低聚葡萄糖氧化酶能够氧化低聚葡萄糖末端残基生成相应的低聚糖酸,在低聚糖酸的制备、食品、化工等方面具有潜在的应用价值。从黑曲霉基因组中发现1个疑似编码低聚葡萄糖氧化酶开放读框,共编码473个氨基酸序列,包含2个保守结构域(FAD结合域和黄素结构域)和4个活性位点(His80、Cys141、Asp377和Try428);在毕赤酵母中表达的重组酶蛋白大小为61 kDa,比酶活0.33 U/mg。重组酶的最适作用温度和pH值分别为75 ℃和9.0;在55~85 ℃和pH 5.0~9.0范围内孵育4 h,仍能保持70%以上的酶活;Mn2+和Fe3+分别对酶活具有最强的激活和抑制作用。该酶的Km和Vmax为0.48 mmol/L和2.22 μmol/(L·min),对麦芽三糖、麦芽四糖和麦芽五糖具有相对较高酶活。
The gluco-oligosaccharide oxidases oxidize the reducing end of glycosyl residues linked by alpha- or beta-1,4 bonds and glucose. Due to their significant application in aldonic acids production, these enzymes have high values in feed, food and chemical industries. A novel potential gluco-oligosaccharide oxidase gene from Aspergillus niger was successfully cloned into and heterologously expressed by Pichia pastoris GS115 followed by enzymatic properties analysis. The gene encoded 473 amino acids, containing 2 conserved domains (flavin adenine dinucleotide (FAD) binding domain and flavin structure domain) and 4 active sites (His80, Cys141, Asp377, and Try428). The mature protein revealed a molecular weight of 61 kDa with specific activity of 0.33 U/mg. The optimum temperature and pH of the recombinant enzyme was 75 ℃ and 9.0, respectively. After 4 hours of incubation at 55-85 ℃, pH 5.0-9.0, more than 70% of the enzyme activity was retained. The ion Mn2+ revealed strong activation on the enzyme activity, and Fe3+ exhibited inhibition. The Km and Vmax on lactose were 0.48 μmol/L and 2.22 μmol/(L·min), respectively. In addition, the enzyme has highest oxidation activity on maltotetraose followed by maltotriose and maltopentose.
[1] SCHOEMAKER H E, MINK D, WUBBOLTS M G. Dispelling the myths-biocatalysis in industrial synthesis[J]. Science, 2003, 299(5 613): 1 694-1 697.
[2] VAN HELLEMOND E W, LEFERINK N G H, HEUTS D P H M, et al. Occurrence and biocatalytic potential of carbohydrate oxidases[J]. Advances in Applied Microbiology, 2006, 60: 17-54.
[3] VUONG T V, FOUMANI M, MACCORMICK B, et al. Direct comparison of gluco-oligosaccharide oxidase variants and glucose oxidase: substrate range and H2O2 stability[J]. Scientific Reports, 2016, 6: 37 356. Doi: 10.1038/srep37356 (2016).
[4] RUEY J Y, VAN SCOTT E J. Oligosaccharide aldonic acids and their topical use: United States of America, US/6335023B1 [P]. 2002-01-01.
[5] 白会钗, 缪铭, 江波, 等. 乳糖酸的研究进展[J]. 食品工业科技, 2012,33(2): 430-432.
[6] GREEN B A, YU R J, VAN S E J. Clinical and cosmeceutical uses of hydroxyacids[J]. Clinics in Dermatology, 2009, 27(5): 495-501.
[7] LUIS F G, HAMOUDI S, BELKACEMI K. Lactobionic acid: a high value-added lactose derivative for food and pharmaceutical applications[J]. International Dairy Journal, 2012, 26(2): 103-111.
[8] LI X, CHOMVONG K, YU V Y, et al. Cellobionic acid utilization: from Neurospora crassa to Saccharomyces cerevisiae[J]. Biotechnology for Biofuels, 2015, 8(1): 120.
[9] GREEN J W. The halogen oxidation of simple carbohydrates [M]. New York: Advances in Carbohydrate Chemistry, 1948: 129-184.
[10] ALHAERI M F. Engineering and production of glucooligosaccharide oxidases for site-specific activation of cellulose and hemicellulose substrates[D]. Toronto: University of Toronto, 2015.
[11] VUONG T V, MASTER E R. Fusion of a xylan-binding module to gluco-oligosaccharide oxidase increases activity and promotes stable immobilization[J]. PLoS One, 2014, 9(4): e95170. doi: 10.1371/journal.pone.0095170
[12] LIN S F, HWANG Y L, TSAI Y C. Immobilization of glucooligosaccharide oxidase of Acremonium strictum for oligosaccharic acid production[J]. Biotechnology Techniques, 1996, 10(1): 63-68.
[13] KIRYU T, NAKANO H, KISO T, et al. Purification and characterization of a carbohydrate:acceptor oxidoreductase from Paraconiothyrium sp. that produces lactobionic acid efficiently[J]. Bioscience, Biotechnology, and Biochemistry, 2008, 72(3): 833-841.
[14] LIN S F, YANG T Y, INUKAI T, et al. Purification and characterization of a novel glucooligosaccharide oxidase from Acremonium strictum T1[J]. Biochimica et Biophysica Acta, 1991, 1 118(1): 41-47.
[15] HUANG C, LAI W, LEE M, et al. Crystal structure of glucooligosaccharide oxidase from Acremonium strictum[J]. Journal of Biological Chemistry, 2005, 280(46): 38 831-38 838.
[16] FOUMANI M, VUONG T V, MASTER E R. Altered substrate specificity of the gluco-oligosaccharide oxidase from Acremonium strictum[J]. Biotechnology and Bioengineering, 2011, 108(10): 2 261-2 269.
[17] 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.
[18] LEE M H, LAI W L, LIN S F, et al. Structural characterization of glucooligosaccharide oxidase from Acremonium strictum[J]. Applied and Environmental Microbiology, 2005, 71(12): 8 881-8 887.
