两种假单胞杆菌协同发酵产脂肪酶优化和酶性质研究

Optimization of Synergetic Fermentation of Two Pseudomonas sp. to Produce Lipase and Study of the Properties of the Lipase

假单胞菌具有现有微生物中丰富的酶库,目前研究单一的假单胞杆菌发酵产脂肪酶的较多,而忽略了假单胞杆菌协同发酵产脂肪酶的能力。由此本研究选取假单胞菌种中具有代表性的荧光假单胞杆菌和绿脓杆菌作为研究对象,优化假单胞杆菌的协同作用产生高活性脂肪酶的条件。通过对菌种接种比例、碳源、氮源、初始p H值、温度、表面活性剂及发酵时间等进行单因素优化和正交试验获得最适发酵条件。荧光假单胞杆菌和绿脓杆菌最佳接种比例(w∶w)为1.3∶1,最适发酵产酶条件为α-乳糖6 g/L、蛋白胨8 g/L、吐温-80 1%、CaCl_20.5 g/L、KH_2PO_46 g/L、Mn SO_40.3 g/L、Na Cl 5 g/L、接种量8%,、初始p H7.5,培养温度32℃、摇床转速150 r/min,在最适发酵条件下,该菌株最大产酶酶活能达到(33.424±0.05)U/m L。荧光假单胞杆菌和绿脓杆菌协同发酵产生的脂肪酶比单一的荧光假单胞杆菌和单一的绿脓杆菌具有较高的单位酶活性催化合成单甘酯能力,具有良好的应用前景。

Microbial lipase is a kind of important industrial enzyme,in which Pseudomonas has abundant enzyme pool in existing microorganisms.The current study mainly focuses on a single Pseudomonas for producing lipase,while ignoring synergistic fermentation of Pseudomonas sp to produce lipase.Therefore,in this study,Pseudomonas fluorescens and Pseudomonas aeruginosa were selected as the research objects to optimize their synergetic fermentation conditions for producing highly-active lipase.The optimum fermentation conditions were obtained by single factor test and orthogonal test on the inoculation ratio,carbon source,nitrogen source,initial pH,temperature,surfactant and fermentation time as follows:the best inoculation ratio of Pseudomonas fluorescens to Pseudomonas aeruginosa was 1.3∶1(w∶w);the optimal fermentation proportioning wereα-lactose 6 g/L,peptone 8 g/L,Tween-80 1%,Ca-Cl2 0.5 g/L,KH2PO4 6 g/L,MnSO4 0.3 g/L,and NaCl 5 g/L;the inoculating quantity was 8%,initial pH=7.5,culture temperature was at 32℃,and shaking speed was 150 r/min.Under the above conditions,the maximum enzyme activity of this strain reached up to(33.424±0.05)U/mL.Lipase produced by co-fermentation of Pseudomonas fluorescens and Pseudomonas aeruginosa had a higher unit enzyme activity to catalyze the synthesis of monoglyceride than that produced by single Pseudomonas fluorescens or Pseudomonas aeruginosa,and it had good application prospect.