CN1304582C - 一种外源添加反丁烯二酸促进微生物合成1,3-丙二醇的方法 - Google Patents
一种外源添加反丁烯二酸促进微生物合成1,3-丙二醇的方法 Download PDFInfo
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Abstract
本发明提供了一种外源添加反丁烯二酸促进微生物合成1,3-丙二醇的方法,属于生物化工技术领域。本发明包括:培养基制备,种子培养,及发酵产1,3-丙二醇的步骤;其特征在于:在发酵培养基中添加反丁烯二酸,浓度为5~25毫摩尔/升;添加时机选择在菌种进行发酵的对数生长期。所添加的反丁烯二酸是一种外源电子受体。本发明适用于1,3-丙二醇的厌氧及有氧发酵过程。本发明的优点在于:可加速菌体对甘油的利用,显著提高1,3-丙二醇浓度和生产强度,降低生产成本。
Description
技术领域
本发明属于生物化工技术领域,特别是提供了一种外源添加反丁烯二酸促进微生物合成1,3-丙二醇的方法。
背景技术
1,3-丙二醇(简称PDO)是一种重要的化工原料,可作为有机溶剂应用于油墨、印染、涂料、润滑剂、抗冻剂等行业。1,3-丙二醇最主要的用途是作为聚酯和聚氨酯合成的单体,特别是与对苯二甲酸聚合生成的聚对苯二甲酸丙二酯(PTT),显示了比以1,2-丙二醇、丁二醇、乙二醇为单体合成的聚合物更优良的性能。目前全球每年消费数千万吨聚对苯二甲酸乙二酯(PET),而PTT的化学稳定性、生物可降解性等与PET相当,但耐污染性、韧性和回弹性及抗紫外性能等更优越。此外PTT纤维还具有耐磨、吸水性低、低静电等优点,可在地毯领域与尼龙竞争。它还可用于具有优良性能的无纺布、工程塑料、服装、家庭装饰、垫衬料、织物等方面。PTT被评为美国98年六大石化新产品之一,被认为将是PET的升级产品。
PTT的优越性能及市场潜力早在50年前就被人们所认识,只因原料1,3-丙二醇生产技术难度大、成本高而导致PTT很难大规模工业化生产,迄今为止,只有Dupont和Shell两家跨国公司采用传统的化学合成路线,以环氧乙烷或丙烯为原料生产仅供它们合成PTT自用的1,3-丙二醇。化学合成法的缺点是副产物多,选择性差,操作条件需高温高压,设备投资巨大,原料为不可再生资源,且环氧乙烷和另一路线的中间产物丙烯醛分别是易燃易爆或剧毒的危险品。由于发酵法生产1,3-丙二醇选择性高,操作条件温和,因此近年来受到特别的重视。
生物合成法生产1,3-丙二醇是利用微生物歧化甘油产生。自然界中可将甘油转化为1,3-丙二醇的微生物主要是厌氧或兼性厌氧菌,其中克雷伯氏肺炎杆菌(Klebsiella pneumoniae)、丁酸梭状芽胞杆菌(Clostridium butyricum)及弗氏柠檬酸菌(Citrobacter freundii)具有较高的1,3-丙二醇转化率,而且对甘油和产物1,3-丙二醇具有较高的耐受力,因此具有较高的开发价值与应用前景。微生物代谢甘油产1,3-丙二醇主要由二羟丙酮(dha)操纵子调控,其代谢路线分为氧化支路和还原支路两条分支:在氧化途径中,甘油在依赖NAD+的甘油脱氢酶(GDH)的催化作用下去氢转变为二羟丙酮(DHA),二羟丙酮在二羟丙酮激酶的作用下磷酸化为磷酸二羟丙酮,进而进入酵解途径产生ATP、NADH以及乙醇、乙酸、乳酸等代谢副产物;在还原途径中,甘油在甘油脱水酶(GDHt)的作用下转变为3-羟基丙醛,然后利用氧化途径产生的NADH,在依赖于NADH的1,3-丙二醇氧化还原酶的作用下生成1,3-丙二醇;通过利用NADH还原3-羟基丙醛,菌体在生理上实现了NAD+的再生。甘油脱氢酶、甘油脱水酶及1,3-丙二醇氧化还原酶是催化1,3-丙二醇转化的关键酶。
目前生物合成法生产1,3-丙二醇存在产物浓度低、甘油转化率低以及生产周期长生产强度低等问题,因而缺乏市场竞争力。
为了促进1,3-丙二醇的生产,目前主要有以下几种方法:
1,采用微氧条件下发酵生产1,3-丙二醇(王剑锋等,克雷伯氏菌微氧发酵生产1,3-丙二醇的研究,现代化工,2001,21(5):28-31。修志龙等,一种微生物微氧发酵生产1,3-丙二醇的方法,中国专利公开号:CN1348007)
2,采用添加葡萄糖作辅底物在厌氧条件下发酵生产1,3-丙二醇(张健等,以葡萄糖为辅助底物发酵生产1,3-丙二醇的研究,现代化工,2002,22(6):32-35。)
3,采用外源添加维生素C或维生素E等还原剂的方法促进1,3-丙二醇生产(李春等,外源添加还原剂促进菌体合成1,3-丙二醇的方法,专利公开号:1446919A)
通过代谢及酶学分析发现,影响1,3-丙二醇合成有两方面关键因素。一是菌体内与1,3-丙二醇合成相关的关键酶活性的大小,包括甘油脱氢酶、甘油脱水酶及1,3-丙二醇氧化还原酶的活性变化。另一个影响因素是菌体内NADH含量或NAD/NADH的比例变化。实验结果表明,1,3-丙二醇发酵过程中添加反丁烯二酸可以有效地提高1,3-丙二醇合成关键酶的活性,同时降低了菌体内NAD+/NADH的比例,从而使得1,3-丙二醇浓度及生产强度显著提高。目前的研究中还未有外源添加反丁烯二酸促进微生物合成1,3-丙二醇的报导。
发明内容
本发明目的在于提供一种外源添加反丁烯二酸促进微生物合成1,3-丙二醇的方法。提高菌体内1,3-丙二醇合成关键酶的活性,同时降低NAD/NADH的比例,从而显著提高1,3-丙二醇的浓度和生产强度。
本发明包括制备发酵培养基,并向发酵培养基中接入种子液,进行厌氧或有氧发酵合成1,3-丙二醇的步骤。其特征在于:在发酵培养基中添加反丁烯二酸,浓度为5~25毫摩尔/升。添加时机选择在菌种进行发酵的对数生长期。
本发明所述的添加的反丁烯二酸是一种外源电子受体。
本发明适用于1,3-丙二醇的厌氧及有氧发酵过程。
本发明的优点在于提高菌体内1,3-丙二醇合成关键酶的活性,同时降低NAD/NADH的比例,从而显著提高1,3-丙二醇的浓度和生产强度。并且,操作简单,不增加额外的设备和人工,仅通过较低的附加投入,显著提高了1,3-丙二醇的生产强度,获得较高的1,3-丙二醇浓度,降低了发酵成本。
具体实施方式
实例1:
(1)菌种:克雷伯氏杆菌(Klebsiella pneumoniae)M5al
(2)培养基
LB斜面培养基:每升体积培养基含酵母粉5.0g,蛋白胨10.0g,琼脂粉15.0g,NaCl 10.0g,pH调节至7.0。
种子及发酵培养基成分见表1
表1
培养基组分 | 种子培养基(/l) | 发酵培养基(/l) | 微量元素溶液(mg/l) | |
甘油K2HPO4·3H2O(NH4)2SO4KH2PO4MgSO4·7H2O酵母粉微量元素溶液CaCO3消泡剂 | 20g4.45g2.0g1.3g0.2g1.0g2ml2.0g | 20g2.225g2.0g0.65g0.2g1.5g2ml0.1ml | ZnCl2MnCl2·4H2OH3BO3CoCl2·6H2ONiCl2·6H2ONiCl2·H2ONa2MoO4·2H2OCuCl2·H2OCuSO4·5H2O浓盐酸(37%) | 70100602002527.64352029.280.9ml |
(3)培养方式:
A.种子活化
挑取甘油管保藏的菌种一环转接至LB斜面活化,30℃下培养12小时可获得活化种子。
B.种子培养
种子培养采用有氧培养,使用500ml三角瓶,装液量100ml。培养温度30℃,摇床转速140rpm,培养时间14-16h。
C.发酵培养
发酵培养使用5L发酵罐,发酵过程中使用含起始甘油浓度为20g/l发酵培养基,装液量4L,培养温度37℃,通入氮气,通气量0.2vvm,进行厌氧发酵,pH值以50%KOH调节为6.85。发酵罐搅拌转速150rpm。其中对照罐不加入反丁烯二酸,实验罐加入反丁烯二酸使之浓度为5mM。
(5)发酵结果
发酵进行8小时,其中对照罐中残余甘油浓度5.48g/L,发酵液中含1,3-丙二醇7.54g/L,丁二酸0.41g/L,乳酸0.88g/L,醋酸2.21g/L,2,3-丁二醇0.03g/L。1,3-丙二醇摩尔得率0.55,甘油消耗速度为2.06g/(Lh),1,3-丙二醇生产强度0.94g/(Lh)。添加5mM反丁烯二酸罐中残余甘油浓度1.01g/L,发酵液中含1,3-丙二醇11.10g/L,丁二酸1.12g/L,乳酸0.97g/L浓度,醋酸3.46g/L,2,3-丁二醇0.07g/L。1,3-丙二醇摩尔得率0.56,甘油消耗速率为2.76g/(Lh),1,3-丙二醇生产强度1.28g/(Lh)。可见,添加5mM反丁烯二酸时甘油消耗速度比对照提高34%,生产强度提高36%,1,3-丙二醇浓度较对照提高47.2%,而1,3-丙二醇得率基本与对照相当。
实例2:
(1)菌种:同实例1
(2)培养基:同实例1
(3)培养方式:
A.种子活化
挑取甘油管保藏的菌种一环转接至LB斜面活化,30℃下培养12小时可获得活化种子。
B.种子培养
种子培养采用有氧培养,使用500ml三角瓶,装液量100ml。培养温度30℃,摇床转速140rpm,培养时间14-16h。
C.发酵培养
发酵培养使用500mL三角瓶,在气浴摇床中进行,温度37℃,摇床转速100rpm,装液量为200mL/500mL瓶,接种量为10%,培养基中加入1.g CaCO3调节pH。摇瓶以六层纱布包扎进行微氧发酵。为了考察反丁烯二酸的影响,分别设置0mM、5mM、15mM、25mM四组反丁烯二酸浓度试验。
(5)发酵结果
发酵共进行4h,结果见表2。
表2添加反丁烯二酸后甘油消耗和产物合成
反丁烯二酸(毫摩尔/升) | 甘油消耗(摩尔/升) | 产物合成(摩尔/升) | 1,3-丙二醇得率(摩尔/摩尔) | |||||
051525 | 甘油64.8106.6135.5143.6 | 丁二酸0.649.516.7 | 乳酸4.17.810.19.8 | 乙酸未检测到未检测到未检测到0.9 | 1,3-丙二醇23.535.94243.1 | 2,3-丁二醇5.66.47.88.2 | 乙醇9.121.13134 | 339.9338.9317.8300.8 |
可见,随着反丁烯二酸添加量的提高,甘油消耗速度及1,3-丙二醇的生产强度都有大幅度的提高。添加5、15、25毫摩尔/升反丁烯二酸时甘油的消耗速度分别比对照提高65%、109%及122%;1,3-丙二醇的生产强度分别提高53%,79%及83%;1,3-丙二醇浓度分别提高53%,79和84%。
Claims (2)
1.一种外源添加反丁烯二酸促进微生物合成1,3-丙二醇的方法,包括:培养基制备,种子培养,及发酵产1,3-丙二醇的步骤;其特征在于:在发酵培养基中添加反丁烯二酸,添加后在发酵体系里的终浓度为5~25毫摩尔/升;添加时机选择在克雷伯氏杆菌进行发酵的对数生长期。
2.如权利要求1或2所述的方法,其特征在于:适用于1,3-丙二醇的厌氧及有氧发酵过程。
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