产γ-内酰胺水解酶菌株的筛选及发酵条件研究

Study on the screening of lactamase and its fermentation conditions

(-)γ-内酰胺是合成两种抗艾滋病药物(-)carbovir和(-)abacavir的重要原料,具有重要的应用价值,微生物酶法拆分(+/-)γ-内酰胺生产(-)γ-内酰胺的方法具有良好的应用前景。以N-乙酰苯丙氨酸为唯一碳源,从土壤中筛选得到了69株具有酰胺水解酶活性的菌株,利用液相色谱分析方法确定了其中20株有较高的酰胺水解酶活性,利用手性色谱分析的方法进一步得到了一株具有较高立体选择性,能拆分(+/-)γ-内酰胺而获得(-)γ-内酰胺的菌株L29-9,对该菌株的产酶培养基的碳、氮源及初始pH值等进行了研究。结果表明:当选择碳源柠檬酸2g/L、氮源酵母提取物5g/Lp、H 7.0、培养时间40h时,通过完整细胞转化,在30℃下经过12h的反应,产物(-)γ-内酰胺产率达40%,ee值99.5%。

（ - ）-7-Lactam is one of the key starting materials for the synthesis （ - ）-cabovir and （ - ）-abacavir, which serve as two powerful antiviral agents. The production of 7-1actam using enzymatic resolution of racemic 7-1actam was attempted in the paper. 20 out of 69 strains capable of producing lactamase were screened from soil samples collected in several districts throughout Beijing; particularly N-actylphenylalanine was used as sole carbon source in the screening method. It was found by chiral HPLC analysis that strain L29-9 was able to enantioselectively hydrolyze the （ ＋ ）-isomer in the racemic lactam, thus giving the desired （ - ）-7-1actam with high enantiomeric excess （ 〉 70%）. Ferment conditions of strain L29-9, including carbon source, nitrogen source, pH, and culturing time, were studied and the optimum conditions were as follows： citric acid 2g/L, yeast extract 5glL, pHT.O, culturing for 40h. Biotransformation using whole cells of the strain was inducted at 30% for 12h, giving （ - ）-7-1actam as enantiopure product with yield up to 40% and 99.5% e.e.