异麦芽酮糖是蔗糖的同分异构体,具有多种优秀的生理功能,广泛应用于食品行业。蔗糖异构酶可以将蔗糖异构为异麦芽酮糖,与其他微生物来源的蔗糖异构酶相比,Pantoea dispersa UQ68J来源的蔗糖异构酶转化蔗糖时异麦芽酮糖产率高,副产物少。为了更好地将异麦芽酮糖应用于食品领域,该研究将P.dispersa来源的蔗糖异构酶在食品安全微生物枯草芽孢杆菌(Bacillus subtilis)中重组表达,研究重组蔗糖异构酶的酶学性质并优化其制备异麦芽酮糖的反应条件。结果表明,重组酶的最适pH值为6.0,最适温度为30 ℃,在pH 5.0~8.0稳定性良好,在45 ℃下的半衰期为68 min。用该重组酶制备异麦芽酮糖,当蔗糖质量浓度为400 g/L,加酶量为20 U/g,在30 ℃、pH 6.0条件下转化10 h时,异麦芽酮糖产率可达90.61%,当蔗糖质量浓度提高到700 g/L,异麦芽酮糖产率仍可达89.20%。该研究提高了蔗糖异构酶的安全特性,实现了异麦芽酮糖的高产率,为工业生产异麦芽酮糖奠定了基础。
Isomaltulose is an isomer of sucrose and has many excellent physiological functions, so it is widely used in the food industry. Sucrose isomerase can isomerize sucrose into isomaltulose. Compared with sucrose isomerase derived from other microorganisms, the sucrose isomerase derived from Pantoea dispersa UQ68J can obtain higher yield of isomaltulose and fewer by-products when converting sucrose. In order to better apply isomaltulose to the food field, P. dispersa sucrose isomerase was recombinantly expressed in food safety microorganism Bacillus subtilis, the enzymatic properties of recombinant sucrose isomerase were studied and the reaction conditions for preparing isomaltulose were optimized in this study. The results showed that the optimal pH and temperature of recombinant sucrose isomerase were 6.0 and 30 ℃, respectively. The recombinant sucrose isomerase had good stability in the range of pH 5.0-8.0, and its half-life at 45 ℃ was 68 min. Using this recombinant sucrose isomerase to prepare isomaltulose, the yield was 90.61% with 400 g/L sucrose as the substrate, when the amount of enzyme was 20 U/g, at 30 ℃ and pH 8.0 for 10 h. When the sucrose concentration increased to 700 g/L, the yield of isomaltulose still reached 89.20%. This research improves the safety characteristics of sucrose isomerase, realizes the high yield of isomaltulose, and lays the foundation for the industrial production of isomaltulose.
[1] SAWALE P D, SHENDURSE A M, MOHAN M S, et al.Isomaltulose (palatinose) :An emerging carbohydrate[J].Food Bioscience, 2017, 18:46-52.
[2] 高学秀, 李宁, 袁卫涛, 等.异麦芽酮糖研究进展[J].中国食品添加剂, 2022, 33(1):26-31.
GAO X X, LI N, YUAN W T, et al.Review of isomaltulose[J].China Food Additives, 2022, 33(1):26-31.
[3] LINA B A R, JONKER D, KOZIANOWSKI G.Isomaltulose (Palatinose®):A review of biological and toxicological studies[J].Food and Chemical Toxicology, 2002, 40(10):1375-1381.
[4] LIU L N, BILAL M, LUO H Z, et al.Studies on biological production of isomaltulose using sucrose isomerase:Current status and future perspectives[J].Catalysis Letters, 2021, 151(7):1868-1881.
[5] 葛亚中, 徐俊.异麦芽酮糖的转基因法生产研究[J].中国食品添加剂, 2004(3):14-18;31.
GE Y Z, XU J.Study on the production of isomultose by transgenic plants[J].China Food Additives, 2004(3):14-18;31.
[6] MU W M, LI W J, WANG X, et al.Current studies on sucrose isomerase and biological isomaltulose production using sucrose isomerase[J].Applied Microbiology and Biotechnology, 2014, 98(15):6569-6582.
[7] 吴琦, 黄思敏, 郭小宇, 等.蔗糖异构酶PalI在解脂耶氏酵母中的表面展示[J].微生物学通报, 2022, 49(8):3094-3106.
WU Q, HUANG S M, GUO X Y, et al.Effective surface display of sucrose isomerase PalI on the cells of Yarrowia lipolytica[J].Microbiology China, 2022, 49(8):3094-3106.
[8] LI S, CAI H, QING Y J, et al.Cloning and characterization of a sucrose isomerase from Erwinia rhapontici NX-5 for isomaltulose hyperproduction[J].Applied Biochemistry and Biotechnology, 2011, 163(1):52-63.
[9] 程胜, 段绪果, 吴敬.重组蔗糖异构酶的制备及应用条件优化[J].食品与发酵工业, 2015, 41(5):41-47.
CHENG S, DUAN X G, WU J.Studies on the preparation of sucrose isomerase and its application for the production of isomaltulose[J].Food and Fermentation Industries, 2015, 41(5):41-47.
[10] WU L G, BIRCH R G.Characterization of the highly efficient sucrose isomerase from Pantoea dispersa UQ68J and cloning of the sucrose isomerase gene[J].Applied and Environmental Microbiology, 2005, 71(3):1581-1590.
[11] 张大伟, 康倩.枯草芽胞杆菌蛋白质表达分泌系统发展及展望[J].微生物学杂志, 2019, 39(1):1-10.
ZHANG D W, KANG Q.Development and prospect of protein expression and secretion systems in Bacillus subtilis[J].Journal of Microbiology, 2019, 39(1):1-10.
[12] YAO D B, ZHANG K, SU L Q, et al.Enhanced extracellular Bacillus stearothermophilus α-amylase production in Bacillus subtilis by balancing the entire secretion process in an optimal strain[J].Biochemical Engineering Journal, 2021, 168:107948.
[13] MA R J, WANG Y H, LIU L, et al.Production enhancement of the extracellular lipase LipA in Bacillus subtilis:Effects of expression system and Sec pathway components[J].Protein Expression and Purification, 2018, 142:81-87.
[14] ZHANG K, SU L Q, WU J.Enhanced extracellular pullulanase production in Bacillus subtilis using protease-deficient strains and optimal feeding[J].Applied Microbiology and Biotechnology, 2018, 102(12):5089-5103.
[15] 裴雯雯, 曾艳, 刘娟娟, 等.β-葡萄糖苷酶的枯草芽孢杆菌表达、酶学性质分析及其在宝藿苷Ⅰ制备中的应用[J].食品工业科技, 2022, 43(6):133-140.
PEI W W, ZENG Y, LIU J J, et al.Expression of recombinant β-glucosidase in Bacillus subtilis and its enzymatic characterization and application in preparation of icariside I[J].Science and Technology of Food Industry, 2022, 43(6):133-140.
[16] 李信志, 卢争辉, 周玉玲, 等.枯草芽孢杆菌SCK6超级感受态的制备和转化条件优化[J].生物工程学报, 2017, 33(4):692-698.
LI X Z, LU Z H, ZHOU Y L, et al.Preparation and transformation optimization for supercompetent B.subtilis SCK6 cells[J].Chinese Journal of Biotechnology, 2017, 33(4):692-698.
[17] 张康. 枯草芽孢杆菌菌株改造、启动子优化和普鲁兰酶的高效制备研究[D].无锡:江南大学, 2018.
ZHANG K.Modification of Bacillus subtilis strain, promoter optimization and high-level expression of pullulanase[D].Wuxi:Jiangnan University, 2018.
[18] YOU C, ZHANG X Z, ZHANG Y H.Simple cloning via direct transformation of PCR product (DNA Multimer) to Escherichia coli and Bacillus subtilis[J].Applied and Environmental Microbiology, 2012, 78(5):1593-1595.
[19] MILLER G L.Use of dinitrosalicylic acid reagent for determination of reducing sugar[J].Analytical Chemistry, 1959, 31(3):426-428.
[20] ZHANG D H, LI X Z, ZHANG L H.Isomaltulose synthase from Klebsiella sp.strain LX3:Gene cloning and characterization and engineering of thermostability[J].Applied and Environmental Microbiology, 2002, 68(6):2676-2682.
[21] 耿梦华, 陈晟, 吴敬, 等.吸附交联法固定化蔗糖异构酶及其在异麦芽酮糖制备中的应用[J].食品与生物技术学报, 2019, 38(4):104-110.
GENG M H, CHEN S, WU J, et al.Immobilization of sucrose isomerase by adsorption and crosslinking method for the synthesis of isomaltulose[J].Journal of Food Science and Biotechnology, 2019, 38(4):104-110.
