Recombinant Expression of NADPH-Dependent Mannitol Dehydrogenase and Transformation Conditions of Mannitol

doi:10.13560/j.cnki.biotech.bull.1985.2017-0168

Abstract

Abstract: This work is to analyze the heterogeneous expression of NADPH-dependent mannitol dehydrogenase and the transformation of fructose for laying a foundation of constructing mannitol synthetic way. The recombinant strain BL21（DE3）/pET28a-mdh of expressing mannitol dehydrogenase was constructed and induced，and the target protein was purified with His-tag. Mannitol production from mannitol dehydrogenase transforming fructose was analyzed by HPLC. As results，the recombinant strain was constructed successfully and the mannitol dehydrogenase was purified. The activity of the purified mannitol dehydrogenase was 270 U/mL. The conditions of fructose transforming to mannitol by purified mannitol dehydrogenase were optimized，and the optimal conditions were as follows：300 g/L substrate fructose，pH5.8，9 mmol/L NADPH and incubation at 40℃. Under the optimized conditions，the transformation rate of mannitol reached 97.4%. In conclusion，the NADPH-dependent mannitol dehydrogenase was expressed successfully in Escherichia coli，providing the basis for studying the metabolism regulation of mannitol synthesis in E. coli.

Key words: mannitol dehydrogenase, NADPH, mannitol, Escherichia coli, purification

FENG Zhi-mei, ZHAO Ya-tong, LIU Ye-xue, LU Fu-ping, LI Yu. Recombinant Expression of NADPH-Dependent Mannitol Dehydrogenase and Transformation Conditions of Mannitol[J]. Biotechnology Bulletin, 2017, 33(8): 186-191.

References

[1] Saha BC, Racine FM. Biotechnological production of mannitol and its applications[J] . Applied Microbiology and Biotechnology, 2011, 89（4）：879-891.
[2] 应伟丽, 韩晓颖, 李媚, 等. 电化学法合成甘露醇[J] . 广西民族大学学报：自然科学版, 2005, 11（3）：85-87.
[3] 张晓卿. 微生物转化制备甘露醇的研究[D] . 南宁：广西大学, 2004.
[4] Gaspar P, Neves AR, Ramos A, et al. Engineering Lactococcus lactis for production of mannitol：high yields from food-grade strains deficient in lactate dehydrogenase and the mannitol transport system[J] . Applied & Environmental Microbiology, 2004, 70（3）：1466.
[5] 王小芳. 利用代谢工程构建D-甘露醇生产菌株[D] . 天津：天津科技大学, 2013.
[6] 陈艳, 田康明, 李玉, 等. 以蔗糖为底物利用重组大肠杆菌合成甘露醇[J] . 微生物学通报, 2014, 41（11）：2182-2190.
[7] Ortiz ME, Fornaguera MJ, Raya RR, et al. Lactobacillus reuteri CRL 1101 highly produces mannitol from sugarcane molasses as carbon source[J] . Applied Microbiology and Biotechnology, 2012, 95（1）：991-999.
[8] Saha BC, Racine FM. Effects of pH and corn steep liquor variability on mannitol production by Lactobacillus intermedius NRRL B-3693[J] . Applied Microbiology and Biotechnology, 2010, 87（2）：553-560.
[9] 王芳. 甘露醇发酵及甘露醇脱氢酶的克隆表达[D] . 石家庄：河北科技大学, 2009.
[10] 程雅韵, 王新, 郑琳, 等. 假肠膜明串珠菌甘露醇脱氢酶基因的克隆及表达[J] . 食品科学, 2016, 37（13）：153-156.
[11] 林谦. 乳酸细菌甘露醇脱氢酶研究进展[J] . 科技传播, 2010, （19）：134-135.
[12] Weckbecker A, Gr?ger H, Hummel W. Regeneration of nicotinamide coenzymes：principles and applications for the synthesis of chiral compounds[J] . Advances in Biochemical Engineering/Biotechnology, 2010, 120（120）：195-242.
[13] Reshamwala SM, Pagar SK, Velhal VS, et al. Construction of an efficient Escherichia coli whole-cell biocatalyst for d-mannitol production[J] . Journal of Bioscience & Bioengineering, 2014, 118（6）：628-631.
[14] Kaup B, Bringer-Meyer S, Sahm H. Metabolic engineering of Escherichia coli：construction of an efficient biocatalyst for D-mannitol formation in a whole-cell biotransformation[J] . Applied Microbiology and Biotechnology, 2016, 68（2）：235-240.
[15] Wasylenko TM, Ahn WS, Stephanopoulos G. The oxidative pentose phosphate pathway is the primary source of NADPH for lipid overproduction from glucose in Yarrowia lipolytica[J] . Metabolic Engineering, 2015, 30：27-39.
[16] Papagianni M, Legi?a M. Increased mannitol production in Lactobacillus reuteri ATCC 55730 production strain with a modified 6-phosphofructo-1-kinase[J] . Journal of Biotechnology, 2014, 181（10）：20-26.