CN105603007B - 一种微生物转化的方法 - Google Patents
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- 229960005190 phenylalanine Drugs 0.000 claims abstract description 42
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Abstract
本发明涉及采用摩根菌属微生物转化L‑苯丙氨酸生产R‑(+)‑2‑羟基‑3‑苯基丙酸。该方法过程简单，具有重要的工业应用价值。
Description
技术领域
本发明采用摩根菌转化L-苯丙氨酸生产D-苯乳酸，属于工业微生物领域。
背景技术
D-苯乳酸，学名R-(+)-2-羟基-3-苯基丙酸，英文名为：R-(+)-2-hydroxy-3-phenylpropanoic acid、D-(+)-3-Phenyllactic acid。
苯乳酸是近年来发现可以由部分乳酸菌分泌产生的一种新型生物防腐剂。它有D-苯乳酸和L-苯乳酸两种对映异体体，能够有效抑制多种引起食物腐败的细菌及产生毒素的真菌。研究表明D-苯乳酸的抑菌能力略高于L-苯乳酸。
作为一种新型生物防腐剂，苯乳酸在食品工业中具重要的应用价值。当前主要通过大肠基因工程菌表达乳酸脱氢酶转化苯丙酮酸生产，如中国专利CN201410818165.X、CN201410393768.X和CN201210338378.3。也有通过各类乳酸菌转化苯丙酮酸或苯丙氨酸生产，如中国专利CN201510089823.0、CN201210005544.8等。也有采用芽孢杆菌转化苯丙酮酸产苯乳酸的方法(Efficient Conversion of Phenylpyruvic Acid to PhenyllacticAcid by Using Whole Cells of Bacillus coagulans SDM，2011，PLoS One.6(4):e19030)。
基于D-苯乳酸的重要应用价值，本专利提出了采用摩根菌属微生物转化L-苯丙氨酸生产D-苯乳酸的方案。L-苯丙氨酸当前主要通过微生物发酵法生产，原料丰富易得。
摩根菌(Morganella)是一类可使苯丙氨酸氧化脱氨的革兰氏阴性细菌。现有2个种和2个亚种：摩根摩根菌(Morganella morganii)、耐冷摩根菌(Morganellapsychrotolerans)、摩根摩根菌摩根亚种(Morganella morganii subsp.morganii)和摩根摩根菌西伯尼亚种(Morganella morganii subsp.Sibonii subsp.)。摩根菌具有强大的氧化脱氨能力(Sherris Medical Microbiology，2004，4th ed.)，L-苯丙氨酸可以被摩根菌氧化脱氨成苯丙酮酸。
相关文献认为这些微生物只能将L-苯丙氨酸氧化成苯丙酮酸，而无法进一步还原成苯乳酸(α-keto acids are novel siderophores in the genera proteus,providencia,and morganella and are produced by amino acid deaminases，1993，Journal of bacteriology,175(9),2727-2733)，这可能与之条件选择不当有关。
本发明可通过摩根菌属微生物将L-苯丙氨酸转化成光学纯的D-苯乳酸。苯丙酮酸虽然是更为直接的底物，但价格较高，因此L-苯丙氨酸是最佳的底物。摩根菌是一种兼性厌氧菌，可以在厌氧条件或好氧条件下转化生产D-苯乳酸，具有高转化率和高纯度的特点。
发明内容
本发明通过培养摩根菌属微生物菌体，转化L-苯丙氨酸生产D-苯乳酸，技术方案如下：
1、菌株
本发明所用的菌株有购自美国ATCC菌种库的Morganella morganiisubsp.sibonii ATCC 49948、Morganella morganii subsp.morganii ATCC 25830以及购自中国工业微生物菌种保藏管理中心的Morganella morganii CICC 21517和购自BCCM/LMG Bacteria Collection的Morganella psychrotolerans LMG 23374。
2、菌体培养
液态发酵培养基组成为：碳源0-50g/L，氮源0-50g/L，磷酸氢二铵0-2g/L，磷酸二氢钾0-1g/L，硫酸镁0-0.5g/L，硫酸亚铁0-0.5g/L，氯化纳0-5g/L，可调节pH至2-8，可也pH自然；接种量为5-20％，发酵温度为20-40℃，发酵周期为24-72小时。种子和发酵均采用此培养基。
用于培养菌种的碳源可以是葡萄糖、果糖、麦芽糖、木糖、蔗糖、半乳糖、甘油。培养用氮源可以是硫酸铵、氯化铵、硝酸铵、蛋白胨、酵母膏、尿素、玉米浆等有机氮源。碳源和氮源可以是一种或多种的组合。
液态培养菌体的过程可以采用厌氧或好氧方式。
3、转化产D-苯乳酸
转化过程采用的方案有2种：
(1)细胞的液态培养过程，将L-苯丙氨酸底物加入上述的培养体系中；L-苯丙氨酸添加量为0-20g/L。
(2)将液态发酵培养物离心去除发酵液得到菌体，将菌体放入含有L-苯丙氨酸底物的水溶液反应体系中进行；转化过程温度20-40℃，pH 2-8，转化时间1-24小时。转化液中L-苯丙氨酸起始浓度为0.1-20g/L。
4、样品的检测分析
转化液采用PerkinElmer Series 200高效液相色谱仪检测分析，色谱条件为：流动相是甲醇-0.1％甲酸水(40:60)、采用汉邦Megres C18色谱柱(4.6×250mm，5μm)，流速0.6ml/min、柱温30℃、进样量20μl、检测器波长200nm。
采用江苏汉邦科技有限公司的DAC-HB50制备色谱柱制备转化样品，制备色谱条件为：流动相50％甲醇、柱温自然、流速3ml/min、进样量5ml。样品达到色谱纯99.9％，反复进样分离得到的产品于50℃下真空旋转蒸干。称取制备得到的样品0.5g，溶于去离子水中并定容至50ml，采用日本爱宕AP-300全自动旋光仪测定放光度。进一步采Varian Gemini2000(VnmrS 600MHz,600/54/ASP)分析样品的核磁数据。样品进一步采用UPLC-QTOF-MS法分析分子量，仪器为Waters MALDI SYNAPT QTOF MS液相色谱串联四极杆飞行时间质谱仪。
具体实施方式
实施例1
转化产物的分析测定。
配制培养基如下：酵母膏5g/L，蛋白胨10g/L，氯化钠5g/L。500ml三角瓶中装液量为100ml，共50瓶，120℃，20分钟灭菌。取Morganella morganii CICC 21517甘油管种子液1mL接种，发酵温度30℃，摇床转速200rpm。培养24后，将发酵液离心(转速10000rpm，时间10min)，去除上清液获得菌体，离心获得的菌体用无菌水清洗两遍。取20g湿菌体放入L-苯丙氨酸浓度为10g/L的1000ml溶液中，混合均匀。于37℃摇床振荡(100rpm)24小时后，100℃水浴加热3分钟杀灭菌体。再离心(转速10000rpm，时间10min)去除菌体，得到转化后的上清液。液相分析D-苯乳酸浓度为2.1g/L，剩余L-苯丙氨酸浓度为5.2g/L。采用制备色谱得到1.3g纯品。
旋光仪分析其旋光度为核磁数据为:1H NMR(CDCl3 600MHz):δ7.31(2H,d J 7.6Hz),7.29(1H,d,J 7.6Hz),7.24(2H,d,J 7.6Hz)，4.491H,dd,J 4.3,7.3Hz)，4.27(2H,OHs,broad)，3.21(1H,dd,J 4.3,14.0Hz)，2.96(1H,dd,J 7.3,14.0Hz)。13C NMR(CDCl3,600MHz):δ177.57，135.93，129.50，129.54，128.56，128.63，127.13，71.15，40.11。转化产物的质谱数据为：(-ESI,negative mode)m/z:165.0[M-1]。
根据以上数据，确定其分子及光学结构与D-苯乳酸完全一致。
实施例2
好氧培养与厌培养的比较。配制培养基：葡萄糖30g/L，蛋白胨20g/L，磷酸氢二铵1g/L，磷酸二氢钾1g/L，硫酸镁0.3g/L，硫酸亚铁0.3g/L；起始pH和发酵过程pH均为自然500ml三角瓶中装液量为100ml，共2瓶，120℃，20分钟灭菌。
取Morganella morganii subsp.sibonii ATCC 49948甘油管种子液1mL接种1瓶，于厌氧培养箱中35℃培养72小时，离心获得的菌体用无菌水清洗两遍。取0.1g湿菌体放入L-苯丙氨酸浓度为2g/L的4ml溶液中，混合均匀，厌氧培养箱中35℃静置转化24小时后，100℃水浴加热3分钟杀灭菌体。再离心(转速10000rpm，时间10min)去除菌体，得到转化后的上清液。液相分析D-苯乳酸浓度为1.1g/L，剩余L-苯丙氨酸浓度为0.3g/L。
取Morganella morganii subsp.sibonii ATCC 49948甘油管种子液1mL接种1瓶，于摇床(200rpm)中35℃培养24小时。将发酵液离心(转速10000rpm，时间10min)，去除上清液获得菌体，离心获得的菌体用无菌水清洗两遍。取0.1g湿菌体放入L-苯丙氨酸浓度为2g/L的4ml溶液中，混合均匀。于35℃摇床振荡(100rpm)12小时后，100℃水浴加热3分钟杀灭菌体。再离心(转速10000rpm，时间10min)去除菌体，得到转化后的上清液。液相分析D-苯乳酸浓度为1.0g/L，剩余L-苯丙氨酸浓度为0.4g/L。
实施例3
培养基组成为：葡萄糖1g/L，尿素1g/L，磷酸氢二铵1g/L，磷酸二氢钾0.1g/L，硫酸镁0.1g/L，硫酸亚铁0.1g/L。250ml三角瓶中装液量为50ml，120℃，20分钟灭菌。取Morganella morganii subsp.sibonii ATCC 49948甘油管种子液0.5mL接种，发酵温度35℃，摇床转速200rpm。培养24后，将发酵液离心(转速10000rpm，时间10min)，去除上清液获得菌体，离心获得的菌体用无菌水清洗两遍。取0.05g湿菌体放入L-苯丙氨酸浓度为0.1g/L的2ml溶液中，并调节pH至6，混合均匀。于厌氧培养箱中静止转化24小时，微孔滤膜过滤转化液，液相色谱分析转化液中D-苯乳酸浓度为0.02g/L及L-苯丙氨酸残留量为0.03g/L。
实施例4
培养基组成为：果糖50g/L，硫酸铵5g/L，磷酸二氢钾1g/L，硫酸镁0.5g/L，硫酸亚铁0.5g/L。250ml三角瓶中装液量为50ml，120℃，20分钟灭菌。取Morganella morganiisubsp.morganii ATCC 25830甘油管种子液0.5mL接种，发酵温度20℃，摇床转速200rpm。培养72后，将发酵液离心(转速10000rpm，时间10min)，去除上清液获得菌体，离心获得的菌体用无菌水清洗两遍。取0.1g湿菌体放入L-苯丙氨酸浓度为2g/L的2ml溶液中，混合均匀。于20℃摇床振荡(100rpm)24小时后，微孔滤膜过滤转化液，液相色谱分析转化液中D-苯乳酸浓度为0.8g/L及L-苯丙氨酸残留量为0.3g/L。
实施例5
培养基组成为：木糖50g/L，硫酸铵1g/L，磷酸氢二铵1g/L，磷酸二氢钾1g/L，硫酸镁0.2g/L。250ml三角瓶中装液量为50ml，120℃，20分钟灭菌。取Morganella morganiisubsp.morganii ATCC 25830甘油管种子液0.5mL接种，发酵温度40℃，摇床转速200rpm。培养48后，将发酵液离心(转速10000rpm，时间10min)，去除上清液获得菌体，离心获得的菌体用无菌水清洗两遍。取0.1g湿菌体放入L-苯丙氨酸浓度为2g/L的2ml溶液中，混合均匀。于40℃摇床振荡(100rpm)24小时后，微孔滤膜过滤转化液，液相色谱分析转化液中D-苯乳酸浓度为0.3g/L及L-苯丙氨酸残留量为1.5g/L。
实施例6
培养基组成为：甘油15g/L，硝酸铵1g/L，磷酸氢二铵1g/L，磷酸二氢钾0.1g/L，硫酸镁0.1g/L，硫酸亚铁0.1g/L。250ml三角瓶中装液量为50ml，120℃，20分钟灭菌。取Morganella psychrotolerans LMG 23374甘油管种子液0.5mL接种，发酵温度30℃，摇床转速200rpm。培养24后，将发酵液离心(转速10000rpm，时间10min)，去除上清液获得菌体，离心获得的菌体用无菌水清洗两遍。取0.1g湿菌体放入L-苯丙氨酸浓度为2g/L的2ml溶液中，混合均匀。于20℃厌氧培养箱中静止转化24小时，微孔滤膜过滤转化液，液相色谱分析转化液中D-苯乳酸浓度为0.9g/L及L-苯丙氨酸残留量为0.7g/L。
实施例7
培养基组成为：麦芽糖15g/L，氯化铵1g/L，磷酸氢二铵1g/L，磷酸二氢钾0.1g/L，硫酸镁0.1g/L，硫酸亚铁0.1g/L，调节pH至5。250ml三角瓶中装液量为50ml，共10瓶，120℃，20分钟灭菌。取Morganella psychrotolerans LMG 23374甘油管种子液0.5mL接种，发酵温度10℃，摇床转速200rpm。培养24后，将发酵液离心(转速10000rpm，时间10min)，去除上清液获得菌体，离心获得的菌体用无菌水清洗两遍。取1g湿菌体放入L-苯丙氨酸浓度为20g/L的2ml溶液中，混合均匀。于40℃厌氧培养箱中静止转化24小时，微孔滤膜过滤转化液，液相色谱分析转化液中D-苯乳酸浓度为7.2g/L及L-苯丙氨酸残留量为9.1g/L。
实施例8
培养基组成为：半乳糖1g/L，尿素1g/L，磷酸氢二铵1g/L，磷酸二氢钾0.1g/L，硫酸亚铁0.1g/L，调节pH至2。250ml三角瓶中装液量为50ml，120℃，20分钟灭菌。取Morganellamorganii CICC 21517甘油管种子液0.5mL接种，发酵温度35℃，摇床转速200rpm。培养24后，将发酵液离心(转速10000rpm，时间10min)，去除上清液获得菌体，离心获得的菌体用无菌水清洗两遍。取0.1g湿菌体放入L-苯丙氨酸浓度为2g/L的2ml溶液中，并调节pH至2，混合均匀。于37℃厌氧培养箱中静止转化24小时，微孔滤膜过滤转化液，液相色谱分析转化液中D-苯乳酸浓度为0.4g/L及L-苯丙氨酸残留量为1.4g/L。
实施例9
培养基组成为：葡萄糖1g/L，蛋白胨1g/L，磷酸氢二铵1g/L，硫酸镁0.1g/L，硫酸亚铁0.1g/L，L-苯丙氨酸20g/L调节pH至8。250ml三角瓶中装液量为50ml，120℃，20分钟灭菌。取Morganella morganii CICC 21517甘油管种子液0.5mL接种，发酵温度35℃，摇床转速200rpm。培养24后，微孔滤膜过滤转化液，液相色谱分析转化液中D-苯乳酸浓度为8.1g/L及L-苯丙氨酸残留量为8.2g/L。
实施例10
培养基组成为：葡萄糖1g/L，尿素1g/L，磷酸氢二铵1g/L，磷酸二氢钾0.1g/L，硫酸镁0.1g/L，硫酸亚铁0.1g/L，调节pH至8。500ml三角瓶中装液量为100ml，120℃，20分钟灭菌，共10瓶。各取Morganella morganii CICC21517甘油管种子液0.5mL接种，发酵温度35℃，摇床转速200rpm。培养24后，将发酵液离心(转速10000rpm，时间10min)，去除上清液获得菌体，离心获得的菌体用无菌水清洗两遍。取5g湿菌体放入L-苯丙氨酸浓度为3g/L的10ml溶液中，并调节pH至5，混合均匀。于35℃摇床振荡(100rpm)1小时后，微孔滤膜过滤转化液，液相色谱分析转化液中D-苯乳酸浓度为0.8g/L及L-苯丙氨酸残留量为1.3g/L。
实施例11
培养基组成为：葡萄糖10g/L，蛋白胨1g/L，磷酸氢二铵1g/L，磷酸二氢钾0.1g/L，硫酸镁0.1g/L，硫酸亚铁0.1g/L，pH至5。250ml三角瓶中装液量为50ml，120℃，20分钟灭菌。取Morganella morganii subsp.sibonii ATCC49948甘油管种子液0.5mL接种，发酵温度35℃，摇床转速200rpm。培养24后，微孔滤膜过滤转化液，液相色谱分析发酵液中D-苯乳酸浓度为0.01g/L。
Claims (4)
1.一种转化L-苯丙氨酸生产R-(+)-2-羟基-3-苯基丙酸的方法，其特征在于，所述方法是利用摩根菌属微生物进行转化生产；所述摩根菌属微生物为保藏编号分别为CICC21517、ATCC 49948、ATCC 25830、LMG 23374的菌株；
转化过程采用的步骤如下：
(1)细胞的液态培养过程，将L-苯丙氨酸底物加入培养体系中；L-苯丙氨酸添加量为0-20g/L；
(2)将液态发酵培养物离心去除发酵液得到菌体，将菌体放入含有L-苯丙氨酸底物的水溶液反应体系中进行；转化过程温度为20-40℃，pH为2-8，转化时间为1-24小时；转化液中L-苯丙氨酸起始浓度为0.1-20g/L。
2.根据权利要求1所述的方法，其特征在于，所述摩根菌属微生物的培养过程能够在厌氧状态也能够在好氧状态进行。
3.根据权利要求2所述的方法，其特征在于，培养得到的菌体，其转化L-苯丙氨酸能够在厌氧状态也能够在好氧状态进行。
4.根据权利要求2所述的方法，其特征在于，菌体培养过程中能够直接加入L-苯丙氨酸边长菌体边转化生产R-(+)-2-羟基-3-苯基丙酸。
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