一种酶活提高的甲壳素酶突变体的制作方法

本发明涉及一种酶活提高的甲壳素酶突变体，属于酶工程和基因工程
技术领域：
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背景技术：
：甲壳素酶(ec3.2.1.14.)又称几丁质酶，能催化不溶性的甲壳素糖链β-1,4糖苷键的断裂，生成水溶性的几丁寡糖。由于甲壳素酶既可作为生物防治剂，用于开发转基因植物，达到抗病的目的；也可用于原生质分离、细胞化学定位以及生产单细胞蛋白，因此，其在农业、生物技术、食品、卫生方面均有很重要的应用。而甲壳素酶的水解产物几丁寡糖能够提高机体免疫力、抑制肿瘤细胞生长、在人体肠道内活化增殖双歧杆菌、抗菌防腐、保湿，因此，其在医药、食品、化妆品等工业中也具有广阔的应用前景。甲壳素酶分为3种类型，内切甲壳素酶、外切甲壳素酶和β-n乙酰氨基葡萄糖苷酶。这三种类型的酶均能以甲壳素为底物，将其切割成不同聚合度的几丁寡糖。内切甲壳素酶能随机切割甲壳素糖链中的β-1,4糖苷键，释放n-乙酰几丁寡糖；外切甲壳素酶从甲壳素糖链的非还原末端进行连续切割，释放(glcnac)2；n-乙酰氨基葡萄糖苷酶能够水解(glcnac)2释放出glcnac。甲壳素酶广泛存在于动物、植物和微生物(包括细菌、真菌、粘菌、原生动物、藻类等)中。动物和植物中的甲壳素酶含量低，提取困难，而微生物法生产甲壳素酶具有易培养、操作、成本低等优点，成为广大学者研究的重点。目前，研究产甲壳素酶的微生物主要包括paenibacillusbarengoltzii、marinebacterium(alteromonassp.strain0-7)、streptomycesthermoviolaceusopc-520和bacilluscereus等，但野生菌株中的甲壳素酶产量很低，仅有0.83-1.13u/ml。目前，已经有研究通过异源表达、酶分子改造等技术手段试图提高甲壳素酶表达量，例如，在大肠杆菌或毕赤酵母中异源表达甲壳素酶基因以及通过定点突变或以随机突变的方式改造甲壳素酶的底物结合结构域和催化结构域以提高酶活，但是，在大肠杆菌中异源表达甲壳素酶基因易形成包涵体；在大肠杆菌以及毕赤酵母中异源表达甲壳素酶基因时，甲壳素酶为胞内分泌，因此，提取甲壳素酶需破壁导致酶活损失；在毕赤酵母中异源表达甲壳素酶基因操作复杂、培养周期长；以随机突变的方式改造甲壳素酶的底物结合结构域和催化结构域以提高酶活具有随机突变的不确定性，因此，会导致筛选困难，上述技术均并不能够真正运用于工业生产。因此，急需找到一种新的可克服表达量低、易形成包涵体、破壁导致酶活损失等缺陷、可大幅度提高甲壳素酶表达量的方法。技术实现要素：本发明的第一个目的是提供一种甲壳素酶突变体，含如seqidno.2-6所示的氨基酸序列。本发明的第二个目的是提供编码上述甲壳素酶突变体的基因。在本发明的一种实施方式中，编码所述甲壳素酶突变体c43d、f273w、e336r、c43d/f273w、c43d/f273w/e336r的基因的核苷酸序列分别如seqidno.10-14所示本发明的第三个目的是提供含上述基因的载体。本发明的第四个目的是提供表达上述甲壳素酶突变体的细胞。本发明的第五个目的是提供一种基因工程菌，是以枯草芽孢杆菌为宿主，表达上述甲壳素酶突变体。在本发明的一种实施方式中，是以bacillus.subtiliswb600为宿主，以pp43nmk为表达载体。本发明的第六个目的是提供一种提高甲壳素酶酶活的方法，是将氨基酸序列如seqidno.1所示的甲壳素酶第43位的半胱氨酸突变为天冬氨酸(氨基酸序列如seqidno.2所示)；或，将第273位的苯丙氨酸突变为色氨酸(氨基酸序列如seqidno.3所示)；或，将第336位的谷氨酸突变为精氨酸(氨基酸序列如seqidno.4所示)；或，将第43位的半胱氨酸突变为天冬氨酸，并将第273位的苯丙氨酸突变为色氨酸(氨基酸序列如seqidno.5所示)；或，将第43位的半胱氨酸突变为天冬氨酸，并将第273位的苯丙氨酸突变为色氨酸，并将第336位的谷氨酸突变为精氨酸(氨基酸序列如seqidno.6所示)。本发明的第七个目的是提供上述基因工程菌在发酵领域的应用。本发明的第八个目的是提供一种生产上述甲壳素酶突变体的方法，是将上述基因工程菌接种到lb液体培养基中，35-40℃培养7-10h后，转接到tb培养基中，接种量为2-5％，35-40℃培养12-14h。本发明的第九个目的是提供上述的甲壳素酶突变体在农业、生物、化妆品、食品或卫生领域的应用。本发明通过定点突变的方式，将甲壳素酶分子催化结构域附近的第43位氨基酸(半胱氨酸)突变为天冬氨酸，第273位氨基酸(苯丙氨酸)突变为色氨酸、第336位氨基酸(谷氨酸)突变为精氨酸，显著提高了菌株表达甲壳素酶的酶活力，其中单突变株f273w和e336r较出发菌株酶活分别提高了1.37倍和1.22倍。改造后的菌株产酶能力和催化效率都得到了提高，通过hplc检测纯化后突变体中几丁寡糖的含量发现，催化3h，f273w突变株产糖量为1.65g/l，其中glcnac为0.12g/l，(glcnac)2为1.53g/l，相比野生菌株提高了49.7％。附图说明图1：突变体酶活。图2：纯化后突变体的蛋白电泳图，m：marker；1：c43d；2：穿透液；3：20％b；4：f273w；5：穿透液；6：15％b；7：20％b；8：e336r；9：穿透液；10：15％b。图3：突变体催化后产物含量。具体实施方式lb培养基：胰蛋白胨10g/l、酵母粉5g/l、nacl10g/l，ph7.0。tb培养基：蛋白胨12g/l、酵母膏24g/l、甘油5g/l、kh2po417mmol/l、k2hpo472mmol/l。采用分光光度法测定甲壳素酶酶活，1个单位甲壳素酶酶活定义为：在60℃反应条件下，每小时释放1μmol还原糖所需的酶量为一个酶活单位(u/ml)。酶活测定条件：60℃条件下，0.1ml1％的胶体几丁质，0.3ml磷酸氢二钠-柠檬酸盐缓冲液(ph5.0)和0.1ml发酵上清液，保温15min，100℃加热5min终止反应，加入1mldns试剂，沸水浴加热10min后迅速冷却，用去离子水定容至5ml。利用分光光度计在540nm处测定吸光值，通过glcnac绘制标准曲线，根据标准曲线计算酶活。实施例1产甲壳素酶菌株构建化学合成甲壳素酶基因片段chisb(核苷酸序列如seqidno.9所示)。pcr条件为：98℃3min，30个循环(98℃10s、55℃15s、72℃1min)，72℃5min。以pp43nmk为模板，p43-f和p43-r为引物(见表1)进行全质粒pcr扩增，获得线性化pp43nmk载体。pcr条件为：98℃5min，25个循环(98℃10s、55℃15s、72℃4min30s)，72℃5min。以上扩增产物经电泳检验后，采用胶回收试剂盒对pcr产物进行纯化和回收。通过一步克隆试剂盒clonexpresstmonestepcloningkit(vazymebiotechco.,ltd.nanjing,china)，将甲壳素酶基因片段与线性化载体pp43nmk进行融合，得到重组质粒pp43nmk-chisb。将融合的重组质粒pp43nmk-chisb转化至感受态e.colijm109，用氨苄青霉素lb平板，挑取阳性菌落。37℃摇床过夜培养后提取质粒，转化子由天霖(无锡)有限责任公司进行测序。表1引物实施例2甲壳素酶生产菌株的验证将实施例1中测序正确的质粒pp43nmk-chisb，转化bacillus.subtiliswb600。挑选转化子接种到lb培养基中，37℃，培养8h；转接到tb培养基中，接种量为2％，37℃培养12h，收集发酵上清液，检测发酵上清酶活，结果显示，甲壳素酶被分泌到胞外，酶活力为28.98u/ml(结果如图1所示)。实施例3突变株的获得利用定点突变试剂盒(购自takara，货号：km101)，设计引物(见表2)，以已构建的pp43nmk-chisb为模板，进行pcr，将甲壳素酶分子催化结构域附近的第43位的半胱氨酸突变为天冬氨酸；第273位苯丙氨酸突变为色氨酸；或将第336位谷氨酸突变为精氨酸，分别命名为c43d、f273w、e336r。pcr反应条件为98℃5min，30个循环(98℃10s、55℃15s、72℃4min30s)，72℃5min。采用胶回收试剂盒对pcr产物进行纯化和回收，电泳检验回收产物。产物转化e.colijm109，由天霖(无锡)有限责任公司进行测序，转化子命名为pp43nmk-chisb-c43d、pp43nmk-chisb-f273w、pp43nmk-chisb-e336r。以测序正确的菌株pp43nmk-chisb-c43d的质粒为模板，将甲壳素酶分子第273位苯丙氨酸进行复合突变，突变为色氨酸，命名为c43d/f273w，转化子由天霖(无锡)有限责任公司进行测序，转化子命名为pp43nmk-chisb-c43d/f273w。以测序正确的菌株pp43nmk-chisb-c43d/f273w的质粒为模板，将甲壳素酶分子第336位谷氨酸进行复合突变，突变为精氨酸，命名为c43d/f273w/e336r，转化子由天霖(无锡)有限责任公司进行测序，转化子命名为pp43nmk-chisb-c43d/f273w/e336r。表2定点突变引物序列引物名称引物序列(5'-3')c43d-fgcatttgccgacattgactgggagggacgcc43d-rgtcaatgtcggcaaatgcatagtttatatgagf273w-fccgaacgcagaaacgtggaatattgagagcf273w-rccacgtttctgcgttcggcacccctgcctce336r-fcgatttttctgatttgagaaagaactatatce336r-rtctcaaatcagaaaaatcgaacactccttt实施例4甲壳素酶生产菌株的验证将测序正确的质粒转化bacillus.subtiliswb600，挑选转化子接种到lb液体培养基中，37℃，培养8h，转接到tb培养基中，接种量为2％，37℃，培养12h。收集发酵上清液，检测发酵上请酶活，结果如图1所示。实验结果表明与出发菌株比较，酶活力有所提高，wt、c43d、f273w、e336r、c43d/f273w、c43d/f273w/e336r酶活力分别为28.98u/ml、32.43u/ml、39.74u/ml、32.46u/ml、34.96u/ml、30.42u/ml；其中单突变株f273w和e336r的酶活较出发菌株分别提高了1.37倍和1.22倍；复合突变株c43d/f273w酶活较出发菌株提高了1.14倍。实施例5突变体的纯化按照实施例4中描述的操作方法，收集发酵12h后的上清。利用甲壳素酶基因chisb的c端所带的his标签进行镍柱纯化。纯化所需缓冲液为a液：40mmtris-hcl、150mmnacl(ph7.5)；b液：40mmtris-hcl、150mmnacl、500mm咪唑(ph7.5)。纯化结果如图2所示。研究结果表明，三株突变体菌株均能用ni柱进行纯化，c43d突变株用20％b液可以完全洗脱，穿透液中没有目的蛋白；f273w和e336r突变株用15％b液可以完全洗脱，穿透液中无目的蛋白。实施例6突变体的催化活性检测将纯化后的酶液与终浓度为2g/l的底物胶体甲壳素在60℃下反应3h，100℃加热5min终止反应，14000rpm离心10min获得上清液，用于hplc检测。反应体系如下(1ml)：450μl缓冲液(磷酸氢二钠-柠檬酸缓冲液，ph5.0)，300μl胶体甲壳素，250μl纯酶液。检测结果如图3所示。产量分别为：出发菌株wt：0.09g/lglcnac和0.74g/l(glcnac)2；c43d：0.09g/lglcnac和1.15g/l(glcnac)2；f273w：0.12g/lglcnac和1.53g/l(glcnac)2；e336r：0.17g/lglcnac和1.31g/l(glcnac)2。对比例以已构建的pp43nmk-chisb为模板，采用相同的方法，分别将甲壳素酶分子的第57位天冬酰胺和第308位脯氨酸突变为组氨酸，突变引物见表3。命名为n57h(氨基酸序列如seqidno.7所示，核苷酸序列如seqid15所示)和p308h(氨基酸序列如seqidno.8所示，核苷酸序列如seqid16所示)，转化子由天霖(无锡)有限责任公司进行测序，转化子命名为pp43nmk-chisb-n57h，pp43nmk-chisb-p308h。n57h和p308h突变菌株的酶活相比出发菌株均下降，分别为13.2u/ml和8.7u/ml。表3突变引物引物名称引物序列(5'-3')n57h-fgatcctacagggccacacccacagaaatggn57h-fgtgtggccctgtaggatcaggattcccatgp308h-ftggagtaattgcgaacatgctgataacggtp308h-ratgttcgcaaattactccaacctctaccgt虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。sequencelisting<110>江南大学<120>一种酶活提高的甲壳素酶突变体<160>30<170>patentinversion3.3<210>1<211>563<212>prt<213>bacillussp.dau101<400>1serserasplyssertyrlysileileglytyrtyrprosertrpgly151015alatyrglyargasppheglnvaltrpaspmetaspalaserlysval202530serhisileasntyralaphealaaspilecystrpgluglyarghis354045glyasnproaspprothrglyproasnproglnlystrpsercysgln505560aspgluasnglyvalileaspvalproasnglyserilevalmetgly65707580aspprotrpileaspvalglnlysserasnalaglyaspthrtrpasp859095gluproileargglyasnphelysglnleuleulysleulyslysasn100105110hisprohisleulysthrpheileservalglyglytrpsertrpser115120125asnargpheseraspvalalaalaaspproalaalaarggluasnphe130135140alaalaseralavalasppheleuarglystyrglypheaspglyval145150155160aspleuasptrpglutyrprovalserglyglyleuproglyasnser165170175thrargprogluasplysargasntyrthrleuleuleuglnaspval180185190argglulysleuaspalaalaglualalysaspglylyslystyrleu195200205leuthrilealaserglyalaserproglutyrvalserasnthrglu210215220leuasplysilealagluthrvalasptrpileasnilemetthrtyr225230235240asppheasnglyglytrpglnserileseralahisasnalaproleu245250255phetyraspprolysalalysglualaglyvalproasnalagluthr260265270pheasnilegluserthrvallysargtyrlysglualaglyvallys275280285alaasplysleuvalleuglythrprophetyrglyargglytrpser290295300asncysgluproalaaspasnglyglutyrglnlyscysglyproval305310315320lysgluglythrtrpglulysglyvalpheasppheseraspleuglu325330335lysasntyrileasnlysasnglytyrlysargtyrtrpasnasparg340345350alalysvalpropheleutyrasnalagluasnglyasnpheilethr355360365tyraspaspgluglusertyrglytyrlysthraspleuileglnser370375380asnglyleuserglyalametphetrpasppheserglyaspserasn385390395400glnthrleuleuasnlysleualaalaaspleuglyphealaprogly405410415glyglyasnprogluproproalaseralaproglyasnleuargval420425430thrglulysthralathrserileserleuvaltrpaspalaproser435440445aspglyalaasnilealaglutyrvalleusertyrgluglyglyala450455460valservallysaspthrseralathrileglyglnleulysproasn465470475480thrthrtyrserphethrvalseralalysaspalaaspglylysleu485490495histhrglyprothrileglualathrthrasnseraspglnthrcys500505510glytyrasnglutrplysaspthralavaltyrthrglyglyasparg515520525valvalpheasnglylysvaltyrglualalystrptrpthrlysgly530535540gluglnproaspglnalaglygluserglyvaltrplysleuilegly545550555560aspcyslys<210>2<211>563<212>prt<213>人工合成<400>2serserasplyssertyrlysileileglytyrtyrprosertrpgly151015alatyrglyargasppheglnvaltrpaspmetaspalaserlysval202530serhisileasntyralaphealaaspileasptrpgluglyarghis354045glyasnproaspprothrglyproasnproglnlystrpsercysgln505560aspgluasnglyvalileaspvalproasnglyserilevalmetgly65707580aspprotrpileaspvalglnlysserasnalaglyaspthrtrpasp859095gluproileargglyasnphelysglnleuleulysleulyslysasn100105110hisprohisleulysthrpheileservalglyglytrpsertrpser115120125asnargpheseraspvalalaalaaspproalaalaarggluasnphe130135140alaalaseralavalasppheleuarglystyrglypheaspglyval145150155160aspleuasptrpglutyrprovalserglyglyleuproglyasnser165170175thrargprogluasplysargasntyrthrleuleuleuglnaspval180185190argglulysleuaspalaalaglualalysaspglylyslystyrleu195200205leuthrilealaserglyalaserproglutyrvalserasnthrglu210215220leuasplysilealagluthrvalasptrpileasnilemetthrtyr225230235240asppheasnglyglytrpglnserileseralahisasnalaproleu245250255phetyraspprolysalalysglualaglyvalproasnalagluthr260265270pheasnilegluserthrvallysargtyrlysglualaglyvallys275280285alaasplysleuvalleuglythrprophetyrglyargglytrpser290295300asncysgluproalaaspasnglyglutyrglnlyscysglyproval305310315320lysgluglythrtrpglulysglyvalpheasppheseraspleuglu325330335lysasntyrileasnlysasnglytyrlysargtyrtrpasnasparg340345350alalysvalpropheleutyrasnalagluasnglyasnpheilethr355360365tyraspaspgluglusertyrglytyrlysthraspleuileglnser370375380asnglyleuserglyalametphetrpasppheserglyaspserasn385390395400glnthrleuleuasnlysleualaalaaspleuglyphealaprogly405410415glyglyasnprogluproproalaseralaproglyasnleuargval420425430thrglulysthralathrserileserleuvaltrpaspalaproser435440445aspglyalaasnilealaglutyrvalleusertyrgluglyglyala450455460valservallysaspthrseralathrileglyglnleulysproasn465470475480thrthrtyrserphethrvalseralalysaspalaaspglylysleu485490495histhrglyprothrileglualathrthrasnseraspglnthrcys500505510glytyrasnglutrplysaspthralavaltyrthrglyglyasparg515520525valvalpheasnglylysvaltyrglualalystrptrpthrlysgly530535540gluglnproaspglnalaglygluserglyvaltrplysleuilegly545550555560aspcyslys<210>3<211>563<212>prt<213>人工合成<400>3serserasplyssertyrlysileileglytyrtyrprosertrpgly151015alatyrglyargasppheglnvaltrpaspmetaspalaserlysval202530serhisileasntyralaphealaaspilecystrpgluglyarghis354045glyasnproaspprothrglyproasnproglnlystrpsercysgln505560aspgluasnglyvalileaspvalproasnglyserilevalmetgly65707580aspprotrpileas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