagctaccg ctatgaaaaa ggcgtcccgg aagaagcaag aacaaaactg 1620
aatgaaatgg gccataaatt tggctcatca ccggttgata ttggcaatgt tcaaagcatt 1680
ctgatcgata gagaaaacgg cacatttaca ggcgttgcag attcatcaag aaatggcgca 1740
gcaattggag tcaatctgaa acagtataaa aagtaatag 1779
<210> 2
<211> 591
<212> PRT
<213> 解淀粉芽孢杆菌(Bacillus amyloliquefaciens)
<400> 2
Met Lys Lys Lys Lys Phe Met Asn Leu Cys Phe Ile Val Leu Leu Ser
1 5 10 15
Thr Leu Leu Ala Ala Gly Ser Ile Pro Tyr His Ala Gln Ala Lys Lys
20 25 30
His Pro Phe Ser Tyr Asp Asp Tyr Lys Gln Val Asp Val Gly Lys Asp
35 40 45
Gly Met Val Ala Thr Ala His Pro Leu Ala Ser Gln Ile Gly Ala Asp
50 55 60
Val Leu Lys Lys Gly Gly Asn Ala Val Asp Ala Ala Val Ala Ile Gln
65 70 75 80
Phe Ala Leu Asn Val Thr Glu Pro Met Met Ser Gly Ile Gly Gly Gly
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Gly Phe Met Met Val Tyr Asp Ala Lys Thr Lys Asp Thr Thr Ile Ile
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Asp Ser Arg Glu Arg Ala Pro Ala Gly Ala Thr Pro Asp Met Phe Leu
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Asp Glu Asn Gly Lys Ala Ile Pro Phe Ser Glu Arg Val Thr Lys Gly
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Thr Ala Val Gly Val Pro Gly Thr Leu Lys Gly Leu Glu Lys Ala Leu
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Asp Lys Trp Gly Thr Arg Ser Met Lys Gln Leu Ile Thr Pro Ser Ile
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Lys Leu Ala Ser Lys Gly Phe Pro Ile Asp Ser Val Leu Ala Asp Ala
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Ile Ser Asp Tyr Lys Asp Lys Leu Ser His Thr Ala Ala Lys Asp Val
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Phe Leu Pro Asp Gly Glu Pro Leu Lys Glu Gly Asp Thr Leu Ile Gln
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Lys Asp Leu Ala Lys Thr Phe Thr Ala Ile Lys Tyr Lys Gly Thr Lys
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Ala Phe Tyr Asp Gly Ala Phe Ser Lys Lys Leu Ala Glu Thr Val Gln
245 250 255
Glu Phe Gly Gly Ser Met Thr Glu Lys Asp Ile Lys Asn Phe Asn Val
260 265 270
Thr Ile Asp Glu Pro Ile Trp Gly Asp Tyr Gln Gly Tyr His Ile Ala
275 280 285
Thr Ala Pro Pro Pro Ser Ser Gly Gly Val Phe Leu Leu Gln Met Leu
290 295 300
Asn Leu Leu Asp Asp Phe Lys Leu Ser Gln Tyr Asp Ile Arg Ser Trp
305 310 315 320
Gln Lys Tyr Gln Leu Leu Ala Glu Thr Met His Leu Ala Tyr Ala Asp
325 330 335
Arg Ala Ala Phe Ala Gly Asp Pro Glu Phe Val Asn Val Pro Leu Lys
340 345 350
Gly Leu Leu Asn Pro Asp Tyr Ile Asn Ala Arg Arg Gln Leu Ile Asp
355 360 365
Ile Asp Lys Val Asn Lys Lys Pro Lys Ala Gly Asp Pro Trp Ala Tyr
370 375 380
Gln Glu Gly Ser Ala Asn Tyr Lys Gln Val Lys Gln Pro Thr Asp Lys
385 390 395 400
Gln Glu Gly Gln Thr Thr His Phe Thr Val Thr Asp Arg Phe Gly Asn
405 410 415
Val Val Ser Tyr Thr Thr Thr Ile Glu Gln Leu Phe Gly Ser Gly Ile
420 425 430
Met Val Pro Gly Tyr Gly Val Val Leu Asn Asn Glu Leu Thr Asp Phe
435 440 445
Asp Ala Val Pro Gly Gly Ala Asn Glu Val Gln Pro Asn Lys Arg Pro
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Leu Ser Ser Met Thr Pro Thr Ile Leu Phe Lys Asn Asn Glu Pro Val
465 470 475 480
Leu Thr Val Gly Ser Pro Gly Gly Ala Thr Ile Ile Ser Ser Val Leu
485 490 495
Gln Thr Ile Leu Asn Lys Val Glu Tyr Gly Met Asp Leu Lys Ala Ala
500 505 510
Val Glu Glu Pro Arg Ile Tyr Thr Asn Ser Met Thr Ser Tyr Arg Tyr
515 520 525
Glu Lys Gly Val Pro Glu Glu Ala Arg Thr Lys Leu Asn Glu Met Gly
530 535 540
His Lys Phe Gly Ser Ser Pro Val Asp Ile Gly Asn Val Gln Ser Ile
545 550 555 560
Leu Ile Asp Arg Glu Asn Gly Thr Phe Thr Gly Val Ala Asp Ser Ser
565 570 575
Arg Asn Gly Ala Ala Ile Gly Val Asn Leu Lys Gln Tyr Lys Lys
580 585 590
<210> 3
<211> 1191
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgaaatcta acaatgcgct catcgtcatc ctcggcaccg tcaccctgga tgctgtaggc 60
ataggcttgg ttatgccggt actgccgggc ctcttgcggg atatcgtcca ttccgacagc 120
atcgccagtc actatggcgt gctgctagcg ctatatgcgt tgatgcaatt tctatgcgca 180
cccgttctcg gagcactgtc cgaccgcttt ggccgccgcc cagtcctgct cgcttcgcta 240
cttggagcca ctatcgacta cgcgatcatg gcgaccacac ccgtcctgtg gatcctctac 300
gccggacgca tcgtggccgg catcaccggc gccacaggtg cggttgctgg cgcctatatc 360
gccgacatca ccgatgggga agatcgggct cgccacttcg ggctcatgag cgcttgtttc 420
ggcgtgggta tggtggcagg ccccgtggcc gggggactgt tgggcgccat ctccttgcat 480
gcaccattcc ttgcggcggc ggtgctcaac ggcctcaacc tactactggg ctgcttccta 540
atgcaggagt cgcataaggg agagcgtcga ccgatgccct tgagagcctt caacccagtc 600
agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt 660
atcatgcaac tcgtaggaca ggtgccggca gcgctctggg tcattttcgg cgaggaccgc 720
tttcgctgga gcgcgacgat gatcggcctg tcgcttgcgg tattcggaat cttgcacgcc 780
ctcgctcaag ccttcgtcac tggtcccgcc accaaacgtt tcggcgagaa gcaggccatt 840
atcgccggca tggcggccga cgcgctgggc tacgtcttgc tggcgttcgc gacgcgaggc 900
tggatggcct tccccattat gattcttctc gcttccggcg gcatcgggat gcccgcgttg 960
caggccatgc tgtccaggca ggtagatgac gaccatcagg gacagcttca aggatcgctc 1020
gcggctctta ccagcctaac ttcgatcact ggaccgctga tcgtcacggc gatttatgcc 1080
gcctcggcga gcacatggaa cgggttggca tggattgtag gcgccgccct ataccttgtc 1140
tgcctccccg cgttgcgtcg cggtgcatgg agccgggcca cctcgacctg a 1191
<210> 4
<211> 396
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Lys Ser Asn Asn Ala Leu Ile Val Ile Leu Gly Thr Val Thr Leu
1 5 10 15
Asp Ala Val Gly Ile Gly Leu Val Met Pro Val Leu Pro Gly Leu Leu
20 25 30
Arg Asp Ile Val His Ser Asp Ser Ile Ala Ser His Tyr Gly Val Leu
35 40 45
Leu Ala Leu Tyr Ala Leu Met Gln Phe Leu Cys Ala Pro Val Leu Gly
50 55 60
Ala Leu Ser Asp Arg Phe Gly Arg Arg Pro Val Leu Leu Ala Ser Leu
65 70 75 80
Leu Gly Ala Thr Ile Asp Tyr Ala Ile Met Ala Thr Thr Pro Val Leu
85 90 95
Trp Ile Leu Tyr Ala Gly Arg Ile Val Ala Gly Ile Thr Gly Ala Thr
100 105 110
Gly Ala Val Ala Gly Ala Tyr Ile Ala Asp Ile Thr Asp Gly Glu Asp
115 120 125
Arg Ala Arg His Phe Gly Leu Met Ser Ala Cys Phe Gly Val Gly Met
130 135 140
Val Ala Gly Pro Val Ala Gly Gly Leu Leu Gly Ala Ile Ser Leu His
145 150 155 160
Ala Pro Phe Leu Ala Ala Ala Val Leu Asn Gly Leu Asn Leu Leu Leu
165 170 175
Gly Cys Phe Leu Met Gln Glu Ser His Lys Gly Glu Arg Arg Pro Met
180 185 190
Pro Leu Arg Ala Phe Asn Pro Val Ser Ser Phe Arg Trp Ala Arg Gly
195 200 205
Met Thr Ile Val Ala Ala Leu Met Thr Val Phe Phe Ile Met Gln Leu
210 215 220
Val Gly Gln Val Pro Ala Ala Leu Trp Val Ile Phe Gly Glu Asp Arg
225 230 235 240
Phe Arg Trp Ser Ala Thr Met Ile Gly Leu Ser Leu Ala Val Phe Gly
245 250 255
Ile Leu His Ala Leu Ala Gln Ala Phe Val Thr Gly Pro Ala Thr Lys
260 265 270
Arg Phe Gly Glu Lys Gln Ala Ile Ile Ala Gly Met Ala Ala Asp Ala
275 280 285
Leu Gly Tyr Val Leu Leu Ala Phe Ala Thr Arg Gly Trp Met Ala Phe
290 295 300
Pro Ile Met Ile Leu Leu Ala Ser Gly Gly Ile Gly Met Pro Ala Leu
305 310 315 320
Gln Ala Met Leu Ser Arg Gln Val Asp Asp Asp His Gln Gly Gln Leu
325 330 335
Gln Gly Ser Leu Ala Ala Leu Thr Ser Leu Thr Ser Ile Thr Gly Pro
340 345 350
Leu Ile Val Thr Ala Ile Tyr Ala Ala Ser Ala Ser Thr Trp Asn Gly
355 360 365
Leu Ala Trp Ile Val Gly Ala Ala Leu Tyr Leu Val Cys Leu Pro Ala
370 375 380
Leu Arg Arg Gly Ala Trp Ser Arg Ala Thr Ser Thr
385 390 395
<210> 5
<211> 1018
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cacatggact gaaacatcat cggcattttt ttctgcctgc aaggcttgaa ataacgttga 60
cattcggcac actccttttc atttatatcg taaccgaaga acgttcaaaa aaccaaatca 120
tcaagccgcc attttcactt cgccggcaca ttgagacaat aatggacaaa tccggtatcc 180
tcttcatagc cgttttgctc atacaagctt cttgccttcc ggttgtggtg ctcagtctga 240
agtgttaaac attttgcccc gttttgccct gcataatcct ttgcggcaga aagcagccgg 300
ccgccggctc cctttgtacg cgcatgagga acgacaaata agtcatttaa tatgtatatc 360
cttttcattg acacagaaga aaacgttgga tagagctggg taaagcctat gaattctcca 420
ttttcttctg ctatcaaaat aacagactcg tgattttcca aacgagcttt caaaaaagcc 480
tctgcccctt gcaaatcgga tgcctgtcta taaaattccc gatattggtt aaacagcggc 540
gcaatggcgg ccgcatctga tgtctttgct tggcgaatgt tcatcttatt tcttcctccc 600
tctcaataat tttttcattc tatccctttt ctgtaaagtt tatttttcag aatactttta 660
tcatcatgct ttgaaaaaat atcacgataa tatccattgt tctcacggaa gcacacgcag 720
gtcatttgaa cgaatttttt cgacaggaat ttgccgggac tcaggagcat ttaacctaaa 780
aaagcatgac atttcagcat aatgaacatt tactcatgtc tattttcgtt cttttctgta 840
tgaaaatagt tatttcgagt ctctacggaa atagcgagag atgatatacc taaatagaga 900
taaaatcatc tcaaaaaaat gggtctacta aaatattatt ccatctatta caataaattc 960
acagaatagt cttttaagta agtctactct gaattttttt aaaaggagag ggtaaaga 1018
<210> 6
<211> 1048
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tagtaaaaag aagcaggttc ctccatacct gcttcttttt atttgtcagc atcctgatgt 60
tccggcgcat tctcttcttt ctccgcatgt tgaatccgtt ccatgatcga cggatggctg 120
cctctgaaaa tcttcacaag caccggagga tcaacctggc tcagccccgt cacggccaaa 180
tcctgaaacg ttttaacagc ggcttctctg ttctctgtca actcgatccc atactggtca 240
gccttattct cctgataacg cgagacagca ttagaaaaag gcgtaaccgc aaagctcaaa 300
acagaaaaca aaagcaataa cagcggaagt gccgcaagat catgccgccc ttctaaatga 360
aacatgctgc gggttaggcg aaccgtccgc ttgtaaagct tatcaatgac ataaaatccg 420
gcgagcgaca cgagcaaata gccagccaga ccgatgtaaa cgtgcttcat gacataatgg 480
cccatttcgt ggcccataat aaacagaatt tctgaatcgt caagtttgtt cagcgtcgta 540
tcccacaata caatccgttt attggcccca attcctgtaa cataggcatt cagcgcattt 600
gttttttctg acatgttcac ttcatataca tggtcagccg gaatattggc ttcatctgcc 660
agctctaaaa ttttgctttc aagctctttg tttttcagcg gataaaaatc attgtataaa 720
ggatcgataa tgaccggctg aataaaaaac agaaacagcg aaaacggcac tgttaacagc 780
caggcgtata accaccattt tttttcatgc cttttgatca gccaataaaa aacgagaacg 840
caaagcgtaa agattggaaa gctgatccaa aagctgataa cctgatcctt agcccagctg 900
gccgttgtct gtgtggaaat gttatagtca agcgatactt gatagcctat ccaatctaaa 960
ggcagcgtca ccaatgttgt aatcagcgaa agcacaaaca caaaaccaac ggtctgcaaa 1020
aaccgaaaag gcacggccgc ttcgatcc 1048
Claims (6)
1.一种枯草芽孢杆菌工程菌，其特征在于，所述的枯草芽孢杆菌工程菌携带有重组表达谷氨酰胺酶基因的表达载体。
2.如权利要求1所述的枯草芽孢杆菌工程菌，其特征在于，所述谷氨酰胺酶基因的核苷酸序列为SEQ ID NO:1，其编码的氨基酸序列为SEQ ID NO:2。
3.一种枯草芽孢杆菌突变菌，其特征在于，所述的枯草芽孢杆菌突变菌是以权利要求2所述的枯草芽孢杆菌工程菌为出发菌，通过紫外诱变获得的。
4.如权利要求3所述的枯草芽孢杆菌突变菌，其特征在于，所述的枯草芽孢杆菌突变菌的保藏编号为CCTCC NO: M20211415。
5.权利要求3或4所述的枯草芽孢杆菌突变菌在生产谷氨酰胺酶中的应用。
6.一种谷氨酰胺酶的生产方法，其特征在于，所述方法是以权利要求4所述的突变菌为发酵菌株。
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| CN111394331A (zh) * | 2020-05-06 | 2020-07-10 | 江南大学 | 一种谷氨酰胺转氨酶及其编码基因、表达载体及重组菌 |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111394331A (zh) * | 2020-05-06 | 2020-07-10 | 江南大学 | 一种谷氨酰胺转氨酶及其编码基因、表达载体及重组菌 |
| CN111394331B (zh) * | 2020-05-06 | 2022-11-25 | 江南大学 | 一种谷氨酰胺转氨酶及其编码基因、表达载体及重组菌 |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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