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.
YUAN Xinyao
,
TIAN Kangming
,
JIN Peng
,
CHENG Lei
,
WANG Zhengxiang
. Molecular cloning and characterization of gluco-oligosaccharide oxidase from Aspergillus niger[J]. Food and Fermentation Industries, 2020
, 46(1)
: 30
-35
.
DOI: 10.13995/j.cnki.11-1802/ts.022274
[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.
