响应面法优化海洋细菌NTa产琼胶酶的发酵条件

Response Surface Optimization of Fermentation Conditions for Agarase Production by Marine Bacterium NTa

利用Plackett-Burman试验、最陡爬坡试验和响应面法,对培养基组成、培养条件对Stenotrophomonas sp.NTa发酵产琼胶酶的影响进行分析并优化,研究该菌株发酵产酶的动态规律,并利用薄层色谱和MALDI-TOF MS进行酶解产物的鉴定。结果显示,在琼脂、酵母浸膏、CaCl_(2)、MgSO_(4)·7H_(2)O、培养基的初始pH值、装液量、接种量几个因素中,酵母浸膏和培养基初始pH值对菌株NTa产琼胶酶具有显著影响,在含有1.03%酵母浸膏、初始pH=8.0的基础发酵培养基(NaCl、KNO_(3)、MgSO_(4)·7H_(2)O、CaCl_(2)、K_(2)HPO_(4)、FeSO_(4)·7H_(2)O、琼脂的质量浓度分别为50,5.0,5.0,0.2,0.1,0.02,2.0 g/L)中可获得最高的琼脂酶活力。菌株在28℃、180 r/min条件下发酵时,对数生长中后期快速合成琼胶酶。发酵48 h,琼胶酶活力高达2.688 U/mL。酶解72 h的产物分析结果显示,菌株NTa琼胶酶水解琼脂主要产生四糖。

Effects of medium composition and culture conditions on agarase production by Stenotrophomonas sp.NTa in liquid culture were investigated by Plackett-Burman design,steepest ascent method and response surface methodology.The dynamic law of enzyme production by this strain was studied,and the enzyme hydrolytic products were analyzed by thin-layer chromatography and MALDI-TOF MS.Among the tested seven factors,including agar,yeast extract,MgSO_(4)·7H_(2)O,CaCl_(2),initial culture pH,liquid medium volume,and inoculum size,yeast extract and initial culture pH had significant effects on agarase production.The highest agarase production was obtained in the basic fermentation medium(NaCl 50.0 g/L,KNO_(3)5.0 g/L,MgSO_(4)·7H_(2)O 5.0 g/L,CaCl_(2)0.2 g/L,K_(2)HPO_(4)0.1 g/L,FeSO_(4)·7H_(2)O 0.02 g/L,agar 2.0 g/L)containing 1.03%yeast extract and with initial pH of 8.0.At the conditions of 25℃and 180 r/min,agarase was rapidly synthesized in the middle and late log phase during the fermentation.After incubation for 48 h,the agarase activity reached 2.688 U/mL.Analysis of the enzyme hydrolytic products after 72 h reaction showed that the hydrolysis of agar by strain NTa agarase mainly produced tetraose.