CN109280656A - 重组布氏白僵菌蛋白酶k突变体pk-m1及制备方法 - Google Patents
重组布氏白僵菌蛋白酶k突变体pk-m1及制备方法 Download PDFInfo
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- CN109280656A CN109280656A CN201811234322.7A CN201811234322A CN109280656A CN 109280656 A CN109280656 A CN 109280656A CN 201811234322 A CN201811234322 A CN 201811234322A CN 109280656 A CN109280656 A CN 109280656A
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Abstract
本发明公开一种重组布氏白僵菌蛋白酶K突变体PK‑M1及其制备方法，本发明从改造重组布氏白僵菌的蛋白酶K分子结构入手，得到活性和稳定性均提高的蛋白酶K突变体PK‑M1，氨基酸序列如SEQ ID NO：1所示；优化表达系统，利用酵母细胞外分泌表达技术，可大规模发酵高效表达重组蛋白酶K突变体，进一步提高蛋白酶K产量；采用高效亲和层析的蛋白纯化方式，提高蛋白酶K纯化过程中的收率，实现了蛋白酶K的规模化生产。
Description
技术领域
本发明涉及基因改造和蛋白质工程技术领域，具体涉及一种酶活力高、稳定性好、操作简便、适用于大规模生产的重组布氏白僵菌蛋白酶K突变体PK-M1及制备方法。
背景技术
蛋白酶K（Proteinase K, EC 3.4.21.64）是一种重要的丝氨酸蛋白酶，于1974年首次在林伯氏白色念球菌（Tritirachium album limber）的提取物中发现。蛋白酶K具有极高的酶活性和广泛的底物特异性，对天然蛋白质具有广谱高效的切割能力。该酶偏好于切割脂肪族氨基酸和芳香族氨基酸的羧基端肽键，能优先分解与疏水性氨基酸、含硫氨基酸、芳香族氨基酸C末端邻接的酯键和肽键。
蛋白酶K在生化实验中应用较为广泛：在核酸提取中，可以除去核酸中的DNA酶和RNA酶；在原位杂交中，具有降解包围靶DNA蛋白质的作用，可用来处理杂交前的样本，提高检测的灵敏性；在生物检测方面，可以用来检测脑组织中的致病性朊病毒。除此之外，蛋白酶K在污水处理、工业制造、造纸、生物加工过程及饲料等领域展现出良好的应用效果。
目前，生产蛋白酶K主要有两条途径：一是由林伯氏白色念球菌经发酵后分离纯化获得，二是通过基因重组表达后获得。林伯氏白色念球菌生长缓慢，难以高密度培养，故蛋白酶K的产量较低；而且林伯氏白色念球菌还会分泌其他的蛋白酶，增加了下游分离纯化的难度，使其生产成本较高，不适合大规模生产。2008年，来源于林伯氏白色念球菌的蛋白酶K成功在毕赤酵母系统中实现了分泌表达；之后来源于布氏白僵菌的蛋白酶K也成功在毕赤酵母系统中实现了分泌表达，并且重组布氏白僵菌蛋白酶K活性较野生型林伯氏白色念球菌蛋白酶K高出50％。但在实际运用中发现，重组布氏白僵菌蛋白酶K溶液在4℃保存时酶活稳定性较差，酶活降低很快，大规模产业化应用受到限制。
发明内容
本发明是为了解决现有技术所存在的上述技术问题，提供一种酶活力高、稳定性好、操作简便、适用于大规模生产的重组布氏白僵菌蛋白酶K突变体PK-M1及制备方法。
本发明的技术解决方案是：一种重组布氏白僵菌蛋白酶K突变体PK-M1，其特征在于氨基酸序列如SEQ ID NO：1所示。
上述重组布氏白僵菌蛋白酶K突变体PK-M1的制备方法，其特征在于按照如下步骤进行：
a. 构建重组布氏白僵菌蛋白酶K突变体表达载体 pPIC9K-M1
合成如SEQ ID NO.2所示的M1编码序列并克隆至pUC57质粒中，得到pUC57/M1质粒；扩增pUC57/M1质粒，分别用限制性内切酶EcoR I、Not I将M1编码序列切下，与进行同样双酶切的pPIC9K载体连接，将连接产物转化至TOP 10感受态细胞，挑取克隆，扩增质粒，得到重组布氏白僵菌蛋白酶K突变体载体 pPIC9K-M1；
b. 将重组布氏白僵菌蛋白酶K突变体表达载体pPIC9K-M1转化到GS115酵母感受态细胞
利用限制性内切酶Sal I酶切重组布氏白僵菌蛋白酶K突变体载体 pPIC9K-M1，再用TE缓冲液溶解至浓度为2μg/μL的线性片段，取10μL线性片段与GS115酵母感受态细胞混匀，转移至冰预冷的电转杯中，冰浴5min后电击；之后向电转杯中加入1mL冰预冷的1M山梨醇溶液，混匀后转至1.5mL EP管中，30℃静置孵育5h；将菌体悬液每200μL涂布一块MD平板上，将MD平板置于30℃环境培养至出现单个菌落；
c. 筛选高拷贝数和Mut+表型的GS115/pPIC9K-M1酵母单菌落
配制浓度5g/L G418的YPD筛选平板，在MD平板上挑取200个长出的菌落，点在所述浓度5g/L G418的YPD筛选平板中，再筛选出直径大于2mm的酵母菌落，并以所筛选出的酵母菌落DNA为模板DNA进行PCR鉴定；
所述PCR引物如下：
F-AOX1：gactggttccaattgacaagc；
R-AOX1：ggcaaatggcattctgacat；
所述PCR体系如下表：
所述PCR反应条件为：94℃预变性5min，94℃变性20s，55℃退火20s，72℃延伸2min，反应进行30个循环；72℃延伸5min；
利用琼脂糖凝胶电泳检测PCR产物，筛选PCR产物为 2200bp、1632bp两条条带的酵母单菌落为高拷贝数和Mut+表型的GS115/pPIC9K-M1；
d. 将筛选得到的高拷贝数和Mut+表型的GS115/pPIC9K-M1的酵母单菌落接种至50mLYPD培养基中，30℃，200rpm振荡培养OD600为3.0时，再按1%的接种量接种至50mL BMGY培养基中，30℃，200rpm振荡培养至OD600为2~6；1500g离心收集菌体，用100mL BMMY培养基重悬菌体，28℃，200rpm继续振荡培养120小时，每24h向发酵液中补加终浓度v/v为0.5%的甲醇；离心培养基，得到含有重组布氏白僵菌蛋白酶K突变体PK-M1的发酵液上清；
e. 将发酵液上清经过8,000rpm离心分离、金属离子螯合亲和层析，用含300 mmol/L咪唑、pH 7.5的洗脱缓冲液洗脱，将收集的洗脱液通过超滤去除咪唑、离子交换柱层析分离、大孔脱色胶脱色和冻干后得到重组布氏白僵菌蛋白酶K突变体PK-M1。
本发明从改造重组布氏白僵菌的蛋白酶K分子结构入手，得到活性和稳定性均提高的蛋白酶K突变体PK-M1；优化表达系统，利用酵母细胞外分泌表达技术，可大规模发酵高效表达重组蛋白酶K突变体，进一步提高蛋白酶K产量；采用高效亲和层析的蛋白纯化方式，提高蛋白酶K纯化过程中的收率，实现了蛋白酶K的规模化生产。
附图说明
图1是本发明实施例的重组布氏白僵菌蛋白酶突变体PK-M1氨基酸序列中突变位点的概要图。
图2是本发明实施例利用SDS-PAGE检测重组布氏白僵菌蛋白酶K突变体PK-M1在GS115酵母细胞中的表达结果示意图。
图3是本发明实施例利用电泳对所得冻干重组布氏白僵菌蛋白酶K突变体PK-M1的纯度进行检测的结果示意图。
图4是本发明实施例的重组布氏白僵菌蛋白酶K突变体PK-M1的稳定性结果示意图。
具体实施方式
本发明实施例中未注明具体条件的实验方法，通常可按常规条件，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进行。对于本发明所涉及的术语及相关测定方法解释如下：
1.蛋白酶酶活测定方法：采用中华人民共和国国家标准蛋白酶制剂测定方法（GB/T25327-2009）。
2.酶活单位的定义：1g固体酶粉（或1mL液体酶），在一定温度和pH值条件下，1min水解酪蛋白产生1μg酪氨酸，即为1个酶活力单位，以U/g（U/mL）表示。
3. 蛋白酶K采用福林法测定蛋白酶的活力，使用到的溶液包括：福林使用溶液（一份市售福林溶液与两份水混合，摇匀），碳酸钠溶液（42.4g/L），三氯乙酸（65.4g/L），梯度pH值缓冲液，酪蛋白溶液（10.0g/L）。反应过程如下：试管中加入1mL酶液，40℃温浴2min，加入酪蛋白溶液1mL，摇匀后40℃温浴10min，加入2mL三氯乙酸溶液，摇匀（空白对照先加入三氯乙酸，再加入酪蛋白溶液）。取出静止10min，慢速定性滤纸过滤。取1mL滤液，加碳酸钠溶液5mL，加福林试剂使用溶液1mL，40℃显色20min，于680nm波长，用10mm比色皿测定吸光度。
本发明的重组布氏白僵菌蛋白酶K突变体PK-M1的制备方法，按照如下步骤进行：
a. 构建重组布氏白僵菌蛋白酶K突变体表达载体 pPIC9K-M1
将氨基酸序列如SEQ ID NO：3所示的野生型布氏白僵菌蛋白酶K的DNA（SEQ ID NO：4所示）编码序列替换为毕赤酵母偏爱密码子组成的DNA序列，在其5’端添加EcoR I酶切位点，3’端顺次添加六聚组氨酸标签、终止密码子TAA及Not I酶切位点，并在此基础上引入相应突变，如图1所示，H101S、R102T、A103S、K104P、N346G、V347D。利用化学方法合成如SEQ IDNO.2所示的M1编码序列（北京六合华大基因科技有限公司）并克隆至pUC57质粒中（北京六合华大基因科技有限公司），得到pUC57/M1质粒；扩增pUC57/M1质粒，分别用限制性内切酶EcoR I、Not I将M1编码序列切下，与进行同样双酶切的pPIC9K载体连接，将连接产物转化至TOP 10感受态细胞，挑取克隆，扩增质粒，得到重组布氏白僵菌蛋白酶K突变体载体pPIC9K-M1；
b. 将重组布氏白僵菌蛋白酶K突变体表达载体pPIC9K-M1转化到GS115酵母感受态细胞
利用限制性内切酶Sal I酶切重组布氏白僵菌蛋白酶K突变体载体 pPIC9K-M1，再用TE缓冲液（pH 8.0）溶解至浓度为2μg/μL的线性片段，取10μL线性片段与GS115酵母感受态细胞混匀，转移至冰预冷的电转杯（两极间隙0.2 cm）中，冰浴5min后电击，电击参数：电压1.5KV，电容25μF，电阻250Ω；之后迅速向电转杯中加入1mL冰预冷的1M山梨醇溶液，混匀后转至1.5mL EP管中，30℃静置孵育5h；将菌体悬液每200μL涂布一块MD平板（13.4g/L 酵母基础氮源；0.4mg/L 生物素 ；20g/L 葡萄糖，1.5% 琼脂）上，将MD平板置于30℃环境培养至出现单个菌落；
c. 筛选高拷贝数和Mut+表型的GS115/pPIC9K-M1酵母单菌落
配制5g/L G418浓度的YPD筛选平板（酵母提取物 10g/L，蛋白胨 20g/L，葡萄糖 20g/L，1.5% 琼脂），在MD平板上挑取200个长出的菌落，点在所述G418浓度的YPD筛选平板中，再筛选出直径大于2mm的酵母菌落。
分别挑取所筛选出的直径大于2mm的酵母单菌落接种至3ml YPD培养基中，30℃，200rpm振荡培养24h，使用酵母基因组提取试剂盒提取基因组DNA，以该基因组DNA作为模板DNA，对所筛选出的酵母菌落进行PCR鉴定。
所述PCR引物如下：
F-AOX1：gactggttccaattgacaagc；
R-AOX1：ggcaaatggcattctgacat；
PCR体系如下表：
| 反应体系 | 体积（μL） |
| 2×Taq Mix | 10 |
| F-AOX1 | 0.4 |
| R-AOX1 | 0.4 |
| 模板DNA | 0.4 |
| ddH<sub>2</sub>O | 8.8 |
PCR反应条件为：94℃预变性5min，94℃变性20s，55℃退火20s，72℃延伸2min，反应进行30个循环；72℃延伸5min；
利用琼脂糖凝胶电泳检测PCR产物，筛选PCR产物为 2200bp、1632bp两条条带的酵母单菌落为高拷贝数和Mut+表型的GS115/pPIC9K-M1；
d. 将筛选得到的高拷贝数和Mut+表型的GS115/pPIC9K-M1的酵母单菌落接种至50mLYPD培养基（酵母提取物 10g/L，蛋白胨 20g/L，葡萄糖 20g/L）中，30℃，200rpm振荡培养OD600为3.0时，再按1%的接种量接种至50mL BMGY培养基（酵母提取物 10g/L，蛋白胨 20g/L，YNB 13.4g/L，0.1mol/L pH 6.0磷酸缓冲液，生物素 0.4mg/L，甘油 10g/L）中，30℃，200rpm振荡培养至OD600为2~6；1500g离心收集菌体，用100mL BMMY培养基（酵母提取物10g/L，蛋白胨 20g/L，YNB 13.4g/L，0.1mol/L pH 6.0磷酸缓冲液，生物素 0.4mg/L，甲醇5mL/L）重悬菌体，28℃，200rpm继续振荡培养120小时，每24h向发酵液中补加终浓度v/v为0.5%的甲醇；离心培养基，得到含有重组布氏白僵菌蛋白酶K突变体PK-M1的发酵液上清，置于-70℃保存；
如在发酵罐扩大培养过程中，菌体生长阶段温度控制在28℃，诱导表达阶段温度控制在25℃，整个发酵过程中pH控制在7.0。甲醇诱导时间为120小时，溶氧量控制在35%。诱导剂甲醇的补加和溶氧量偶联，当溶氧量高于35%时补加甲醇，重组布氏白僵菌蛋白酶K突变体PK-M1的表达量可以达到2g/L发酵液。
该产率优于目前现有的蛋白酶K工业生产方法的产率（0.3~0.5g/L发酵液），从而使得后期纯化、冻干的高收率成为可能，为规模化工业生产提供了条件。
e. 将发酵液上清经过8,000rpm离心分离、金属离子螯合亲和层析，用含300mmol/L咪唑、pH 7.5的洗脱缓冲液洗脱，将收集的洗脱液通过超滤去除咪唑、离子交换柱层析分离、大孔脱色胶脱色和冻干后得到重组布氏白僵菌蛋白酶K突变体PK-M1，综合收率相比现有技术提高了80%。
对所得重组布氏白僵菌蛋白酶K突变体PK-M1氨基酸序列分析，其氨基酸序列如SEQ ID NO：1所示，即氨基酸序列第101位的His突变为Ser、第102位的Arg突变为Thr、第103位的Ala突变为Ser、第104位的Lys突变为Pro、第346位的Asn突变为Gly、第347位的Val突变为Asp；所述“101位”……“329位”并不是表示从N端起的绝对位置，而是表示与SEQ ID NO.3的氨基酸序列相比的相对位置。
实验：
1.利用SDS-PAGE检测含有重组布氏白僵菌蛋白酶K突变体PK-M1在酵母细胞GS115/pPIC9K-M1中的表达：
SDS-PAGE配方如下：
| 12%分离胶（mL） | 5%浓缩胶（mL） | |
| 超纯水 | 3.3 | 3.4 |
| 30%聚丙烯酰胺溶液（29:1） | 4.0 | 0.83 |
| 1.5mol/L Tris溶液（pH 8.8） | 2.5 | 0.63 1.5mol/L Tris溶液（pH 6.8） |
| 10% SDS | 0.1 | 0.05 |
| 10%过硫酸铵溶液 | 0.1 | 0.05 |
| TEMED | 0.004 | 0.005 |
在小烧杯中依次加入12％分离胶所需溶液成分，灌入预先装配好的双层玻璃板的间隙中，室温放置20min以上，直至凝胶聚合完全。再在小烧杯中依次加入5％浓缩胶所需溶液成分，灌入双层玻璃板间已凝聚的分离胶上方的间隙，室温放置20min以上，直至凝胶聚合完全。
2×SDS蛋白上样buffer配方：1.5M Tris-HCL pH 6.8 1ml，SDS 0.4g，溴酚蓝0.02g，甘油4ml，加双蒸水定容至10ml。
将GS115/pPIC9K-M1的发酵液上清由-70℃取出，冰上融化后取10μL上清液混合等体积的2×SDS蛋白上样buffer，以沸水浴处理10min，12000g离心1min，吸取上清加入到如上制备的凝胶的样品孔道中，上样量为10μL。同时加入蛋白质分子量标准，120V恒压电泳1h。
卸下凝胶。按下述配方配制染色液和脱色液，进行考马斯亮蓝染色，以观察样品中的目的蛋白条带。
染色液配方：双蒸水650ml；异丙醇250ml；醋酸100ml；1g考马斯亮蓝R-250。
脱色液配方：双蒸水850ml；乙醇50ml；醋酸100ml。
结果如图2所示，摇瓶条件下在发酵液上清中观察到分子量29KD的明显目的蛋白条带。其中，蛋白质分子量标准（MAKER）的3条可见的蛋白条带分别表示48kD、35kD及25kD。结果表明，本发明的重组布氏白僵菌蛋白酶K突变体PK-M1能够在酵母细胞中有效表达。
2. 重组布氏白僵菌蛋白酶K突变体PK-M1酶活测定
采用中华人民共和国国家标准蛋白酶制剂测定方法(GB/T 25327-2009)，在pH 7.5条件下，分别检测蛋白酶ProK和本发明实施例所制备的重组布氏白僵菌蛋白酶K突变体PK-M1冻干粉的蛋白酶K酶活。
所述蛋白酶ProK是按照本发明实施例的制备方法，将野生型布氏白僵菌蛋白酶K的DNA编码序列ProK（SEQ ID NO.4）重组克隆纯化冻干得到的冻干粉。
结果如下所示：
| PK-M1 | ProK | |
| 酶活（U/mg） | 48.7 | 30.3 |
可见，本发明制备得到的重组布氏白僵菌蛋白酶K突变体PK-M1酶活性明显优于野生型蛋白酶K。
3. 利用SDS-PAGE检测纯化冻干后的重组布氏白僵菌蛋白酶K突变体PK-M1的纯度。
结果如图3所示。观察到分子量29KD的明显目的蛋白条带。其中，蛋白质分子量标准（MAKER）的3条可见的蛋白条带分别表示48kD、35kD及25kD。结果表明，纯化冻干后的重组布氏白僵菌蛋白酶K突变体PK-M1具有极高的纯度（99%）。
4. 重组布氏白僵菌蛋白酶K突变体PK-M1的稳定性
对以溶液形式存放于-20℃的重组布氏白僵菌蛋白酶K突变体PK-M1和以冻干粉形式存放于4℃的重组布氏白僵菌蛋白酶K突变体PK-M1的酶活稳定性进行了检测。
结果如图4所示。重组布氏白僵菌蛋白酶K突变体PK-M1冻干粉的酶活力在一年内基本保持不变；溶液形式的重组布氏白僵菌蛋白酶K突变体PK-M1在一年后也保留了95%以上的酶活，表明利用本发明方法制备的重组布氏白僵菌蛋白酶K突变体PK-M1具有极高的稳定性。
序列表
<110> 大连博格林生物科技有限公司
<120> 重组布氏白僵菌蛋白酶K突变体PK-M1及制备方法
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Gly Thr Ile Gly Ser Lys Thr Tyr Gly Val Ala Lys Lys Val Ser Ile
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Glu Cys Thr Lys Gly Ala Ile Ala Ser Met Ser Leu Gly Gly Gly Tyr
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gaattcgctc cagttgttga accagctcca ttgattgaag ctagaggcca aactattgct 60
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aaggtttcta agccaaagtt tgtctacact gacgtttttc caggttacgc tgcttctttg 180
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gctatagtct ccattaacgc cattgtcaga caaccaggtg ctccatgggg tttgggtaga 300
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ggtgtttttg ttgctgttgc tgctggtaac gataacagag atgctgctaa cacttctcca 780
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ttttctaact acggtaaggt cttggatatt tttgctccag gtaccggtat tttgtctact 900
tggatcaacg gtggtactaa cactatttct ggtacctcta tggctactcc acatattgct 960
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Claims (2)
1.一种重组布氏白僵菌蛋白酶K突变体PK-M1，其特征在于氨基酸序列如SEQ ID NO：1所示。
2.一种权利要求1所述重组布氏白僵菌蛋白酶K突变体PK-M1的制备方法，其特征在于按照如下步骤进行：
a. 构建重组布氏白僵菌蛋白酶K突变体表达载体 pPIC9K-M1
合成如SEQ ID NO.2所示的M1编码序列并克隆至pUC57质粒中，得到pUC57/M1质粒；扩增pUC57/M1质粒，分别用限制性内切酶EcoR I、Not I将M1编码序列切下，与进行同样双酶切的pPIC9K载体连接，将连接产物转化至TOP 10感受态细胞，挑取克隆，扩增质粒，得到重组布氏白僵菌蛋白酶K突变体载体 pPIC9K-M1；
b. 将重组布氏白僵菌蛋白酶K突变体表达载体pPIC9K-M1转化到GS115酵母感受态细胞
利用限制性内切酶Sal I酶切重组布氏白僵菌蛋白酶K突变体载体 pPIC9K-M1，再用TE缓冲液溶解至浓度为2μg/μL的线性片段，取10μL线性片段与GS115酵母感受态细胞混匀，转移至冰预冷的电转杯中，冰浴5min后电击；之后向电转杯中加入1mL冰预冷的1M山梨醇溶液，混匀后转至1.5mL EP管中，30℃静置孵育5h；将菌体悬液每200μL涂布一块MD平板上，将MD平板置于30℃环境培养至出现单个菌落；
c. 筛选高拷贝数和Mut+表型的GS115/pPIC9K-M1酵母单菌落
配制浓度5g/L G418的YPD筛选平板，在MD平板上挑取200个长出的菌落，点在所述浓度5g/L G418的YPD筛选平板中，再筛选出直径大于2mm的酵母菌落，并以所筛选出的酵母菌落DNA为模板DNA进行PCR鉴定；
所述PCR引物如下：
F-AOX1：gactggttccaattgacaagc；
R-AOX1：ggcaaatggcattctgacat；
所述PCR体系如下表：
所述PCR反应条件为：94℃预变性5min，94℃变性20s，55℃退火20s，72℃延伸2min，反应进行30个循环；72℃延伸5min；
利用琼脂糖凝胶电泳检测PCR产物，筛选PCR产物为 2200bp、1632bp两条条带的酵母单菌落为高拷贝数和Mut+表型的GS115/pPIC9K-M1；
d. 将筛选得到的高拷贝数和Mut+表型的GS115/pPIC9K-M1的酵母单菌落接种至50mLYPD培养基中，30℃，200rpm振荡培养OD600为3.0时，再按1%的接种量接种至50mL BMGY培养基中，30℃，200rpm振荡培养至OD600为2~6；1500g离心收集菌体，用100mL BMMY培养基重悬菌体，28℃，200rpm继续振荡培养120小时，每24h向发酵液中补加终浓度v/v为0.5%的甲醇；离心培养基，得到含有重组布氏白僵菌蛋白酶K突变体PK-M1的发酵液上清；
e. 将发酵液上清经过8,000rpm离心分离、金属离子螯合亲和层析，用含300 mmol/L咪唑、pH 7.5的洗脱缓冲液洗脱，将收集的洗脱液通过超滤去除咪唑、离子交换柱层析分离、大孔脱色胶脱色和冻干后得到重组布氏白僵菌蛋白酶K突变体PK-M1。
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| CN109207460A (zh) * | 2018-10-23 | 2019-01-15 | 大连博格林生物科技有限公司 | 重组布氏白僵菌蛋白酶k突变体pk-m2及制备方法 |
| CN110106160A (zh) * | 2019-05-29 | 2019-08-09 | 中国农业科学院饲料研究所 | 催化活性提高的碱性蛋白酶突变体prok-g及其编码基因和应用 |
| CN113337492A (zh) * | 2021-06-02 | 2021-09-03 | 武汉瀚海新酶生物科技有限公司 | 一种蛋白酶k耐热突变体 |
| CN113913412A (zh) * | 2021-10-13 | 2022-01-11 | 湖北大学 | 蛋白酶k突变体及其制备方法 |
| WO2023096513A1 (en) * | 2021-11-26 | 2023-06-01 | Blirt S.A. | Tritirachium album proteinase k mutant and its zymogen, expression plasmid, recombinant pichia pastoris strain and method of producing the mature form of proteinase k mutant |
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| CN109207460A (zh) * | 2018-10-23 | 2019-01-15 | 大连博格林生物科技有限公司 | 重组布氏白僵菌蛋白酶k突变体pk-m2及制备方法 |
| CN109207460B (zh) * | 2018-10-23 | 2021-06-15 | 大连博格林生物科技有限公司 | 重组布氏白僵菌蛋白酶k突变体pk-m2及制备方法 |
| CN110106160A (zh) * | 2019-05-29 | 2019-08-09 | 中国农业科学院饲料研究所 | 催化活性提高的碱性蛋白酶突变体prok-g及其编码基因和应用 |
| CN113337492A (zh) * | 2021-06-02 | 2021-09-03 | 武汉瀚海新酶生物科技有限公司 | 一种蛋白酶k耐热突变体 |
| CN113913412A (zh) * | 2021-10-13 | 2022-01-11 | 湖北大学 | 蛋白酶k突变体及其制备方法 |
| CN113913412B (zh) * | 2021-10-13 | 2023-10-03 | 湖北大学 | 蛋白酶k突变体及其制备方法 |
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