CN111778097A - 一种用固定化的新型磷脂酶b于油脂脱胶的方法 - Google Patents
一种用固定化的新型磷脂酶b于油脂脱胶的方法 Download PDFInfo
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Abstract
本发明涉及一种用固定化的新型磷脂酶B于油脂脱胶的方法,特征在于先以介孔二氧化钛颗粒为载体,固定游离的磷脂酶B突变体,得到具有高活性和稳定性的固定化磷脂酶B突变体;然后对欲脱胶的植物油酸处理调pH到5.0‑6.0,再加入固定化磷脂酶B突变体和去离子水,在45℃下脱胶1‑4h,将脱胶的植物油升温至100℃,对酶灭活10min后,进行胶质分离,得到脱胶油。方法科学且合理,工艺简单且脱脂效率高,条件温和、无污染且油质量好,固定化磷脂酶B突变体的酶活较野生型磷脂酶B提高了30%,固定化磷脂酶B突变体脱胶后植物油的磷含量降至2mg/kg,且固定化磷脂酶B可以回收和重复使用,生产成本较低,易于实现连续化反应和自动化生产,有很强实用性。
Description
技术领域
本发明涉及酶的基因应用工程技术领域,特别是一种用固定化的新型磷脂酶B于油脂脱胶的方法。
背景技术
脱胶是油脂精炼过程中的一个重要步骤,在传统的油脂脱胶工艺中,较多用的是水化脱胶法,由于其经济成本较高、物料能耗大和环境污染严重等原因而日趋淘汰。近些年来,业界致力于将酶法脱胶用于油脂精炼的脱胶环节中,有显著成效。酶法脱胶相较于传统的水化脱胶,具有条件温和、无污染、油质量高等优势,证明了该项技术在提高油脂工业的经济和环保效益方面均具有巨大的应用价值和潜力。但是由于来源于动物、植物、微生物的游离的磷脂酶B 资源有限、价格昂贵,而且在应用游离的磷脂酶B 进行反应时,只能应用一次、不能回收,故导致产物成本较高,从而限制了其在工业化生产中的应用。继后问世了一种“固定化酶”,它是指用物理或化学方法将酶绑定在一定空间范围内(俗称“固定化”)仍具有催化活性作用的酶制剂。事实证明,酶通过固定化后还可以提高活力和稳定性,更易于实现连续化反应和自动化生产,可重复利用,有利于提高利用率和降低生产成本,有一定的市场应用前景;同时,选择合适的固定化方法和选择合适的固定载体对酶的固定化过程及固定化酶的性能均影响较大和相当重要,例如固定化载体的几何结构(如比表面积、孔径、孔容等)、表面性质(亲疏水性)、表面活性(官能团种类和密度)、理化性质(物理、化学稳定性)对酶活性及稳定性均有直接影响。随着材料科学领域理论与技术的发展,人们掌握了介孔二氧化钛(TiO2)材料良好的生物相容性,并应用到了生物传感、生物催化以及药物传输等领域的实践中,二氧化钛(TiO2)的等电点在6.0-6.4左右,与细胞生理环境较为接近,因而是一种酶固定化的优良载体,但在实践中如何充分利用介孔二氧化钛(TiO2)良好的生物相容性于油脂脱胶中,目前还在探索研究阶段,是业界正在致力攻克的一大难题。
发明内容
本发明的目的是要充分利用介孔二氧化钛(TiO2)良好的生物相容性、以它为载体制备一种高活性及稳定性的固定化磷脂酶B突变体,并使之应用于油脂脱胶的方法。
本发明的一种用固定化的新型磷脂酶B于油脂脱胶的方法,特征在于以介孔二氧化钛(TiO2)颗粒为载体固定游离的磷脂酶B突变体,制备得到固定化的新型磷脂酶B突变体,然后将所述的固定化的新型磷脂酶B突变体应用到油脂脱胶中,方法步骤如下:
步骤(1) 制备介孔二氧化钛(TiO2)颗粒
以水合二氧化钛TiO2·nH2O和碳酸钾为原料,控制水合温度为80℃、处理12h,得到水合中间物K2Ti2O5晶须,将所述的水合中间物K2Ti2O5置于水中超声分散,静置后,加入0.5M的盐酸进行搅拌,直到全部K+全部交换出来,抽滤、水洗至中性,干燥后得到介孔TiO2的前驱体水合H2Ti2O5,然后将得到的介孔TiO2的前驱体水合H2Ti2O5置于马弗炉中在500℃下煅烧2h,制备得到锐钛矿型介孔二氧化钛(TiO2)颗粒;
步骤(2)以所述的介孔二氧化钛(TiO2)颗粒为载体、固定游离的磷脂酶B突变体,制备固定化的新型磷脂酶B突变体,操作流程如下:
(2a)取10 mL游离的磷脂酶B突变体酶液,在酶液中加入0.1g介孔二氧化钛颗粒,在摇床中25℃、150rpm吸附固定16h,得到固定化酶液;
(2b)将得到的固定化酶液高速离心,再用缓冲液冲洗2-3次,过滤后得到由介孔二氧化钛固定化的新型磷脂酶B突变体;
步骤(3) 油脂脱胶
先对欲脱胶的植物油进行调pH到5.0-6.0的酸处理,再加入步骤(1)得到的固定化的新型磷脂酶B突变体和去离子水,在45℃下脱胶1-4h,然后将脱胶的植物油升温至100℃,对酶灭活10min后进行胶质分离,得到脱胶油。
基于上述构思的本发明用固定化的新型磷脂酶B于油脂脱胶的方法,由于利用二氧化钛(TiO2)的等电点在6.0-6.4左右,与细胞生理环境较为接近,因而是一种酶固定化的优良载体的机理,在合理地制备得到介孔二氧化钛(TiO2)颗粒的基础上,将它作为载体,用以固定游离的磷脂酶B突变体,制备得到活性和稳定性优良的固定化的新型磷脂酶B突变体,并使之参与到油脂脱胶工序中,促进植物油的高效、高质量脱胶。本发明的方法科学且合理,工艺简单且脱脂效率高,条件温和、无污染且油质量好,固定化磷脂酶B突变体的酶活较野生型磷脂酶B提高了30%,固定化磷脂酶B突变体脱胶后植物油的磷含量降至2mg/kg。固定化磷脂酶B可以回收和重复使用,生产成本较低,易于实现连续化反应和自动化生产,有很强实用性。
附图说明
图1是本发明实施例中固定化酶与现有游离酶应用范围比较示意图;
图2是本发明实施例中固定化酶与游离酶最适温度比较示意图。
具体实施方式
下面结合实施例对本发明作进一步说明。
实施例1:克隆和表达
编码磷脂酶的基因是通过常规技术从以上所指示出的菌株进行克隆的,或者作为合成基因订购并且插入到适合的质粒中。
选择一个正确重组基因序列克隆入表达载体pUC702中,转化至链霉菌Streptomyces lividans TK24宿主细胞中表达该基因构建体。
实施例2:定点突变
以包含整合的表达构建体的重组链霉菌的野生型假定蛋白基因序列为模板,设计突变引物,依次将第71位丝氨酸突变为甘氨酸,将第180位天冬氨酸突变为谷氨酸,得到了具有改进的磷脂酶B活性的突变体。然后将包含野生型假定蛋白的重组菌以及测序正确的突变体分别从平板接种到250mL的锥形瓶在摇床上中进行培养,每个锥形瓶包含50ml 的3%(w/v)TSB液体培养基(含5µg/ml的硫链丝菌素)。在28℃下培养36h后,4℃下18 800g离心20min后, 收集上清液获得粗酶液。
实施例3:酶活力检测
(1)磷脂酶B酶活测定方法
①设置100µl标准检测体系,包含50mM Tris-HCl pH=7.0缓冲液、0.5%(w/v)1, 2-dimyristoyl-sn-glycero-3-phosphate, monosodium salt (DMPA)底物、0.5%(w/v)Ttiton X-100、10mM EDTA以及5%(v/v)磷脂酶B,于40℃下水浴反应5min后,100℃水浴反应5min终止反应。
②反应结束后,21 800g离心5min,按照试剂盒NEFA C Kit (Wako Pure ChemicalIndustries Ltd) 的使用说明,以油酸为标品,检测上述酶反应体系释放出的游离脂肪酸含量。
(3)酶活力定义为
以DMPA为底物,在40℃,pH=7.0的条件下,每分钟产生1µmol游离脂肪酸的酶量定义为一个酶活力单位,记为U/mL。
(4)磷脂酶B酶活的测定
测定野生型磷脂酶B和磷脂酶B突变体的粗酶液活力,突变体的酶活比突变前提高了30%,分别为29.1U/ml和41.6U/ml。
实施列4:固定化磷脂酶B突变体制备
取实施例2中得到的磷脂酶B突变体酶液10mL,称0.1g介孔二氧化钛颗粒加入到酶液中,在摇床中25℃、150rpm吸附固定8h;将得到的固定化酶1 2000rpm高速离心,用pH7.0Tris-HCl缓冲液冲洗2-3次,过滤得到介孔二氧化钛固定化磷脂酶B突变体。
实施例5:固定化磷酶最适pH和最适温度
按照实施例3所述的方法设置100µl标准检测体系,包含50mM pH=4.0-8.0缓冲液、0.5%(w/v)1, 2-dimyristoyl-sn-glycero-3-phosphate, monosodium salt (DMPA)底物、0.5%(w/v)Ttiton X-100、10mM EDTA以及5%(v/v)游离的或固定化磷脂酶B突变体,于30-60℃下水浴反应5min后,100℃水浴反应5min终止反应。
酶活测定结果,以酶活最高的实验组作为相对酶活100%,比较相对酶活。
在图1中,固定化酶相比游离酶具有更好的应用范围,游离脂肪酶的最适pH在5,而固定化脂肪酶pH在6,这可能是载体表面羟基微环境对酶活产生的影响,在带负电荷载体表面会使酶最适pH增加。
图2是游离酶和固定化酶最适温度比较,可见游离磷脂酶B突变体酶和固定化磷脂酶B突变体的最适温度分别为40℃、45℃,且在60℃下,固定化磷脂酶B仍保留了80%活性,而固定化酶对温度耐受性好于游离酶。
实施列5:游离磷脂酶B和固定化磷脂酶B应用于油脂脱胶
称取100g植物油放于250ml的具塞锥形瓶中,水浴加热到75℃,加入130ul浓度为45%的柠檬酸溶液,10000rpm均质1min,在75℃条件下继续搅拌(500rpm) 反应30min,随后将温度降至50℃,加入浓度为4% 的氢氧化纳溶液,将反应体系的pH调至5.0,在500rpm搅拌5min后,加入3ml去离子水和1ml实施例2制备的磷脂B突变体粗酶液或30mg实施例4制备的固定化磷脂酶B突变体,10000r/min均质1min,而后在温度为45℃条件下继续搅拌(500rmp)反应4h,酶反应结束后将反应体系温度升至100℃,酶灭活10min,随后迅速转入离心机10000rpm离心10min,收集上层油层进行磷含量及脱磷率分析。
其中游离磷脂酶B突变体和固定化磷脂酶B突变体使植物油的磷含量分别降至7mg/kg 、2mg/kg。
实施列6:固定化PLD的重复性
将实施例5中反应结束后体系中的固定化磷脂酶B突变体过滤出来,用pH7.0 Tris-HCl缓冲清洗三次,按照实施例5的实验过程,重复进行油脂脱胶,结果发现该固定化磷脂酶B突变体在循环使用10次仍能可以将植物油的磷含量降低至3mg/kg。
在此描述并且要求保护的本发明不限于在此披露的特定方面的范围,因为这些方面旨在作为本发明若干方面的说明。预期任何等效方面都处于本发明的范围内。实际上,除在此所示和描述的那些之外,本发明的不同修改对于本领域普通技术人员而言从前述描述将变得清楚。此类修改也旨在落入所附权利要求书的范围内。在有冲突的情况下,以包括定义的本披露为准。
Claims (1)
1.一种用固定化的新型磷脂酶B于油脂脱胶的方法,特征在于以介孔二氧化钛(TiO2)颗粒为载体固定游离的磷脂酶B突变体,制备得到固定化的新型磷脂酶B突变体,然后将所述的固定化的新型磷脂酶B突变体应用到油脂脱胶中,方法步骤如下:
步骤(1) 制备介孔二氧化钛(TiO2)颗粒
以水合二氧化钛TiO2·nH2O和碳酸钾为原料,控制水合温度为80℃、处理12h,得到水合中间物K2Ti2O5晶须,将所述的水合中间物K2Ti2O5置于水中超声分散,静置后,加入0.5M的盐酸进行搅拌,直到全部K+全部交换出来,抽滤、水洗至中性,干燥后得到介孔TiO2的前驱体水合H2Ti2O5,然后将得到的介孔TiO2的前驱体水合H2Ti2O5置于马弗炉中在500℃下煅烧2h,制备得到锐钛矿型介孔二氧化钛(TiO2)颗粒;
步骤(2)以所述的介孔二氧化钛(TiO2)颗粒为载体、固定游离的磷脂酶B突变体,制备固定化的新型磷脂酶B突变体,操作流程如下:
(2a)取10 mL游离的磷脂酶B突变体酶液,在酶液中加入0.1g介孔二氧化钛颗粒,在摇床中25℃、150rpm吸附固定16h,得到固定化酶液;
(2b)将得到的固定化酶液高速离心,再用缓冲液冲洗2-3次,过滤后得到由介孔二氧化钛固定化的新型磷脂酶B突变体;
步骤(3) 油脂脱胶
先对欲脱胶的植物油进行调pH到5.0-6.0的酸处理,再加入步骤(1)得到的固定化的新型磷脂酶B突变体和去离子水,在45℃下脱胶1-4h,然后将脱胶的植物油升温至100℃,对酶灭活10min后进行胶质分离,得到脱胶油。
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