摘要
木薯α 羟腈酶是一种催化羟腈化合物分解和合成的裂解酶 ,它在不对称合成手性羟腈化合物和手性药物等方面具有广泛的应用前景。通过RT PCR从木薯叶组织总RNA中扩增出α 羟腈酶基因的cDNA序列 ,并将其克隆于质粒 pPIC9K。序列分析表明 ,克隆的基因和文献报道的 3个木薯羟腈酶的cDNA序列不完全一致 ,可能是木薯羟腈酶基因家族的新成员。通过一系列步骤 ,将获得的α 羟腈酶基因cDNA序列插入表达载体 pET30a,在大肠杆菌获得高效表达。每升发酵液获得 2 10 0单位的羟腈酶 ,粗酶液的比活为 8 5u/mg蛋白质 ,并首次利用热变性法纯化重组羟腈酶。重组羟腈酶催化合成的 (S) 扁桃腈的ee值达 95 2 % ,转化率为 98 2 %。
Hydroxynitrile lyase (ME HNLs,E.C.4.1.2.3.37) from the cyanogenic crop cassava(\%Manihot esculentz,\%Crantz) catalyze the condensation of hydrocyanic acid and aldehydes or ketone into (s)\|cyanohydrins, which are valuable starting material for various optically active compounds, such as pharmaceuticals and agrochemicals. The cDNA of a ME HNL were obtained by RT\|PCR and cloned. The sequencing result for the cDNA showed that the sequence encoded for the ME HNL was inconsistent with all those which are published, such as \%hnl10,hnl24,hnl4.\% The full sequence analysis demonstrated that the cloned cDNA was about 75.2%,79.8%,99.2% homologous to other three reported HNL genes from cassava, respectively, among which the last was the same to the cloned gene except the five base substitution at the site 142,337,476,634 and 636,respectively. The two base substitutions lead to change the amino acid sequence, i.e. , Ser113→Gly113,Phe158→Tyr158.To construct the recombinant plasmid pET30a\|hnl, the cDNA was inserted into an expression vector pET30a. After transformation of pET30a\|hnl and induction with IPTG, the ME HNL was efficiently expressed in \%E.coli\%. BL21(DE3) and reached over 2100units/L of culture with the specific activity 8.5u/mg protein. By one simple treatment, incubating 10 minutes at 70℃, the recombinant ME HNL may be used as an catalyst for production of (S)\|mandelonitrile with enantiomeric excess of 95.2% and 98.2% yield.
出处
《生物工程学报》
CAS
CSCD
北大核心
2001年第1期78-78,共1页
Chinese Journal of Biotechnology
基金
国家自然科学基金支持的重大项目!(2 9790 12 8)&&
