CN113862244A - 一种高比活的碱性蛋白酶突变体及其在液体洗涤剂中的应用 - Google Patents
一种高比活的碱性蛋白酶突变体及其在液体洗涤剂中的应用 Download PDFInfo
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- CN113862244A CN113862244A CN202111167372.XA CN202111167372A CN113862244A CN 113862244 A CN113862244 A CN 113862244A CN 202111167372 A CN202111167372 A CN 202111167372A CN 113862244 A CN113862244 A CN 113862244A
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Abstract
本发明属于碱性蛋白酶的蛋白质工程改造技术领域,提供一种洗涤用的碱性蛋白酶突变体JD125及其在液体洗涤剂中的应用。所述碱性蛋白酶突变体JD109(SEQ ID NO:2)的亲本蛋白酶为枯草芽孢杆菌PB92的碱性蛋白酶或者诺维信的碱性蛋白酶产品Savinase(SEQ ID NO:1)。所述碱性蛋白酶突变包含以下氨基酸的置换:N74D,N85R,G116R,S126L,P127Q,S128A,其中所述位置对应于氨基酸序列SEQ ID NO:1的多肽的氨基酸位置(根据SEQ ID NO:1进行的编号)。本发明的碱性蛋白酶突变体适合于在清洁或者洗涤剂组合物中使用。本发明的碱性蛋白酶突变体在碱性条件下的酶活比亲本蛋白酶Savinase有显著的提高,同时在不同洗涤剂中的稳定性也有所提高。为其更好的适应洗涤剂行业的发展奠定了技术基础。
Description
技术领域
本发明属于蛋白质工程改造技术领域,具体涉及一种高比活的碱性蛋白酶突变体及其在液体洗涤剂中的应用。
背景技术
碱性蛋白酶(Alkaline Protease)属丝氨酸蛋白水解酶类,丝氨酸蛋白水解酶类是具有启动蛋白质肽键水解的活性位点丝氨酸的酶(EC3.4.21),主要有枯草杆菌蛋白酶类(EC3.4.21.62),这类酶属于MEROPS分类方案的S8肽酶家族,肽酶S8家族的成员在其氨基酸序列中具有酶催化活性位点为Asp、His和Ser 的催化三联体。碱性蛋白酶在中性至碱性条件下水解蛋白质肽键、酯键和酰胺键,其最适作用pH一般为8~11。该酶种最早在猪的胰脏中发现。碱性蛋白酶广泛存在于微生物、植物和动物中。碱性蛋白酶主要应用领域包括洗涤剂、饲料、药物、皮革、大豆加工、酿酒厂、肉类嫩化、废物管理、摄影和诊断等轻工业领域。产碱性蛋白酶的微生物主要从盐碱湖、深海、沙地等碱性环境中分离得到。近几十年,随着新的碱性蛋白酶生产菌株的不断分离和纯化,碱性蛋白酶的研究有了很大的发展。迄今,芽孢杆菌(Bacillus)、放线菌(Actinomyces)以及真菌均有报道可以产碱性蛋白酶。目前用于工业化生产的菌种主要为地衣芽孢杆菌、枯草芽孢杆菌、嗜碱性芽孢杆菌、解淀粉芽孢杆菌等(Tekin N et al . Pol J. Microbiol , 2017 , 66(1): 39-56)。碱性蛋白酶单独占全球酶市场的25%(MikkelsenM L et al . Food and Chemical Toxicology , 2015: 07-21)。其在酶工业中占有很大的比重,全球酶制剂市场中蛋白酶的销售额达到 60%,而碱性蛋白酶就占35%。
碱性蛋白酶是最早应用于洗涤剂产品的酶制剂。污渍中血、奶、蛋、果汁、汗渍、咖啡等蛋白质大分子物质的水溶性不好,普通的表面活性剂和其他助洗剂都很难去除,碱性蛋白酶可将其分解成易溶于水的小分子肽键,然后再分解成氨基酸,从而很容易被洗去。洗涤剂产品中的碱性蛋白酶在没有稳定剂和抑制剂的情况下会自酶解而逐渐丧失酶活性。为提高洗涤剂中蛋白酶的稳定性,诺维信公司开发了高效的蛋白酶抑制剂4-FPBA(4-formyl-phenyl-boronic acid)用于稳定洗涤剂碱性蛋白酶产品,随后又致力于开发以肽醛为基础的抑制剂。
可替代地,氨基酸改变具有这样的性质:改变多肽的物理化学特性。例如,氨基酸改变可以改进多肽的热稳定性、改变底物特异性、改变最适pH等。可以根据本领域中已知的程序,如定点诱变或丙氨酸扫描诱变来鉴定多肽中的必需氨基酸(Cunningham et al .Science 1989, 244.4908: 1081-1085)。在后一项技术中,在该分子中的每个残基处引入单个丙氨酸突变,并且对所得突变体分子的蛋白酶活性进行测试以鉴定对于该分子的活性至关重要的氨基酸残基,参考希尔顿等人研究结果(Hilton et al. Journal ofBiological Chemistry, 1996, 271.9: 4699-4708.)。酶或其他生物学相互作用的活性部位还可通过对结构的物理分析来确定,如由下述技术确定:核磁共振晶体学(crystallography)、电子衍射、或光亲和标记,连同对推定的接触位点(contract site)氨基酸进行突变,参考德沃斯等人(de vos et al. Science, 1992, 255.5042: 306-312.)。迭代饱和突变也是高效筛选蛋白酶突变的良好方法,参考(Reetz et al. Natureprotocols, 2007, 2.4: 891-903),使用迭代饱和突变的方法,Reetz等人对乙醇脱氢酶进行高效蛋白工程改造(Sun, Zhoutong, et al. ACS Catalysis, 2016, 6.3: 1598-1605.)。对于BPN (SEQIDNO:2),包含氨基酸S221、H64以及D32的催化三联体对于酶的蛋白酶活性是必需的。
越来越多的商用蛋白酶是天然存在的野生型蛋白酶的蛋白质工程化变体,例如Everlase®、Relase®、Ovozyme®、Polarzyme®、Liquanase®、Liquanase Ulta®和Kannase®(诺维信公司(Novozymes a/s))、Purafast®、PurafoctOXP®、FN3®、FN4®和Eraser®*、Excellase®,(杰能科国际公司(Genencor International,Inc.))。目前野生型的碱性蛋白酶无法满足不同条件下的洗涤要求,碱性蛋白酶的性能有待进一步提高,如温度和pH等洗涤条件会随时间而发生变化,并且许多污渍在传统的洗涤条件下仍然难以完全去除,此外,洗涤中条件可以导致酶失活(例如pH,温度或螯合不稳定性),从而导致在洗涤循环过程中洗涤性能的损失。因此,洗涤稳定性高和洗涤性能良好的碱性蛋白酶依然有较大需求。
我国碱性蛋白酶的市场需求量约为15亿元,主要被酶制剂巨头诺维信和杜邦公司垄断,主要原因是我们国内碱性蛋白酶生产菌种的生产能力较差、酶的发酵活力低、酶比活低以及洗涤应用效果差。因此,高活力、高稳定性、高效价的碱性蛋白酶的蛋白质工程改造和筛选及其高产工程菌的构建成为国内外的研究热点。
通过蛋白质工程手段可以有效提高酶的催化活性、酸碱稳定性、热稳定性、底物特异性和表达效价(Johannes TW et al . Curr . Microbiol , 2006 , 9:261-267)。蛋白工程质改造为酶的功能改善和应用开辟了崭新的途径,在工业、农业和医学等领域取得巨大的成功。
发明内容
有鉴于此,本发明提供一种高比活的碱性蛋白酶突变体及其在液体洗涤剂中的应用,旨在获得突变体蛋白,改善其耐碱性、耐表面活性剂和稳定性,使其能更好的适用于洗涤剂行业。本发明提供了一种高比活的碱性蛋白酶突变体的蛋白工程质改造及其突变体,使碱性蛋白酶在碱性pH条件下的洗涤液中的酶活有所提高,从而洗涤剂的洗涤效果。为其更好的适应工业化生产奠定了基础。从而有利于碱性蛋白酶在洗涤领域的广泛应用。
本发明由如下技术方案实现的:一种高比活的碱性蛋白酶突变体,所述碱性蛋白酶突变体的亲本蛋白酶为嗜碱芽孢杆菌(Bacillusalcalophilus)的碱性蛋白酶PB92,对应于氨基酸序列SEQIDNO:1。与SEQIDNO:1相比,Lederbergialentus碱性蛋白酶SubtilisinSavinase具有99.7%同一性的氨基酸序列(N85S,根据SEQIDNO:1进行的编号,269个氨基酸中有268个氨基酸相同);Alkalihalobacillusclausii碱性蛋白酶具有98.9%同一性的氨基酸序列(N85S,S253N,N255K,根据SEQIDNO:1进行的编号,266/269氨基酸);BacillusclausiiKSM-K16碱性蛋白酶具有98.5%同一性的氨基酸序列(N85S,A224V,S250G,S253N,根据SEQIDNO:1进行的编号,265/269氨基酸);Bacilluscirculans碱性蛋白酶具有97.7%同一性的氨基酸序列(N85S,S97D,S99R,S101A,V102I,根据SEQIDNO:1进行的编号,263/269氨基酸);AlkalihalobacillusclausiiTH2019碱性蛋白酶具有96.6%同一性的氨基酸序列(N85S,A224V,S250G,S253N,T254P,S259N,G260R,A266T,T268R,根据SEQIDNO:1进行的编号,260/269氨基酸)。
本发明提出的一种高比活的碱性蛋白酶突变体,其包含与SEQIDNO:1具有至少90%同一性的氨基酸序列,且与SEQIDNO:1相比在选自下组中的位置上包含氨基酸的取代:74,85,116,126,127,128。
在本发明的一些实施例中,所述突变体的氨基酸序列与SEQIDNO:2相比具有至少91%,92%,93%,94%,95%,96%,97%,98%,或至少99%的同一性。
在一些更具体的实施例中,所述突变体的氨基酸序列与SEQIDNO:2相比具有至少99.1%,99.2%,99.3%,99.4%,99.5%,99.6%,99.7%,99.8%,或至少99.9%的同一性。
本发明还提供一种液体洗涤剂组合物,包含上述的高比活的蛋白酶突变体。
本发明所制备的液体洗涤剂组合使用MGDA和STPP标准。
本发明还提供了上述的高比活的碱性蛋白酶突变体的制备方法,包括:
步骤1:获取编码碱性蛋白酶突变体的DNA分子,所述碱性蛋白酶突变体包含与SEQIDNO:1具有至少90%同一性的氨基酸序列。
步骤2:将步骤1获得的所述DNA分子与表达载体融合,构建重组表达载体,转化宿主细胞;
步骤3:诱导含重组表达载体的宿主细胞表达融合蛋白,分离纯化表达的融合蛋白。
在本发明的一些实施例中,步骤1所述的碱性蛋白酶突变体包含下组中的氨基酸的取代:N74D,N85R,G116R,S126L,P127Q,S128A。
在本发明的一些实施例中,步骤2所述的宿主细胞为枯草芽孢杆菌(Bacillussubtilis)。
在本发明的一些实施例中,步骤2所述的宿主细胞为地衣芽孢杆菌(Bacilluslicheniformis)。
本发明还提供了上述的高比活的碱性蛋白酶突变体在洗涤中的应用。
本发明以碱性蛋白酶PB92为基础,提供了包含N74D,N85R,G116R,S126L,P127Q,S128A中6个突变位点的组合突变体,该碱性蛋白酶突变体命名为JD125。突变体JD125在作为洗涤剂使用时比亲本蛋白酶在极端条件下有更好的保留活性,可以在更高的温度下以及更强的碱性环境下使用该蛋白酶,试验显示:在不加任何保护剂和稳定剂的前提下,碱性蛋白酶及其突变体50℃保温半小时,可以明显看出碱性蛋白酶突变体JD125剩余活力明显高于野生菌,即使保温更长时间,始终突变体的残余活力高于野生型。在不加任何保护剂和稳定剂的前提下,碱性蛋白酶及其突变体在不同pH下保温1小时,可以明显看JD125突变体(N74D,N85R,G116R,S126L,P127Q,S128A)剩余活力在pH大于9后高于野生酶。在不加任何保护剂和稳定剂的前提下,碱性蛋白酶及其突变体JD125相同的添加量,添加在MGDA和STPP洗涤体系中,碱性蛋白酶突变体JD125相比野生型有更好的稳定性,碱性蛋白酶突变体JD125的良好性能有助于拓展碱性蛋白酶的使用范围,为其更好的适应工业化生产奠定了基础。
附图说明
图1为碱性蛋白酶SubtilisinSavinase(SEQIDNO:1)及碱性蛋白酶突变体JD125(SEQIDNO:2)的氨基酸序列比对图;
图2为碱性蛋白酶SubtilisinSavinase(SEQIDNO:1)不同发酵时间蛋白SDS-PAGE电泳图;
图3为碱性蛋白酶突变体JD125(SEQIDNO:2)不同发酵时间蛋白SDS-PAGE电泳图。
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,2001)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例所记载的具体方法步骤。
以下实例是为了更好地说明阐述本发明内容,本领域相关的技术人员可以借助实例更好地理解和掌握本发明。但是,本发明的保护和权利要求范围不限于所提供的案例。
碱性蛋白酶突变体的标注:采用“原来的氨基酸位置取代的氨基酸”来表示碱性蛋白酶突变体中突变的氨基酸。如S259K,表示位置259的氨基酸由原来的碱性蛋白酶的Ser(S)替换为lys(K),位置的编号与附件序列表SEQIDNO:1中的编号相对应。
本发明中,用于限定氨基酸位置的命名法基于以编号PB92保藏于Genbank的芽孢杆菌的碱性蛋白酶的氨基酸序列,其作为SEQIDNO:1在序列表中给出(SEQIDNO:1的氨基酸1-269)。因此,在本上下文中,用于位置编号的基础SEQIDNO:1,始于A1(Ala1)并且止于R269(Arg269)。SEQIDNO:1作为位置编号的标准,并因此作为命名的基础。
对于本发明实施例中所涉及的培养基,具体配方如下:
LB液体培养基:胰蛋白胨1%,酵母粉0.5%,NaCl1%;
LB平板:胰蛋白胨1%,酵母粉0.5%,NaCl1%,琼脂2%;
溶液A:0.4g酵母提取物,0.08g酪蛋白水解物,溶于40mL水中。
溶液B:5g葡萄糖,溶于10mL水中。
溶液C:4.8gKH2PO4,11.2gK2HPO4,0.16gMgSO4•7H2O,0.8g柠檬酸三钠,1.6g(NH4)2SO4,溶于200mL水中。
溶液D:0.9gMnCl2•4H2O,1.415g硼酸,0.68gFeSO4•7H2O,13.45mgCuCl2•2H2O,23.5mgZnSO4•7H2O,20.2mgCoCl2•6H2O,12.6mg高钼酸钠,0.855g酒石酸钠,溶于500mL水中。
溶液E:2.16gMgCl2•6H2O,溶于20mL水中。
溶液F:147mgCaCl2溶于20mL水中。
溶液G:36.5g山梨醇,溶于100mL水中。
GMⅠ:10mL溶液A,1.5mL溶液B,25mL溶液C,100uL溶液D,25mL溶液G,加灭菌水至100mL。
GMII:98mLGMⅠ,1mL溶液E,1mL溶液F,混匀。
GMⅢ:9mLGMⅡ,1mL甘油。
种子培养基:酵母浸粉0.5%,胰蛋白胨0.5%,葡萄糖1%,K2HPO41.8%;
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%,CaCl20.1~0.5%,MgSO40.1~0.5%,K2HPO40.5~2%。
本发明实施中所述碱性蛋白酶的酶活测定方法可采用下述方法:
碱性蛋白酶的酶活测定方法I:
碱性蛋白酶酶活测定方法:参照GB/T23527-2009附录B福林法进行,具体反应过程如下:
蛋白酶在一定的温度与pH条件下,水解酪蛋白底物,产生含有酚基的氨基酸(如:酪氨酸、色氨酸等),在碱性条件下,将福林试剂(Folin)还原,生成钼蓝与钨蓝,用分光光度计于波长680nm下测定溶液的吸光度。酶活力与吸光度成正比例,由此可以计算产品的酶活力。蛋白酶活力定义,即蛋白酶活力为蛋白酶活力单位表示,定义为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μ/g(μ/ml)表示。
试剂和溶液
(1)福林(Folin)试剂(福林︰水=1︰2);(2)42.4g/L碳酸钠溶液;(3)0.5mol/L氢氧化钠溶液;(4)硼酸缓冲液(pH10.5);(5)10.0g/L酪蛋白溶液;(6)100μg/mL和1mg/ml的L-酪氨酸标准溶液;(7)6.54%三氯乙酸。
酶活测定
(1)标准曲线的制定:配置浓度分别为0μg/mL,10μg/mL,20μg/mL,30μg/mL,40μg/mL和50μg/mL的L-酪氨酸标准溶液。分别取标准液各1.00ml,各加0.4mol/L碳酸钠溶液5.00ml、福林试剂使用液1.00ml,振荡均匀,置于40℃水浴中显色20min,取出用分光光度计于波长680nm,10mm比色皿,以不含酪氨酸的0管为空白,分别测定其吸光度。以吸光度A为纵坐标,酪氨酸的浓度C为横坐标,绘制标准曲线(此线应通过零点)。
(2)酶活测定
取预稀释适量酶液,然后加入等体积的40℃预热10%酪蛋白,40℃反应10min;然后加入与反应体系等体积三氯乙酸(浓度6.54%),混匀后室温静置10min以终止反应。取出1ml已终止的反应液,然后加入5ml42.4g/L碳酸钠溶液,接着加入1ml福林(Folin)试剂,40℃显色反应20min;最后测定OD608值。
(3)计算
从标准曲线上读出样品最终稀释液的酶活力,单位为μ/mL。样品的酶活力按下面的公式计算:
X=A×K×4/10×n=2/5×A×K×n
式中:X——样品的酶活力(μ/g或μ/ml)
A——样品平行试验的平均吸光度
K——吸光常数
4——反应试剂的总体积(ml)
10——反应时间10min,以1min计
n——稀释倍数。
碱性蛋白酶的酶活测定方法II:
蛋白水解活性可以通过采用Suc-AAPF-PNA底物的方法来确定。Suc-AAPF-PNA是N琥珀酰基-丙氨酸-丙氨酸-脯氨酸-苯丙氨酸-对硝基苯胺的缩写,并且它是可以通过内切蛋白酶切割的一种封闭肽在切割后,一个游离PNA分子被释放出来,并且它具有黄色颜色并且因此可以通过可见光分光光度法在波长405nm进行测量。Suc-AAPF-PNA底物是通过巴亨(Bachem)(目录号L1400溶解在DMSO中)制造。
待分析的蛋白酶样品被稀释在残余活性缓冲液中(100mMTrispH8.6)。通过转移30μl的稀释的酶样品至96孔微量滴定板并添加70μl底物工作溶液(0.72mg/mI于100mMTrispH86中)进行该测定。将该溶液在室温混合并且在0D405nm经5分钟每20秒测量吸收。
在一组给定条件下,时间相关吸收曲线的斜率(每分钟的吸光度)与所讨论的蛋白酶的活性成正比例。应该将蛋白酶样品稀释至其中斜率为线性的水平。
实施例1:碱性蛋白酶突变体JD125的构建和表达:本发明的碱性蛋白酶突变体的构建可通过本领域技术人员熟知的方法构建和表达。
碱性蛋白酶突变体JD125的氨基酸序列为SEQIDNO:2。根据SEQIDNO:2的氨基酸序列,获得其优化后的核酸序列SEQIDNO:3,由生工生物工程(上海)股份有限公司合成SEQIDNO:3。使用GibsonAssembly的方法将SEQIDNO:3连接到pBE2R载体上,用热激法转入大肠杆菌DH5α,提质粒进行测序确定,获得碱性蛋白酶重组质粒pBE2R-AP(JD125)。
将测序正确的重组质粒pBE2R-AP(JD125)转入感受态细胞WB600中,具体转化过程如下:用枪头挑取在LB平板上生长的WB600单菌落于2mLGMⅠ中,培养12h;将过夜培养的菌液加到98mLGMⅠ中,37℃,200rpm培养约4h;取10mL菌液加入90mLGMII中,37℃,200rpm培养约1.5h;菌体冰水浴30min,4000rpm,4℃离心30min,去上清;加10mLGMⅢ,混匀,即为感受态细胞WB600。然后在500μL感受态细胞中加入5μLpBE2R-AP(JD125)质粒,直接将感受态细胞置于37℃,200rpm摇床培养1.5h,低速离心3min,弃部分上清,均匀涂布于含40μg/mL卡那霉素的脱脂奶粉培养基平板上,37℃恒温培养箱培养12h。次日平板上的单菌落即为含碱性蛋白酶突变体AP(JD125)的重组菌株WB600/pBE2R-AP(JD125)。碱性蛋白酶突变体枯草杆菌重组工程菌接种于5mLLB液体培养基(蛋白胨1%,NaCl1%,酵母粉0.5%)中,37℃,200rpm振荡培养12h,将菌液按2%的接菌量分别转接于发酵产酶培养基中,37℃,200rpm振荡培养84h。
实施例2:碱性蛋白酶突变体的收集和电泳检测:发酵完成后,发酵液13000r/min离心15min,然后上清液用0.22μm膜在正压滤器上过滤去除残留枯草芽孢杆菌。上清液用于电泳检测和后续的酶活检测。电泳检测方法如下:将100%的三氯乙酸(1kg三氯乙酸溶于454ml水)加入蛋白样品中,使三氯乙酸的终浓度为13%,混匀后放置冰上30分钟。15000g,4℃离心15分钟,弃上清,倒置使其干燥,获得蛋白沉淀。加入Tris-HCl缓冲液(50mMTris-HCl,100mMNaCl,pH8)重悬,然后按照SDS-PAGE方法进行电泳检测。SDS-PAGE电泳,结果显示为单一条带的蛋白样品。
实施例3:碱性蛋白酶突变体的热稳定性和pH稳定性分析:碱性蛋白酶及其突变体JD125进行酶活测定,野生型碱性蛋白酶及其突变体JD125的蛋白浓度0.2mg/ml,蛋白缓冲液为50mMTris-HCl,100mMNaCl,pH8.0。酶活测定方法参照方法I或方法II进行,在50℃保温2.5h,每隔0.5h取样测定酶活性。测试结果如表1。
表1野生型碱性蛋白酶与突变体JD125在50℃保温不同时间的蛋白酶残余活性
在不加任何保护剂和稳定剂的前提下,野生型碱性蛋白酶及其突变体JD125在50℃保温半小时,可以明显看出突变体JD125剩余活力明显高于野生菌,即使保温更长时间,始终突变体JD125的残余活力高于野生型。
实施例4:野生型碱性蛋白酶、碱性蛋白酶突变体JD125pH稳定性的比较:配制一系列pH梯度、浓度为0.2M的缓冲溶液:Na2HPO4-NaH2PO4(pH6.0-7.0)、Tris-HCl(pH8.0-9.0)、Gly-NaOH(pH10.0-12.0),将酶液(浓度为0.2mg/ml)分别在一系列pH梯度缓冲液系统中25℃保存lh,参照GB/T23527-2009附录B福林法测定酶活,结果如表2所示。
表2野生型碱性蛋白酶与突变体在不同pH条件下的活性
在不加任何保护剂和稳定剂的前提下,野生型碱性蛋白酶及其突变体JD125在不同pH下保温1小时,可以明显看出突变体JD125剩余活力在pH大于9后高于野生酶。
实施例5:碱性蛋白酶突变体在液体洗涤剂中的活性测定:
液体洗涤剂配方制备如表3所示。
表3液体洗涤剂配方
将两种洗涤剂都溶解在50mMCHES缓冲液(N-环己基-2-氨基乙磺酸)中以确保在实验过程中而且在添加蛋白酶样品后pH维持在10.0。
取0.2mg/ml蛋白酶液10μl与190μl标准洗涤剂溶液在1.5mlEP管中混合,参照GB/T23527-2009附录B福林法测定酶活,结果如表4所示。
表4蛋白酶及其突变体JD125在STPP和MGDA标准洗涤中在稳定性数据
在不加任何保护剂和稳定剂的前提下,野生型碱性蛋白酶及其突变体JD125相同的添加量,添加在MGDA和STPP洗涤体系中,在这两种洗涤体系中,碱性蛋白酶突变体JD125相比野生型碱性蛋白酶有更好的稳定性,具有更高的酶活。
序列表
<110> 上海佶凯星生物科技有限公司
<120> 一种高比活的碱性蛋白酶突变体及其在液体洗涤剂中的应用
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Claims (6)
1.一种高比活的碱性蛋白酶突变体,其特征在于,所述高比活的碱性蛋白酶突变体的亲本蛋白酶为枯草芽孢杆菌PB92的蛋白酶,所述高比活的碱性蛋白酶突变体包含以下氨基酸的置换:N74D,N85R,G116R,S126L,P127Q,S128A,其中所述位置对应于氨基酸序列SEQ IDNO :1的多肽的氨基酸位置。
2.根据权利要求1所述的高比活的碱性蛋白酶突变体,其特征在于,所述高比活的碱性蛋白酶突变体还包含N74D+N85R+G116R+S126L+P127Q+S128A置换组合。
3.根据权利要求1所述的高比活的碱性蛋白酶突变体,其特征在于,所述亲本蛋白酶与氨基酸序列SEQ ID NO:1 具有至少95%序列一致性的氨基酸序列。
4.根据权利要求1所述的高比活的碱性蛋白酶突变体,其特征在于,所述亲本蛋白酶具有SEQ ID NO :2表示的氨基酸序列。
5.根据权利要求4所述的高比活的碱性蛋白酶突变体,其特征在于,所述亲本蛋白酶与氨基酸序列SEQ ID NO:2具有至少95%的序列一致性的氨基酸序列。
6.一种液体洗涤剂组合物,其特征在于,包含权利要求1~5所述的蛋白酶突变体。
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CN116426509A (zh) * | 2023-04-27 | 2023-07-14 | 上海佶凯星生物科技有限公司 | 一种碱性蛋白酶组合突变体及其应用 |
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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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 |
CN116218818A (zh) * | 2022-09-15 | 2023-06-06 | 上海佶凯星生物科技有限公司 | 一种高比活碱性木聚糖酶突变体及其应用 |
CN116426509A (zh) * | 2023-04-27 | 2023-07-14 | 上海佶凯星生物科技有限公司 | 一种碱性蛋白酶组合突变体及其应用 |
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