Dataset Information

Cloning, expression, and characterization of a porcine pancreatic ?-amylase in Pichia pastoris.

ABSTRACT: Pancreatic ?-amylase (?-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 ?-amylase (PPA). The full-length cDNA encoding the PPA was isolated from pig pancreas by RT-PCR and cloned into the pPICZ?A vector. After the resultant pPICZ??-PPA plasmid was transferred into Pichia pastoris, 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 (?-amylase from porcine pancreas). The rePPA exhibited a molecular mass of approximately 58 kDa and showed optimal temperature (50 °C), optimal pH (7.5), Km (47.8 mg/mL), and Vmax (2,783 U/mg) similar to those of the natural enzyme. The recombinant enzyme was stable at 40 °C but lost 60% to 90% (P < 0.05) after exposure to heating at ?50 °C for 30 min. The enzyme activity was little affected by Cu2+ or Fe3+, but might be inhibited (40% to 50%) by Zn2+ at concentrations in pig digesta. However, Ca2+ exhibited a dose-dependent stimulation of the enzyme activity. In conclusion, the present study successfully cloned the porcine pancreatic ?-amylase gene and over-expressed the gene in P.pastoris as an extracellular, functional enzyme. The biochemical characterization of the over-produced enzyme depicts its potential and future improvement as an animal feed additive.

SUBMITTER: Sun LH

PROVIDER: S-EPMC6104570 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Cloning, expression, and characterization of a porcine pancreatic α-amylase in <i>Pichia pastoris</i>.

Sun Lv-Hui LH Qin Tao T Liu Yan Y Zhao Hua H Xia Xinjie X Lei Xingen X

Animal nutrition (Zhongguo xu mu shou yi xue hui) 20180102 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 <i>Pichia pastoris</i>, Ni Sepharose affinity c ...[more]

PMID: 30140765

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Project description:BackgroundFungal amylase, mainly constitute of fungal α-amylase and glucoamylase, are utilized in a broad range of industries, such as starch hydrolysis, food and brewing. Although various amylases have been found in fungi, the amylases from Aspergillus dominate the commercial application. One of main problems exist with regard to these commercial use of amylases is relatively low thermal and acid stability. In order to maximize the efficiency of starch process, developing fungal amylases with increased thermostability and acid stability has been attracting researchers' interest continually. Besides, synergetic action of glucoamylase and α-amylase could facilitate the degradation of starch. And co-expressing glucoamylase with α-amylase in one host could avoid the need to ferment repeatedly and improves cost-effectiveness of the process.ResultsA novel fungal glucoamylase (RpGla) gene encoding a putative protein of 512 amino acid residues was cloned from Rhizomucor pusillus. BLAST analysis revealed that RpGla shared highest identity of 51% with the Rhizopus oryzae glucoamylase (ABB77799.1). The fungal glucoamylase RpGla was expressed in Pichia pastoris (KM71/9KGla) with maximum activity of 1237 U ml(-1). The optimum pH and temperature of RpGla were pH 4.0 and 70 °C, respectively. Fungal α-amylase (RpAmy) gene was also cloned from R. pusillus and transformed into KM71/9KGla, resulted in recombinant yeast KM71/9KGla-ZαAmy harboring the RpGla and RpAmy genes simultaneously. The maximum saccharogenic activity of KM71/9KGla-ZαAmy was 2218 U ml(-1), which improved 79% compared to KM71/9KGla. Soluble starch hydrolyzed by purified RpGla achieved 43% glucose and 34% maltose. Higher productivity was achieved with a final yield of 48% glucose and 47% maltose catalyzed by purified enzyme preparation produced by KM71/9KGla-ZαAmy.ConclusionsA novel fungal glucoamylase and fungal α-amylase genes were cloned from Rhizomucor pusillus. The two enzymes showed good thermostability and acid stability, and similar biochemical properties facilitated synergetic action of the two enzymes. A dramatic improvement was seen in amylase activity through co-expressing RpGla with RpAmy in Pichia pastoris. This is the first report of improving activity through co-expression glucoamylase with α-amylase in P. pastoris. Besides, fungal glucoamylase and α-amylase from R. pusillus were shown as promising candidates for further application in starch hydrolysis.

Project description:BACKGROUND: Laccases are blue multi-copper oxidases and catalyze the oxidation of phenolic and non-phenolic compounds. There is considerable interest in using these enzymes for dye degradation as well as for synthesis of aromatic compounds. Laccases are produced at relatively low levels and, sometimes, as isozymes in the native fungi. The investigation of properties of individual enzymes therefore becomes difficult. The goal of this study was to over-produce a previously reported laccase from Cyathus bulleri using the well-established expression system of Pichia pastoris and examine and compare the properties of the recombinant enzyme with that of the native laccase. RESULTS: In this study, complete cDNA encoding laccase (Lac) from white rot fungus Cyathus bulleri was amplified by RACE-PCR, cloned and expressed in the culture supernatant of Pichia pastoris under the control of the alcohol oxidase (AOX)1 promoter. The coding region consisted of 1,542 bp and encodes a protein of 513 amino acids with a signal peptide of 16 amino acids. The deduced amino acid sequence of the matured protein displayed high homology with laccases from Trametes versicolor and Coprinus cinereus. The sequence analysis indicated the presence of Glu 460 and Ser 113 and LEL tripeptide at the position known to influence redox potential of laccases placing this enzyme as a high redox enzyme. Addition of copper sulfate to the production medium enhanced the level of laccase by about 12-fold to a final activity of 7200 U L-1. The recombinant laccase (rLac) was purified by ~4-fold to a specific activity of ~85 U mg(-1) protein. A detailed study of thermostability, chloride and solvent tolerance of the rLac indicated improvement in the first two properties when compared to the native laccase (nLac). Altered glycosylation pattern, identified by peptide mass finger printing, was proposed to contribute to altered properties of the rLac. CONCLUSION: Laccase of C. bulleri was successfully produced extra-cellularly to a high level of 7200 U L(-1) in P. pastoris under the control of the AOX1 promoter and purified by a simple three-step procedure to homogeneity. The kinetic parameters against ABTS, Guaiacol and Pyrogallol were similar with the nLac and the rLac. Tryptic finger print analysis of the nLac and the rLac indicated altered glycosylation patterns. Increased thermo-stability and salt tolerance of the rLac was attributed to this changed pattern of glycosylation.

Project description: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.

Dataset Information

Cloning, expression, and characterization of a porcine pancreatic ?-amylase in Pichia pastoris.

Publications

Cloning, expression, and characterization of a porcine pancreatic α-amylase in <i>Pichia pastoris</i>.

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