氧化葡萄糖酸杆菌产2-酮基-D-葡萄糖酸的发酵过程优化
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摘要
2-酮基-D-葡萄糖酸(2-keto-D-gluconic acid,2-KGA)可作为食品添加剂、照片显影剂、洗涤剂等,也是合成D-异抗坏血酸的重要前体。为提高2-KGA的产量及转化率,对2-KGA发酵生产工艺条件进行优化。从5株野生菌株中筛选出最优菌株Gluconobacter japonicus CGMCC 1. 49,在摇瓶上进行培养基以及培养条件的优化。在此基础上,在3 L发酵罐中进行发酵优化实验,提高2-KGA的产量。当葡萄糖质量浓度为100 g/L,玉米浆粉质量浓度为20 g/L,发酵温度为30℃时,摇瓶水平发酵产量为76. 3 g/L,较优化前提高了120. 0%。在3 L发酵罐上控制恒定p H值为6、溶氧浓度30%条件下,一次性补料发酵产量最高为122. 1 g/L,较摇瓶水平提高了26. 7%,转化率达75. 5%。研究表明,发酵过程优化对提高野生菌株产2-KGA是一种行之有效的方法,优化结果可为后续的放大实验节省发酵成本,为氧化葡萄糖酸杆菌产2-KGA的工业化生产奠定了基础。
In order to increase the yield of 2-keto-D-gluconic acid( 2-KGA),a strain Gluconobacter japonicus CGMCC 1. 49 with the best performance was selected from five wild strains. The culture medium and conditions of G.japonicus CGMCC 1. 49 were optimized in shaking flasks,followed by optimizing its fermentation condition in a 3 L fermenter. With 100 g/L glucose,20 g/L corn syrup powder,and fermented at 30 ℃,the yield of 2-KGA in shaking flasks reached 76. 3 g/L,which was 120. 0% higher than that before optimization. In the 3 L fermenter with constant p H of 6 and 30% dissolved oxygen,disposable feed fermentation resulted in the highest yield of 2-KGA( 122. 1 g/L),which was 26. 7% higher than that in shaking flasks,and the conversion rate was 75. 5%. Therefore,fermentation optimization was an effective method to improve 2-KGA production from wild type strains. The results can lower the cost of scale-up fermentation and lay a foundation for industrializing the production of 2-KGA by Gluconobacter.
In order to increase the yield of 2-keto-D-gluconic acid( 2-KGA),a strain Gluconobacter japonicus CGMCC 1. 49 with the best performance was selected from five wild strains. The culture medium and conditions of G.japonicus CGMCC 1. 49 were optimized in shaking flasks,followed by optimizing its fermentation condition in a 3 L fermenter. With 100 g/L glucose,20 g/L corn syrup powder,and fermented at 30 ℃,the yield of 2-KGA in shaking flasks reached 76. 3 g/L,which was 120. 0% higher than that before optimization. In the 3 L fermenter with constant p H of 6 and 30% dissolved oxygen,disposable feed fermentation resulted in the highest yield of 2-KGA( 122. 1 g/L),which was 26. 7% higher than that in shaking flasks,and the conversion rate was 75. 5%. Therefore,fermentation optimization was an effective method to improve 2-KGA production from wild type strains. The results can lower the cost of scale-up fermentation and lay a foundation for industrializing the production of 2-KGA by Gluconobacter.
引文
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[17]陈吉铭,堵国成,陈坚,等.普通生酮基古龙酸菌2-酮基-L-古龙酸合成途径在氧化葡萄糖酸杆菌中的整合表达与强化[J].食品与生物技术学报,2016,35(6):611-616.
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[21]胡于东.氧化葡萄糖酸杆菌梯度强度组成型启动子的筛选与应用[D].无锡:江南大学,2015.
[22]张欢.氧化葡萄糖酸杆菌膜结合乙醇脱氢酶亚基Ⅲ的功能鉴定及提高菌株羟基酸合成能力的研究[D].上海:华东理工大学,2016.
[23]李翎,许琳,魏淼,等.氧化葡萄糖酸杆菌中5-葡萄糖酸脱氢酶基因的克隆、表达及酶学性质分析[J].生物技术通报,2014(9):157-163.
[24]杨燕花,吕永坤,陈吉铭,等. 2-酮基-L-古龙酸一步发酵生产菌株发酵过程优化[J].食品与发酵工业,2016,42(7):60-64.
[25]翟兵兵,董秀涛,丁明珠,等. VC三菌种一步发酵方法的构建与研究[J].中国生物工程杂志,2016,36(12):72-78.
[2]周强,魏转,孙文敬.等. D-异抗坏血酸生产技术研究进展[J].食品科学,2008,29(8):647-651.
[3]石岗,颜方贵.维生素C重要前体2,5-二酮基-D-葡萄糖酸发酵研究.[J].食品与发酵工业,2002,38(5):18-23.
[4]陈萍,苗晓燕,张筱梅.等.利用荧光假单胞菌固定化细胞生产2-酮基-D-葡萄糖酸[J].食品科学,2010,31(21):258-261.
[5]王贝贝,李昊聪,孙文敬,等.粘质沙雷氏菌膜结合葡萄糖酸脱氢酶的分离纯化及其酶学特性[J/OL].食品科学,1-14[2019-05-16].
[6]余泗莲,汪美生,孙文敬,等.荧光假单胞菌JD1202利用大米淀粉水解糖连续发酵生产2-酮基-D-葡萄糖酸[J].中国食品添加剂,2012(6):191-197.
[7]张炜,谢志鹏,罗玮,等.沙雷氏菌Serratia sp. BK-98发酵生产2-酮基-D-葡萄糖酸的工艺优化及动力学研究[J].化工学报,2011,62(5):1 371-1 376.
[8]姚瑞苗.氧化葡萄糖酸杆菌的抑制物耐受性及全糖转化探究[D].上海:华东理工大学,2017.
[9] PHILIPPE C,KRUPOVIC M,JAOMANJAKA F,et al.Bacteriophage GC1, a novel tectivirus infecting Gluconobacter cerinus,an acetic acid bacterium associated with wine-making[J]. Viruses-Basel,2018,10(1):39.
[10] LI L,CLEENWERCK I,DE VUYST L,et al. Identification of acetic acid bacteria through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and report of Gluconobacter nephelii Kommanee et al. 2011and Gluconobacter uchimurae Tanasupawat et al. 2012 as later heterotypic synonyms of Gluconobacter japonicus Malimas et al. 2009 and Gluconobacter oxydans(HENNEBERG 1897)De Ley 1961(Approved Lists 1980)emend Gossele et al. 1983,respectively[J]. Systematic and Applied Microbiology,2017,40(3):123-134.
[11] SIEMEN A,KOSCIOW K,SCHWEIGER P,et al. Production of 5-ketofructose from fructose or sucrose using genetically modified Gluconobacter oxydans strains[J].Applied Microbiology and Biotechnology,2018,102(4):1 699-1 710.
[12] WAN Hui,KANG Zhen,LI Jianghua,et al. Effect of high2-KLG concentration on expression of pivotal genes involved in 2-KLG synthesis in Gluconobacter oxydans WSH-003[J]. Acta microbiologica Sinica,2016,56(10):1 656-1 663.
[13]薛庆. 2-酮基-D-葡萄糖酸产生菌膜结合葡萄糖脱氢酶的分离纯化及性质[D].石家庄:河北师范大学,2015.
[14]李博义,潘海峰,孙伟荣,等. 5-酮基-D-葡萄糖酸发酵生产的工艺条件优化[J].生物工程学报,2014,30(9):1 486-1 490.
[15]李文婧,徐慧,刘建军,等.玉米浆粉预处理对粘质沙雷氏菌发酵产2-酮基-D-葡萄糖酸的影响[J].食品工业科技,2018,39(5):130-133.
[16]陈吉铭.氧化葡萄糖酸杆菌中2-酮基-L-古龙酸合成途径的整合表达与强化[D].无锡:江南大学,2015.
[17]陈吉铭,堵国成,陈坚,等.普通生酮基古龙酸菌2-酮基-L-古龙酸合成途径在氧化葡萄糖酸杆菌中的整合表达与强化[J].食品与生物技术学报,2016,35(6):611-616.
[18]王雪娇,程晓志,冯晨龙,等.一株2-酮基-D-葡萄糖酸(2KGA)产生菌的发酵研究[J].中国食品添加剂,2014(3):109-114.
[19]陈鸿胜,李克非,舒行宙,等.基于Gluconobacter oxydans膜结合脱氢酶的静息细胞催化合成2-酮基-D-葡萄糖酸[J].食品工业科技,2012,33(19):177-181.
[20]袁翠娟.氧化葡萄糖酸杆菌621H中双组分蛋白组氨酸激酶应答调节子杂合蛋白质与细胞内其他蛋白质相互作用的研究[D].上海:华东理工大学,2017.
[21]胡于东.氧化葡萄糖酸杆菌梯度强度组成型启动子的筛选与应用[D].无锡:江南大学,2015.
[22]张欢.氧化葡萄糖酸杆菌膜结合乙醇脱氢酶亚基Ⅲ的功能鉴定及提高菌株羟基酸合成能力的研究[D].上海:华东理工大学,2016.
[23]李翎,许琳,魏淼,等.氧化葡萄糖酸杆菌中5-葡萄糖酸脱氢酶基因的克隆、表达及酶学性质分析[J].生物技术通报,2014(9):157-163.
[24]杨燕花,吕永坤,陈吉铭,等. 2-酮基-L-古龙酸一步发酵生产菌株发酵过程优化[J].食品与发酵工业,2016,42(7):60-64.
[25]翟兵兵,董秀涛,丁明珠,等. VC三菌种一步发酵方法的构建与研究[J].中国生物工程杂志,2016,36(12):72-78.