基因工程菌EGs2产β-葡聚糖酶条件优化及产酶特性

摘要：用乳清粉作为主要培养基组成，采用正交试验对重组菌EGs2的发酵条件进行了优化，并对粗酶液的酶学性质进行了研究。重组菌EGs2的最佳发酵条件为：摇床转速170 r?min-1，接种量1 ％，发酵培养基初始pH值6.0，种龄12 h。重组菌EGs2β－葡聚糖酶的表达与菌体生长呈正相关，发酵14h后，菌体生物量最高可达1.71 g?L -1，β－葡聚糖酶活力最高可达321.56 U?ml-1。所得粗酶液的最适反应温度和pH值分别为60 ℃和6.0，在70 ℃以下保温20 min后，残余酶活力均在80 %以上。在pH 5.0-9.0 放置48 h后仍能保持均90 %以上的残余酶活力。

Optimization of Recombinant Escherichia coli EGs2 Fermentation Condition Affecting β-glucanase Production and Its Characteristics of Enzyme

Abstract: The fermentation conditions of mutant EGs2 (obtained from directed evolution for thermostable β-glucanase) for β-glucanase production were investigated with orthogonal experiment.Fermentation was conducted in 250 mL flask, each containing 30 mL of medium contained Whey 10 g?L-1,Yeast extract 5 g?L-1,NaCl 10 g?L-1,the temperature was 37 ℃.The optima culture conditions were as following: initial pH value 6.0, shaking speed 170 r?min-1,inoculation volume 1 %,inoculation time 12 h.β-glucanase production by mutant EGs2 was associated with cell growth and biomass,β-glucanase activity was increased significantly when cells entered growth phase.The bacterium began the stable phase after cultured for 14 h with the highest biomass 1.71 g?L -1 and β-glucanase activity 321.56 U?ml-1.When β-glucan was used as a substrate,the optimum temperature and pH were respectively 60 ℃ and 6.0.After 20 min incubation at 40 ℃,50 ℃,55 ℃,60 ℃,65 ℃,70 ℃, the residual activity remained at least 80%.After 48 h conservation at pH 5.0-9.0,the residual activity remained at least 90%.The enzyme was stable below 70 ℃ and at pH 5.0-9.0.