CN104774824B - 一种新型蛋白酶及其在酪蛋白磷酸肽制备中的应用 - Google Patents
一种新型蛋白酶及其在酪蛋白磷酸肽制备中的应用 Download PDFInfo
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Abstract
本发明涉及一种新型微生物源中性蛋白酶及其在酪蛋白磷酸肽制备中的应用。通过对米曲霉固态发酵产物分离纯化得到一种中性蛋白酶，分子量为58kD左右，适宜反应pH为7.0‑8.0，对‑Ser‑His‑、‑Val‑Glu‑、‑Lys‑Tyr‑和‑Arg‑Gly‑等肽键选择性较强。该酶可以水解酪蛋白产生酪蛋白磷酸肽，产率和纯度均高于同等条件下胰蛋白酶制备的酪蛋白磷酸肽，为目前酪蛋白磷酸肽的生产提供有利的参考。
Description
所属技术领域：
本发明涉及一种微生物源中性蛋白酶及其在酪蛋白磷酸肽制备中的应用，属于微生物蛋白酶领域。
背景技术：
蛋白酶是一种重要的工业酶制剂，占酶制剂市场的65％以上，广泛来源于动物内脏、植物根茎和微生物中。而微生物蛋白酶以其生产成本低廉、便于控制、产量高以及生产周期短等优势成为蛋白酶制剂的主要生产方式。在食品上应用的蛋白酶要考虑到安全性，目前通过基因工程改造的菌株产蛋白酶应用于食品行业在很多国家是遭到拒绝的，因此需要从公认无害的菌株中获得安全蛋白酶。
蛋白酶具有特异的识别位点，切割蛋白底物的特定肽键，因此不同蛋白酶作用于同一底物，水解液中的成分各不相同。蛋白水解液中包含各种小肽和氨基酸，很多小肽具有各种不同的生理功能，如抗氧化、抗高血压、增强免疫力等。酪蛋白磷酸肽(CPP)是以酪蛋白为原料，经过蛋白酶水解而得到的功能性小肽。它可作为载体促进肠道内对钙铁锌硒等微量元素的吸收，是目前唯一成功应用于功能性食品的活性肽，具有广阔的应用前景。
目前CPP的制备主要依靠胰蛋白酶水解酪蛋白，并经过进一步的纯化得到纯度较高的产品。胰蛋白酶来源于动物胰脏，生产成本较高，工艺复杂，在一定程度上限制了CPP在食品领域的应用。近年来相对廉价的微生物蛋白酶成为CPP制备的研究热点，应用较多的是地衣芽孢杆菌产的碱性蛋白酶。然而由于酶解时为了达到其最适pH，经常要加入NaOH维持pH大于9的碱性环境，使 得Na+的含量大幅升高，对生产不利。另外考虑到酪蛋白的溶解性，最适生产pH应为7.0-8.0。因此，获得更适合于CPP工业生产的微生物蛋白酶具有重要的商业价值。
发明内容：
本发明的目的提供一种新型微生物中性蛋白酶，从安全菌株米曲霉的固态发酵所产酶系中分离纯化获得。所用米曲霉于2013年5月13日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.7586，保藏地址为北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所。
本发明的另一目的是将获得的中性蛋白酶应用于CPP的制备，从而降低CPP生产成本，并获得产率和纯度较高的CPP，推进CPP在食品行业更广泛的应用。
所述新型中性蛋白酶的纯化方法如下：
(1)米曲霉固态发酵后，加入无菌生理盐水振荡得到粗酶液，利用硫酸铵沉淀去除大部分杂质，进一步用等电点沉淀得到粗纯蛋白酶。
(2)上一步样品脱盐并调节pH后上样于RESOURCE-Q层析柱，高盐洗脱，收集活性组分。
(3)收集到的样品脱盐后超滤，低温保存。
所述蛋白酶的酶学性质如下：
蛋白酶PE的最适反应温度为55℃，最适pH 8.0；40℃放置120min，酶活在95％以上；pH 5-10条件下40℃放置60min，酶活维持在90％以上，酶活稳定性较好。PE不耐高温，55℃条件下，PE酶活半衰期为30min，60℃放置30min已完全失活。金属离子对酶活影响的实验表明，Fe3+对PE有一定的抑制 作用，Mn2+对PE有激活作用。以酪蛋白为底物时，PE的表观米氏常数Km＝0.36g·L-1，最大反应速率Vm＝18.18μg·mL-1·min-1。Km值反映了酶与底物的亲和力，较低的Km值说明蛋白酶PE与酪蛋白较高的亲和力。
所述蛋白酶酶切位点及酶切方式如下：
用蛋白酶水解牛胰岛素氧化B链，水解液通过MALDI-TOF-MS检测酶解肽段分子量，与牛胰岛素氧化B链的随机片段比对，得到酶解肽段的氨基酸序列，与牛胰岛素氧化B链氨基酸序列对比。结果表明，该蛋白酶对牛胰岛素B链的-Ser-His-、-Val-Glu-、-Lys-Tyr-和-Arg-Gly-组成的肽键有较强的切割能力，对Val、Leu及Gly等脂肪族氨基酸组成的肽键选择性较强。在酪蛋白的氨基酸序列中，磷酸丝氨酸基团附近存在很多Val和Gly组成的肽键，容易被PE切割而形成纯度较高的CPP。另外，相比较胰蛋白酶、胃蛋白酶等专一性较高的蛋白酶，纯化到的米曲霉蛋白酶PE具有较多的切割位点，
所述利用该蛋白酶制备CPP的方法如下：
配制20g/L酪蛋白溶液(pH 8.0)，按照1000U/g底物分别加入蛋白酶，40℃保温3h后80℃灭酶10min，调节pH 4.6，离心去除沉淀；上清液调节pH 6.8，加入1.5mL氯化钡溶液，混匀后加入无水乙醇至50mL，4℃沉淀过夜，离心后去除上清液，收集沉淀即为CPP粗品，于50℃烘箱烘干备用。此方法制备的CPP产率为30.26％，r(N/P)为5.17。
本发明提供的蛋白酶应用于CPP的制备具有一定的优势：
(1)其来源于安全菌株米曲霉，可利用廉价原料发酵获得，有利于降低CPP的生产成本。
(2)蛋白酶最适pH为7.0-8.0，在此弱碱性条件下，酪蛋白溶解度较高，有利于酪蛋白的酶解，同时可以减少钠盐50％以上。
(3)本发明利用米曲霉蛋白酶水解酪蛋白得到的CPP，产率和纯度均明显高于相同条件下胰蛋白酶制备的CPP。
附图说明：
图1.蛋白酶的SDS-PAGE电泳图和酶谱
1、蛋白酶酶谱；2、蛋白酶SDS-PAGE图谱；3、标准蛋白
图2.蛋白酶酶解酪蛋白制备的CPP持钙能力曲线
具体实施方式：
实施例1：米曲霉蛋白酶的分离纯化
(1)接种米曲霉孢子悬浮液于麸皮豆粕固态培养基，30℃恒温培养36h，每隔6h扣瓶一次。发酵完成后加入无菌生理盐水，摇床震荡1h，过滤离心得到粗酶液。用40％饱和度的硫酸铵去除杂质，70％饱和度的硫酸铵沉淀蛋白酶。
(2)上一步样品用20mmol/L Tris-Hcl(pH 8.5)缓冲液复溶后利用HCl-NaOH
溶液调节pH至蛋白酶等电点，离心后复溶沉淀。
(3)样品脱盐后上样于用20mmol/L Tris-Hcl(pH 8.5)平衡过的RESOURCE-Q层析柱，待穿透蛋白充分洗下后，用含0.5mol/L Nacl的缓冲液线性洗脱，检测波长为280nm，收集酶活组分，脱盐超滤后得到纯化蛋白酶。
SDS-PAGE检测蛋白酶PE的纯度与大小，非变性胶检测蛋白酶PE的酶谱，结果见图1.
实施例2：蛋白酶酶切位点的确定
配制1mg/mL的牛胰岛素氧化B链，加入蛋白酶后，于40℃保温1h，80℃灭酶10min。取少量水解液，用MALDI-TOF-MS检测水解液中小肽分子量，通过与牛胰岛素氧化B链的随机肽段比较得到肽段序列，所得结果与牛胰岛素氧化B链全长作对比，以此确定蛋白酶的酶切位点，结果如下表：
注：表中箭头表示酶切位点
实施例3：利用蛋白酶酶解酪蛋白制备CPP
配制的20g/L的酪蛋白溶液(pH 8.0)，按照1000U/g底物分别加入中性蛋白酶和胰蛋白酶，40℃保温3h，80℃灭酶10min。加入去离子水至15mL，调节pH 4.6，离心去除沉淀。上清调节至pH 6.8，加入1.5mL氯化钡溶液，混匀后加入无水乙醇至50mL，4℃沉淀过夜，离心后去除上清，沉淀于50℃烘箱烘干。利用中性蛋白酶制备的CPP产率为30.26％，r(N/P)为5.17，利用胰蛋白酶在相同条件下酶解酪蛋白所产CPP产率为18.46％，r(N/P)为7.35。
实施例4：CPP持钙能力检测
在弱碱性条件下，NaH2PO4与CaCl2形成Ca3(PO4)2会释放H+，在维持体系pH不变的同时，NaOH溶液的加入量与Ca3(PO4)2形成量一致。在100mL的反应体系中加入NaH2PO4和CPP，使二者在反应体系中的浓度分别为0.008mol/L和0.1g/L。将溶液加热至37℃保温，然后加入CaCl2，使反应体系中CaCl2的浓度为0.008mol/L。立即加入0.05mol/L的NaOH溶液，在2min内将反应体系的pH值调节至8.0，随后不断加入0.05mol/L的NaOH，使反应体系的pH维 持在8.0。从第2min开始，每隔5min记录一次0.05mol/L NaOH的消耗量，持续1h(图2)。制备的CPP含量为0.1g/L时，可以延迟钙沉淀55min。
以上所述的仅是本发明的优选实施方式，本发明不限于以上实施例。可以理解，本领域技术人员在不脱离本发明的基本构思的前提下直接导出或联想到的其他改进和变化，均应认为包含在本发明的保护范围之内。
Claims (2)
1.一种微生物源中性蛋白酶，其特征在于，所述蛋白酶可以酶解酪蛋白产生酪蛋白磷酸肽(CPP)，所述蛋白酶是从安全菌株米曲霉固态发酵所产蛋白酶酶系中分离纯化得到，其大小为58kD，最适pH 7.0-8.0，所述蛋白酶对-Val-Glu-和-Arg-Gly-肽键具有很强的切割能力，所述米曲霉于2013年5月13日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.7586，保藏地址为北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所；所述固态发酵时使用的固态发酵培养基成分为麸皮6g、脱脂大豆蛋白3g、豆粕1g、水10ml。
2.权利要求1所述蛋白酶在制备酪蛋白磷酸肽中的应用，其特征在于该蛋白酶酶解酪蛋白获得的酪蛋白磷酸肽产率和纯度均高于相同条件下胰蛋白酶制备的酪蛋白磷酸肽。
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| 米曲霉HDF-7蛋白酶的分离纯化及酶学性质研究;于艳颖等;《黑龙江大学工程学报》;20110505;第2卷(第2期);第74-79页 * |
| 米曲霉蛋白酶的分离纯化及酶学性质研究;马俊阳等;《微生物学通报》;20131011;第41卷(第1期);摘要，第84页左栏第3段，第84-85页1材料与方法 * |
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