β-半乳糖苷酶是广泛使用的食品添加剂,主要用于降解乳制品中的乳糖,缓解乳糖不耐受症状。该研究对实验室保藏菌株马克斯克鲁维酵母的发酵条件进行了优化。在20 g/L半乳糖、20 g/L玉米浆干粉、40 ℃、初始pH 6.5、150 r/min的条件下,粗酶液的酶活力为26.3 U/mL,表明该菌株具有利用廉价碳氮源高效生产β-半乳糖苷酶的潜力。通过DEAE阴离子交换层析进一步纯化得到比活力为124.09 U/mg的纯化酶。纯化后酶的最适温度40 ℃,最适pH 6,Km为5.28 mmol/L,kcat为4.74 s-1。此外,发现该酶受Mg2+的促进,在20~40 ℃表现出优异的热稳定性,40 ℃下30 min仍能保持95.6%的活力。因此,马克斯克鲁维酵母β-半乳糖苷酶比乳酸克鲁维酵母的热稳定性更好,具有潜在的工业应用潜力。
β-Galactosidase is a widely used food additive, primarily used to degrade lactose in dairy products and alleviate symptoms of lactose intolerance. In this study, we optimized the fermentation conditions of Kluyveromyces marxianus, a strain preserved in our laboratory. Under the conditions of 20 g/L galactose, 20 g/L corn steep powder, 40 ℃, initial pH 6.5, and 150 r/min, the crude enzyme displayed an enzyme activity of 26.3 U/mL, indicating that the strain had the potential to efficiently produce β-galactosidase using cheap carbon and nitrogen sources. Further purification via DEAE anion exchange chromatography resulted in a purified enzyme with a specific activity of 124.09 U/mg. The purified enzyme exhibited an optimal temperature of 40 ℃, an optimal pH of 6, a Michaelis constant of 5.28 mmol/L, and a catalytic constant of 4.74 s-1. Additionally, the enzyme was found to be promoted by Mg2+ and exhibited excellent thermal stability between 20-40 ℃, retaining 95.6% activity at 40 ℃ for 30 min. As K. marxianus, which belongs to the same edible strain as Kluyveromyces lactis, its β-galactosidase has better thermal stability than common K. lactis, and has potential industrial application potential.
[1] 董艺凝, 陈海琴, 张灏, 等.β-半乳糖苷酶的研究现状与进展[J].食品与生物技术学报, 2018, 37(4):337-343.
DONG Y N, CHEN H Q, ZHANG H, et al.Research status and progress on β-galactosidase[J].Journal of Food Science and Biotechnology, 2018, 37(4):337-343.
[2] 曹晓聪, 张朝军, 苟浩琦, 等.棉花β-半乳糖苷酶基因家族的全基因组鉴定及分析[J].分子植物育种, 2021, 19(24):8032-8047.
CAO X C, ZHANG C J, GOU H Q, et al.Genome-wide identification and analysis of cotton β-galactosidase gene family[J].Molecular Plant Breeding, 2021, 19(24):8032-8047.
[3] 范琴, 曾璐瑶, 杨金茹, 等.粪便宏基因组来源低分子量碱性β-半乳糖苷酶的异源表达及酶学性质[J].微生物学通报, 2022, 49(5):1594-1607.
FAN Q, ZENG L Y, YANG J R, et al.Heterologous expression and enzyme properties of low-molecular-weight and basic β-galactosidase derived from the metagenome of faeces[J].Microbiology China, 2022, 49(5):1594-1607.
[4] JUMA A A, BADAWY A S, MOHAMMED S B.Isolation and purification of β-galactosidase enzyme from local lactic acid bacteria isolates to overcome the phenomenon of non-degradation of milk lactose[J].IOP Conference Series:Earth and Environmental Science, 2021, 910(1):012075.
[5] POP C, SPOREA I, SANTOS J, et al.Efficacy and safety of a novel therapeutic of natural origin (NTN) in adult patients with lactose intolerance:A multicenter, randomized, crossover, double-blind, placebo-controlled study[J].Foods, 2022, 11(17):2600.
[6] KOLEV P, ROCHA-MENDOZA D, RUIZ-RAMÍREZ S, et al.Screening and characterization of β-galactosidase activity in lactic acid bacteria for the valorization of acid whey[J].JDS Communications, 2022, 3(1):1-6.
[7] 段小果, 李博, 贺银凤.乳酸菌与酵母菌共生机理的研究进展[J].微生物学通报, 2017, 44(8):1988-1995.
DUAN X G, LI B, HE Y F.Progress in symbiotic mechanism between lactic acid bacteria and yeast[J].Microbiology China, 2017, 44(8):1988-1995.
[8] ZHAO J C, MU Y L, GU X Y, et al.Site-directed mutation of β-galactosidase from Streptococcus thermophilus for galactooligosaccharide-enriched yogurt making[J].Journal of Dairy Science, 2022, 105(2):940-949.
[9] INTANON M, ARREOLA S L, PHAM N H, et al.Nature and biosynthesis of galacto-oligosaccharides related to oligosaccharides in human breast milk[J].FEMS Microbiology Letters, 2014, 353(2):89-97.
[10] ZHOU Z H, HE N N, HAN Q, et al.Characterization and application of a new β-galactosidase Gal42 from marine bacterium Bacillus sp.BY02[J].Frontiers in Microbiology, 2021, 12:742300.
[11] 卜建斌, 云战友, 邢黎明.β-半乳糖苷酶的应用及固定化[J].农产品加工(学刊), 2006(2):4-6;10.
BU J B, YUN Z Y, XING L M.Application and immobilization of β-galactosidase[J].Academic Periodical of Farm Products Processing, 2006(2):4-6;10.
[12] DEUTCH CHARLES E, FARDEN AMY M, DICESARE EMILY S.Characterization of β-galactosidase and α-galactosidase activities from the halophilic bacterium Gracilibacillus dipsosauri[J].Annals of Microbiology, 2021, 71(1):1-15.
[13] KARIM A, GERLIANI N, AÏDER M.Kluyveromyces marxianus:An emerging yeast cell factory for applications in food and biotechnology[J].International Journal of Food Microbiology, 2020, 333:108818.
[14] LEDERBERG J.The β-D-galactosidase of Escherichia coli, strain K-12[J].Journal of Bacteriology, 1950, 60(4):381-392.
[15] 郑义, 张健, 曹永强, 等.产β-D-半乳糖苷酶马克斯克鲁维酵母的分离鉴定及其产酶特性[J].食品科学, 2020, 41(2):158-165.
ZHENG Y, ZHANG J, CAO Y Q, et al.Isolation, identification and enzymatic characteristics of Kluyveromyces marxianus producing β-D-galactosidase[J].Food Science, 2020, 41(2):158-165.
[16] 剧淑君, 缪铭, 江波.克鲁维酵母SK16.001 β-半乳糖苷酶的纯化工艺研究[J].食品工业科技, 2011, 32(7):182-185.
JU S J, MIAO M, JIANG B.Study on purification process of β－galactosidase from Kluyveromyces SK16.001[J].Science and Technology of Food Industry, 2011, 32(7):182-185.
[17] DUAN F Y, ZHAO R F, YANG J Y, et al.Integrated utilization of dairy whey in probiotic β-galactosidase production and enzymatic synthesis of galacto-oligosaccharides[J].Catalysts, 2021, 11(6):658.
[18] 谢毅, 江培翝, 郭杰炎.乳酸克鲁维酵母(Kluveromces lactis)β-半乳糖苷酶的稳定性研究[J].复旦学报(自然科学版), 1999,38(5):523-528.
XIE Y, JIANG P H, GUO J Y.Studies on stability of K.lactis β-galactosidase[J].Journal of Fudan University (Natural Science), 1999,38(5):523-528.
[19] 黄倩, 李洁, 郑晓春, 等.奶酪中高产β-半乳糖苷酶菌株的筛选及发酵条件的优化[J].中国调味品, 2021, 46(2):23-30.
HUANG Q, LI J, ZHENG X C, et al.Screening of high-yield β-galactosidase strains in cheese and optimization of fermentation conditions[J].China Condiment, 2021, 46(2):23-30.
