CN102533692A - 一种酸性条件下具有降解脂肪能力的脂肪酶 - Google Patents

一种酸性条件下具有降解脂肪能力的脂肪酶 Download PDF

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CN102533692A
CN102533692A CN2010105773690A CN201010577369A CN102533692A CN 102533692 A CN102533692 A CN 102533692A CN 2010105773690 A CN2010105773690 A CN 2010105773690A CN 201010577369 A CN201010577369 A CN 201010577369A CN 102533692 A CN102533692 A CN 102533692A
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lipase
acidic condition
lypase
under acidic
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刘刚
方井霞
张柏松
苏东风
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Abstract

本发明公开了一种经过改造可以在酸性条件下具有降解油脂能力脂肪酶的核苷酸序列和氨基酸序列，可以克服现有技术中，需要在酸性条件下使用的脂肪酶，酶活力下降快、实用性差的缺陷，以实现脂肪酶在使用过程中耐酸性能高、稳定性好、降低成本、减少工艺与实用性好的优点。

Description

一种酸性条件下具有降解脂肪能力的脂肪酶
技术领域
本发明属于分子生物学、酶学、生理学领域。具体的说，涉及一种经过分子生物学手段改造、可以耐酸性的脂肪酶DNA序列及其对应的氨基酸序列。
背景技术
脂肪酶(lipase，EC3.1.1.3)是一类催化三酰甘油酯分解、合成和酯交换的酶类，具有极强的选择性和专一性，并具有在水相和有机相界面催化反应的独特性能，已被广泛应用于食品、油脂化工等行业。随着应用领域的拓宽，已涉及到单细胞蛋白生产、化妆品生产、环境保护、新型生物材料、生物传感器、生物医学及生物能源等新兴领域，有着广阔的应用前景。
脂肪酶广泛存在于自然界的动、植物和微生物体内。如：细菌有28个属、放线菌4个属、酵母菌10个属、其它真菌23个属均能产生脂肪酶。鉴于微生物脂肪酶具有重要的工业应用价值和潜在的巨大需求，许多工业化实验室与科研单位长期从事着繁琐的高产脂肪酶微生物筛选工作，但因为微生物生长条件的复杂性，使得具有特殊性质的脂肪酶资源开发受到微生物生长的限制。
发明内容
本发明的目的是提供了一种新型耐酸性脂肪酶及其结构基因。该基因表达产物脂肪酶在强酸条件下显示活性，可应用于饲料添加剂和其他酸性条件的工业过程中。
为了达到本发明的目的，实现本发明的具体技术步骤如下：由GenBank数据库获得变形斑沙雷菌S.proteamaculans脂肪酶基因序列附图1，合成此基因序列并与质粒pUC18相连(不仅限于此克隆载体)，以含脂肪酶基因的质粒DNA作为模板，a(5‘-CTT GAA TTC GAT TAGAGT CGT ATA AGA TG-3，EcoR I)和b(5-CTT GGT ACC TTA ATT CGT ATT CTG TCC TC-3KpnI)为引物进行易错PCR扩增。每100ul反应体系为：10ul 10x易错PCR缓冲液(500mmol/L KCl，70mmol/L MgCl2，100mmol/L Tris-HCI pH8.3，0.1％(w/v明胶)；10ul 10X dNTP混合物(2mmol/L dGTP，2mmol/L dATP，10mmol/LdCTP和10mmol/LdTTP)；引物a和b各30pmol；10ul的5mmol/L MnCl2；质粒DNA模板20pmol；Taq DNA聚合酶为5U，再加入灭菌的超纯水至总体积为100ul。PCR程序为：95℃，5min；94℃30S，52℃1min，72℃1min，35个循环；72℃10min。将PCR产物以1％(w/v)的琼脂糖凝胶电泳，用PCR纯化回收试剂盒进行纯化回收。纯化后的易错PCR产物与pMD18-T载体连接(不限于此载体)，转化大肠杆菌DH5a超级感受态细胞(不限于此受体，但受体应于质粒载体对应)，涂布LB(含100ug/mL的Amp)平板，挑选具有插入片段的克隆，组成突变体库。将所有转化子挑取至RB(1000mL LB培养基中加入20mL 0.2％(W/V)罗丹明B和40mL橄榄油乳化液，pH自然)平板，于37℃培养24h后，选取有变色现象的转化子。分别接种到3mL LB培养基(含100u g/mL氨苄青霉素)，37℃剧烈振摇至OD600约0.6，各吸取3ul菌液滴至罗丹明B平板，37℃培养12～24h后可见明显变色现象。选取变色快和变色圈大的克隆进行液体发酵，制备足够的菌液量，低温下经IPTG诱导表达后超声波破碎细胞，离心收集上清液，用pNPP法测定脂肪酶活力。分组、分别调节pH为3，各保持2小时，期间半小时测定一次脂肪酶活力见图2，选择一份酶活力降低不足10％的样品，(此后采用相同方法对此转化子的脂肪酶酶液在pH＝4、pH＝5时进行测定，酶活力稳定)，将其对应转化子DNA测序(其核苷酸序列与氨基酸序列见SEQ NO.1和SEQ NO.2)。测序结果表明，结构基因中共有4个碱基发生了突变，分别是g178t；g372a；c271g；t692c。由此造成的4个氨基酸发生变化(A60S、E124K、R91A、L231P)，形成本发明涉及的一种新的酸性条件下具有降解脂肪能力的脂肪酶。
本发明涉及的脂肪酶是一种新型脂肪酶，其一级结构由于4个氨基酸的突变与已知的脂肪酶性质大不相同，从而造成具有更高的耐受酸性的能力，适合于饲料添加剂和其他相关工业。
附图说明
图1变形斑沙雷菌S.proteamaculans原脂肪酶基因序列；
图2低pH下脂肪酶活力相对变化
具体实施方式
以下实施例仅用于说明本发明而非用于限制本发明的范围。
实例中未注明具体条件的实验方法，通常按照常规条件，如《分子克隆》(1989)中所述条件进行。
在本发明的实施例中使用的E.coli BL21和载体pET28购自Novagen公司
实施例1：
酸性脂肪酶酶液的获得
依照SEQ NO.1合成酸性脂肪酶基因，PCR扩增合成产物与质粒pET28-a经酶切后连接并转化E.coli BL21(不仅限于此质粒与对应受体)。37。C液体发酵培养，经IPTG诱导表达后超声波破碎细胞，离心收集上清液，即为含此酸性脂肪酶酶液。
实施例2：
耐酸性脂肪酶水解油脂
向40％的植物油溶液中(pH3)中，加入适量酶液，以每分钟100转搅拌2小时，色谱法测得油相含有脂肪酸，水相含有甘油，说明脂肪酶可水解油脂产生甘油和脂肪酸。
Figure ISA00000376787000011
Figure ISA00000376787000021

Claims (7)

1.一种耐酸性的脂肪酶，其氨基酸序列由SEQ NO.2所示的氨基酸序列组成。
2.一种编码权利1所述的酸性脂肪酶的基因。
3.根据权利要求2所述的基因，其核苷酸序列由SEQ NO.1所示的核苷酸序列组成。
4.含有权利3所述的基因的重组质粒。
5.含有权利要求4所述的重组菌。
6.利用权利要求1、2或3制备的脂肪酶。
7.通过权利要求1、2、3或5得到的酸性脂肪酶在化工、纺织、食品、饲料或医药工业方面的应用
CN2010105773690A 2010-12-08 2010-12-08 一种酸性条件下具有降解脂肪能力的脂肪酶 Pending CN102533692A (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106135648A (zh) * 2015-04-17 2016-11-23 兰瑛 脂肪酶组合物及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1625563A (zh) * 2002-02-19 2005-06-08 宝洁公司 新的真菌脂肪酶
CN101506359A (zh) * 2006-06-15 2009-08-12 法国米奥里大药厂 用于生产脂肪酶的方法、能产生所述脂肪酶的转化解脂耶氏酵母细胞以及它们的应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1625563A (zh) * 2002-02-19 2005-06-08 宝洁公司 新的真菌脂肪酶
CN101506359A (zh) * 2006-06-15 2009-08-12 法国米奥里大药厂 用于生产脂肪酶的方法、能产生所述脂肪酶的转化解脂耶氏酵母细胞以及它们的应用

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106135648A (zh) * 2015-04-17 2016-11-23 兰瑛 脂肪酶组合物及其应用
CN106135648B (zh) * 2015-04-17 2020-01-14 深圳市汇尚科科技有限公司 脂肪酶组合物及其应用

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Application publication date: 20120704