Haas M J, Allen J, Berka T R
Eastern Regional Research Center, U.S. Department of Agriculture, Philadelphia, PA 19118.
Gene. 1991 Dec 20;109(1):107-13. doi: 10.1016/0378-1119(91)90594-2.
A lambda gt11 cDNA library was constructed in Escherichia coli using poly(A)-selected mRNA from the fungus, Rhizopus (Rp.) delemar. Lipase-producing members of the library were identified by means of a phenotypic score wherein the release of fatty acids by lipase causes a characteristic color change in the growth medium. One such isolate contained a 1287-bp insert (LIP cDNA) which hybridizes to 1.25- to 1.35-kb mRNA species from Rp. delemar. The lipase produced in E. coli containing the LIP cDNA exhibits the same substrate selectivity as the authentic fungal enzyme, hydrolyzing ester bonds at the stereospecific numbering (sn) sn-1 and sn-3, but not the sn-2, positions of triglycerides. The complete nucleotide sequence of the LIP cDNA was determined. By reference to the N-terminal sequence of authentic Rp. delemar lipase, the lipase-encoding region was identified within this fragment. The LIP cDNA encodes a putative preprolipase consisting of a 26-amino-acid(aa) signal sequence, a 97-aa propeptide, and a 269-aa mature enzyme. The predicted mature lipase has the same molecular weight and aa composition as that of Rp. delemar, is highly homologous to that produced by the fungus Rhizomucor miehei, and contains the consensus pentapeptide (Gly-Xaa-Ser-Yaa-Gly) which is conserved among lipolytic enzymes. It is concluded that the LIP cDNA is an essentially full-length analogue of the lipase-encoding gene of Rp. delemar. The lipase encoded by the LIP cDNA occupies a cytoplasmic location when synthesized in E. coli. Unprocessed forms of the lipase accumulate in E. coli.
利用来自真菌德氏根霉（Rhizopus (Rp.) delemar）的聚腺苷酸（poly(A)）选择的信使核糖核酸（mRNA），在大肠杆菌中构建了一个λgt11 cDNA文库。通过表型评分来鉴定文库中产生脂肪酶的成员，其中脂肪酶释放脂肪酸会导致生长培养基中出现特征性颜色变化。一个这样的分离株包含一个1287碱基对（bp）的插入片段（LIP cDNA），它与德氏根霉的1.25至1.35千碱基（kb）的mRNA种类杂交。在含有LIP cDNA的大肠杆菌中产生的脂肪酶表现出与天然真菌酶相同的底物选择性，水解甘油三酯的立体专一编号（sn）sn-1和sn-3位置的酯键，但不水解sn-2位置的酯键。确定了LIP cDNA的完整核苷酸序列。通过参考天然德氏根霉脂肪酶的N端序列，在该片段内鉴定出脂肪酶编码区域。LIP cDNA编码一个假定的前原脂肪酶，由一个26个氨基酸（aa）的信号序列、一个97个aa的前肽和一个269个aa的成熟酶组成。预测的成熟脂肪酶与德氏根霉的分子量和aa组成相同，与米黑根毛霉（Rhizomucor miehei）产生的脂肪酶高度同源，并且包含在脂解酶中保守的共有五肽（甘氨酸-Xaa-丝氨酸-Yaa-甘氨酸）。得出的结论是，LIP cDNA是德氏根霉脂肪酶编码基因的一个基本全长类似物。当在大肠杆菌中合成时，LIP cDNA编码的脂肪酶位于细胞质中。未加工形式的脂肪酶在大肠杆菌中积累。
