5肌苷酸生产菌构建及发酵工艺研究发酵工程专业论文.docxVIP

ABSTRACTInosine ABSTRACT Inosine 5-Monophosphate(5-IMP)plays an important role in food area since it is an indispensable component for the new generation of flavor enhancer．It is often added into infant formula，fodder or health care products for its functional feature．Although Japanese scientists have started to study the production of 5-IMP by fermentation long time ago，the disadvantages such as overlong fermentation period and complicated control technology are still inevitable．This paper focused on an acid phosphotransferase gene from Morganella morganii，and several E coli and B．subtilis engineering strains were constructed for 5-IMP production．Furthermore，fermentation process for strain culture and a two—step method for 5-IMP production were investigated．All those studies would provide new insights into 5-IMP production． The catalyst AP／PTase and AP／PTaseM are encoded by gene sequences ofphoCY and phoCYM，their optimum conditions were pH 4．5，35。C and pH 5．2，35。C respectively．The result showed that AP／PTaseM was more efficient for 5-IMP production than AP／PTasea． Following studies showed that AP／PTaseM was stimulated by M92+，Mn2+，Fe2+，Zn2+at 5-1 0 mmol／L，but inhibited by high concentrations of these ions．Activity was not affected by Ca2+or Tween 80，but surfactants Tween 60 and Triton X．1 00 were promoters．The conversion rates were improved by 49．98％and 40．57％at 8．0％of Tween 60 and 5．0％of Triton X一1 00 respectively． The cultivation process for AP／PTaseM expression was investigated to increase the enzyme activity of XLl一Blue+pQE30-phoC∥．The optimal addition time of IPTG Was early-logphase(OD60022．0~2．5)，andmaximumtotalenzymeactivity(1983．07U／L)was achieved after induction for 8 h at 32。C．The optimum substrates concentration were 1 20 mmol／L of inosine and 200 mmol／L of TSPP,with which the conversion rate reached to 99．32％． On the other hand，two plasmids were constructed and tranferred into inosine-producing strain to produce 5-IMP by丑subtilis two-step fermentation．2