CN111206025B - 一种比活提高的溶菌酶突变体 - Google Patents
一种比活提高的溶菌酶突变体 Download PDFInfo
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Abstract
本发明公开了一种比活提高的溶菌酶突变体，属于生物工程技术领域。本发明公开了一种利用基因工程在人源溶菌酶C末端添加疏水短肽Val‑Ile‑Pro‑Leu‑Phe而得到的人源溶菌酶突变体，其比活提高了126％。本发明还公开了一种含突变人源溶菌酶基因的重组质粒，以及经过该重组质粒转化毕赤酵母后获得的高效表达人源溶菌酶突变体的重组毕赤酵母基因工程菌。本发明获得的人源溶菌酶突变体具有比活高，制备简单的特点，具有潜在的临床应用价值，在饲料、食品工业上也具有广泛的用途。
Description
技术领域
本发明涉及一种比活提高的溶菌酶突变体，属于生物工程技术领域。
背景技术
溶菌酶又称胞壁质酶或N-乙酰胞壁质聚糖水解酶，能够水解细菌细胞壁中β-1，4糖苷键，破坏细胞壁肽聚糖的结构，从而保护宿主细胞免受细菌感染。人源溶菌酶属于c型溶菌酶，由130个氨基酸组成，相对分子量为14700。人源溶菌酶还具有抗病毒、增强免疫力和抗肿瘤的功效。溶菌酶作为一种天然蛋白质，能在胃肠内作为营养物质被消化和吸收，对人及动物无毒害作用，也不会在体内残留，是一种安全性很高的药品、饲料和食品添加剂。在畜牧业溶菌酶可用作饲料防腐剂和杀菌剂。在食品行业，溶菌酶可作为抗菌防腐剂添加到食品中，且对人体无任何毒副作用，也可利用其具有一定甜味的特点，作为低卡路里的食品甜味剂。
目前，市场上销售的溶菌酶主要是从鸡蛋清、动物脏器等中提取获得的，这种溶菌酶热稳定性差，活力只有人源溶菌酶活力的一半。但人源溶菌酶受限于原料来源、纯化精制成本等因素，制备量较少，无法满足各领域的需求。因此，提供一种低成本的高产溶菌酶的方法，具有广阔的应用前景。
发明内容
为了解决上述问题，本发明通过基因工程技术在人源溶菌酶的C末端添加疏水短肽Val-Leu-Phe、Val-Ile-Pro-Leu-Phe或Val-Ile-Pro-Leu-Phe，显著提高人源溶菌酶的比活，有效提升人源溶菌酶的杀菌效果。
本发明的第一个目的是提供一种比活提高的人源溶菌酶，所述人源溶菌酶的氨基酸序列如SEQ ID NO:4或SEQ ID NO:6或SEQ ID NO:8所示。
本发明的第二个目的是提供一种编码上述人源溶菌酶的基因，所述基因的核苷酸序列如SEQ ID NO:3或SEQ ID NO:5或SEQ ID NO:7所示。
本发明的第三个目的是提供一种含有上述基因的载体。
在本发明的一种实施方式中，所述载体是pPIC9K。
本发明的第四个目的是提供一种表达上述人源溶菌酶的基因工程菌。
本发明的第五个目的是提供一种提高人源溶菌酶比活的方法，是在氨基酸序列如SEQ ID NO:2所示的人源溶菌酶的C末端添加疏水短肽Val-Leu-Phe、Val-Ile-Pro-Leu-Phe或Val-Ile-Pro-Ala-Ala-Ile-Pro。
在本发明的一种实施方式中，所述基因工程菌以毕赤酵母细胞为宿主。
在本发明的一种实施方式中，所述基因工程菌以毕赤酵母KM71或者GS115为宿主。
本发明还提供了上述人源溶菌酶在制备饲料添加剂、食品防腐方面的应用。
本发明还提供了上述人源溶菌酶在食品或饲料领域的应用。
与现有的技术相比，本发明的优点和积极效果是：
本发明利用毕赤酵母表达系统来作为生产人源溶菌酶的表达系统，具有生产成本低、操作简单、生长迅速、表达效率高、发酵与分泌性能良好等优点，本发明优化后的编码人源溶菌酶m5HLM(氨基酸序列如SEQ ID NO:6所示，编码其的核苷酸序列如SEQ ID NO:5所示)的基因经毕赤酵母表达系统产生的人源溶菌酶比活为21043U/g，较野生型提高了约126％。
本发明通过基因工程技术在人源溶菌酶的C末端添加疏水短肽Val-Leu-Phe、Val-Ile-Pro-Leu-Phe或Val-Ile-Pro-Leu-Phe，分别获得人源溶菌酶m3HLM、m5HLM、m7HLM，其比活相对于野生型分别提高了40.32％、126.15％、79.80％，有效提升了人源溶菌酶的杀菌效果，对将其用于生产具有高活力抑菌功能的饲料添加剂具有积极作用。本发明所述的基因工程改造的人源溶菌酶不仅对致病菌具有较强的杀灭和抑制作用，同时还具有广谱抗菌的优点，由于抑菌的高效性和对人体的安全性高，也适合于各种食品的防腐。
本发明采用毕赤酵母表达系统可以获得高表达量、高活性的人源溶菌酶，不受原材料来源的限制，克服了以其它方式获取溶菌酶的成本高、表达量低和比活低的缺点。高比活、低成本溶菌酶的生产与推广应用，不仅可对饲料畜牧业的发展产生可观的经济效益和社会效益，同时也可产生巨大的生态效益。
附图说明
图1：pPIC9K-m5HLM构建图谱。
图2：本发明所述的人源溶菌酶SDS-PAGE电泳考马斯亮蓝染色结果示意图。
具体实施方式
人源溶菌酶及突变体酶活测定方法为：利用管蝶法鉴定人源溶菌酶的生物活性，并通过比浊法定量测定人源溶菌酶酶活力大小。
管碟法是效仿抗物素的生物学鉴定方法，指示菌为1.5％溶壁微球菌(OD600＝1)，双蝶制备好后静置10min，在牛津杯中加入样品及阴性对照，24h后观察透明圈的大小。
比浊法以溶壁微球菌作为底物，通过菌悬液OD450处吸光度值的变化进行酶活力测定。溶壁微球菌活化及培养步骤参照国标GB/T 30990-2014。称取一定量的溶菌酶标品(100000U/mg)，用缓冲液稀释成一定的浓度梯度(50-250U/mL)，取0.5mL的酶液加入2.5mL的菌悬液混匀，记录在450nm处反应1min时的读数A1，反应2min时的读数A2，计算△E＝|A1－A2|的值，以酶活力为纵坐标，△E为横坐标作酶活力标准曲线。同样测定发酵上清液的△E(1min内的变化范围在0.025-0.125)，根据酶活力标准曲线计算出发酵上清液的酶活力。
发酵液上清的蛋白质含量使用Bradford方法进行测定，用牛血清蛋白作为标样制作标准曲线。将发酵上清稀释到一定倍数，在已加入5mL考马斯亮蓝G-250溶液的试管中加入100μL稀释的发酵上清液，振荡混匀，室温静置5-10min，于波长在595nm的分光光度计测定吸光值，依据制作的标准曲线算出蛋白浓度，单位mg·mL-1。
比活计算为上清液酶活力除以蛋白质含量。
下面通过具体实施例对本发明所述的耐热人源溶菌酶的基因工程改造过程进行具体说明。
实施例1构建融合五肽的溶菌酶表达载体pPIC9K-m5HLM
溶菌酶在自然界中大量存在且具有较显著的杀菌效果，为了进一步拓展溶菌酶的抗菌谱和抑菌功能，有必要对溶菌酶进行分子改造以开发出更加适应现代生产需求的新型溶菌酶。对鸡蛋清溶菌酶的研究显示，通过重组技术将疏水短肽连接于蛋清溶菌酶C末端，发现改造的溶菌酶对大肠杆菌杀菌活性显著增强。人源溶菌酶和蛋清溶菌酶都属于c型溶菌酶，因此也对人源溶菌酶C端进行疏水性改造以提高其抗菌效果。选用三种不同长度的疏水短肽进行C末端融合，分别是三肽Val-Leu-Phe，五肽Val-Ile-Pro-Leu-Phe和七肽Val-Ile-Pro-Ala-Ala-Ile-Pro。
野生型人源溶菌酶基因HLM根据Pichia pastoris密码子偏好性优化后再通过人工合成获得，具体核苷酸序列见SEQ ID NO:1，并整合在pUC57 Simple质粒上，得到pUC57Simple-HLM。设计引物F1：ggcGGATCCAAGGTTTTCGAAAGATGTGAACT和引物R1：ggcGCGGCCGCTTAgaacaaagggataccCACACCACATCCTTGAACATA，以pUC57 Simple-HLM为模板，扩增获得731bp基因条带(条带1)。用BamHI和NotI双酶切pPIC9K原始质粒，胶回收9000bp左右条带(条带2)；用BamHI和NotI双酶切条带1，胶回收该条带后，将条带1和条带2用T4连接酶连接起来，连接产物转化大肠杆菌JM109感受态，涂LB抗性平板(LB固体培养基：蛋白胨10g·L-1，酵母膏5g·L-1，氯化钠10g·L-1，氨苄50μg·mL-1，琼脂20g·L-1)于37℃培养12h左右。挑取平板上2个单菌落接种至100mL液体LB培养基中(蛋白胨10g·L-1，酵母膏5g·L-1，氯化钠10g·L-1，氨苄50μg·mL-1)，于37℃摇床220转/分钟培养15h后提取重组质粒。重组质粒用XbaI和SacI酶切，分别得到2253bp和7478bp的基因片段的，即为正确重组质粒pPIC9K-m5HLM(质粒图谱如图1所示)，并用引物ACAGAAGGAAGCTGCCCTG测序验证。添加五肽后的溶菌酶核苷酸序列如SEQ ID NO:5所示，氨基酸序列如SEQ ID NO:6所示。
实施例2构建表达m5HLM溶菌酶的重组毕赤酵母基因工程菌KM71-pPIC9K-m5HLM
将质粒pPIC9K-m5HLM用SacI酶切线性化后电转化毕赤酵母KM71，涂布YPD抗性平板(蛋白胨20g·L-1，酵母膏10g·L-1，葡萄糖20g·L-1，20g·L-1琼脂粉，G418 1000μg·mL-1)于30℃培养48h，从平板上挑取单菌落，得到重组菌株KM71-pPIC9K-m5HLM。将单菌落接种于含25mL无抗生素的YPD培养基的250mL摇瓶中，于30℃、200转/分钟培养48小时，离心收集菌体后加入到25mL YP培养基(蛋白胨20g·L-1，酵母膏10g·L-1)，28℃、200转/分钟培养72小时，每12h补加甲醇终浓度为1％(v/v)，诱导结束后离心获取的发酵上清液即为溶菌酶突变体m5HLM酶液。酶液的SDS-PAGE电泳如图2所示。
实施例3重组菌株KM71-pPIC9K-m5HLM的发酵培养
将重组菌株KM71-pPIC9K-m5HLM分别进行发酵罐扩大培养。将甘油管中的菌液吸取100μL接种100mL的YPD培养基中培养18h左右作为种子培养液，然后将种子培养液接种于3L发酵罐中的1L基础盐发酵液培养基(K2SO4 18.2g·L-1，MgSO4·7H2O 14.9g·L-1，CaSO4·2H2O 0.93g·L-1，KOH 4.13g·L-1，85％H3PO4 26.7mL·L-1，甘油30g·L-1，PTM1 4.35mL·L-1)中进行发酵培养。
3L罐培养分为2个阶段：第一阶段是甘油相培养，此时流加50％甘油为碳源，温度30℃，pH 5.5，溶氧20％以上，培养到OD600大约等于100时开始进入第二阶段甲醇诱导相；第二阶段的甲醇浓度用甲醇流加仪控制在1％左右，温度28℃，pH 5.0，溶氧20％以上培养120h后放罐，发酵液离心去菌体后的发酵上清液即为酶液。
测定上罐发酵得到突变体酶m5HLM酶液的比活，m5HLM的比活为9305U/g，突变体酶m5HLM的比活最高为21043U/g，较野生型提高了约126％。
实施例4融合三肽和七肽的溶菌酶的比活测定
以原始溶菌酶(氨基酸序列如SEQ ID NO:2所示，编码其的核苷酸序列如SEQ IDNO:1所示)为基础，构建表达添加三肽的溶菌酶(氨基酸序列如SEQ ID NO:4所示，编码其的核苷酸序列如SEQ ID NO:3所示)和添加七肽的溶菌酶(氨基酸序列如SEQ ID NO:8所示，编码其的核苷酸序列如SEQ ID NO:7所示)，改造步骤同实施例。
得到的原始溶菌酶、添加三肽溶菌酶和添加七肽溶菌酶的比活为9305U/g，13057U/g和16730U/g。
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。
SEQUENCE LISTING
<110> 江南大学
<120> 一种比活提高的溶菌酶突变体
<160> 11
<170> PatentIn version 3.3
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<211> 390
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agagcaacaa actacaatgc tggtgataga tctactgatt atggtatctt ccaaatcaat 180
tcaaggtatt ggtgtaatga tggaaagact ccaggtgctg tcaacgcttg ccatttgtct 240
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Asp Arg Ser Thr Asp Tyr Gly Ile Phe Gln Ile Asn Ser Arg Tyr Trp
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Cys Asn Asp Gly Lys Thr Pro Gly Ala Val Asn Ala Cys His Leu Ser
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agagcaacaa actacaatgc tggtgataga tctactgatt atggtatctt ccaaatcaat 180
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Cys Asn Asp Gly Lys Thr Pro Gly Ala Val Asn Ala Cys His Leu Ser
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Lys Val Phe Glu Arg Cys Glu Leu Ala Arg Thr Leu Lys Arg Leu Gly
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Claims (10)
1.一种比活提高的人源溶菌酶，其特征在于，所述人源溶菌酶的氨基酸序列如SEQ IDNO:4或SEQ ID NO:6或SEQ ID NO:8所示。
2.一种编码权利要求1所述人源溶菌酶的基因，其特征在于，所述基因的核苷酸序列如SEQ ID NO:3或SEQ ID NO:5或SEQ ID NO:7所示。
3.一种含有权利要求2所述人源溶菌酶基因的载体。
4.如权利要求3所述的载体，其特征在于，所述载体是pPIC9K。
5.一种表达权利要求1所述人源溶菌酶的基因工程菌。
6.如权利要求5所述的基因工程菌，其特征在于，所述基因工程菌以毕赤酵母(Pichiapastoris)KM71或者GS115为宿主。
7.一种提高人源溶菌酶比活的方法，其特征在于，在氨基酸序列如SEQ ID NO:2所示的人源溶菌酶的C末端添加疏水短肽Val-Leu-Phe、Val-Ile-Pro-Leu-Phe或Val-Ile-Pro-Ala-Ala-Ile-Pro。
8.权利要求1所述的人源溶菌酶在制备饲料添加剂中的应用。
9.权利要求1所述的人源溶菌酶在食品防腐方面的应用。
10.权利要求1所述的人源溶菌酶在食品或饲料领域的应用。
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| CN113637598A (zh) * | 2021-03-18 | 2021-11-12 | 上海复华兴生物技术有限公司 | 一种整合高拷贝人源溶菌酶基因的重组毕赤酵母工程菌及构建方法 |
| CN113185614A (zh) * | 2021-04-22 | 2021-07-30 | 江南大学 | 一种抗蛋白酶降解的猪源抗菌肽突变体及其制备方法与应用 |
| CN113425836A (zh) * | 2021-06-08 | 2021-09-24 | 广州奇龙生物科技有限公司 | 重组人溶菌酶的新应用和祛痰药物 |
| CN113801864B (zh) * | 2021-08-13 | 2023-09-26 | 青岛农业大学 | 一种用于编码溶酶菌lysin6的基因及其应用 |
| CN114149986B (zh) * | 2022-02-08 | 2022-05-06 | 中国科学院天津工业生物技术研究所 | 一种地衣芽孢杆菌溶菌酶突变体及其在虹鳟鱼保鲜中的应用 |
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