Burkholderia sp.JXJ-16低温耐有机溶剂脂肪酶产酶条件优化及粗酶酶学性质

Optimization of enzyme production conditions and enzymatic properties of cold-adapted and organic solvent-tolerant lipase from Burkholderia sp.JXJ-16

以Burkholderia sp.JXJ-16为出发菌株,对其低温耐有机溶剂脂肪酶的产酶条件进行单因素实验并研究其粗酶酶学性质。该菌株的最佳产酶条件为:蔗糖3.75 g/L、尿素11.25 g/L、K_2HPO_42 g/L、(NH4)2SO_41 g/L、Mn SO_40.25 g/L、猪油乳化液体积分数2.5%,初始p H9.0、培养温度30℃、装样量20 m L/250 m L、接种量3%、发酵时间20 h。JXJ-16脂肪酶粗酶对中链对硝基苯酚酯有最大水解活力,最适底物为对硝基苯酚辛酸酯;该粗酶在35℃、p H8.5~9.0时酶活力最高,且具有较好的温度(30~60℃)和p H(5.0~10.5)稳定性;Na^+、Mg^(2+)、Ca^(2+)、Mn^(2+)、EDTA对粗酶活力具有激活作用,Zn^(2+)、Cu^(2+)、Fe^(3+)对粗酶活力具有抑制作用,K^+对粗酶活力没有显著性影响;除乙醇和乙腈外,该粗酶在一定体积分数的异丙醇、甲醇、丙酮、乙酸乙酯、三氯甲烷、二甲苯和正己烷中具有良好耐受性,且处于激活状态。综上,该菌株能利用廉价易得的培养基原料达到最佳产酶效果,其所产脂肪酶为低温碱性脂肪酶,具有较好的温度和p H稳定性,对多数供试有机溶剂具有良好耐受性。

Enzyme production conditions for Burkholderia sp. JXJ- 16 producing cold- adapted and organic solvent- tolerant lipase were optimized by signal factor analysis to maximize lipase production.A maximum lipase production was obtained after20 h fermentation of 20 m L of medium containing sucrose 3.75 g / L,urea 11.25 g / L,K_2HPO_42 g / L,（ NH4）2SO_41 g / L,Mn SO40.25 g / L,and lard emulsion 2.5%（ v / v） at an initial p H9.0 in 250 m L Erlenmeyer flask with 3% inoculum at 30 ℃.The crude JXJ- 16 lipase preferentially catalyzed the p- nitrophenyl esters of different fatty acids with medium chains,and its best substrate was p- nitrophenyl caprylate.The optimal reaction temperature for the crude lipase was 35 ℃,and it remained better stability at temperatures ranging from 30 to 60 ℃.The optimum hydrolytic activity of the crude lipase was observed at p H8.5 to9.0,and it exhibited better stability at p H5.0 to 10.5.The activity of the crude lipase was activated by Na~＋,Mg^2＋,Ca^2＋,Mn^2＋,and EDTA,respectively,whereas inhibited by Zn^2＋,Cu^2＋and Fe^3＋,respectively.Moreover,K＋had no remarkable effect on its activity.The crude lipase had high tolerance and enhanced activity in the presence of different concentration of isopropyl alcohol,methanol,acetone,ethyl acetate,chloroform,benzene,and n- hexane,respectively,except ethanol and acetonitrile. In a word,the strain JXJ-16 could utilize cheap raw materials as a fermentation medium to achieve the optimal lipase production,and its cold- adapted alkaline lipase had better temperature and p H stability and showed high tolerance to most of the tested organic solvents.