嗜糖假单胞菌麦芽四糖淀粉酶的枯草芽孢杆菌表达及酶学性质分析

Properties of the enzyme expressed by the optimized Pseudomonas saccharophila maltotetraohydrolase gene(mta) in Bacillus subtilis

麦芽四糖淀粉酶是淀粉酶中第3种外切型酶,可以顺序地从淀粉非还原性末端依次切割第4个α-1,4糖苷键,产物为麦芽四糖,广泛应用于食品、医疗保健等领域。该研究采用含有P43启动子和SacB信号肽的pWB980枯草芽孢杆菌表达载体,连接嗜糖假单胞菌来源的麦芽四糖淀粉酶基因,获得了重组菌pWB980-G4/1A747,实现了麦芽四糖淀粉酶在枯草芽孢杆菌中的外分泌表达,并具有生物学活性。对表达产物进行酶学性质分析,结果表明麦芽四糖淀粉酶可广泛作用于7种不同来源的淀粉,生成单一产物麦芽四糖。最适反应温度为50℃,最适pH为7.0。进一步对pWB980-G4/1A747菌株进行发酵条件优化,得到其最佳活化时间为5 h,接种量为5%(v/v),通气量为20 mL/150 mL,培养时间为24 h,培养温度为37℃。

G4-amylase catalyses hydrolysis of 1,4-α-D-glucosidic linkages in amylaceous polysaccharides to remove successive maltotetraose residues from the non-reducing chain ends. As a new type of exo-amylase,it is widely used in food,medical care and other fields. The gene sequence of this enzyme from Pseudomonas saccharophila was optimized,and the gene was synthesized and then cloned into expression vector pWB980. The protein with the activity of G4-amylase has been detected in the culture medium of Bacillus subtilis pWB980-G4 /1A747. Hydrolysis product analysis of seven sources of starch showed that only maltotetraose were presented. By DNS determination,the optimum temperature was 50 ℃,the optimum pH was 7. 0. The further research indicated that the optimal fermentation conditions were activation time 5 h,inoculum 5%( vol),loading liquid amount 20 mL in 150 mL,culture time 24 h and culture temperature 37 ℃.