Sun Lv-Hui, Qin Tao, Liu Yan, Zhao Hua, Xia Xinjie, Lei Xingen
Department of Animal Nutrition and Feed Science, Huazhong Agricultural University, Wuhan 430070, China.
International Center of Future Agriculture for Human Health, Sichuan Agricultural University, Chengdu 611134, China.
Anim Nutr. 2018 Jun;4(2):234-240. doi: 10.1016/j.aninu.2017.11.004. Epub 2018 Jan 2.
Pancreatic α-amylase (α-1, 4-glucan-4-glucanohydrolase, EC.3.2.1.1) plays a primary role in the intestinal digestion of feed starch and is often deficient in weanling pigs. The objective of this study was to clone, express, and characterize porcine pancreatic α-amylase (PPA). The full-length cDNA encoding the PPA was isolated from pig pancreas by RT-PCR and cloned into the pPICZαA vector. After the resultant pPICZαΑ-PPA plasmid was transferred into , Ni Sepharose affinity column was used to purify the over-expressed extracellular recombinant PPA protein (rePPA) that contains a His-tag to the C terminus and was characterized against the natural enzyme (α-amylase from porcine pancreas). The rePPA exhibited a molecular mass of approximately 58 kDa and showed optimal temperature (50 °C), optimal pH (7.5), (47.8 mg/mL), and (2,783 U/mg) similar to those of the natural enzyme. The recombinant enzyme was stable at 40 °C but lost 60% to 90% ( < 0.05) after exposure to heating at ≥50 °C for 30 min. The enzyme activity was little affected by Cu or Fe, but might be inhibited (40% to 50%) by Zn at concentrations in pig digesta. However, Ca exhibited a dose-dependent stimulation of the enzyme activity. In conclusion, the present study successfully cloned the porcine pancreatic α-amylase gene and over-expressed the gene in as an extracellular, functional enzyme. The biochemical characterization of the over-produced enzyme depicts its potential and future improvement as an animal feed additive.
胰腺α-淀粉酶（α-1,4-葡聚糖-4-葡聚糖水解酶，EC.3.2.1.1）在饲料淀粉的肠道消化中起主要作用，且在断奶仔猪中常常缺乏。本研究的目的是克隆、表达和鉴定猪胰腺α-淀粉酶（PPA）。通过RT-PCR从猪胰腺中分离出编码PPA的全长cDNA，并将其克隆到pPICZαA载体中。将所得的pPICZαΑ-PPA质粒转入[具体宿主菌名称缺失]后，使用镍琼脂糖亲和柱纯化在C末端含有His标签的过表达细胞外重组PPA蛋白（rePPA），并针对天然酶（猪胰腺α-淀粉酶）对其进行鉴定。rePPA的分子量约为58 kDa，其最佳温度（50°C）、最佳pH（7.5）、[具体底物亲和力数值缺失]（47.8 mg/mL）和[具体催化活性数值缺失]（2,783 U/mg）与天然酶相似。重组酶在40°C下稳定，但在≥50°C加热30分钟后损失60%至90%（P<0.05）。酶活性受铜或铁的影响较小，但在猪消化道中的锌浓度下可能会被抑制（40%至50%）。然而，钙对酶活性表现出剂量依赖性刺激。总之，本研究成功克隆了猪胰腺α-淀粉酶基因，并在[具体宿主菌名称缺失]中作为细胞外功能性酶进行了过表达。过量产生的酶的生化特性描述了其作为动物饲料添加剂的潜力和未来改进方向。
