Cloning,Heterologous Expression And Characterization Of Gluco-oligosaccharide Oxidase

Gluco-oligosaccharide oxidase(GOOX)can specifically oxidize glucose and heptasaccharlde or shorter oligosaccharides derived from glucose linked by ?-1,4 or ?-1,4 glycosidic bonds,producing related aldonic acids.GOOX has important applications in the fields of sugar acid preparation and biological assay,but only three kinds of GOOX have been reported.The development of new GOOX was practically valuable for related industries.In this study,gluco-oligosaccharide oxidase genes from Aspergillus niger and Bacillus subtilis were cloned,expressed and characterized.An open reading frame suspected to encode gluco-oligosaccharide oxidase was screened from the genome of Bacillus subtilis.Two genes of different lengths,bsgoox-L and bsgoox-S,were cloned with two pairs of primers.The lengths of the two genes were 1443 bp and 1356 bp,respectively.Likewise,an open reading frame was screened from the genome of Aspergillus niger.Amino acid sequence alignment revealed that Angoox and Bsgoox were highly similar to oligosaccharide oxidases derived from A.piperis CBS 112811 and Bacillus velezensis,with similarities of 97.26%and 79.27%,respectively.Both enzymes possessed a FAD binding domain and a flavin domain,belonging to the oligosaccharide oxidase family.Heterologous expression of angoox,bsgoox-L and bsgoox-S in Pichia Pastoris was successfully conducted,and enzyme activity of 5,3.9,3.5 mU/mL was obtained,respectively,through shaking flask fermentation.The enzyme proteins were purlfied through ammonium sulfate precipitation,desalting and gel filtration.The purification multiples and yields were 19.2 and 42.2%for Angoox,14.1 and 39.8%for Bsgoox-L,and 18.7 and 33.2%for Bsgoox-S.The enzymatic properties and substrate specificities were further analyzed.Angoox maitained good stability at 55?-85? or pH5.0-10.0,with optimum temperature and pH of 75? and 9.0,respectively.Bsgoox-L and Bsgoox-S showed same enzymatic properties with optimum temperature and pH value of 80? and 8.0,respectively,and maitained good stability at 50?-85? or pH 5.0-9.0.Ca2+ and EDTA inhibited the activity of the two enzymes,while Na+,Cu2,K+ promoted the activity of the two enzymes.Angoox and Bsgoox possessed relatively broader substrate range,as well as different substrate preferences,of which Angoox showed best efficiency on maltose oligosaccharides but Bsgoox on sucrose.