CN113584005B - 一种氨肽酶的制备及其在蛋白脱苦中的应用 - Google Patents
一种氨肽酶的制备及其在蛋白脱苦中的应用 Download PDFInfo
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- CN113584005B CN113584005B CN202110997938.5A CN202110997938A CN113584005B CN 113584005 B CN113584005 B CN 113584005B CN 202110997938 A CN202110997938 A CN 202110997938A CN 113584005 B CN113584005 B CN 113584005B
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Abstract
本发明公开了一种氨肽酶的制备及其在蛋白脱苦中的应用,属于酶工程技术领域。本发明提供了一种氨基酸序列如SEQ ID No.1所示的氨肽酶在酵母菌表达,其酶活达545U/mL。此氨肽酶对蛋白的脱苦能力强,将此蛋白水解酶以700U/g蛋白的添加量就能够消除大豆蛋白、鱼蛋白和虾蛋白的苦味,反应条件温和、并且不改变肽的其他特性。因此,本发明的氨肽酶在蛋白脱苦中具备极高的应用前景。
Description
技术领域
本发明涉及一种氨肽酶的制备及其在蛋白脱苦中的应用,属于酶工程技术领域。
背景技术
蛋白是在酸、碱、热处理或酶解的作用下得到的多肽混合物,具有良好的生物活性。蛋白里的部分肽由于分子量较小,容易被人体吸收,其中某些低分子肽还能够起到独特的生理功能。大豆蛋白有降血压,血脂和胆固醇的功效,可以调节血糖促进血液循环,改善人体吸收功能,增强免疫力,抗氧化等。鱼蛋白有滋养皮肤,缓解疲劳,提高机体免疫力,美白的功能。南极磷虾蛋白氨基酸种类齐全,营养很高,可以为人体提供很多微量元素,还可以预防骨质疏松和心血管疾病,抑制癌细胞,通常作为功能性保健产品或特殊医疗用品。但蛋白中存在大量N末端为疏水氨基酸残基的肽(分子量在500-1000Da之间),这些疏水性氨基酸带有不同程度的苦味,会直接影响食品的品质,一定程度上限制其在食品领域中的应用。因此,对于蛋白进行脱苦对于提高食品的口味和品质十分必要。为了提高肽的口味和市场价值,必须对蛋白进行脱苦处理。目前,蛋白脱苦主要有物理法、化学法及生物法。生物脱苦酶法因绿色、环保、过程可控等优势,广泛运用到蛋白脱苦中,其中氨肽酶是最为常用的脱苦酶之一,它通过水解肽的N末端疏水氨基酸残基来缓解蛋白苦味。
发明内容
鉴于目前还未有有效能降低苦味的氨肽酶,本发明筛选得到了来源于Aspergillus oryzae并将其应用于蛋白脱苦中,大大降低了蛋白的苦味。
本发明提供了一种使蛋白脱苦的方法,所述方法为将氨基酸序列如SEQ ID NO.1所示的氨肽酶添加至含有蛋白的反应体系中进行脱苦反应。
在一种实施方式中,氨肽酶的添加量为400~900U/g蛋白。
在一种实施方式中,所述反应的温度为55~65℃、pH为6.5~7.5、时间为4~6小时。
优选的,反应温度为50℃、pH为7,反应时间为5h。
本发明提供了一种降低蛋白中苦味肽含量的方法,所述方法是将氨基酸序列如SEQ ID NO.1所示的氨肽酶添加至含有蛋白的反应体系中进行脱苦反应。
在一种实施方式中,将氨肽酶以每克蛋白添加400-900U的量添加至反应体系中。
在一种实施方式中,在55~65℃、pH 6.5~7.5的条件下反应4~6小时。
优选的,反应温度为50℃、pH为7,反应时间为5h。
在一种实施方式中,将反应后的反应液离心取上清液,上清液中含有脱苦反应后得到的蛋白。
本发明提供了一种蛋白脱苦的产品,所述产品含有氨基酸序列如SEQ ID NO.1所示的氨肽酶。
本发明提供了氨基酸序列如SEQ ID NO.1所示的氨肽酶、编码氨基酸序列如SEQID NO.1所示的氨肽酶的基因、携带所述氨肽酶的基因的重组质粒、含有所述氨肽酶的基因的微生物细胞和/或所述产品在蛋白脱苦中的应用。
在一种实施方式中,编码所述氨肽酶的基因的核苷酸序列如SEQ ID NO.2所示。
在一种实施方式中,所述蛋白来自于动物或植物,包括但不限于大豆蛋白,鱼蛋白和虾蛋白和/或乳蛋白。
本发明的有益效果:
本发明提供了一种重组酵母菌氨肽酶制备的工艺。本发明将氨肽酶与底物的用量比为700U/g分别应用于大豆蛋白,鱼蛋白和南极磷虾蛋白中,水解温度为50℃,150rpm,酶解5h,沸水浴10min,冷却一段时间后,可使蛋白的苦味几乎降为零,提供了氨肽酶对大豆蛋白,鱼蛋白和南极磷虾蛋白的脱苦工艺。氨肽酶能显著性地降低蛋白的苦味,提升其风味和品质,该方法相对于其他方法脱苦条件温和,易控,不会使肽营养成分丢失,且不改变肽各种重要特性,将在功能性食品深加工中有极大的应用前景。
附图说明
图1为重组酵母菌发酵上清液中的氨肽酶的SDS-PAGE分析图。
具体实施方式
下面结合具体实施例对本发明进行进一步的阐述。
1、下述实施例中涉及的大豆蛋白、鱼蛋白、虾蛋白购买于山东海龙元生物科技有限公司。氨肽酶1-4均为市售的商品氨肽酶。
2、下述实施例中涉及的培养基如下:
LB固体培养基(g/L):蛋白胨10、酵母粉5、氯化钠10、琼脂13,pH 7.0。
LB液体培养基(g/L):蛋白胨10、酵母粉5、氯化钠10,pH 7.0。
BMMY培养基(g/L):YNB 13.4,酵母提取物10.0,胰蛋白胨20.0,(NH4)2SO4 10.0,K2HPO4 2.29,KH2PO4 11.8。
BMGY培养基(g/L):在BMMY培养基中加入甘油10.0。
YPD液体培养基(g/L):葡萄糖20.0,胰蛋白胨20.0,酵母粉10.0,固体培养基中加入琼脂粉15.0~20.0。
MD固体培养基(g/L):YNB 13.4,琼脂粉20.0,葡萄糖20.0。
3、下述实施例中涉及的检测方法如下:
(1)氨肽酶酶活测定方法:
Tris-HCl缓冲液(50mM pH 8.0):准确称取Tris 6.055g、NaCl 2.92g、加入约800mL去离子水,充分搅拌溶解,用HCl调节pH至8.0,定容至1000mL。
底物(200mmol/L的L-亮氨酸-4-硝基苯胺(Leu-pNA):准确称取0.5026g Leu-pNA,用纯乙醇定容至10mL,常温避光保存。
对硝基苯酚标准曲线的制作:称取一定量的Leu-pNA用100%乙醇完全溶解到终浓度为100μg·mL-1,以其为母液稀释不同倍数得到100、95、90、85、80、75、70、65、60、55、50、45、40、35、30、25、20μg·mL-1的Leu-pNA溶液,将上述不同浓度pNA溶液在酶标仪405nm波长处检测吸光值,以Leu-pNA的浓度为横坐标,吸光值为纵坐标,作出线性图,所做标准曲线为:Y=aX+b
一个单位的反应总体系为1.5mL,在反应体系中加入600μL的三羟甲基氨基,然后加入稀释适当倍数的50μL酶,(对照中加入50μL灭活酶)混合均匀后放置于50℃预热5min,然后加入50μL 200mmol·L-1的Leu-pNA,混合均匀在50℃反应10min后加入800μL乙酸终止反应,在波长405nm处检测反应液吸光值。
酶活力定义:在特定条件下,每分钟释放的1μg pNA的酶的量为一个单位酶活(U)。
计算:
酶活力(U/mL)=ΔOD×1.5×N/(K×10×0.05),
式中:ΔOD:反应液测定前后变化值;
1.5:反应体积总体积(mL);
N:稀释倍数;
K:标准曲线斜率;
10:反应时间(min);
0.05:酶体积(mL)。
(2)苦味的检测方法:
酶解液经8000rpm离心10min,取上清80mL送电子舌测苦味,使用装配传感器C00和传感器AE1的日本Insent公司的SA402B味觉检测系统检测苦味。苦味传感器C00可响应样品中不同的苦味物质并产生电信号,电信号最终可以通过计算转化成苦味值,样品与Reference溶液(2.24g·L-1氯化钾和0.045g·L-1酒石酸)之间苦味值的差值即为最终输出的苦味值。检测过成重复四次,最终取后三次结果的平均值。一般苦味值小于2则认为无苦味或者味蕾感知不出。
(3)蛋白疏水性测定方法:
将蛋白用浓度0.01mM/L,pH7.0的磷酸缓冲液配成浓度范围为0.01-0.1%的溶液,取10μL8 mM/L的ANS(1-苯胺基-8萘基磺酸盐)溶液分别加入到2mL不同浓度的蛋白肽溶液中,振荡,取混合样品于比色皿中,用荧光分光光度计在激发波长390nm,发射波长为470nm,激发和发射狭缝校正宽度均为5nm的条件下,测定ANS-蛋白复合物的荧光强度,每个浓度重复测定3次,以荧光强度对蛋白浓度作图,其斜率为蛋白溶液的表面疏水性指数。数值越大,即代表疏水性越好,疏水肽含量越高。
(4)不同肽段肽含量的测定方法:
酶解液经8000rpm离心10min,取上清液和标准品过0.22μm滤膜,滤液进行高效液相(HPLC)分析;色谱条件:色谱柱:TSKgel2000SWXL300 mm×7.8mm,流动相:乙腈/水/三氟乙酸,40/60/0.1(v·v-1),检测:UV220 nm,流速:0.5mL·min-1,柱温:30℃。分子量校正曲线所用标准品:1、细胞色素C(MW 12384Da),2、杆菌酶(MW 1422Da),3、乙氨酸-乙氨酸-酪氨酸-精氨酸(MW 451Da),4、乙氨酸-乙氨酸-乙氨酸(MW 189Da),5、抑肽酶(MW 6500Da)。
计算由Waters GPC软件自动进行,根据分子量大小划分成不同区域,分子量小于180Da的部分即为游离氨基酸,分子量大于10000Da部分视为大分子蛋白质。分子量在500-1000Da之间的肽的苦味最为显著,这区间肽含量越少,则苦味越低。
实施例1:表达氨肽酶的重组菌的构建
将来源于Aspergillus oryzae、Aspergillus flavus、Aspergillus sojae、Aspergillus minisclerotigenes、Aspergillus transmontanensis、Aspergillusnovoparasiticus、Aspergillus arachidicola氨肽酶(所述氨肽酶对应的NCBI上的登录号或基因号分别为NCBI登录号:XP_001825745.1、GenBank:RAQ51025.1、NCBI登录号:Q8J2N2、GenBank:KAB8272165.1、GenBank:KAE8313457.1、GenBank:KAB8217617.1、GenBank:KAE8342201.1),分别构建重组菌,具体实施步骤如下:
(1)重组质粒的构建
利用化学方法将核苷酸序列如SEQ ID NO.2所示的编码氨肽酶AoAPase的基因合成到载体pPIC9K上,直接获得重组质粒pPIC9K-AoAPase,将重组质粒转化大肠杆菌(Escherichia coli)JM109,得到转化产物;将转化产物涂布在LB固体培养基(含有40μg/mL卡那霉素)上,于37℃恒温培养箱中倒置培养8~12h,得到转化子;挑取转化子接种至LB液体培养基中,于37℃、200rpm的条件下摇瓶培养8~12h后提取质粒进行测序验证,验证正确即获得重组质粒pPIC9K-AoAPase;
按照上述方法,将来源于A.flavus、A.sojae、A.minisclerotigenes、A.transmontanensis、A.novoparasiticus、A.arachidicola的氨肽酶编码基因连接至载体pPIC9K上,构建得到重组质粒pPIC9K-AfAPase、pPIC9K-AsAPase、pPIC9K-AmAPase、pPIC9K-AtAPase、pPIC9K-AnAPase、pPIC9K-AaAPase。
(2)重组质粒的转化
将重组质粒pPIC9K-AoAPase线性化,反应体系置于37℃温浴2h后,取线性化质粒转化毕赤酵母P.pastoris KM71,得到转化产物;将转化产物涂布在MD固体培养基上,于30℃恒温培养箱中倒置培养1.5~2天,得到转化子;挑取转化子在新的MD平板上,于30℃恒温培养箱中倒置培养1.5~2天,编号保菌。
将重组质粒pPIC9K-AfAPase、pPIC9K-AsAPase、pPIC9K-AmAPase、pPIC9K-AtAPase、pPIC9K-AnAPase、pPIC9K-AaAPase按照上述方法线性化,并转化至毕赤酵母P.pastoris KM71中,分别构建得到重组菌。
实施例2:重组菌表达氨肽酶
(1)利用实施例1中构建得到的重组菌,摇瓶发酵产酶。
从MD平板上挑选大的单菌落到已标号的小管里,每个小管加入4mL的BMGY培养基,培养2-3天。当小管底部已有明显菌体沉淀后,5000rpm离心5min,在超净台中倒掉上清液,加入2mL的BMMY培养基,重悬之后加入1%的甲醇,30℃培养2-3天,每24h补加1%体积的甲醇。测菌液的酶活和蛋白含量,挑选5-10个酶活的转化子,再进行摇瓶筛选。
将5-10个酶活高的转化子,加入50mL的BMGY培养基,培养2-3天。当摇瓶底部已有明显菌体沉淀后,5000rpm离心5min,在超净台中倒掉上清液,加入100mL的BMMY培养基,重悬之后加入1%的甲醇,30℃培养3-4天,每24h补加1%体积的甲醇,获得发酵液。
(2)酶活的测定
将发酵液于5000rpm的条件下离心5min取上清液,加入50%(w·v-1)固体硫酸铵盐析过夜。硫酸铵盐析后的酶液于4℃,10,000rpm离心20min,取沉淀物用适量pH 8.0,50mM磷酸盐缓冲液复溶后装入透析袋,透析袋提前用缓冲液浸润,透析12h以上,中途需要换透析液3-4次,最后收集透析干净的液体即为纯品氨肽酶。对氨肽酶进行酶活测定(表1),发现均有酶活,证明重组氨肽酶成功重组表达。其中,氨肽酶AoAPase在41kDa条带出存在蛋白带(如图1所示),且酶活最大为545U/mL。
表1氨肽酶的酶活测定
氨肽酶 | 酶活(U/mL) |
AoAPase | 545 |
AfAPase | 432 |
AsAPase | 456 |
AmAPase | 342 |
AtAPase | 378 |
AnAPase | 410 |
AaAPase | 391 |
实施例3:不同氨肽酶在蛋白脱苦中的应用
实施例2获得不同来源的氨肽酶对蛋白进行脱苦:
称取大豆蛋白、鱼蛋白、虾蛋白配制成浓度为20g/L蛋白溶液100mL,向蛋白溶液中分别添加氨肽酶400U/g底物,利用恒温水浴摇床维持水解温度为50℃,150rpm酶解5h,沸水浴10min,冷却一段时间,将酶解液8000rpm离心10min,取上清液测苦味。
结果如表2所示:实施例2获得不同来源的氨肽酶对不同类型蛋白的脱苦效果具有显著差异。其中,氨肽酶AoAPase对大豆蛋白、鱼蛋白、虾蛋的脱苦效果较为明显。
表2不同氨肽酶对不同蛋白脱苦的应用
实施例4:氨肽酶AoAPase对不同蛋白脱苦的应用
具体步骤如下:
准确称取大豆蛋白、鱼蛋白、虾蛋白配制成浓度为20g/L蛋白溶液100mL,按氨肽酶与底物的用量比为0U/g,400U/g,500U/g,600U/g,700U/g,800U/g和900U/g加入AoAPase于大豆蛋白溶液中,利用恒温水浴摇床维持水解温度为50℃,150rpm酶解5h,沸水浴10min,冷却一段时间,将酶解液8000rpm离心10min,取上清液测苦味。
由表3所示,结果显示氨肽酶AoAPase能够显著降低蛋白的苦味。当添加量达到700U/g时,苦味几乎为零。当加酶量大于700U/g时,蛋白苦味反而有所上升,综合而言,最佳加酶量为700U/g。
表3氨肽酶AoAPase对不同蛋白脱苦的应用
实施例5:不同氨肽酶对大豆蛋白的处理效果
具体步骤如下:
准确称取大豆蛋白配制成浓度为20g/L蛋白溶液100mL,按氨肽酶与底物的用量比为700U/g加入不同氨肽酶于蛋白溶液中,利用恒温水浴摇床维持水解温度为50℃,150rpm酶解5h,沸水浴10min,冷却一段时间,将酶解液8000rpm离心10min,取上清液测苦味及疏水性。
由表4所示,结果显示不同氨肽酶的脱苦效果差异显著。其中,氨肽酶AoAPase对大豆蛋白的脱苦效果尤为显著,疏水性明显的降低,且苦味肽(500-1000Da)含量仅为20%,则表明经氨肽酶AoAPase处理的大豆蛋白中疏水性肽含量降低,从而改善了风味。
表4不同氨肽酶对大豆蛋白的处理效果
实施例6:不同氨肽酶对鱼蛋白的处理效果
具体步骤如下:
准确称取鱼蛋白配制成浓度为20g/L蛋白溶液100mL,按氨肽酶与底物的用量比为700U/g加入不同氨蛋白酶于蛋白溶液中,利用恒温水浴摇床维持水解温度为50℃,150rpm酶解5h,沸水浴10min,冷却一段时间,将酶解液8000rpm离心10min,取上清液测苦味及疏水性。
由表5所示,结果显示不同氨肽酶的脱苦效果差异显著。其中,氨肽酶AoAPase对鱼蛋白的脱苦效果尤为显著,疏水性明显的降低,且苦味肽(500-1000Da)含量仅为22%,则表明经氨肽酶AoAPase处理的鱼蛋白中疏水性肽含量降低,从而改善了风味。
表5不同氨肽酶对鱼蛋白的处理效果
实施例7:不同氨肽酶对虾蛋白的处理效果
具体步骤如下:
准确称取虾蛋白配制成浓度为20g/L蛋白溶液100mL,按氨肽酶与底物的用量比为700U/g加入不同氨肽酶于蛋白溶液中,利用恒温水浴摇床维持水解温度为50℃,150rpm酶解5h,沸水浴10min,冷却一段时间,将酶解液8000rpm离心10min,取上清液测苦味及疏水性。
由表6所示,结果显示不同氨肽酶的脱苦效果差异显著。其中,氨肽酶AoAPase对虾蛋白的脱苦效果尤为显著,疏水性明显的降低,且苦味肽(500-1000Da)含量仅为27%,则表明经氨肽酶AoAPase处理的虾蛋白中疏水性肽含量降低,从而改善了风味。
表6不同氨肽酶对虾蛋白的处理效果
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> 一种氨肽酶的制备及其在蛋白脱苦中的应用
<130> BAA211114A
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Claims (6)
1. 一种使蛋白脱苦的方法,其特征在于,所述方法为将氨基酸序列如SEQ ID NO.1所示的氨肽酶添加至含有蛋白的反应体系中进行脱苦反应,所述反应体系中,氨肽酶的添加量为400~900 U/g蛋白;所述蛋白包括大豆蛋白,鱼蛋白和虾蛋白和/或乳蛋白。
2.如权利要求1所述的方法,其特征在于,所述反应的温度为55~65℃、pH为6.5~7.5、时间为4~6小时。
3. 一种降低蛋白中苦味肽含量的方法,其特征在于,将氨基酸序列如SEQ ID NO.1所示的氨肽酶添加至含有蛋白的反应体系中进行脱苦反应,将氨肽酶以每克蛋白添加400-900 U的量添加至反应体系中;所述蛋白包括大豆蛋白,鱼蛋白和虾蛋白和/或乳蛋白。
4. 如权利要求3所述的方法,其特征在于,在55~65℃、pH 6.5~7.5的条件下反应4~6小时。
5.如权利要求4所述的方法,其特征在于,将反应后的反应液离心取上清液,上清液中含有脱苦反应后得到的蛋白。
6. 氨基酸序列如SEQ ID NO.1所示的氨肽酶、编码氨基酸序列如SEQ ID NO.1所示的氨肽酶的基因、携带所述氨肽酶的基因的重组质粒或含有所述氨肽酶的基因的微生物细胞在蛋白脱苦中的应用;所述蛋白包括大豆蛋白,鱼蛋白和虾蛋白和/或乳蛋白。
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