CN1285730C - β-丙氨酸的生物合成方法 - Google Patents
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Abstract
本发明提供了一种β-丙氨酸的生物合成方法,特别是在微生物酶的作用下合成β-丙氨酸的合成方法,所述的方法是用可产生氨基化酶的微生物分别在含丙烯酸的种子培养基和含丙烯酸的发酵培养基中培养出相应的氨基化酶,将所述的氨基化酶加入转化液中,所述的转化液为含有1%~50%(g/ml)丙烯酸的氨水溶液,在酶的作用下合成β-丙氨酸,经脱氨、纯化得β-丙氨酸产品。这种方法用微生物酶作催化剂,反应效率高,且无污染;反应原料易得,步骤简单,成本低;产物纯度高。
Description
(一)技术领域
本发明涉及β-丙氨酸的生物合成方法,特别是在微生物酶的作用下合成β-丙氨酸的合成方法。
(二)背景技术
β-丙氨酸,又名β-氨基丙酸。1972年由Ross和Monroe在尿嘧啶的降解产物中发现。它是一种非蛋白质的氨基酸,是自然界中唯一的β型氨基酸。β-丙氨酸主要的生理活性作用是合成泛酸、辅酶A。现代医学研究发现,在哺乳动物的神经***中,它可作为大脑中神经传递者;具离子通道的激活作用;可治疗由组织缺氧引起的肝损伤。
在精细化工领域β-丙氨酸用于合成聚合β-丙氨酸,电镀缓冲剂,染料等生产。聚合β-丙氨酸在水处理工业中用作絮凝剂;在医药工业中,β-丙氨酸是可用作许多药物的中间体,例泛酸钙,系维生素b族物质,是多种代谢环节中,包括碳水化合物、蛋白质、脂肪及上皮功能维持正常所必需的辅酶a的组分部分。肌肽是一种十分有效的伤口愈合剂。N-(2,5-二氯-4-氰硫基苯)-β-丙氨酸是一种有效的抗真菌剂,β-丙氨酸具有较宽广的应用领域和市场前景。
目前β-丙氨酸采用化学合成法生产。化学合成法有三种工艺:(1)丙稀腈法:此法丙稀腈经与氨在二苯胺和叔丁醇溶液中反应,生产β-氨基丙腈,再进行碱解即得;此法的缺点是产物中存在有40%以上的NaCl和杂质,需要反复纯化,反应过程中产生的NH3污染大气;(2)β-氨基丙腈法:β-氨基丙腈与氢氧化钡反应生成β-氨基丙酸钡和氮气,通入CO2,钡盐析出,产生β-氨基丙酸,钡离子用树脂去除,此法的缺点是生产成本高;(3)琥珀酰亚胺降解法:琥珀酰亚胺在碱性氯酸钠溶液中生成β-氨基丙酸,反应液用盐酸调pH,用3倍量的95%乙醇使无机盐析出,滤液用4倍量的蒸馏水稀释,离子交换树脂交换,交换液脱色,浓缩结晶,此法需大量的乙醇,生产成本高,生产场地安全性要求高。总之化学合成法的原料、中间体及副产物有毒,对环境污染严重。
(三)发明内容
本发明的目的是提供一种β-丙氨酸的生物合成方法,以克服现有β-丙氨酸的化学合成方法对环境易造成污染且成本高的不足。
本发明达到发明目的所采用的技术方案是:
一种β-丙氨酸的生物合成方法,所述的方法是用可产生氨基化酶的微生物分别在含丙烯酸的种子培养基和含丙烯酸的发酵培养基中培养出相应的氨基化酶,将所述的氨基化酶加入转化液中,所述的转化液为含有1%~50%(g/ml)丙烯酸的氨水溶液,在酶的作用下合成β-丙氨酸,经脱氨、纯化得β-丙氨酸产品。转化液的pH保持在2.0~9.6,在20~80℃条件下搅拌转化反应2-40小时,然后转化液经脱氨、离子交换、纯化得到β-丙氨酸产品。
所述的转化反应器在密闭环境下进行。
所述的微生物为藤黄八叠球菌或大肠杆菌。
所述的种子培养基为:终浓度百分含量(g/ml)0.01~10%丙烯酸,0.1~10%牛肉膏,0.1~2.0%玉米浆,0.1~10%蛋白胨,0.01~0.1%KH2PO4,0.011~0.15%MgSO4,0.05~0.5%NaCl,调pH3.0~9.5;
所述的发酵培养基为:终浓度百分含量(g/ml)0.01~10%丙烯酸,0.1~2.0%玉米浆,0.1~10%蛋白胨,0.01~0.1%KH2PO4,0.011~0.15%MgSO4,0.05~0.5%NaCl,调pH3.0~9.5。
所述的转化液可以是氨水及下式之一:
①磷酸缓冲液;②柠檬酸缓冲液;③醋酸缓冲液;④蒸馏水。
通常选用0.2M磷酸缓冲液,pH5.7~8.0;0.1M柠檬酸缓冲液,pH3.0~6.6;0.2M醋酸缓冲液,pH3.6~5.8。
所述的方法按如下步骤进行:
(1)将藤黄八叠球菌或大肠杆菌菌种接入种子培养基中,25~40℃,150~300r/min摇床培养16~40小时,然后以1%~20%的接种量转入发酵培养基,20~60℃,100~300r/min发酵16~60小时;
(2)发酵液按1%~50%的比例加入转化液,转化液中丙稀酸浓度1%~50%(g/ml),用氨水控制转化液的pH为2.0~9.6,25~40℃下搅拌转化4~20小时,用高锰酸钾滴定丙烯酸含量,当丙烯酸量保持1~4小时不变时终止反应;
(3)反应后转化液经脱氨、离子交换,纯化得β-丙氨酸产品。
所述的所述的方法中的步骤(3)具体为:反应后转化液3000r/min25分钟,离心去除菌体,上清液减压脱氨浓缩,进行离子交换分离纯化,分步收集的液体用茚三酮显色,显色的收集液再脱色浓缩结晶,即可得β-丙氨酸产品。
所述的转化液为终浓度(g/ml)为0.03的丙烯酸,0.22×10-3的MgSO4.7H2O,余量为蒸馏水,用氨水调pH7.2~7.5。
所述的所述的方法按如下步骤进行:
(1)将藤黄八叠球菌或大肠杆菌菌种接入种子培养基中,30~35℃,200~300r/min摇床培养16~20小时,然后以1%~20%的接种量转入发酵培养基,30~35℃,150~300r/min发酵16~20小时;
(2)发酵液按1%~50%的比例加入转化液,转化液中丙稀酸浓度(g/ml)1%~50%,用氨水控制转化液的pH为4.0~8.0,30~35℃下搅拌转化6~10小时,用高锰酸钾滴定丙烯酸含量,当丙烯酸量保持1~4小时不变时终止反应。
所述方法按如下步骤进行:
(1)将藤黄八叠球菌或大肠杆菌菌种接入种子培养基中,30℃,200r/min摇床培养24小时,然后以1%~20%的接种量转入发酵培养基,30~35℃,200r/min发酵16~20小时;
(2)发酵液按1%~50%的比例加入转化液,30~35℃下搅拌转化8小时,每2小时测一次pH值,用氨水调至pH7.2~7.5,用高锰酸钾滴定丙烯酸含量,当丙烯酸量保持1~4小时不变时终止反应;
(3)反应后转化液3000r/min 25分钟,离心去除菌体,上清液减压脱氨浓缩,进行离子交换分离纯化,分步收集的液体用茚三酮显色,显色的收集液再脱色浓缩结晶,即可得β-丙氨酸产品。
上述步骤中,所述的种子培养基为:终浓度(g/ml)为1.1%的玉米浆,0.02%的MgSO4.7H2O,0.1%的KH2PO4,0.145%的氯化钠,0.5%的牛肉膏,121℃灭菌20min,0.8%的丙烯酸,余量为水,用氨水调pH7.0;所述的发酵培养基为:终浓度(g/ml)为1.1%的玉米浆,0.02%的MgSO4.7H2O,0.1%的KH2PO4,0.145%的氯化钠,121℃灭菌20min,0.8%的丙烯酸,余量为水,用氨水调pH7.0;所述的转化液为终浓度(g/ml)为0.03的丙烯酸,0.22×10-3的MgSO4.7H2O,余量为水,用氨水调pH7.2~7.5。
培养基中采用MgSO4.7H2O,可提高菌体产酶的活性。
本发明所述的β-丙氨酸的生物合成方法的有益效果主要体现在:(1)用微生物酶做催化剂,反应效率高,且无污染;(2)反应原料易得,步骤简单,成本低;(3)产物纯度高。
(四)附图说明
图1为α-丙氨酸标准样品高效液相色谱图;
图2为β-丙氨酸标准样品高效液相色谱图;
图3为实施例1制得β-丙氨酸样品高效液相色谱图;
图4为实施例1所得样品红外图谱;
图5为Aldrich Condensed Phase数据库对样品红外图谱的匹配图;
图6为β-丙氨酸标准样品核磁共振图谱;
图7为实施例1所得样品核磁共振图谱;
图8、图9为纸层析结果示意图。其中:a1、a2分别代表大肠杆菌产生的酶转化的结果;d1、d2分别代表藤黄八叠球菌产生的酶转化的结果;d3为实施例1发酵液离心后的上清液;α代表α-丙氨酸标样;β代表β-丙氨酸标样。
(五)具体实施方式
下面结合具体实施例对本发明作进一步描述:
实施例1:β-丙氨酸的生物合成
种子培养基:玉米浆 1.1g,MgSO4.7H2O 0.02g,KH2PO4 0.1g,氯化钠 0.145g,牛肉膏 0.5g,水 100mL 121℃灭菌 20min,丙烯酸 0.8g,氢氧化钠调 pH7.0;
发酵培养基:玉米浆 1.1g,MgSO4.7H2O 0.02g,KH2PO4 0.1g,氯化钠 0.145g,水 100mL 121℃灭菌 20min,丙烯酸 0.8g,氢氧化钠调pH7.0;
转化液:丙烯酸 15g,MgSO4.7H2O 0.11g,氨水调 pH7.5,0.2M磷酸缓冲液定容 500mL。
藤黄八叠球菌斜面菌种接入种子培养基,200r/min,30℃摇床培养24h。以20%的接种量转入发酵培养基,30℃,200r/min摇床培养18小时得发酵液。发酵液按50%的比例加入转化液,转化液中丙烯液浓度3%用氨水控制转化液的pH为7.5,为防止丙烯酸和氨水的挥发,转化反应器密闭,在30℃条件下搅拌转化8小时,反应后转化液3000r/min25分钟,离心去除菌体,上清液减压脱氨浓缩,进行离子交换分离纯化,分步收集的液体用茚三酮显色,显色的收集液再脱色浓缩结晶,得纯度90%的β-丙氨酸样品。转化率达60%。
实施例2:β-丙氨酸的生物合成
种子培养基:玉米浆 0.5g,MgSO4.7H2O 0.02g,KH2PO4 0.05g,氯化钠 0.10g,牛肉膏 0.8g,水 100mL 121℃灭菌 20min,丙烯酸 0.08g,盐酸调 pH6.5;
发酵培养基:玉米浆 0.5g,MgSO4.7H2O 0.02g,KH2PO4 0.05g,氯化钠 0.10g,水 100mL 121℃灭菌 20min,丙烯酸 0.08g,盐酸调 pH6.5;
转化液:丙烯酸 10g,MgSO4.7H2O 0.10g,氨水调 pH4.8,0.2M醋酸缓冲液定容 500mL。
藤黄八叠球菌斜面菌种接入种子培养基,100r/min,25℃摇床培养20h。以15%的接种量转入发酵培养基,25℃,100r/min摇床培养16小时。发酵液按40%的比例加入转化液,转化液中丙烯液浓度2%,用氨水控制转化液的pH为4.8,为防止丙烯酸和氨水的挥发,转化反应器密闭,在25℃条件下搅拌转化10小时,反应后转化液3000r/min 25分钟,离心去除菌体,上清液减压脱氨浓缩,进行离子交换分离纯化,分步收集的液体用茚三酮显色,显色的收集液再脱色浓缩结晶,得纯度95%的β-丙氨酸样品。转化率达54%。
实施例3:β-丙氨酸的生物合成
种子培养基:玉米浆 1.8g,MgSO4.7H2O 0.02g,KH2PO4 0.05g,氯化钠 0.45g,牛肉膏 6.5g,水 100mL 121℃灭菌 20min,丙烯酸 7.5g,盐酸调 pH5.5;
发酵培养基:玉米浆 1.8g,MgSO4.7H2O 0.02g,KH2PO4 0.05g,氯化钠 0.45g,水 100mL 121℃灭菌 20min,丙烯酸 7.5g,盐酸调 pH5.5;
转化液:丙烯酸 200g,MgSO4.7H2O 0.12g,氨水调 pH6.0,0.1M柠檬酸缓冲液定容 500mL。
大肠杆菌斜面菌种接入种子培养基,300r/min,55℃摇床培养18h。以15%的接种量转入发酵培养基,55℃,300r/min摇床培养16小时。发酵液按50%的比例加入转化液,转化液中丙烯液浓度45%,用氨水控制转化液的pH为6.0,为防止丙烯酸和氨水的挥发,转化反应器密闭,在55℃条件下搅拌转化3小时,反应后转化液3000r/min25分钟,离心去除菌体,上清液减压脱氨浓缩,进行离子交换分离纯化,分步收集的液体用茚三酮显色,显色的收集液再脱色浓缩结晶,得纯度80%的β-丙氨酸样品。转化率达50%。
实施例4:β-丙氨酸的生物合成
种子培养基:玉米浆 1.0g,MgSO4.7H2O 0.02g,KH2PO4 0.04g,氯化钠 0.30g,牛肉膏 8.5g,121℃灭菌 20min,丙烯酸 4.5g,水 100mL氢氧化钠调 pH9.0;
发酵培养基:玉米浆 1.0g,MgSO4.7H2O 0.02g,KH2PO4 0.04g,氯化钠 0.30g,121℃灭菌 20min,丙烯酸 4.5g,氢氧化钠调 pH9.0;
转化液:丙烯酸 90g,MgSO4.7H2O 0.10g,氨水调 pH9.0 蒸馏水定容 500mL。
大肠杆菌斜面菌种接入种子培养基,250r/min,20℃摇床培养50h。以20%的接种量转入发酵培养基,20℃,250r/min摇床培养45小时。发酵液按30%的比例加入转化液,转化液中丙烯液浓度18%,用氨水控制转化液的pH为8.5,为防止丙烯酸和氨水的挥发,转化反应器密闭,在20℃条件下搅拌转化36小时,反应后转化液3000r/min 25分钟,离心去除菌体,上清液减压脱氨浓缩,进行离子交换分离纯化,分步收集的液体用茚三酮显色,显色的收集液再脱色浓缩结晶,得纯度98%的β-丙氨酸样品。转化率达58%。
实施例5:β-丙氨酸含量测定
(1)高效液相色谱:
分别取实施例1所得冷冻干燥结晶样品、β-丙氨酸标准样品(1mg/ml)、a-丙氨酸标准样品,进行高效液相色谱分析:
柱型号:SB-804
柱温:30℃
梯度方式:恒流0.5ml/min
检测器:蒸发光
计算方法:面积归一法
实施例1所得冷冻干燥结晶样品色谱图如图3所示,β-丙氨酸标准样品色谱图如图2所示,a-丙氨酸标准样品色谱图如图1所示。结果分析分别见表1、表2、表3:
表1 a-丙氨酸标准样品高效液相色谱图分析结果
峰号 | 保留时间 | 峰高 | 峰面积 | 含量 |
1234567 | 0.1150.1980.4320.6400.7730.8650.965 | 1700.2502242.0833032.4172788.0002755.3332226.7503270.750 | 9173.48317262.81321173.76621139.2329991.2008519.96721364.021 | 0.1080.2030.2490.2490.1170.1000.251 |
89101112131415161718192021222324252627282930313233343536373839404142434445464748495051 | 1.1821.2481.3571.4071.5401.6151.6731.8901.9822.0482.1402.2232.6732.8403.0233.3233.4323.6483.9154.1824.3904.6825.2655.8486.0156.2326.5326.8156.8987.0987.2327.3827.7157.8488.0328.2328.5828.9159.2829.3989.6159.7829.98210.165 | 3619.9173740.5833751.1673987.6673523.0003364.2504364.3334975.5005831.9175970.5835566.0005668.83315945.03820433.11512299.4009029.1399028.48910352.18814876.5128518.8354813.432358473.06348932.3365542.6055505.6825706.3825929.1215376.6526045.6905652.1835115.8444447.7135312.8684530.5294402.8143960.3063259.6683930.8224098.3914061.8453826.5453313.6223736.1153137.399 | 29223.50012792.46718715.04516780.63721033.5189596.65031716.25034103.24629109.28730176.57818443.57046070.26699602.148258071.953195660.09446897.67256445.047119731.188313796.50078775.02315380.4414979341.000871589.50068417.61724397.49220933.06130912.62577384.39147495.65643641.21140899.23454353.41465990.92240760.83235346.09472598.39125392.61539040.23463817.64138422.93835873.44134588.53921026.47954667.484 | 0.3440.1500.2200.1970.2470.1130.3730.4010.3420.3550.2170.5421.1713.0342.3000.5510.6641.4083.6890.9260.18158.54410.2480.8040.2870.2460.3630.9100.5580.5130.4810.6390.7760.4790.4160.8540.2990.4590.7500.4520.4220.4070.2470.643 |
5253545556 | 10.63210.89811.53211.69812.065 | 2731.2151963.5382010.4312279.5081601.778 | 29320.20725650.38732923.19520862.55518898.500 | 0.3450.3020.3870.2450.222 |
表2 β-丙氨酸标准样品高效液相色谱图分析结果
峰号 | 保留时间 | 峰高 | 峰面积 | 含量 |
123456789101112131415161718192021222324252627282930313233343536 | 0.1480.2820.3400.4320.6070.7320.8150.9071.0981.1821.2651.4901.6401.7151.8401.9902.0482.1902.7983.1983.2653.3733.5323.8654.2324.3654.5824.8154.9155.1825.7655.9326.1656.4326.6826.848 | 1620.6051628.5601543.1653639.2593168.0752634.6582313.3803570.4743372.0343394.7564131.4783742.5264657.2264494.5753825.1584783.8576351.4625200.79025601.00011669.00012421.0009851.00015168.00052416.00013667.0009453.00020791.00013829.00013089.000359788.00018524.00019685.00018268.00016684.00016339.00015023.000 | 7669.3914879.6404407.23026543.02915925.93113466.7668296.31824290.53920468.03716484.27932089.50429959.57025467.47329704.07218772.08425627.81148346.85933594.852542744.81356640.89877564.29736196.500186700.500893984.62590115.20332023.500341163.59469644.89857102.0005718041.000145309.000253335.094267904.906136319.203219523.094182350.703 | 0.0510.0330.0290.1770.1060.0900.0550.1620.1370.1100.2140.2000.1700.1980.1250.1710.3230.2243.6230.3780.5180.2421.2465.9670.6010.2142.2770.4650.38138.1670.9701.6911.7880.9101.4651.217 |
373839404142434445464748495051525354555657 | 7.0987.4157.6827.8828.1488.4828.6488.8329.1159.3329.5489.74810.06510.49810.76510.93211.19811.56513.13213.26515.098 | 13854.00011235.0008414.0007673.0006974.0005290.0004604.0005878.0005856.0005525.0005619.0006327.0007273.0007218.0007322.0008172.0007430.0008074.00024336.00025553.0003757.000 | 281462.406132027.79758167.602102299.79787284.89870697.79742188.89862213.89882054.20341471.60265698.39861195.699133415.703194430.59453504.602112392.89875472.703147993.2031533847.3751915890.50035430.199 | 1.8790.8810.3880.6830.5830.4720.2820.4150.5480.2770.4390.4080.8911.2980.3570.7500.5040.98810.23812.7880.236 |
表3 实施例1所得冷冻干燥结晶样品色谱图分析结果
峰号 | 保留时间 | 峰高 | 峰面积 | 含量 |
1234567891011121314151617181920 | 0.1480.2480.3650.5400.6230.7320.8480.8731.0401.1821.3151.3981.5231.6151.7821.8821.9482.0402.1072.182 | 1773.279263.1172013.0942494.0602865.7593419.1662730.1433493.8534184.2494889.9363437.0533782.7514666.2984304.5663579.9624538.8004830.3585244.6265127.1854870.313 | 4766.188611.05315522.6439551.31511462.49217714.9734609.99217039.36329474.18825402.73219189.89315066.44637167.72728442.96118023.63522384.16019291.04522746.88322914.97338164.719 | 0.1510.0190.4930.3030.3640.5620.1460.5410.9360.8060.6090.4781.1800.9030.5720.7110.6120.7220.7271.211 |
2122232425262728293031323334353637383940414243444546474849505152535455565758596061 | 2.6652.8153.1233.2153.3323.5573.8903.9574.1404.3574.5984.9235.1985.6485.8325.9986.1326.4156.7486.9487.2157.4657.6827.9828.1158.3158.4488.5988.8159.0829.2159.4159.69810.03210.26510.46510.66511.13211.63211.99812.132 | 17708.12121484.36110663.0788990.6698201.96810006.32819144.75217696.6379008.8205270.94518404.7774441.96640545.7385116.4623115.6453035.3572960.1262715.1363114.5602982.2143223.7533376.8212475.9462393.4282250.1972179.8512509.6211820.8612087.9872143.5262031.2952903.9492247.9591954.3831828.980752.6341785.2881058.7141392.5711212.1541277.385 | 120517.172304087.43880574.32059610.18055128.859130379.930196313.453144151.93877547.38322487.828298696.21918031.266652815.18842712.15215211.27126278.14525354.78347526.85221373.90039188.75827249.03935140.61345255.88711981.14316028.44115155.61310761.12512903.80328550.7649677.63011242.43725617.55329371.54322529.95318145.2755603.71516379.52315166.05716915.74213116.9235894.876 | 3.8269.6532.5581.8921.7504.1396.2324.5762.4620.7149.4820.57220.7231.3560.4830.8340.8051.5090.6781.2440.8651.1151.4370.3800.5090.4810.3420.4100.9060.3070.3570.8130.9320.7150.5760.1780.5200.4810.5370.4160.187 |
(2)红外光谱
取实施例1所得样品进行红外光谱分析,红外图谱如图4所示,图5为Aldrich Condensed Phase数据库对样品红外图谱的匹配图;分析结果见表4:
表4 实施例1所得样品红外分析结果
指数(index) | 配合度(match) | 化合物名称(Compound Name) | |
1 | 9821 | 86.55 | β-丙氨酸 |
2 | 9399 | 54.13 | 醋酸铯 |
3 | 9918 | 51.48 | L-胱氨酸 |
4 | 2092 | 48.91 | 醋酸四乙基铵 |
5 | 9916 | 47.98 | 甲硫氨酸 |
6 | 9730 | 46.31 | 巯基醋酸钠盐 |
7 | 9834 | 46.13 | L-亮氨酸 |
8 | 9783 | 45.64 | 硫代苹果酸钠 |
9 | 9836 | 45.60 | L-异亮氨酸 |
10 | 9833 | 45.60 | D-亮氨酸 |
(3)核磁共振谱
分别取实施例1所得样品、β-丙氨酸标准样品进行核磁共振谱检测;β-丙氨酸标准样品核磁共振图谱如图6所示,实施例1所得样品核磁共振图谱如图7所示。
(4)纸层析
分别取实施例1~4所得样品进行纸层析,以α-丙氨酸和β-丙氨酸标准样品为对照,实验结果见图8、图9。其中:a1、a2分别代表实施例3、4大肠杆菌产生的酶转化的结果;d1、d2分别代表实施例1、2藤黄八叠球菌产生的酶转化的结果;d3为实施例1发酵液离心后的上清液,α代表α-丙氨酸标样;β代表β-丙氨酸标样。结果表明a1、a2中含β-丙氨酸,d1、d2中也含β-丙氨酸,而d3中不含β-丙氨酸。
(5)结论:
根据以上检测数据,分析表明所制得产物成分为β-丙氨酸。
Claims (5)
1.一种β-丙氨酸的生物合成方法,其特征在于所述的方法按如下步骤进行:
(1)将藤黄八叠球菌或大肠杆菌菌种接入种子培养基中,25~40℃,150~300r/min摇床培养16~40小时,然后以1~20%的接种量转入发酵培养基,20~60℃,100~300r/min发酵16~60小时,得发酵液;所述的种子培养基为:终浓度百分含量以g/ml计为:0.01~10%丙烯酸,0.1~10%牛肉膏,0.1~2.0%玉米浆,0.1~10%蛋白胨,0.01~0.1%KH2PO4,0.011~0.15%MgSO4,0.05~0.5%NaCl,调pH 3.0~9.5;所述的发酵培养基为:终浓度百分含量以g/ml计为:0.01~10%丙烯酸,0.1~2.0%玉米浆,0.1~10%蛋白胨,0.01~0.1%KH2PO4,0.011~0.15%MgSO4,0.05~0.5%NaCl,调pH 3.0~9.5;
(2)发酵液按1%~50%的比例加入转化液,转化液中丙稀酸浓度1%~50%g/ml,用氨水控制转化液的pH为2.0~9.6,25~40℃下搅拌转化4~20小时,用高锰酸钾滴定丙烯酸含量,当丙烯酸量保持1~4小时不变时终止反应,所述的转化反应在密闭环境下进行;
(3)反应后转化液经脱氨、离子交换,纯化得β-丙氨酸产品。
2.如权利要求1所述的β-丙氨酸的生物合成方法,其特征在于所述的方法中的步骤(3)为:反应后转化液3000r/min 25分钟,离心去除菌体,上清液减压脱氨浓缩,进行离子交换分离纯化,分步收集的液体用茚三酮显色,显色的收集液再脱色浓缩结晶,即得β-丙氨酸产品。
3.如权利要求1所述的β-丙氨酸的生物合成方法,其特征在于所述的转化液为终浓度为0.03g/ml的丙烯酸,0.22×10-3的MgSO4.7H2O,余量为蒸馏水,用氨水调pH7.2~7.5。
4.如权利要求1所述的β-丙氨酸的生物合成方法,其特征在于所述的方法按如下步骤进行:
(1)将藤黄八叠球菌或大肠杆菌菌种接入种子培养基中,30~35℃,200~300r/min摇床培养16~20小时,然后以1~20%的接种量转入发酵培养基,30~35℃,150~300r/min发酵16~20小时;
(2)发酵液按1%~50%的比例加入转化液,转化液中丙稀酸浓度1%~50%g/ml,用氨水控制转化液的pH为4.0~8.0,30~35℃下搅拌转化6~10小时,用高锰酸钾滴定丙烯酸含量,当丙烯酸量保持1~4小时不变时终止反应。
5.如权利要求3所述的β-丙氨酸的生物合成方法,其特征在于所述方法按如下步骤进行:
(1)将藤黄八叠球菌或大肠杆菌菌种接入种子培养基中,30℃,200r/min摇床培养24小时,然后以1%~20%的接种量转入发酵培养基,30~35℃,200r/min发酵16~20小时;
(2)发酵液按1%~50%的比例加入转化液,30~35℃下搅拌转化8小时,每2小时测一次pH值,用氨水调至pH7.2~7.5,用高锰酸钾滴定丙烯酸含量,当丙烯酸量保持1~4小时不变时终止反应;
(3)反应后转化液3000r/min 25分钟,离心去除菌体,上清液减压脱氨浓缩,进行离子交换分离纯化,分步收集的液体用茚三酮显色,显色的收集液再脱色浓缩结晶,即可得β-丙氨酸产品;
所述的种子培养基为:终浓度以g/ml计为1.1%的玉米浆,0.02%的MgSO4.7H2O,0.1%的KH2PO4,0.145%的氯化钠,0.5%的牛肉膏,121℃灭菌20min,0.8%的丙烯酸,余量为水,用氨水调pH7.0;
所述的发酵培养基为:终浓度以g/ml计为1.1%的玉米浆,0.02%的MgSO4.7H2O,0.1%的KH2PO4,0.145%的氯化钠,121℃灭菌20min,0.8%的丙烯酸,余量为水,用氨水调pH7.0;
所述的转化液为终浓度为0.03g/ml的丙烯酸,0.22×10-3的MgSO4.7H2O,余量为水,用氨水调pH7.2~7.5。
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