重组枯草芽孢杆菌表达4-木糖醇脱氢酶的发酵和反应条件优化
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摘要
L-木酮糖(L-苏式-戊酮糖)是一种昂贵的稀有戊糖,而4-木糖醇脱氢酶(xylitol-4-dehydrogenase,XDH)是生物合成L-木酮糖(L-xylulose)的关键酶。为了促进L-木酮糖的生产,对枯草芽孢杆菌外源表达4-木糖醇脱氢酶的发酵条件进行优化,并对以木糖醇为底物静息细胞催化反应合成L-木酮糖的反应条件进行研究。通过单因素实验确定了最佳培养基的成分(g/L)为:蔗糖25,尿素6,MgSO_40. 05,MnSO_40. 01,FeSO_40. 01,初始pH值8. 0。最佳发酵条件为:装液量40 mL(250 mL锥形瓶),接种量1. 67%,发酵培养温度28℃。在优化条件下4-木糖醇脱氢酶酶活为5. 183 U/L,与初始酶活相比提高了231. 2%。最优反应条件:底物质量浓度20 g/L,50mmol/L甘氨酸-氢氧化钠缓冲溶液(pH 10. 0),反应温度45℃,优化反应条件下转化率达到17. 74%。通过对菌株发酵和反应工艺条件的优化,为后续L-木酮糖工业化生产奠定了基础。
L-xylulose( L-threo-pentulose) is one of the rare sugars,and xylitol-4-dehydrogenase( XDH) is a key enzyme to biosynthesize L-xylulose. To accelerate the production of L-xylulose,conditions to express XDH in Bacillus subtilis and to catalyze L-xylulose formation from xylitol with resting cell were optimized. The optimal medium had an initial pH of 8 and contained 25 g/L sucrose,6 g/L urea,0. 05 g/L MgSO_4,0. 01 g/L Mn SO_4,and 0. 01 g/L FeSO_4. The optimal fermentation condition was as follows: 40 mL liquid volume in a 250 mL conical flask,1. 67% inoculum size and cultured at 28 ℃. Under the optimized condition,the XDH activity improved by 231. 2% compared against its initial activity and reached 5. 183 U/L. Moreover,the optimal reaction condition was: 20 g/L substrate,50 mmol/L glycine-NaOH buffer solution( pH = 10. 0) and reacted at 45 ℃,which resulted in the yield of 17. 74%L-xylulose. In conclusion,this study lays a foundation for industrial production of L-xylulose by optimizing the fermentation and reaction conditions of a strain.
L-xylulose( L-threo-pentulose) is one of the rare sugars,and xylitol-4-dehydrogenase( XDH) is a key enzyme to biosynthesize L-xylulose. To accelerate the production of L-xylulose,conditions to express XDH in Bacillus subtilis and to catalyze L-xylulose formation from xylitol with resting cell were optimized. The optimal medium had an initial pH of 8 and contained 25 g/L sucrose,6 g/L urea,0. 05 g/L MgSO_4,0. 01 g/L Mn SO_4,and 0. 01 g/L FeSO_4. The optimal fermentation condition was as follows: 40 mL liquid volume in a 250 mL conical flask,1. 67% inoculum size and cultured at 28 ℃. Under the optimized condition,the XDH activity improved by 231. 2% compared against its initial activity and reached 5. 183 U/L. Moreover,the optimal reaction condition was: 20 g/L substrate,50 mmol/L glycine-NaOH buffer solution( pH = 10. 0) and reacted at 45 ℃,which resulted in the yield of 17. 74%L-xylulose. In conclusion,this study lays a foundation for industrial production of L-xylulose by optimizing the fermentation and reaction conditions of a strain.
引文
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[19]蔡威,张建志,江正强,等.梅利斯丛梗孢酵母发酵生产赤藓糖醇的条件优化[J].食品科学,2013,34(21):259-263.
[20]潘龙,靳魁奇,刘宇鹏.静息细胞转化赤藓糖醇生产L-赤藓酮糖条件优化[J].食品与发酵工业,2014,40(12):72-76.
[21]张玉宝.转化木糖醇生物L-木酮糖菌株的筛选[D].泰安:山东农业大学,2013.
[22] DISCHE Z,BORENFREUND E. A new spectrophotometric method for the detection and determination of keto sugars and trioses[J]. J Biol Chem,1951,192(2):583.
[23] DISCHE Z L,RUM B S. A new spectrophotometric test for the detection of methylpentose[J]. J Biol Chem,1951(192):579-582.
[24]蒋新龙.发酵工程[M].杭州:浙江大学出版社,2011:92
[25]张丹,朱可丽,张海宏,等. Vc二步发酵中Na+、Ca2+及p H对巨大芽胞杆菌生长作用的影响[J].微生物学杂志,1998,18(3):24-26.
[2]林清泉,刘有才,李丽峰,等.稀有糖的制备及应用最新进展[J].食品与发酵工业,2013,39(6):146-151.
[3] DOTEN R C,MORTLOCK R P. Production of D-and Lxylulose by mutants of Klebsiella pneumoniae and Erwinia uredovora[J]. Applied&Environmental Microbiology,1985,49(1):158.
[4] MUNIRUZZAMAN S,PAN Y T,ZENG Y,et al. Inhibition of glycoprotein processing by L-fructose and L-xylulose[J]. Glycobiology,1996,6(8):795-803.
[5] HEINZ F,HERTEL S,VOGEL M. Composition to reduce the level of sugar in the blood:German,WO98/20882[P]. 1998-05-22.
[6] PARK C S,YEOM S J,LIM Y R,et al. Substrate specificity of a recombinant ribose-5-phosphate isomerase from Streptococcus pneumoniae and its application in the production of L-lyxose and L-tagatos[J]. World J Microbiol Biotechnol,2011(27):743-750.
[7] PASTINEN O,SCHOEMAKER H S,LEISOLA M. Xylose isomerase catalyzed novel hexose epimerization[J]. Biocatal Biotransform,1999(17):393-400.
[8] GRANSTROM T B. L-Xylose and L-lyxose production from xylitol using Alcaligenes 701B strain and immobilized Lrhamnose isomerase enzyme[J]. Enzyme&Microbial Technology,2005,36(7):976-981.
[9] TAKATA G,POONPERM W,MORIMOTO K,et al. Cloning and overexpression of the xylitol dehydrogenase gene from Bacillus pallidus and its application to L-xylulose production[J]. Journal of the Agricultural Chemical Society of Japan,2010,74(9):1 807-1 813.
[10] DOTENR C,MORTLOCK R P. Characterization of xylitol-utilizing mutants of Erwinia uredovora[J]. Journal of Bacteriology,1985,161(2):529-533.
[11] KHAN A R,TOKUNAGA H,YOSHIDA K,et al. Conversion of xylitol to L-xylulose by Alcaligenes sp. 701Bcells[J]. Journal of Fermentation&Bioengineering,1991,72(6):488-490.
[12] POONPERM W,TAKATA G,MORIMOTO K,et al.Production of L-xylulose from xylitol by a newly isolated strain of Bacillus pallidus Y25 and characterization of its relevant enzyme xylitol dehydrogenase[J]. Enzyme&Microbial Technology,2007,40(5):1 206-1 212.
[13]张玉宝,赵祥颖,杨丽萍,等.一株产L-木酮糖菌株的分离筛选及鉴定[J].食品科学,2014,35(1):199-203.
[14] MENG Q,ZHANG T,JIANG B,et al. Advances in applications,metabolism,and biotechnological production of L-xylulose[J]. Applied Microbiology and Biotechnology,2016,100(2):535-540.
[15] USVALAMPI A,KIVIHARJU K,LEISOLA M,et al.Factors affecting the production of L-xylulose by resting cells of recombinant Escherichia coli[J]. Journal of Industrial Microbiology&Biotechnology,2009,36(10):1 323-1 330.
[16] AARNIKUNNAS J. Cloning and expression of a xylitol-4-dehydrogenase gene from Pantoea ananatis[J]. Applied&Environmental Microbiology,2006,72(1):368.
[17]陈振,陈献忠,张利华,等.代谢改造热带假丝酵母发酵木糖母液生产木糖醇[J].中国生物工程杂志,2017(5):66-75.
[18]张超,张涛,江波,等. 1株产木糖醇菌株的筛选鉴定[J].食品与发酵工业,2016,42(5):114-119.
[19]蔡威,张建志,江正强,等.梅利斯丛梗孢酵母发酵生产赤藓糖醇的条件优化[J].食品科学,2013,34(21):259-263.
[20]潘龙,靳魁奇,刘宇鹏.静息细胞转化赤藓糖醇生产L-赤藓酮糖条件优化[J].食品与发酵工业,2014,40(12):72-76.
[21]张玉宝.转化木糖醇生物L-木酮糖菌株的筛选[D].泰安:山东农业大学,2013.
[22] DISCHE Z,BORENFREUND E. A new spectrophotometric method for the detection and determination of keto sugars and trioses[J]. J Biol Chem,1951,192(2):583.
[23] DISCHE Z L,RUM B S. A new spectrophotometric test for the detection of methylpentose[J]. J Biol Chem,1951(192):579-582.
[24]蒋新龙.发酵工程[M].杭州:浙江大学出版社,2011:92
[25]张丹,朱可丽,张海宏,等. Vc二步发酵中Na+、Ca2+及p H对巨大芽胞杆菌生长作用的影响[J].微生物学杂志,1998,18(3):24-26.