葡枝根霉(Rhizopus stolonifer)YF6脂肪酶基因的克隆及其序列分析

筛选并鉴定了1株高产脂肪酶菌株葡枝根霉YF6,并采用简并PCR等分子技术从中克隆到1个新的脂肪酶基因lipRs及其对应的全长cDNA,序列分析表明,该基因编码区全长1173bp,不含内含子序列,编码1段由26个氨基酸残基组成的信号肽和1个由365个氨基酸残基组成的成熟蛋白,该成熟蛋白计算分子量(Mr)为39268.27,等电点为pH7.66,有5个可能的N-糖基化位点,含有脂肪酶特征序列GHSLGGA,与来自米根霉(Rhizopus oryzae)的脂肪酶(AAF32408)同源性最高,一致性为83%,相似性为89%.该基因序列已提交GenBank,登录号为DQ139862.

Cloning and Sequence Analysis of a Novel Lipase Gene from Rhizopus stolonifer YF6

A novel lipase-encoding cDNA from Rhizopus stolonifer YF6 was cloned by using degenerate PCR, rapid amplification of cDNA ends (RACE) and RT-PCR, and its structural gene was amplified by PCR. No intron was found in this lipase gene by nucleotide sequences alignment between the structural gene and its corresponding cDNA. The cDNA of the putative lipase gene (lipRs) consisted of 1 173 bp, including an open reading frame encoding a 26-amino acid signal peptide at the N-terminal end and a 365-amino acid mature protein with a predicted molecular mass of 39 268, a pI value of 7.66 and 5 potential N-glycosylation sites (N-X-T/S). The 7 conserved amino acid residues, GHSLGGA, required to the active site of the lipase from Rhizopus sp. were all found in this lipase, RSL. The amino acid sequences alignment result showed that the RSL had a high degree of identity with other Rhizopus lipases, and the highest one from Rhizopus oryzae (AAF32408) was 83%. This lipase gene sequence has been submitted to GenBank and its accession number is DQ139862.