运用鸟枪克隆术,从环状芽胞杆菌B2301基因组中克隆出乳糖酶编码基因,其完整读框大小为5 133 bp,编码1 710个氨基酸残基,不含典型细菌信号肽序列,与已有报道的β-半乳糖苷酶的最高一致性为93.6%。初步表达和纯化了此乳糖酶,重组乳糖酶在50 ℃(ONPG)/60 ℃(乳糖)和pH 6.0~6.5下表现出最高催化活性,Zn2+、Fe2+、Cu2+、EDTA和SDS对重组酶表现出不同程度的抑制作用;该酶在50和55 ℃下催化合成低聚半乳糖的Vmax分别为2.21和2.47 g/(L·h),Km分别为10.46和14.37 g/L。所获得的乳糖酶具有以乳糖为底物酶法合成低聚半乳糖的优良应用属性,可为后续针对此酶的高效表达与工业化应用奠定基础。
By shotgun cloning,a complete open reading frame encoding a lactase was cloned and sequenced from Bacillus circulans B2301. It was composed of 5 133 nucleotides,which encoded a sequence of 1 710 amino acid residues without typical bacterial signal peptide. The mature peptide shared a maximum of 93.6% sequence identity with the reported β-galactosidases. It was fundamentally expressed,and the recombinant lactase was purified. The recombinant enzyme displayed the maximum activity at pH 6.0-6.5 and 50 ℃ on ONPG or 60 ℃ on lactose. Its activity was inhibited by Zn2+,Fe2+,Cu2+,EDTA or SDS in different degrees. For galacto-oligosaccharides synthesis,its Vmax was 2.21 g/(L·h) at 50 ℃ and 2.47 g/(L·h) at 55 ℃,its Km was 10.46 g/L at 50 ℃ and 14.37 g/L at 55℃. This study revealed the excellent biochemical properties of the lactase for galacto-oligosaccharides preparation from lactose,which lays a foundation for its high-efficient expression and industrial application in the future.
[1] KUMARI S,PANESAR P S,PANESAR R.Production of β-galactosidase using novel yeast isolate from whey[J]. International Journal of Dairy Science,2011,6(2): 150-157.
[2] PANESAR P S,PANESAR R,SINGH R S,et al.Microbial production,immobilization and applications of β-D-galactosidase[J]. Journal of Chemical Technology & Biotechnology,2006,81(4): 530-543.
[3] HUSAIN Q.β-Galactosidases and their potential applications: A review[J]. Critical Reviews in Biotechnology,2010,30(1): 41-62.
[4] COELHO D A B,SARAIVA M T L F,RENARD G,et al. Microbial β-galactosidases of industrial importance: Computational studies on the effects of point mutations on the lactose hydrolysis reaction[J]. Biotechnology Progress,2020Feb 21. DOI: 10.1002/btpr. 2982[Epub ahead of print].
[5] SCHAAFSMA G.Lactose and lactose derivatives as bioactive ingredients in human nutrition[J]. International Dairy Journal,2008,18(5): 458-465.
[6] GOSLING A,STEVENS G W,BARBER A R,et al.Recent advances refining galactooligosaccharide production from lactose[J]. Food Chemistry,2010,121(2): 307-318.
[7] RODRIGUEZ-COLINA B,FERNANDEZ-ARROJO L,BALLESTEROS A O,et al.Galactooligosaccharides formation during enzymatic hydrolysis of lactose: Towards a prebiotic-enriched milk[J]. Food Chemistry,2014,145: 388-394.
[8] TORRES D,MARIA F,CALVES G,et al.Galacto-oligosaccharides: Production,properties,applications,and significance as prebiotics[J]. Comprehensive Reviews in Food Science and Food Safety,2010,9(5): 438-454.
[9] BODE L.Human milk oligosaccharides: Every baby needs a sugar mama[J]. Glycobiology,2012,22(9): 1 147-1 162.
[10] 赵继华,牛丹丹,MCHUNU N P,等. 高转苷活性乳糖酶快速筛选方法的建立与初步应用[J]. 食品与发酵工业,2020,46(7):17-20.
[11] 牛丹丹,靳晓,吴海洋,等. 解淀粉芽胞杆菌α-淀粉酶高活力突变体的创建与性质研究[J]. 食品与发酵工业,2017,43(10): 24-29.
[12] 诸葛健,王正祥. 工业微生物实验技术手册[M]. 北京: 中国轻工业出版社,1994.
[13] 王正祥,诸葛健. 产甘油假丝酵母胞浆3-磷酸甘油脱氢酶编码基因的克隆[J]. 微生物学报,1999,39(4): 321-326.
[14] BRAFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dyebinding[J]. Analytical Biochemistry,1976,72: 248-254.
[15] GB/T 33409—2016B/T 33409—2016. β-半乳糖苷酶活性检测方法分光光度法[S]. 北京: 中国标准出版社,2016.
[16] NIU D,TIAN K M,MCHUNU N P,et al.Biochemical characterization of three Aspergillus niger β-galactosidases[J]. Electronic Journal of Biotechnology,2017,27: 37-43.
[17] RING M,ARMITAGE I M,HUBER R E. m-Fluorotyrosine substitution in β-galactosidase; Evidence for the existence of a catalytically active tyrosine[J]. Biochemical and Biophysical Research Communications,1985,131(2): 675-680.
[18] LI L,LI G,CAO L C,et al.Characterization of the cross-linked enzyme aggregates of a novel β-galactosidase,a potential catalyst for the synthesis of galacto-oligosaccharides[J]. Journal of Agricultural and Food Chemistry,2015,63(3): 894-901.
[19] 王君,李玮,王雪荣,等. 固定化酶法高效制备低聚异麦芽糖[J]. 中国食品学报,2017,17(8): 101-108.
[20] SONG J Y,IMANAKA H,IMAMURA K,et al.Cloning and expression of a β-galactosidase gene of Bacillus circulans[J]. Bioscience,Biotechnology,and Biochemistry,2011,75(6): 1 194-1 197.
[21] ISHIKAWA K,KATAOKA M,YANAMOTO T,et al.Crystal structure of β-galactosidase from Bacillus circulans ATCC 31382 (BgaD) and the construction of the thermophilic mutants[J]. FEBS Journal,2015,282(13): 2 540-2 552.
[22] BULTEMA J B,KUIPERS B J H,DIJKHUIZEN L. Biochemical characterization of mutants in the active site residues of the β-galactosidase enzyme of Bacillus circulans ATCC 31382[J]. FEBS Open Bio,2014(4): 1 015-1 020.
[23] BRADY D,MARCHANT R,MCHALE L,et al.Isolation and partial characterization of β-galactosidase activity produced by a thermotolerant strain of Kluyveromyces marxianus during growth on lactose-containing media[J]. Enzyme and Microbial Technology,1995,17(8): 696-699.
[24] SHAIKH S A,KHIRE J M,KHAN M I. Characterization of a thermostable extracellular β-galactosidase from a thermophilic fungus Rhizomucor sp.[J]. Biochimica et Biophysica Acta,1999,1 472: 314-322.
