Department of Biotechnology, Faculty of Biological Sciences , Alzahra University, Tehran, Iran.
Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran.
Iran Biomed J. 2022 Mar 1;26(2):153-9. doi: 10.52547/ibj.26.2.153.
Lipase enzymes are of great importance in various industries. Currently, extensive efforts have been focused on exploring new lipase producer microorganism as well as genetic and protein engineering of available lipases to improve their functional features.
For screening lipase-producing lactobacilli, isolated strains were inoculated onto tributyrin agar plates. Molecular identification of lipase-producing Lactobacilli was performed by sequencing the 16Sr DNA gene, and a phylogenetic tree was constructed. The LAF_RS05195 gene, encoding lipase protein in L. fermentum isolates, was identified using specific primers, amplified by PCR (918 bp) and cloned into the pET28a (+) vector. The recombinant proteins were expressed 2, 4, 6, and 12 hours after induction with IPTG and assessed using the SDS polyacrylamide gel electrophoresis (SDS-PAGE). Enzymatic activity of the purified recombinant protein was measured at 410 nm in the presence of ρ-NPA and ρ-NPP.
Among five identified native lipase-producing isolates, one isolate showed 98% similarity with Enterococcus species. The other four isolates indicated 98% similarity to L. fermentum. After purification steps with Ni-NTA column, a single protein band of about 34 kDa was detected on SDS-PAGE gel. The enzymatic activity of purified recombinant protein alongside ρ-NPA and ρ-NPP was measured to be 0.6 U/ml and 0.2 U/ml, respectively.
In the present research, a novel lipase/esterase from L. fermentum was cloned and expressed. The novel lipase/esterase has the merit to be further studied due to its substrate specificity.
脂肪酶在各个行业都具有重要意义。目前，人们广泛致力于探索新的脂肪酶产生微生物，并对现有脂肪酶进行遗传和蛋白质工程改造，以改善其功能特性。
为了筛选产脂肪酶的乳杆菌，将分离的菌株接种到三丁酸甘油酯琼脂平板上。通过测序 16SrDNA 基因对产脂肪酶的乳杆菌进行分子鉴定，并构建系统发育树。使用特异性引物鉴定编码发酵乳杆菌分离株脂肪酶蛋白的 LAF_RS05195 基因，通过 PCR（918bp）扩增并克隆到 pET28a（+）载体中。在 IPTG 诱导后 2、4、6 和 12 小时表达重组蛋白，并使用 SDS 聚丙烯酰胺凝胶电泳（SDS-PAGE）进行评估。在存在 ρ-NPA 和 ρ-NPP 的情况下，测量纯化重组蛋白的酶活性在 410nm 处。
在鉴定的五个天然产脂肪酶的分离株中，有一个分离株与肠球菌属具有 98%的相似性。其他四个分离株与发酵乳杆菌具有 98%的相似性。经过 Ni-NTA 柱纯化步骤后，在 SDS-PAGE 凝胶上检测到约 34kDa 的单一条带。用 ρ-NPA 和 ρ-NPP 测量纯化重组蛋白的酶活性分别为 0.6U/ml 和 0.2U/ml。
在本研究中，克隆并表达了来自发酵乳杆菌的新型脂肪酶/酯酶。由于其底物特异性，新型脂肪酶/酯酶具有进一步研究的价值。
