1株产烟酸羟基化酶海洋细菌H9的分离鉴定及培养条件优化

摘要

利用96孔板和紫外显影相结合的定向快速筛选方法,从烟台渤海湾采集的腐败鱼尸上分离筛选得到1株高产烟酸羟基化酶的菌株H9,通过形态、生理生化特征和16S rDNA系统发育分析鉴定为恶臭假单胞菌(Pseudomonas purida),对其产酶培养条件进行优化,最适培养基和培养条件为:可溶性淀粉15g/L、玉米浆15g/L、烟酸10g/L、K2HPO4 1.0 g/L、初始pH 7.0、培养温度25℃、接种量7％、500mL三角瓶装液量50 mL.优化后烟酸羟基化酶活力达到0.37 U/mL,比优化前提高了68％,采用静息细胞转化生产6-羟基烟酸产率达到124.77g/L,具有工业化生产6-羟基烟酸的应用前景.%A nicotinic acid hydroxylase-producing strain from spoiled fish from Bohai Bay in Yantai,designated H9,was rapidly and directionally screened using a 96-well microplate and ultraviolet imaging.Strain H9 was identified as Pseudomonas putida based on its morphological,physiological and biochemical characteristics,and 16S rDNA sequence.Our results showed that the highest activity of nicotinic acid hydroxylase of 0.37 U/mL was obtained by culturing strain H9 with 50 mL of a medium consisting of 15 g/L soluble starch,15 g/L corn steep liquor,10 g/L nicotinic acid and,1.0 g/L K2HPO4 at an initial pH of 7.0 in a 500-mL flask at 25 ℃ with an inoculum amount of 7％,which was increased by 68％ as compared to that before optimization.The yield of 6-hydroxynicotinic acid (6-HNA) produced by resting cell biotransformation reached 124.77 g/L.Accordingly,strain H9 had a promising application potential in the industrial production of 6-hydroxynicotinic acid.