类芽孢杆菌来源D-甘露醇氧化酶的性质及制备D-甘露糖的应用

Characterization of a D-mannitol oxidase from Paenibacillus sp.and its application in the preparation of D-mannose

D-甘露糖具有多种功能活性,在食品、医药、农业等行业应用广泛。D-甘露醇氧化酶可以高效地将D-甘露醇转化为D-甘露糖,在D-甘露糖的酶法制备中具有应用潜力。从类芽孢杆菌(Paenibacillussp.)HGF5中发掘出一个D-甘露醇氧化酶(PsOX),与天蓝链霉菌(Streptomyces coelicolor)来源的D-甘露醇氧化酶(AldO)氨基酸序列相似性为50.94%,分子量约为47.4 kDa,构建了重组表达质粒pET-28a-PsOX并在大肠杆菌BL21(DE3)中表达,PsOX对D-甘露醇的Km、kcat/Km值分别为5.6 mmol/L、0.68 L/(s·mmol),最适pH和温度分别为7.0和35℃,在60℃以下保持稳定。Ps OX对400 mmol/L D-甘露醇的摩尔转化率为95.2%。利用PsOX与AldO全细胞分别催化73 g/L D-甘露醇,PsOX反应9 h后反应完全,生成70 g/L D-甘露糖,相较于AldO具有更高的催化效率。PsOX作为新型D-甘露糖氧化酶为D-甘露糖的酶法制备提供了依据。

D-mannose has many functional activities and is widely used in food,medicine,agriculture and other industries.D-mannitol oxidase that can efficiently convert D-mannitol into D-mannose has potential application in the enzymatic preparation of D-mannose.A D-mannitol oxidase(PsOX)was found from Paenibacillus sp.HGF5.The similarity between PsOX and the D-mannitol oxidase(AldO)from Streptomyces coelicolor was 50.94%.The molecular weight of PsOX was about 47.4 kDa.A recombinant expression plasmid pET-28a-PsOX was constructed and expressed in Escherichia coli BL21(DE3).The Km and kcat/Km values of PsOX for D-mannitol were 5.6 mmol/L and 0.68 L/(s∙mmol).Further characterization of PsOX showed its optimal pH and temperature were 7.0 and 35℃,respectively,while its enzyme activity could be stably remained below 60℃.The molar conversion rate of 400 mmol/L D-mannitol by PsOX was 95.2%.The whole cells of PsOX and AldO were used to catalyze 73 g/L D-mannitol respectively.The reaction catalyzed by PsOX completed in 9 h and 70 g/L D-mannose was produced.PsOX showed a higher catalytic efficiency compared to that of AldO.PsOX may facilitate the enzymatic preparation of D-mannose as a novel D-mannose oxidase.