CN114807099B - 普鲁兰酶突变体、工程菌及其应用 - Google Patents
普鲁兰酶突变体、工程菌及其应用 Download PDFInfo
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- CN114807099B CN114807099B CN202210272672.2A CN202210272672A CN114807099B CN 114807099 B CN114807099 B CN 114807099B CN 202210272672 A CN202210272672 A CN 202210272672A CN 114807099 B CN114807099 B CN 114807099B
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Abstract
本发明公开了一种普鲁兰酶突变体、工程菌及其应用，所述普鲁兰酶突变体是将SEQ ID NO.1所示氨基酸序列的第365位、第401位、第504位或第499位氨基酸进行单突变或多突变获得的。本发明构建的普鲁兰酶突变体的催化活性、热稳定性与耐酸稳定性均得到了提高，其中突变体PulAR‑A365V‑401C‑H499A‑T504V的催化效率较野生型普鲁兰酶PulAR在pH5.0、6.0，60℃条件下分别提高了6.6倍与9.6倍；在60℃，65℃的半衰期分别比野生型普鲁兰酶提高了2.6和3.1倍，且在pH 4.5与pH 5.0的半衰期分别比野生型普鲁兰酶提高了1.6和1.8倍。
Description
(一)技术领域
本发明涉及一种普鲁兰酶，特别涉及一种通过定点突变与组合突变的方法构建普鲁兰酶PulAR突变体，并用于制备热稳定性和耐酸性提高的普鲁兰酶。
(二)背景技术
普鲁兰酶(Pullulanase,EC 3.2.1.41)是一种淀粉脱支酶，能够专一性地断开普鲁兰糖、可溶性淀粉、支链淀粉和一些寡聚糖中的α-1,6糖苷键。普鲁兰酶广泛应用于糖化步骤，与糖化酶或β-淀粉酶协同作用，提高糖化效率，减少糖化酶或β-淀粉酶的使用量，从而提高生产量，降低生产成本的目的。
为了最大化发挥普鲁兰酶和糖化酶或β-淀粉酶之间的协同效应，普鲁兰酶与糖化酶、β-淀粉酶需具有一致的作用温度。工业生产中为了达到糖化酶等的最大催化效率、减少染菌的机率，糖化过程温度和pH一般控制在60℃、pH 4.5-5.0，反应时间达到48小时以上。因此，耐热耐酸的普鲁兰酶亟需开发。然而大部分的从自然界中挖掘到的普鲁兰酶通常不具备在高温、酸性的条件下能够保持高活性和高稳定性。而且目前国内的普鲁兰酶市场被美国杰能科公司垄断，因此仍需对普鲁兰酶加大开发力度，摆脱对进口普鲁兰酶产品的依赖。
得益于生物信息学、基因组学和蛋白质组学等前沿生物技术的快速发展，以及随着人们对普鲁兰酶晶体结构与功能关系的解析、酶催化机制等的深入研究。发明人前期从嗜热菌厌氧芽孢杆菌中克隆并表达了I型普鲁兰酶PulAR，该酶的最适作用条件为55℃、pH6.0，但催化效率较低。本发明通过蛋白质工程技术，构建催化温度、热稳定性、耐酸稳定性与催化效率显著改善的普鲁兰酶PulAR突变体，促进淀粉酶法水解技术工艺发展。
(三)发明内容
本发明目的是提供一种普鲁兰酶PulAR突变体、工程菌及其应用，提升普鲁兰酶PulAR的催化活性、热稳定性与耐酸稳定性，解决野生型普鲁兰酶PulAR在高温、酸性条件下的催化活性低、稳定性差的问题。
本发明采用的技术方案：
本发明提供一种普鲁兰酶PulAR突变体，所述普鲁兰酶PulAR突变体是将SEQ IDNO.1所示氨基酸序列的第365位、第401位、第504位或第499位氨基酸进行单突变或多突变获得的。本发明所述普鲁兰酶PulAR突变体是对普鲁兰酶PulAR基因(SEQ ID NO.2)进行定点突变与组合突变，将获得的突变质粒以热击的方式转入E.coli BL21(DE3)感受态细胞，对获得菌株进行接种、转接、诱导，获得普鲁兰酶PulAR突变体蛋白。
本发明首先将野生型普鲁兰酶PulAR的重组菌E.coli BL21(DE3)/pET32a(+)-PulAR活化并提取质粒，并保存于-20℃。其次通过同源建模，获得待改造的普鲁兰酶的三维结构，选择位于活性中心附近的氨基酸残基，同时根据PulAR与耐酸普鲁兰酶的序列比对，确定了7个关键性氨基酸Q355、A365、T399、V401、Y491、H499与T504。以pET32a-PulAR基因为模板质粒，进行定点突变，获得突变质粒后，并转化，进行优势突变菌的筛选，获得正突变并进行叠加突变，筛选优势突变体。
优选的，所述普鲁兰酶PulAR突变体是将SEQ ID NO.1所示氨基酸序列突变为下列之一：(1)第365位丙氨酸突变为缬氨酸(A365V)；(2)第401位缬氨酸突变为半胱氨酸(V401C)；(3)第504位苏氨酸突变为缬氨酸(T504V)；(4)第499位组氨酸突变为丙氨酸(H499A)；(4)第365位丙氨酸突变为缬氨酸、第401位缬氨酸突变为半胱氨酸(A365V/V401C)；(5)第365位丙氨酸突变为缬氨酸、第401位缬氨酸突变为半胱氨酸、第504位苏氨酸突变为缬氨酸(A365V/V401C/T504V)；(6)第365位丙氨酸突变为缬氨酸、第401位缬氨酸突变为半胱氨酸、第504位苏氨酸突变为缬氨酸、第499位组氨酸突变为丙氨酸(A365V/V401C/T504V/H499A，氨基酸序列SEQ ID NO.3，核苷酸序列SEQ ID NO.4)。
本发明还涉及所述普鲁兰酶PulAR突变体编码基因，含编码基因的重组载体，含所述重组载体的重组基因工程菌。所述重组载体的基础载体可以是pET 32a(+)，所述重组基因工程菌的宿主菌可以是E.coli BL21(DE3)。
优选的，所述重组基因工程菌按如下方法构建：将普鲁兰酶PulAR突变体连入pET32a(+)的EcoR V与Xho I位点，转入E.coli BL21(DE3)感受态细胞，获得所述的重组基因工程菌。
本发明还提供一种所述普鲁兰酶PulAR突变体在制备普鲁兰酶蛋白中的应用，所述的应用方法为：将普鲁兰酶PulAR突变体重组基因工程菌诱导培养后，取湿菌体用缓冲液重悬，超声破碎，破碎液再进行镍柱纯化，获得普鲁兰酶蛋白。
优选的，所述普鲁兰酶PulAR突变体重组基因工程菌诱导培养方法为：将普鲁兰酶PulAR突变体重组基因工程菌接种到含有50μg/mL卡钠霉素(Kan)与100μg/mL氨苄抗生素(Amp)的LB液体培养基中，37℃，220rpm活化培养过夜，获得种子液；将种子液以体积浓度1-5％(优选1％)转接到含50μg/mL Kan与100μg/mL Amp的LB液体培养基中，37℃、220rpm培养2.0-2.5小时，至发酵液OD值达到0.6-0.8后，加入终浓度为0.15mM的IPTG，低温16℃继续诱导培养20小时；发酵液4℃、12，000rpm离心10min，收集湿菌体。
优选的所述普鲁兰酶蛋白按如下方法制备：(1)将湿菌体悬浮于PBS缓冲液(50mM、pH 7.5)，利用超声破碎仪破碎细胞10min，破碎条件：功率为350W，破碎1s，暂停1s；然后4℃、12，000rpm离心30min，收集上清，制得粗酶液；(2)将粗酶液在4℃、8000rpm条件下离心10min，去沉淀，上清液经0.22μm的滤膜过滤后，滤液作为上样液，以0.25mL/min速度上样镍柱(40×12.6mm，Bio-Rad，USA)，上样量为有效柱体积的2％；先用冲洗缓冲液(20mM，pH7.0的磷酸钠缓冲液，含0.3M NaCl，20mM咪唑)洗脱杂蛋白，流速1mL/min，洗脱量为5-10个柱体积，洗脱至基线平衡，使得杂蛋白完全被冲洗掉；再用洗脱缓冲液(Elution buffer，20mM，pH 7.0的磷酸钠缓冲液，含0.3M NaCl，500mM咪唑)洗脱目的蛋白，流速为1mL/min，通过观察检测器紫外吸收值进行监测，当紫外吸收值相对于基线向上扬起时用试管收集洗脱液，当紫外吸收值回到基线时停止收集，将收集到的洗脱液装入透析袋(截留分子量MD 34(3500))，置于pH 7.0、20mM的PBS溶液中4℃透析12h，透析后截留液即为普鲁兰酶蛋白纯酶液。
优选的，所述镍柱纯化前先用超纯水冲洗管路中的杂质与空气，并除去镍柱中的20％无水乙醇；再用5-10倍柱体积的结合缓冲液(Binding buffer，20mM，pH 7.0的磷酸钠缓冲液，含0.3M的NaCl)平衡镍柱，使基线平衡。
本发明还涉及所述普鲁兰酶突变体在淀粉糖化中的应用，例如高麦芽糖浆糖等的生产。
本发明普鲁兰酶突变体基因工程菌的接种、转接、诱导、菌体回收，培养基可为本领域任何可使菌体生长，优选LB培养基：胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，蒸馏水溶解，调节pH 7.0。培养方法和培养条件没有特殊的限制，培养方法和培养条件没有特殊的限制，培养方法和条件可以根据宿主类型和培养方法等因素的不同进行适当的选择。
与现有的技术相比，本发明有益效果主要体现在：本发明构建的普鲁兰酶突变体PulAR-A365V、PulAR-V401C、PulAR-A365V/V401C、PulAR-A365V/V401/T504V、PulAR-A365V/V401C/T504V/H499A在60℃的热稳定性分别较野生型普鲁兰酶PulAR提高了0.23倍、0.25倍、1.06倍、1.75倍、2.65倍；在65℃的热稳定性分别较野生型普鲁兰酶PulAR提高了0.64倍、0.64倍、1.68倍、2.68倍、3.12倍；上述突变体在pH 4.5的稳定性分别较野生型增加了0.30倍、0.31倍、0.74倍、1.06倍、1.57倍；在pH 5.0的稳定性分别较野生型增加了0.39倍、0.38倍、0.77倍、1.23倍、1.84倍。此外，突变体在pH 6.0、60℃的催化效率较野生型提高了0.50倍、0.69倍、2.56倍、3.42倍、6.55倍；在pH 5.0，60℃的催化效率较野生型提高了0.63倍、0.85倍、2.75倍、4.30倍、9.56倍。综上所述，本发明构建的普鲁兰酶PulAR突变体比野生型PulAR具有更好的催化性能和工业属性。
(四)附图说明
图1是普鲁兰酶PulAR野生型及其突变体纯化酶的SDS-PAGE电泳图，泳道M为蛋白Marker，泳道1代表野生型PulAR突变体的纯化蛋白，泳道2代表PulAR-A365V的纯化蛋白，泳道3代表PulAR-V401C的纯化蛋白，泳道4代表PulAR-A365V/V401C的纯化蛋白，泳道5代表PulAR-A365V/V401C/T504V，泳道6代表PulAR-A365V/V401C/T504V/H499A。
图2是普鲁兰酶PulAR野生型及突变体纯化酶的最适催化温度曲线图。
图3是普鲁兰酶PulAR野生型及突变体纯化酶的最适pH曲线图。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此：
实施例1：野生型普鲁兰酶重组菌的构建
根据Genbank中来源于嗜热厌氧芽孢杆菌(Anoxybacillus sp.)AR-29的I型普鲁兰酶的基因(KY273924)片段，设计普鲁兰酶的克隆引物如表1所示，人工合成该片段并插入到载体pET-32a(+)的EcoR V与Xho I酶切位点之间，得到重组质粒pET-32a(+)-PulAR(核苷酸序列如SEQ ID NO.2所示，编码蛋白的氨基酸序列如SEQ ID NO.1所示)，将测序正确的重组质粒pET-32a(+)-PulAR转化到E.coli BL21(DE3)，挑取单菌落进行测序，得到重组菌E.coli BL21(DE3)/pET-32a(+)-PulAR，即为野生型E.coli BL21(DE3)/pET-32a(+)-PulAR。
SEQ ID NO.1
MYEVFSSLILKTNEKMGLFILGGANLLTVHRTFEAYLDTMTVITILIPKSYHSGMVGNFIIEKPNGERCQLQVAKREDLWTSIKYECVIDFAVEIGRRYLIYDDHGAFTDLQIGAVIRTAEFDEQFYYEGNDLGITYTPEATTFKLWAPTATEVKVKLLDEAEGKQEQIPLQRMEKGVWMTTVSGDLEGRYYTFLVCVNLVWREAVDPYAMAVSVNGEYGVVVDLAKTHVPKPTLSPLSSPTDAIIYEVHIRDFTIHGDSGVAHKGLYLGLAELGTSGPNNTTTGLSYLAQLGVTHVELLPFNDFAGVDEKAPLKEYNWGYNPLHYNAPEGSYATNPFDPYARIQELKQAIRALQAQGIRVIMDAVYNHVYIREQSSFEKIVPGYYFRHDLYGMPSNGTGVGNDIASERRMVRKFIVDSVRFWLTEYGVDGFRFDLMGVLDIETMREVEAVVHALDPSALLLGEGWDLPTPLPAEQKATMNNADKLPCIAYFNDRFRDHVKGSTFAIHEKGFALGNMAFREQAMRAIQGNVRIKKEAGMFLNPTQAVNYVESHDNHTFWDKMSVSNADESEEIRQKRQKLATAFVILSQGIPFLHSGQEFYRTKQGIENSYNAPDAINQLDWRQKSLYEKDVRYVAGLIQLRKLHRAFRFSTSAEIEKHLRLVEETPPSVIAYHLQSVQEYGPWSDILVIHHNQEATERLPLPDEEEWHVVCDHTASGTTPLYTVKQEIEVQGISTFVLTKMTDLTKKANHI.
SEQ ID NO.2
gtgtatgaggtcttttcctccttgatcctcaaaacaaatgaaaagatgggtttattcatattgggaggtgccaatttgttaactgttcatcgaacgtttgaagcgtatctggatacgatgacggtgattacaattttaatcccgaagtcgtatcattctggaatggtcgggaattttattatcgaaaagccaaatggagaacgatgtcagcttcaagtggcaaaacgagaagatttatggacaagtattaagtatgaatgtgtaatcgattttgctgtcgagatcgggcggaggtatctcatttatgatgatcacggtgcttttaccgatttgcaaatcggggcagtcattcgcaccgcagaatttgatgaacagttttattacgaagggaatgaccttggtatcacctatactccagaagcaaccacttttaagctttgggctcctacggcgacggaagtgaaggtgaaattgctcgatgaggcggaaggaaagcaggagcaaattccgctgcagcgcatggaaaagggagtatggatgactacagtttccggagaccttgaaggaagatattatacgtttttagtgtgtgtgaaccttgtttggcgtgaggctgttgatccatacgcaatggctgtttcagtcaatggagagtacggggttgtcgtcgatttggcgaaaacacatgtgccgaagccaacgctgtcgccgttatcgtccccgacggatgcgattatttatgaagtgcatattcgcgattttaccattcacggcgatagcggggtggctcacaaagggttgtatttaggccttgctgagctcggaacaagcgggccaaataatacaaccacaggcctttcctatttagcgcagctaggagtaacgcatgtagagttgcttccatttaacgattttgctggagtagatgaaaaagccccgctgaaggaatacaactggggctataacccgctgcattataacgctcctgaagggagctatgccactaatccgtttgatccatatgcgcggattcaggagctgaaacaagcgatccgtgcgttgcaggcacagggcattcgcgtgattatggatgctgtttataatcatgtctatattcgcgagcagtcatcatttgaaaaaattgtccctggttattattttcgacatgatctatatggaatgccatccaatggaacaggagtggggaatgacatagcatcggaacggcggatggtgcgaaaatttattgttgactctgtgcgtttttggctgactgagtatggggtggacggatttcgttttgacttaatgggagttttagatatagagacgatgagagaagtggaggccgtcgtccatgcgctcgatccgtccgcacttttgcttggcgagggatgggatttgccgacccctcttcctgccgagcaaaaagctacgatgaacaacgctgacaagctgccttgtattgcgtattttaatgacagatttcgcgatcatgtcaaaggaagtacctttgctattcatgaaaaggggtttgcattaggaaatatggctttccgtgagcaagcgatgcgagcgatccaaggcaatgtgcgaattaaaaaagaagcagggatgtttctgaacccaacgcaagcggtcaattatgttgaatcacatgataatcacacgttctgggataaaatgagcgtgtccaatgcggatgaaagtgaagagattcgtcagaaacggcaaaagctggccaccgcttttgttattctctcgcaaggcattccgtttttgcatagcggccaagaattttatcggacgaagcaagggatagaaaacagctataatgctccggatgccattaaccagcttgattggaggcaaaaaagcctgtatgagaaggatgtgcggtatgttgcaggattgattcaactgcgcaagcttcatcgggctttccgcttttcaacgtctgccgaaatcgaaaaacatcttcgattggttgaagagactccgccatctgtcattgcttatcatcttcaatccgtgcaagaatatgggccatggagcgatattttggtcattcatcataatcaagaagcaacagagcgcttaccccttccggatgaggaggaatggcatgtcgtgtgcgatcacacagcgagcggaacgactcctttatacaccgtgaaacaagagattgaagtccaggggatcagtacgtttgttcttacgaaaatgactgacttgacgaaaaaagcaaatcacatataa.
实施例2：普鲁兰酶突变体的构建与筛选
1、普鲁兰酶突变体的构建
根据SEQ ID NO.2所示的普鲁兰酶PulAR的基因序列，设计并合成引入突变Q355、A365、T399、V401、Y491、H499与T504的突变引物(表1)，利用实施例1中获得的重组质粒pET-32a(+)-pulAR为模板，进行全质粒PCR扩增，转化至大肠杆菌E.coli BL21(DE3)，进行测序鉴定，获得单突变体E.coli BL21(DE3)-PulAR-Q355H、E.coli BL21(DE3)-PulAR-A365V、E.coli BL21(DE3)-PulAR-T399S、E.coli BL21(DE3)-PulAR-V401T、E.coli BL21(DE3)-PulAR-V401C、E.coli BL21(DE3)-PulAR-Y491V、E.coli BL21(DE3)-PulAR-H499A与E.coliBL21(DE3)-PulAR-T504V。
PCR扩增体系：：1μL正向引物(100μM)，1μL反向引物(100μM)，12.5μL 2×Phanta缓冲液，0.5μL dNTP混合物(各10mM)，1μL质粒模板，0.5μL DNA聚合酶和8.5μL超纯水。
PCR扩增条件：95℃预变性5min，然后30个循环(95℃变性15s，55℃退火15s，72℃延伸6min)，72℃终延伸10min。
表1突变引物设计
注：下划线为突变位点。
2、单突变诱导培养
将步骤1获得的普鲁兰酶单突变体分别转接到10mL含有50μg/mL卡钠霉素(Kan)与100μg/mL氨苄抗生素(Amp)的LB液体培养基中，37℃，220rpm活化培养过夜，获得种子液；分别将1mL种子液转接到装有100mL含50μg/mL Kan与100μg/mL Amp的LB液体培养基的摇瓶中，37℃、220rpm培养2.0-2.5小时，至发酵液OD值达到0.6后加入终浓度为0.15mM的IPTG，低温16℃继续诱导培养20小时；发酵液4℃、12，000rpm离心10min，收集菌体，并悬浮于1mLPBS缓冲液(50mM，pH 7.5)，利用超声破碎仪破碎细胞10min，破碎条件：功率为350W，破碎1s，暂停1s；然后，4℃、12，000rpm离心30min，收集上清，制得突变体粗酶液。
3、酶活测定
普鲁兰酶酶活性的测定采用3,5-二硝基水杨酸方法。普鲁兰酶在一定条件下，催化水解普鲁兰糖生成还原糖。3,5-二硝基水杨酸与还原糖溶液共热后被还原为显棕红色的氨基络合物，在一定范围内其颜色的深浅与还原糖的量成正比，故可以在540nm的波长下进行显色，计算酶活。酶活单位(U)，每分钟催化产生1μmol葡萄糖所需的酶量即为一个酶活力单位。
酶活测定反应体系为0.5mL：50μL质量浓度5％的普鲁兰多糖水溶液(购自Sigma公司)分别溶解在440μL pH 6.0、pH 5.0 100mM NaAc/HAC缓冲液，10μL步骤2制备的突变体粗酶液。55℃下反应10分钟，加入DNS液终止反应，100℃水浴10min，测定OD540光吸收值。以葡萄糖的DNS显色OD540光吸收值绘制标准曲线进行标定。
初筛阶段耐酸性强化的正突变标准是：突变体pH 5.0的酶活/pH 6.0的酶活(ApH5/ApH6比率)比野生型ApH5/ApH6比率(pH 5.0的酶活/pH 6.0的酶活)提高10％。初筛的结果如表2所示，突变体A365V、V401C、T504V、H499A的ApH5/ApH6比野生型PulAR的ApH5/ApH6比率分别提高了0.29、0.55、0.09、0.30；突变体Q355H、T399S、Y491V的ApH5/ApH6较野生型PulAR的ApH5/ApH6降低了0.06、0.08、0.01，而突变体V401T完全失去活性。
表2普鲁兰酶PulAR及其突变体的pH 5.0的活性/pH 6.0的活性的比例
实施例3：组合突变与普鲁兰酶蛋白的纯化
1、组合突变
将实施例2中获得的普鲁兰酶的有益突变A365V、V401C、T504V与H499A进行组合突变研究，以实施例1中突变方法分别构建组合突变体PulAR-A365V/V401C、PulAR-A365V/V401C/T504V、PulAR-A365V/V401C/T504V/H499A，具体如下：
(1)组合突变体PulAR-A365V/V401C构建
以实施例2单突变体E.coli BL21(DE3)-PulAR-A365V的质粒pET-32a-PulAR-A365V为模板，以表2中Q355H(F)、Q355H(R)为引物，采用实施例2方法进行全质粒PCR，筛选获得突变体PulAR-A365V/V401C。
(2)组合突变体PulAR-A365V/V401C/T504V构建
以上述已构建组合突变体E.coli BL21(DE3)-PulAR-A365V/V401C的质粒pET-32a-PulAR-A365V/V401C为模板，以表2中V401C(F)、V401C(R)为引物，采用实施例2方法进行全质粒PCR，筛选获得突变体PulAR-A365V/V401C/T504V。
(3)组合突变体PulAR-A365V/V401C/T504V/H499A构建
以上述已构建组合突变体E.coli BL21(DE3)-PulAR-A365V/V401C/T504V的质粒pET-32a-PulAR-A365V/V401C/T504为模板，以表2中T504V(F)、T504V(R)为引物，采用实施例2方法进行全质粒PCR，筛选获得突变体PulAR-A365V/V401C/T504V/H499A。
2、纯酶
(1)采用实施例2步骤2方法对步骤1制备的各个组合突变体、实施例2制备的突变体PulAR-A365V、PulAR-V401C及实施例1野生型进行诱导培养，收集的湿菌体按50g/L的量用(pH 7.0、100mM)磷酸钾缓冲液重悬，置于冰水混合物中，破碎10min；破碎条件：功率为350W，破碎1s，暂停1s，破碎混合液即为粗酶液；
(2)将粗酶液在4℃、8000rpm条件下离心10min，去沉淀，上清液经0.22μm的滤膜过滤后，滤液作为上样液使用镍柱(40×12.6mm，Bio-Rad，USA)纯化酶蛋白，其步骤为：
先用超纯水冲洗管路中的杂质与空气，并除去镍柱中的20％无水乙醇；
平衡：用5-10倍柱体积的结合缓冲液(Binding buffer，20mM，pH 7.0的磷酸钠缓冲液，含0.3M的NaCl)平衡镍柱，使基线平衡。
样品上样：将先前收集到的滤液上样，设置流速为0.25mL/min，总上样量为10mL。
洗脱杂蛋白：用5-10倍柱体积的冲洗缓冲液(20mM，pH7.0的磷酸钠缓冲液，含0.3MNaCl，20mM咪唑)洗脱杂蛋白，流速1mL/min，直至基线平衡，使得杂蛋白完全被冲洗掉。
洗脱目的蛋白：用洗脱缓冲液(Elution buffer，20mM，pH 7.0的磷酸钠缓冲液，含0.3M NaCl，500mM咪唑)洗脱目的蛋白，流速为1mL/min；通过观察检测器紫外吸收值进行监测，当紫外吸收值相对于基线向上扬起时用试管收集洗脱液，当紫外吸收值回到基线时停止收集。将收集到的洗脱液(即含目的蛋白洗脱液)，置于冰上保存。
透析：将含目的蛋白洗脱液装入透析袋(截留分子量MD 34(3500))，置于pH 7.0、20mM的PBS溶液中4℃透析12h，透析后截留液即为纯酶液，将纯酶液用pH 7.0、20mM的PBS调整至蛋白含量为2.6mg/mL。将各个纯酶液进行琼脂糖凝胶电泳检测，结果见图1所示，结果表明突变体蛋白与野生型的蛋白的表达量一致，并无明显差异。
实施例4：普鲁兰酶及突变体的最适作用条件及稳定性的测定
1、最适温度及热稳定性表征
(1)最适温度
将表3中野生型及突变体按实施例3方法制备的纯酶，在不同温度(40、50、55、60、65、70与80℃)采用实施例2方法测定酶活力，并以最适反应温度下酶活力为100％绘制温度对酶的影响曲线，结果如图2所示，图2可以看出野生型PulAR与突变体PulAR-A365V、PulAR-V401C、PulAR-A365V-V401C、PulAR-A365V-V401C-T504V、PulAR-A365V-V401C-T504V-H499A的最适温度分别为55、55、60、60、60与65℃。表明突变体PulAR-V401C、PulAR-A365V-V401C、PulAR-A365V-V401C-T504V、PulAR-A365V-V401C-T504V-H499A最适温度显著提高，分别比野生型PulAR提高了5、5、5、10℃。
(2)热稳定性
半衰期(t1/2)，是指在特定温度下酶活性降低一半时所需的时间，是表征酶热稳定性的重要参数。
将表3中野生型及突变体按实施例3方法制备的纯酶，分别用pH 6.0的100mM乙酸盐缓冲液将其稀释至蛋白浓度0.1μg/μL，在60、65℃保温一定时间，每隔2h，取样，采用实施例2方法测定残余酶活，结果见表3所示。
由表3可以看出，突变体PulAR-A365V、PulAR-V401C、PulAR-A365V-V401C、PulAR-A365V-V401C-T504V、PulAR-A365V-V401C-T504V-H499A比野生型PulAR在60℃的半衰期分别提高了0.23、0.25、1.06、1.75、2.65倍。而在65℃，突变体PulAR-A365V、PulAR-V401C、PulAR-A365V-V401C、PulAR-A365V-V401C-T504V、PulAR-A365V-V401C-T504V-H499A半衰期分别比野生型提高了1.6、1.6、1.68、2.68、3.12倍。
表3普鲁兰酶PulAR及其突变体在60、65℃的热稳定性测定
kd：表示酶的失活率常数
2、最适pH及pH稳定性表征
(1)最适pH
将表4中野生型及突变体按实施例3方法制备的纯酶，采用实施例2方法在60℃，pH4.5-6.0(4.5、5.0、5.5、6.0)的100mM乙酸盐缓冲液，pH 6.5-8.0(6.5、7.0、7.5、8.0)的100mM磷酸盐缓冲液中测定反应的最适pH。结果见图3所示，野生型PulAR及突变体的最适pH并未发生明显改变。
(2)pH稳定性
将表4中野生型及突变体按实施例3方法制备的纯酶，用pH 4.5或者pH 5.0的100mM乙酸盐缓冲液将其稀释至蛋白浓度0.1μg/μL，25℃孵育一定时间，每隔1h，取样，采用实施例2方法检测残余酶活，以未孵育的样品酶活为100％，结果如表4所示，表4可以看出突变体在pH 4.5的半衰期比野生型PulAR提高了0.30倍、0.31倍、0.74倍、1.06倍、1.57倍。而在pH 5.0的半衰期比野生型PulAR提高了0.39倍、0.38倍、0.77倍、1.23倍、1.84倍。
表4普鲁兰酶PulAR及其突变体在pH 4.5、5.0的稳定性测定
kd：表示酶的失活率常数
实施例5：普鲁兰酶及突变体的动力学参数测定
分别用pH 6.0、5.0的100mM乙酸盐缓冲液配制成一系列浓度普鲁兰糖溶液(1.0、1.25、1.33、2.0、2.5、3.33与5.0mg/mL)。在500μL普鲁兰糖溶液的加入以蛋白含量计1μg纯酶(野生型及突变体按实施例3方法制备获得纯酶)，在60℃，pH 6.0与60℃，pH 5.0条件下反应10min，加入DNS液终止反应，100℃水浴10min，测定OD540光吸收值，根据实施例2酶活测定部分计算产物麦芽三糖的量。使用origin 9.1的米氏方程进行拟合，计算得出普鲁兰酶及其突变体对底物的米氏常数Km，周转数kcat以及催化效率kcat/Km。结果如表5所示，可以看出在60℃，pH 6.0的条件下普鲁兰酶突变体PulAR-A365V、PulAR-V401C、PulAR-A365V-V401C、PulAR-A365V-V401C-T504、PulAR-A365V-V401C-T504V-H499A的催化效率比野生型普鲁兰酶PulAR分别提高了0.50、0.69、2.56、3.42、6.55倍，而在60℃，pH 5.0的条件下PulAR-A365V、PulAR-V401C、PulAR-A365V-V401C、PulAR-A365V-V401C-T504、PulAR-A365V-V401C-T504V-H499A的催化效率分别比野生型PulAR分别提高了0.63，0.85，2.75，4.30，9.56倍(表6)。
表5普鲁兰酶PulAR及其突变体动力学参数的测定(60℃，pH 6.0)
表6普鲁兰酶PulAR及其突变体动力学参数的测定(60℃，pH 5.0)
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序列表
<110> 浙江工业大学
<120> 普鲁兰酶突变体、工程菌及其应用
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ccgacggatg cgattattta tgaagtgcat attcgcgatt ttaccattca cggcgatagc 780
ggggtggctc acaaagggtt gtatttaggc cttgctgagc tcggaacaag cgggccaaat 840
aatacaacca caggcctttc ctatttagcg cagctaggag taacgcatgt agagttgctt 900
ccatttaacg attttgctgg agtagatgaa aaagccccgc tgaaggaata caactggggc 960
tataacccgc tgcattataa cgctcctgaa gggagctatg ccactaatcc gtttgatcca 1020
tatgcgcgga ttcaggagct gaaacaagcg atccgtgcgt tgcaggcaca gggcattcgc 1080
gtgattatgg atgtggttta taatcatgtc tatattcgcg agcagtcatc atttgaaaaa 1140
attgtccctg gttattattt tcgacatgat ctatatggaa tgccatccaa tggaacagga 1200
tgcgggaatg acatagcatc ggaacggcgg atggtgcgaa aatttattgt tgactctgtg 1260
cgtttttggc tgactgagta tggggtggac ggatttcgtt ttgacttaat gggagtttta 1320
gatatagaga cgatgagaga agtggaggcc gtcgtccatg cgctcgatcc gtccgcactt 1380
ttgcttggcg agggatggga tttgccgacc cctcttcctg ccgagcaaaa agctacgatg 1440
aacaacgctg acaagctgcc ttgtattgcg tattttaatg acagatttcg cgatgcagtc 1500
aaaggaagtg tttttgctat tcatgaaaag gggtttgcat taggaaatat ggctttccgt 1560
gagcaagcga tgcgagcgat ccaaggcaat gtgcgaatta aaaaagaagc agggatgttt 1620
ctgaacccaa cgcaagcggt caattatgtt gaatcacatg ataatcacac gttctgggat 1680
aaaatgagcg tgtccaatgc ggatgaaagt gaagagattc gtcagaaacg gcaaaagctg 1740
gccaccgctt ttgttattct ctcgcaaggc attccgtttt tgcatagcgg ccaagaattt 1800
tatcggacga agcaagggat agaaaacagc tataatgctc cggatgccat taaccagctt 1860
gattggaggc aaaaaagcct gtatgagaag gatgtgcggt atgttgcagg attgattcaa 1920
ctgcgcaagc ttcatcgggc tttccgcttt tcaacgtctg ccgaaatcga aaaacatctt 1980
cgattggttg aagagactcc gccatctgtc attgcttatc atcttcaatc cgtgcaagaa 2040
tatgggccat ggagcgatat tttggtcatt catcataatc aagaagcaac agagcgctta 2100
ccccttccgg atgaggagga atggcatgtc gtgtgcgatc acacagcgag cggaacgact 2160
cctttataca ccgtgaaaca agagattgaa gtccagggga tcagtacgtt tgttcttacg 2220
aaaatgactg acttgacgaa aaaagcaaat cacatataa 2259
Claims (9)
1.一种普鲁兰酶突变体，其特征在于，所述普鲁兰酶突变体是将SEQ ID NO.1 所示氨基酸序列突变为下列之一：（1）第365位丙氨酸突变为缬氨酸；（2）第401位缬氨酸突变为半胱氨酸；（3）第504位苏氨酸突变为缬氨酸；（4）第499位组氨酸突变为丙氨酸；（4）第365位丙氨酸突变为缬氨酸、第401位缬氨酸突变为半胱氨酸；（5）第365位丙氨酸突变为缬氨酸、第401位缬氨酸突变为半胱氨酸、第504位苏氨酸突变为缬氨酸；（6）第365位丙氨酸突变为缬氨酸、第401位缬氨酸突变为半胱氨酸、第504位苏氨酸突变为缬氨酸、第499位组氨酸突变为丙氨酸。
2.一种含权利要求1所述普鲁兰酶突变体的编码基因的重组载体。
3.一种含权利要求2所述重组载体的重组基因工程菌。
4.如权利要求3所述的重组基因工程菌，其特征在于，所述重组基因工程菌按如下方法构建：将普鲁兰酶突变体连入pET 32a(+)的EcoR V 与 Xho I位点，转入E. coli BL21(DE3) 感受态细胞，获得所述的重组基因工程菌。
5.一种权利要求1所述普鲁兰酶突变体在制备普鲁兰酶中的应用。
6.如权利要求5所述的应用，其特征在于，所述的应用为：将普鲁兰酶突变体重组基因工程菌诱导培养后，取噬菌体用缓冲液重悬，超声破碎，破碎液再进行镍柱纯化，获得普鲁兰酶蛋白。
7.如权利要求6所述的应用，其特征在于，所述普鲁兰酶突变体重组基因工程菌诱导培养方法为：将普鲁兰酶突变体重组基因工程菌接种到含有50 μg/mL卡钠霉素与100 μg/mL氨苄抗生素的LB液体培养基中，37 ℃，220 rpm活化培养过夜，获得种子液；将种子液以体积浓度1-5%转接到含50 μg/mL卡钠霉素与100 μg/mL 氨苄抗生素的LB液体培养基中，37℃、220 rpm培养至发酵液OD值达到0.6-0.8后，加入终浓度为0.15 mM的IPTG，低温16 ℃继续诱导培养20小时；发酵液4 ℃、12，000 rpm离心10 min，收集噬菌体。
8.如权利要求6所述的应用，其特征在于，所述普鲁兰酶蛋白按如下方法制备：（1）将噬菌体悬浮于50 mM、pH 7.5的PBS缓冲液，利用超声破碎仪破碎细胞10min，破碎条件：功率为350 W，破碎1 s，暂停1 s；然后4 ℃、12，000 rpm离心30 min，收集上清，制得粗酶液；（2）将粗酶液在4 oC、8000 rpm 条件下离心10 min，去沉淀，上清液经0.22 μm的滤膜过滤后，滤液作为上样液，以0.25 mL/min速度上样镍柱，上样量为有效柱体积的2 %；先用冲洗缓冲液洗脱杂蛋白，流速 1 mL/min，洗脱量为5-10个柱体积，洗脱至基线平衡，使得杂蛋白完全被冲洗掉；再用洗脱缓冲液洗脱目的蛋白，流速为1 mL/min，通过观察检测器紫外吸收值进行监测，当紫外吸收值相对于基线向上扬起时用试管收集洗脱液，当紫外吸收值回到基线时停止收集，将收集到的洗脱液装入透析袋，置于pH 7.0、20 mM的PBS溶液中4 oC透析12 h，透析后截留液即为普鲁兰酶蛋白纯酶液；所述冲洗缓冲液为含0.3 M NaCl和20 mM咪唑的20 mM，pH7.0的磷酸钠缓冲液；所述洗脱缓冲液为含0.3 M NaCl和500 mM咪唑的20 mM，pH7.0的磷酸钠缓冲液。
9.一种权利要求1所述普鲁兰酶突变体在淀粉糖化中的应用。
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