Zhao Hua, Chen Dan, Tang Jiayong, Jia Gang, Long Dingbiao, Liu Guangmang, Chen Xiaoling, Shang Haiying
Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
Chongqing Academy of Animal Science, Chongqing, 402460, China.
PLoS One. 2014 Dec 29;9(12):e114385. doi: 10.1371/journal.pone.0114385. eCollection 2014.
Pancreatic lipase plays a key role in intestinal digestion of feed fat, and is often deficient in young animals such as weaning piglets. The objective of this study was to express and characterize a partial codon optimized porcine pancreatic lipase (opPPL). A 537 bp cDNA fragment encoding N-terminus amino acid residue of the mature porcine pancreatic lipase was synthesized according to the codon bias of Pichia pastoris and ligated to the full-length porcine pancreatic lipase cDNA fragment. The codon optimized PPL was cloned into the pPICZαA (Invitrogen, Beijing, China) vector. After the resultant opPPL/pPICZαΑ plasmid was transformed into P. pastoris, the over-expressed extracellular opPPL containing a His-tag to the C terminus was purified using Ni Sepharose affinity column (GE Healthcare, Piscataway, NJ, USA), and was characterized against the native enzyme (commercial PPL from porcine pancreas, Sigma). The opPPL exhibited a molecular mass of approximately 52 kDa, and showed optimal temperature (40°C), optimal pH (8.0), Km (0.041 mM), and Vmax (2.008 µmol x mg protein(-1) x min(-1)) similar to those of the commercial enzyme with p-NPP as the substrate. The recombinant enzyme was stable at 60°C, but lost 80% (P<0.05) of its activity after exposure to heat ≥60°C for 20 min. The codon optimization increased opPPL yield for ca 4 folds (146 mg x L(-1) vs 36 mg x L(-1)) and total enzyme activity increased about 5 folds (1900 IU x L(-1) vs 367 IU x L(-1)) compared with those native naPPL/pPICZαΑ tranformant. Comparison of gene copies and mRNA profiles between the two strains indicated the increased rePPL yields may partly be ascribed to the increased protein translational efficiency after codon optimization. In conclusion, we successfully optimized 5-terminal of porcine pancreatic lipase encoding gene and over-expressed the gene in P. pastoris as an extracellular, functional enzyme. The recombination enzyme demonstrates a potential for future use as an animal feed additive for animal improvement.
胰脂肪酶在饲料脂肪的肠道消化中起关键作用，且在诸如断奶仔猪等幼龄动物中常缺乏。本研究的目的是表达并鉴定一种部分密码子优化的猪胰脂肪酶（opPPL）。根据巴斯德毕赤酵母的密码子偏好性，合成了编码成熟猪胰脂肪酶N端氨基酸残基的537 bp cDNA片段，并将其连接到猪胰脂肪酶全长cDNA片段上。将密码子优化的PPL克隆到pPICZαA（Invitrogen，中国北京）载体中。将所得的opPPL/pPICZαΑ质粒转化到巴斯德毕赤酵母中后，使用Ni Sepharose亲和柱（GE Healthcare，美国新泽西州皮斯卡塔韦）纯化在C端含有His标签的过表达细胞外opPPL，并与天然酶（来自猪胰腺的商业PPL，Sigma）进行特性比较。opPPL的分子量约为52 kDa，以对硝基苯磷酸酯（p-NPP）为底物时，其显示出与商业酶相似的最适温度（40°C）、最适pH（8.0）、米氏常数（Km，0.041 mM）和最大反应速度（Vmax，2.008 μmol·mg蛋白⁻¹·min⁻¹）。重组酶在60°C时稳定，但在≥60°C加热20分钟后失去80%（P<0.05）的活性。与天然的naPPL/pPICZαΑ转化体相比，密码子优化使opPPL产量提高了约4倍（146 mg·L⁻¹对36 mg·L⁻¹），总酶活性提高了约5倍（1900 IU·L⁻¹对367 IU·L⁻¹）。两种菌株之间基因拷贝数和mRNA谱的比较表明，opPPL产量的增加可能部分归因于密码子优化后蛋白质翻译效率的提高。总之，我们成功地优化了猪胰脂肪酶编码基因的5'端，并在巴斯德毕赤酵母中过表达该基因，使其成为一种细胞外功能性酶。该重组酶显示出未来用作动物饲料添加剂以改善动物性能的潜力。
