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

摘要:

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(Paenibacillus sp.) 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K_m、k_cat/K_m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。PsOX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。

Abstract:

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 K_m and k_cat/K_m 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.