CN114081120A - 一种乳酸菌多铜氧化酶的制备及其在降解生物胺中的应用 - Google Patents
一种乳酸菌多铜氧化酶的制备及其在降解生物胺中的应用 Download PDFInfo
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- CN114081120A CN114081120A CN202111359832.9A CN202111359832A CN114081120A CN 114081120 A CN114081120 A CN 114081120A CN 202111359832 A CN202111359832 A CN 202111359832A CN 114081120 A CN114081120 A CN 114081120A
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- oxidase
- copper
- biogenic amine
- degradation
- tyramine
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Abstract
本发明公开了一种乳酸菌多铜氧化酶的制备及其在降解生物胺中的应用，属于生物工程技术领域。本发明所提供的多铜氧化酶来源于清酒乳杆菌(Lactobacillus sakei)，将其在大肠杆菌中异源表达生产得到。该酶具有较好的降解生物胺的性能，在低浓度下即可降解40.90％的组胺及41.24％的酪胺。在高盐条件下，其降胺性能均可以进一步提升，对组胺和酪胺的降解率分别为75.95％和66.81％。并且在高乙醇条件下也能保持部分降胺能力。将其应用于腌鱼、葡萄酒等发酵食品模拟体系中均有较好的降胺效果，可广泛应用于工业化生产。
Description
技术领域
本发明涉及一种乳酸菌多铜氧化酶的制备及其在降解生物胺中的应用，尤其涉及一种来源于清酒乳杆菌的多铜氧化酶Ls1b的基因、制备方法与其在降解生物胺中的应用，属于生物工程技术领域。
背景技术
生物胺是一类具有生物活性的小分子含氮有机化合物，它们广泛存在于食品，尤其是蛋白质含量较高的发酵食品中。主要由相应的氨基酸通过微生物的脱羧作用形成，或由醛、酮类物质在氨基酸转氨酶作用下产生。适量的生物胺可以促进人体正常的生理活动，但摄入过量外源生物胺则会引起人体诸多不良反应，严重时可能危及生命。其中酪胺和组胺对人体的潜在危害最大。
对于食品中的生物胺，化学法、酶法、微生物法被认为是降低发酵食品生物胺积累的有效方式。中国计量大学2019年申请了“一种采用杨梅叶提取物控制酱油中生物胺含量的方法”(申请号：201910058795.4)，中国海洋大学2020年申请了“库德毕赤酵母菌(Pichiakudriavzevii)在降解生物胺方面的应用”(申请号：202010332776.9)。以上方法具有各自不可避免的局限性，如降解生物胺种类少、降解效果差、营养成分的流失、风味的改变及安全性存疑等。与上述控制技术不同的是，通过向发酵食品中添加生物胺降解酶，不造成食品营养物质损失，不产生新的毒性物质，可以有效控制食品中的生物胺积累。
蓝色多铜氧化酶家族可催化氧化多种底物，其中部分酶可以催化氧化生物胺生成对应的醛、氨和水。此类酶广泛存在于植物、真菌和细菌中，细菌多铜氧化酶的部分特征如不需要糖基化、耐盐、稳定性好等，相较于真菌酶在某些应用上更具优势。江南大学2018年申请了“新型多铜氧化酶及其在降解生物胺中的应用”(申请号：201811383747.4)，其中公开了利用来源于Weissellacibaria的多铜氧化酶。但目前针对能降解生物胺的细菌多铜氧化酶的研究还是较少，因此筛选能降解生物胺，尤其是降解毒性较大的组胺和酪胺能力强的多铜氧化酶是具有非常广阔应用前景的。
发明内容
本发明的目的是提供一种来源于清酒乳杆菌的重组多铜氧化酶Ls1b，并且将其应用于食品生物胺的降解。
本发明提供了一种降解食品中生物胺的方法，所述方法是利用来源于清酒乳杆菌(Lactobacillus sakei)的多铜氧化酶降解生物胺，所述多铜氧化酶的氨基酸序列如SEQID NO.1所示。
在一种实施方式中，将所述多铜氧化酶添加至含有组胺、酪胺、腐胺、色胺、苯乙胺、亚精胺、尸胺和精胺的体系中，所述生物胺的含量为50～200mg/L。
优选的，所述酪胺、腐胺、色胺、苯乙胺、亚精胺、尸胺和精胺的含量为50～100mg/L。
更优选的，所述组胺和酪胺的含量为50～100mg/L，并且所述腐胺、色胺、苯乙胺、亚精胺、尸胺和精胺的含量为50mg/L。
在一种实施方式中，所述多铜氧化酶按照体系中每毫克生物胺添加不少于0.14U的量添加。
优选的，所述多铜氧化酶按照体系中每毫克生物胺添加0.14～1.32U的量添加。
更优选的，所述多铜氧化酶按照体系中每毫克生物胺添加0.33U、1.32U、0.25U、0.14U的量添加。
在一种实施方式中，反应体系中的乙醇含量为0～20％、氯化钠浓度为0～10％。
在一种实施方式中，在pH4.0～7.0、15～30℃下反应。
在一种实施方式中，反应时间不少于20h。
优选的，反应时间为20～24h。
在一种实施方式中，所述食品包括但不限于腌鱼、葡萄酒、酱油、豆瓣酱。
本发明提供了氨基酸序列如SEQ ID NO.1所示的多铜氧化酶在降解生物胺中的应用。
在一种实施方式中，所述生物胺包括组胺、酪胺、腐胺、色胺、苯乙胺、亚精胺、尸胺和精胺。
在一种实施方式中，降解生物胺的反应体系中的生物胺含量为50～200mg/L。
本发明提供了含有如SEQ ID NO.2所示的核苷酸序列的表达载体、表达氨基酸序列如SEQ ID NO.1所示的多铜氧化酶的微生物细胞、或氨基酸序列如SEQ ID NO.1所示的多铜氧化酶在制备降解生物胺的产品中的应用。
在一种实施方式中，所述产品包括生物酶复合剂。
在一种实施方式中，所述微生物细胞可以为大肠杆菌、酿酒酵母、枯草芽孢杆菌。
在一种实施方式中，所述微生物细胞以包括pET系列、pUC系列、pWB系列载体为表达载体。
本发明的有益效果：本发明提供的重组多铜氧化酶降解生物胺的效果较好，在低浓度下即可降解40.90％的组胺及41.24％的酪胺。在高浓度乙醇条件下仍能保持20％以上的组胺、酪胺降解率。在高盐条件下降解生物胺的能力可进一步提升，在本发明的条件下组胺降解率最多可达75.95％，酪胺最多可达66.81％，在高盐度的条件下对于酪胺和组胺的降解率显著优于现有报道的多铜氧化酶，在高盐食品中有较大应用潜力。并且，将本发明的多铜氧化酶应用在不同食品基质的模拟体系中均有较好的降胺效果，可应用的范围广泛。
附图说明
图1是L.sakei中多铜氧化酶基因的验证图；其中，M泳道为Marker；1-3泳道为L.sakei中多铜氧化酶基因；4泳道为阴性对照；
图2是重组多铜氧化酶添加量对组胺降解率的影响；
图3是盐度及pH对重组多铜氧化酶对组胺降解率的影响；
图4是乙醇含量对重组多铜氧化酶对组胺降解率的影响；
图5是重组多铜氧化酶添加量对酪胺降解率的影响；
图6是盐度及pH对重组多铜氧化酶对酪胺降解率的影响；
图7是乙醇含量对重组多铜氧化酶对酪胺降解率的影响；
图8是以ABTS为底物时，pH对重组多铜氧化酶酶活的影响；
图9是重组多铜氧化酶在含食品基质的模拟体系中对生物胺的降解率。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。
下述各例中采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
以下实施例所述乳酸菌多铜氧化酶的名称为Ls1b。
多铜氧化酶酶活测定方法如下：
采用可见光吸收法测定多铜氧化酶活力：以2,2'-联氮-双-3-乙基苯并噻唑啉-6-磺酸(ABTS)为底物，通过检测酶氧化ABTS的量计算多铜氧化酶的酶活。反应时间为3min，反应体系为1mL，包括50μL酶液、100μL 5mmol/L ABTS溶液、350μL去离子水和500μL0.2mol/L的磷酸氢二钠-柠檬酸缓冲液。将每分钟氧化1μmol ABTS所需的酶量定义为一个酶活力单位(U)。酶活计算公式如下所示：
式中
ε：ABTS在420nm下的摩尔吸光系数，ε＝3.6×104M-1cm-1
Δt：反应时间
ΔOD：420nm处吸光度的变化值
V1：反应总体积
V2：反应使用酶量
实施例1：清酒乳杆菌的细胞破碎液对生物胺的降解
将本研究室保藏的清酒乳杆菌(Lactobacillus sakei)接种于液体MRS培养基中，于37℃、200r/min条件下过夜培养。将菌液于4℃、10000r/min离心7min，收集菌体沉淀物，用20mmol/LpH 7.0的磷酸盐缓冲溶液洗涤菌体，之后用磷酸盐缓冲液将菌体重悬。加入终浓度0.5mg/ml的溶菌酶，冰上30min，随后液氮反复冻融3-5次，使其裂解。将裂解液于4℃、10000r/min离心15min收集上清液，后收集溶液过0.45μm水系滤膜即得所需粗酶液。以ABTS为底物，测得其产多铜氧化酶酶活水平为4.63U/L。在生物胺溶液(组胺50mg/L、酪胺50mg/L、腐胺50mg/L、尸胺50mg/L，pH为7.0)中添加2U/L的粗酶液，25℃反应24h，检测生物胺含量。结果表明清酒乳杆菌的粗酶液可以降解酪胺、组胺和腐胺，降解率分别为9.84％、15.24％、5.77％。
实施例2：多铜氧化酶基因的克隆表达与纯化
(1)多铜氧化酶基因的克隆与表达
以本研究室保藏的清酒乳杆菌(Lactobacillus sakei)基因组DNA为模板，扩增目的基因，扩增得到的产物片段，电泳图如图1所示，条带大小在1500bp左右，核苷酸序列如SEQ ID NO.2所示。
将扩增得到的片段通连接至质粒pET-28a的SacⅠ酶与Xho I酶酶切位点间，构建得到重组载体。将重组载体转化至E.coli DH5α后培养、提取质粒进行筛选测序，确定得到具有正确编码框的重组载体。
提取测序正确的重组质粒转化至大肠杆菌BL21(DE3)感受态细胞中，转化液涂布于含50μg/mL硫酸卡那霉素的LB培养基平板，37℃过夜培养，提取阳性菌落质粒，进行菌落PCR鉴定，获得重组菌，命名为E.coli BL21(DE3)-pET28a-Ls1b。
将重组菌E.coli BL21(DE3)-pET28a-Ls1b接种到含50μg/mL硫酸卡那霉素的LB液体培养基中，于37℃、200r/min条件下过夜培养。按2％的接种量进行扩大培养，于37℃、200r/min条件下培养至OD600为0.5～0.7，加入终浓度为1mM的CuCl2及终浓度为0.1mM的IPTG，16℃振荡培养20h。将所得发酵液于4℃、10000r/min离心7min，收集菌体沉淀物，用20mmol/LpH 7.0的磷酸盐缓冲溶液洗涤菌体，之后用磷酸盐缓冲液将菌体重悬。加入终浓度0.5mg/ml的溶菌酶，冰上30min，随后液氮反复冻融3-5次，使其裂解。将裂解液于4℃、10000r/min离心15min收集上清液，后收集溶液过0.45μm水系滤膜即得所需粗酶液。粗酶液的酶活为306.84U/L，与野生酶相比提高了129.47倍。
(2)Ls1b酶蛋白的纯化
使用超纯水清洗镍柱的填料；用5-10个柱体积的结合缓冲液(20mM磷酸盐缓冲液，0.5MNaCl，20-40mM咪唑，pH 7.4)平衡镍柱；上样；上样后用五倍柱体积的结合缓冲液再次平衡镍柱；用洗脱缓冲液(20mM磷酸盐缓冲液，0.5M NaCl，100-500mM咪唑，pH 7.4)使用阶梯梯度进行洗脱，收集洗脱液。使用截留量为10kDa的Millipore超滤管脱盐并浓缩，得到纯蛋白。纯化后的蛋白进行蛋白浓度、酶活及SDS-PAGE蛋白电泳检测。结果显示，蛋白浓度为0.987mg/mL，酶活为469.89U/L。
实施例3：重组多铜氧化酶在不同条件下对组胺的降解效果
(1)重组多铜氧化酶添加量对组胺降解率的影响
反应体系为pH 7.0的磷酸盐缓冲液，组胺终含量为100mg/L；纯化后的重组酶Ls1b按不同的添加量(7U/L、17U/L、33U/L、66U/L、132U/L)加入至反应体系中；于25℃反应24h；通过超高效液相色谱质谱联用仪测定生物胺含量。组胺降解结果如图2所示，随着重组酶Ls1b含量的增加，24h内组胺的降解率从12.16％提升至65.5％。
(2)盐度及pH对重组多铜氧化酶对组胺降解率的影响
反应体系为含100mg/L组胺的磷酸盐缓冲液(pH 4.0/7.0)，其含有不同质量分数的NaCl(0％、2％、4％、6％、8％、10％)；将纯化重组酶Ls1b以终浓度33U/L加到体系中25℃反应24h，检测组胺含量，组胺降解结果如图3所示，NaCl的存在对酶降解组胺有激活作用，且含量越高，所述酶对组胺的降解效果越好，当pH为4或7时，存在10％的NaCl可使组胺降解率均达到70％以上，具体的，在pH4时，NaCl浓度为0％、2％、4％、6％、8％、10％时，降解率分别为0％、36.62％、43.75％、67.69％、68.28％、74.06％；在pH7时，NaCl浓度为0％、2％、4％、6％、8％、10％时，降解率分别为40.9％、61.72％、68.57％、72.03％、72.24％、75.95％。表明所述酶在盐度高的食品体系内降解组胺具有潜力。
(3)乙醇含量对重组多铜氧化酶对组胺降解率的影响
反应体系为含100mg/L组胺的磷酸盐缓冲液(pH 7.0)，其含有不同体积分数的乙醇(0％、10％、15％、20％)；将纯化重组酶Ls1b以终浓度33U/L加到体系中25℃反应24h，检测组胺含量，组胺降解结果如4所示，组胺降解率分别为40.9％、27.95％、21.57％、23.76％。
实施例4：重组多铜氧化酶在不同条件下对酪胺的降解效果
(1)重组多铜氧化酶添加量对酪胺降解率的影响
反应体系为pH 7.0的磷酸盐缓冲液，酪胺终含量为100mg/L；重组酶Ls1b按不同的添加量(7U/L、17U/L、33U/L、66U/L、132U/L)加入至反应体系中；于25℃反应24h；通过超高效液相色谱质谱联用仪测定生物胺含量。酪胺降解结果如图5所示，随着重组酶Ls1b含量的增加，24h内酪胺的降解率从29.82％提升至41.24％。
(2)盐度及pH对重组多铜氧化酶对酪胺降解率的影响
反应体系为含100mg/L酪胺的磷酸盐缓冲液(pH 4.0/7.0)，其含有不同质量分数的NaCl(0％、2％、4％、6％、8％、10％)；将重组酶Ls1b以终浓度33U/L加到体系中25℃反应24h，检测酪胺含量，酪胺降解结果如图6所示，NaCl的存在同样对酶降解酪胺有激活作用，且含量越高，所述酶对酪胺的降解效果越好；当pH为4或7时，存在10％的NaCl可使酪胺降解率均达到60％以上，具体的，在pH4时，NaCl浓度为0％、2％、4％、6％、8％、10％时，降解率分别为12.01％、31.87％、47.87％、60.73％、62.71％、62.39％；在pH7时，NaCl浓度为0％、2％、4％、6％、8％、10％时，降解率分别为32.89％、45.63％、49.62％、61.64％、65.54％、66.82％。
(3)乙醇含量对重组多铜氧化酶对酪胺降解率的影响
反应体系为含100mg/L酪胺的磷酸盐缓冲液(pH 7.0)，其含有不同体积分数的乙醇(0％、10％、15％、20％)；将重组酶Ls1b以终浓度33U/L加到体系中25℃反应24h，检测酪胺含量，酪胺降解结果如7所示，乙醇会抑制部分酶活，在乙醇浓度为0％、10％、15％、15％时，酪胺的降解率分别为32.89％、26.15％、26.41、27.18％。
实施例5：重组多铜氧化酶在不同条件下对其他生物胺的降解效果
(1)按照实施例3或4的实施方式，分别在含有100mg/L色胺、苯乙胺、亚精胺、精胺的反应体系中，调节反应体系的pH为4.0或7.0，调节NaCl的质量分数为0％、2％、4％、6％、8％、10％。
反应24h后，结果如下表所示：
表1：盐度及pH对重组酶Ls1b降解生物胺的影响
(2)按照实施例3或4的实施方式，分别在含有100mg/L色胺、苯乙胺、亚精胺、精胺的反应体系中，pH为7、盐度为0，调节乙醇的体积分数为0％、10％、15％、20％。
反应24h后，结果如下表所示：
表2：乙醇含量对重组酶Ls1b降解生物胺的影响
实施例6：不同条件下的重组多铜氧化酶酶活
采用可见光吸收测定法，以ABTS为底物进行纯化后多铜氧化酶的酶活测试
(1)pH对重组多铜氧化酶酶活的影响
在重组酶Ls1b的最适反应温度下(25℃)，测定酶在不同pH条件下的酶活，以最高的酶活为100％，计算各pH条件下的相对酶活，确定最适反应pH。所述不同pH条件分别是指①20mmol/L磷酸氢二钠-柠檬酸缓冲液，pH 2.2～8.0；②20mmol/L甘氨酸-氢氧化钠缓冲液pH 9.0～10.0。
结果如图8所示，重组酶Ls1b的最适pH为3.0。
(2)氯化钠及乙醇对重组多铜氧化酶酶活的影响
将纯化后的多铜氧化酶与不同浓度的氯化钠溶液混合，在最适温度(25℃)、最适pH(pH为3.0)条件下测定酶活力。以未添加氯化钠时测得的酶活为100％，测定其相对酶活，以确定氯化钠对酶的活力的影响。
将纯化后的多铜氧化酶与不同体积分数的乙醇溶液混合，在最适温度(25℃)、最适pH(pH为3.0)条件下测定酶活力。以未添加乙醇溶液时测得的酶活为100％，测定其相对酶活，以确定对酶的活力的影响。
表3表明在以ABTS为底物时，NaCl对酶活的抑制作用明显，而在乙醇含量达到10％时，仍能保持70.6％的相对酶活。
表3：氯化钠及乙醇对重组酶Ls1b酶活的影响
实施例7：重组多铜氧化酶在含鱼肉的模拟体系中对生物胺的降解
模拟体系中新鲜鱼肉的质量分数为16％，NaCl的质量分数为6％，pH为7.0，添加生物胺的比例接近于腌鱼中的生物胺，其中，组胺200mg/L、尸胺200mg/L，酪胺、腐胺、色胺、苯乙胺、亚精胺和精胺均为50mg/L；将重组酶Ls1b以终浓度100U/L加至体系中15℃反应24h，检测生物胺含量，降解结果如图9所示，其中组胺降解效果最好，其余生物胺均有不同程度的降低，对组胺、酪胺、腐胺的降解率分别为28.1％、3.15％、9.86％。
实施例8：重组多铜氧化酶在含葡萄汁的模拟体系中对生物胺的降解
模拟体系中葡萄汁的体积分数为26％，乙醇的体积分数为10％，pH为4.0，添加生物胺的比例接近于葡萄酒中的生物胺，含量均为50mg/L；将重组酶Ls1b以终浓度100U/L加至体系中30℃反应24h，检测生物胺含量，降解结果如图9所示，酪胺的降解效果最好，降解率达到31.18％，其余胺有不同程度的降低，对酪胺、腐胺、尸胺的降解率分别为31.18％、17.07％、7.81％。
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
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Claims (10)
1.一种降解食品中生物胺的方法，其特征在于，利用来源于清酒乳杆菌(Lactobacillus sakei)的多铜氧化酶降解生物胺，所述多铜氧化酶的氨基酸序列如SEQID NO.1所示。
2.根据权利要求1所述的方法，其特征在于，将所述多铜氧化酶添加至含有组胺、酪胺、腐胺、色胺、苯乙胺、亚精胺、尸胺和精胺的体系中，所述生物胺的含量为50～200mg/L；所述酪胺、腐胺、色胺、苯乙胺、亚精胺、尸胺和精胺的含量优选为50～100mg/L。
3.根据权利要求1或2所述的方法，其特征在于，所述多铜氧化酶按照体系中每毫克生物胺添加不少于0.14U的量添加。
4.根据权利要求1～3任一所述的方法，其特征在于，反应体系中的乙醇含量为0～20％、氯化钠浓度为0～10％。
5.根据权利要求1～4任一所述的方法，其特征在于，在pH4.0～7.0、15～30℃下反应，反应时间不少于20h。
6.根据权利要求1～5任一所述的方法，其特征在于，所述食品包括但不限于腌鱼、葡萄酒、酱油、豆瓣酱。
7.氨基酸序列如SEQ ID NO.1所示的多铜氧化酶在降解生物胺中的应用。
8.根据权利要求7所述的应用，其特征在于，所述生物胺包括组胺、酪胺、腐胺、色胺、苯乙胺、亚精胺、尸胺和精胺；降解生物胺的反应体系中的生物胺含量为50～200mg/L。
9.含有如SEQ ID NO.2所示的核苷酸序列的表达载体、表达氨基酸序列如SEQ ID NO.1所示的多铜氧化酶的微生物细胞或氨基酸序列如SEQ ID NO.1所示的多铜氧化酶在制备降解生物胺的产品中的应用。
10.根据权利要求9所述的应用，其特征在于，所述产品包括但不限于生物酶复合剂。
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