肌醇作为一种生物生长不可缺少的生长因子,在医药、食品、饲料、化妆品等行业具有十分广泛的应用。目前肌醇的制备方法包括水解法、化学法和生物法。生物法,尤其是体外多酶催化制备工艺,因其高转化率和环境友好性而备受关注。然而,这一工艺仍然存在酶制剂用量大和成本高的问题。该课题组前期重组表达了来源于闪烁古生球菌(Archaeoglobus fulgidus)的肌醇-1-磷酸合酶(inositol-1-phosphate synthase,IPS)IPS-Af,但发现其酶活力低,是多酶催化转化过程中酶用量最大的部分。针对上述问题,该文筛选并重组表达了来源于嗜热古菌(Caldivirga maquilingensis)的IPS-Cm和嗜热新芽孢杆菌(Novibacillus thermophilus)的IPS-Nt,对比了不同来源IPS的活力以及在体外多酶级联催化制备肌醇中的应用效果。结果表明,IPS-Cm和IPS-Nt酶活力均高于IPS-Af,其中IPS-Cm制备肌醇的转化率最高。在此基础上,该研究进一步优化了加酶量、缓冲液浓度、金属离子、底物浓度、pH值、温度和反应时间等条件。结果表明,在最佳转化条件下,肌醇产量可达到42.3 g/L,转化率为84.6%,是优化前的2.1倍。
Inositol, as an indispensable growth factor for biological growth, is widely used in medicine, food, feed, cosmetics, and other industries.At present, the preparation methods of inositol include hydrolysis, chemical, and biological methods.Biological processes, especially in vitro multi-enzyme catalytic processes, have attracted much attention due to their high conversion rate and environmental friendliness.However, this process still has the problem of large enzyme dosage and high cost.Inositol-1-phosphate synthase (IPS) IPS-AF, derived from Archaeoglobus fulgidus, was recombinant in the previous stage, but it was found that its enzyme activity was low, and it was the part with the largest amount of enzyme in the process of multi-enzyme catalyzed conversion.To solve these problems, this study screened and expressed IPS-Cm from Caldivirga maquilingensis and IPS-Nt from Novibacillus thermophilus.The activity of IPS from different sources and its application in the preparation of inositol by multi-enzyme cascade catalysis in vitro were compared.Results showed that the enzyme activity of IPS-Cm and IPS-Nt was higher than that of IPS-Af, and the conversion rate of IPS-Cm was the highest.On this basis, the conditions of enzyme addition, buffer concentration, metal ion, substrate concentration, pH, temperature and reaction time were further optimized.Results showed show that under the optimal conversion conditions, the yield of inositol could reach 42.3 g/L and the conversion rate was 84.6%, which was 2.1 times of that before optimization.
[1] 张芹, 王芳, 陈雅蕾, 等.肌醇生产及应用研究进展[J].中国稻米, 2012, 18(3):19-21.
ZHANG Q, WANG F, CHEN Y L, et al.Research progress on inositol production and application[J].China Rice, 2012, 18(3):19-21.
[2] SHAFIE N H, MOHD ESA N, ITHNIN H, et al.Preventive inositol hexaphosphate extracted from rice bran inhibits colorectal cancer through involvement of Wnt/β-catenin and COX-2 pathways[J].BioMed Research International, 2013,2013:681027.
[3] 孙灵霞, 陈锦屏.肌醇生产, 应用研究及前景展望[J].粮食与油脂, 2004, (11):6-8.
SUN L X, CHEN J P.The production, application and prospect of inositol[J].Journal of Cereals & Oils, 2004, (11):6-8.
[4] YOU R, WANG L, SHI C R, et al.Efficient production of myo-inositol in Escherichia coli through metabolic engineering[J].Microbial Cell Factories, 2020, 19(1):109.
[5] GUNASHREE B S, VENKATESWARAN G.Enhanced phytase production through interspecific protoplast fusion of Aspergillus niger CFR 335 and Aspergillus ficuum SGA 01 auxotrophic mutants[J].Enzyme and Microbial Technology, 2010, 46(7):562-567.
[6] 潘声龙. 由玉米浸渍水制取肌醇的工艺研究[D].天津:天津大学, 2008.
PAN S L.Study on technology of producing inositol from corn impregnated water[D].Tianjin:Tianjin University, 2008.
[7] 刘文宝, 金玉坤, 游松.肌醇制备方法的现状与进展[J].沈阳药科大学学报, 2012, 29(3):234-240.
LIU W B, JIN Y K, YOU S.Current situation and development of the study on preparation methods of myo-inositol[J].Journal of Shenyang Pharmaceutical University, 2012, 29(3):234-240.
[8] 魏欣蕾, 游淳.体外多酶分子机器的现状和最新进展[J].生物工程学报, 2019, 35(10):1870-1888.
WEI X L, YOU C.In vitro multi-enzyme molecular machines-a review[J].Chinese Journal of Biotechnology, 2019, 35(10):1870-1888.
[9] MENG D D, WEI X L, ZHANG Y H P J, et al.Stoichiometric conversion of cellulosic biomass by in vitro synthetic enzymatic biosystems for biomanufacturing[J].ACS Catalysis, 2018, 8(10):9550-9559.
[10] ZHANG Y H.Production of biocommodities and bioelectricity by cell-free synthetic enzymatic pathway biotransformations:Challenges and opportunities[J].Biotechnology and Bioengineering, 2010, 105(4):663-677.
[11] LIU M X, HAN P P, ZHANG L L, et al.Biofilm-mediated immobilization of a multienzyme complex for accelerating inositol production from starch[J].Bioconjugate Chemistry, 2021, 32(9):2032-2042.
[12] SAHEKI S, TAKEDA A, SHIMAZU T.Assay of inorganic phosphate in the mild pH range, suitable for measurement of glycogen phosphorylase activity[J].Analytical Biochemistry, 1985, 148(2):277-281.
[13] YOU C, SHI T, LI Y J, et al.An in vitro synthetic biology platform for the industrial biomanufacturing of myo-inositol from starch[J].Biotechnology and Bioengineering, 2017, 114(8):1855-1864.
[14] LI Y J, HAN P P, WANG J, et al.Production of myo-inositol:Recent advance and prospective[J].Biotechnology and Applied Biochemistry, 2022, 69(3):1101-1111.
[15] 程琨. 肌醇体外合成途径的构建及其对小麦抗逆性影响的研究[D].河南:河南农业大学, 2019.
CHENG K.Construction of in vitro synthesis pathway of inositol and its effect on wheat stress resistance[D].Henan:Henan Agricultural University, 2019.
[16] YE X H, ROLLIN J, ZHANG Y H P.Thermophilic α-glucan phosphorylase from Clostridium thermocellum:Cloning, characterization and enhanced thermostability[J].Journal of Molecular Catalysis B:Enzymatic, 2010, 65(1-4):110-116.
[17] CHEN L, ZHOU C, YANG H, et al.Inositol-1-phosphate synthase from Archaeoglobus fulgidus is a class II aldolase[J].Biochemistry, 2000, 39(40):12415-12423.
[18] MENG Q L, ZHANG Y F, JU X Z, et al.Production of 5-aminolevulinic acid by cell free multi-enzyme catalysis[J].Journal of Biotechnology, 2016, 226:8-13.
[19] RAIGOND P, EZEKIEL R, RAIGOND B.Resistant starch in food:A review[J].Journal of the Science of Food and Agriculture, 2015, 95(10):1968-1978.
[20] ZHOU W, YOU C, MA H W, et al.One-pot biosynthesis of high-concentration α-glucose 1-phosphate from starch by sequential addition of three hyperthermophilic enzymes[J].Journal of Agricultural and Food Chemistry, 2016, 64(8):1777-1783.
[21] CHENG K, ZHANG F, SUN F F, et al.Doubling power output of starch biobattery treated by the most thermostable isoamylase from an archaeon Sulfolobus tokodaii[J].Scientific Reports, 2015, 5:13184.
[22] OVISSIPOUR M, ABEDIAN A, MOTAMEDZADEGAN A, et al.The effect of enzymatic hydrolysis time and temperature on the properties of protein hydrolysates from Persian sturgeon (Acipenser persicus) viscera[J].Food Chemistry, 2009, 115(1):238-242.
