Dataset Information

Expression of novel acidic lipase from Micrococcus luteus in Pichia pastoris and its application in transesterification.

ABSTRACT:

Background

Lipases are promising biocatalysts for industrial applications and attract attention to be explored. A novel acidic lipase has been isolated from the lipolytic bacteria Micrococcus luteus EMP48-D (LipEMP48-D) screened from tempeh. The lipase gene had previously been overexpressed in Escherichia coli BL21, but the expression level obtained was relatively low. Here, to improve the expression level, the lipase gene was cloned to Pichia pastoris. We eliminated the native signal sequence of M. luteus and replaced it with α-mating factor (α-MF) signal sequence. We also optimized and synthesized the lipase gene based on codon preference in P. pastoris.

Results

LipEMP48-D lipase was expressed as an extracellular protein. Codon optimization has been conducted for 20 codons, with the codon adaption index reaching 0.995. The highest extracellular lipase activity obtained reached 145.4 ± 4.8 U/mg under AOX1 promoter in P. pastoris KM71 strain, which was 9.7-fold higher than the previous activity in E. coli. LipEMP48-D showed the highest specific activity at pH 5.0 and stable within the pH range 3.0-5.0 at 40 °C. LipEMP48-D also has the capability of hydrolyzing various long-chain triglycerides, particularly olive oil (100%) followed by sunflower oil (88.5%). LipEMP48-D exhibited high tolerance for various polar organic solvents with low log P, such as isopropanol (115.7%) and butanol (114.6%). The metal ions (Na⁺, K⁺, Ca²⁺, Mg²⁺, Mn⁺) decreased enzyme activity up to 43.1%, while Fe²⁺ increased relative activity of enzymes up to 200%. The conversion of free fatty acid (FFA) into fatty acid methyl ester (FAME) was low around 2.95%.

Conclusions

This study was the first to report overexpression of Micrococcus lipase in yeast. The extracellular expression of this acidic lipase could be potential for biocatalyst in industrial fields, especially organic synthesis, food industry, and production of biodiesel.

SUBMITTER: Adina SR

PROVIDER: S-EPMC8026790 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

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.

Project description:BackgroundInducible high-level expression is favoured for recombinant protein production in Pichia pastoris. Therefore, novel regulated promoters are desired, ideally repressing heterologous gene expression during initial growth and enabling it in the production phase. In a typical large scale fed-batch culture repression is desired during the batch phase where cells grow on a surplus of e.g. glycerol, while heterologous gene expression should be active in the feed phase under carbon (e.g. glucose) limitation.ResultsDNA microarray analysis of P. pastoris wild type cells growing in glycerol-based batch and glucose-based fed batch was used for the identification of genes with both, strong repression on glycerol and high-level expression in the feed phase. Six novel glucose-limit inducible promoters were successfully applied to express the intracellular reporter eGFP. The highest expression levels together with strong repression in pre-culture were achieved with the novel promoters P(G1) and P(G6). Human serum albumin (HSA) was used to characterize the promoters with an industrially relevant secreted protein. A P(G1) clone with two gene copies reached about 230% of the biomass specific HSA titer in glucose-based fed batch fermentation compared to a P(GAP) clone with identical gene copy number, while P(G6) only achieved 39%. Two clones each carrying eleven gene copies, expressing HSA under control of P(G1) and P(G6) respectively were generated by post-transformational vector amplification. They produced about 1.0 and 0.7 g L(-1) HSA respectively in equal fed batch processes. The suitability in production processes was also verified with HyHEL antibody Fab fragment for P(G1) and with porcine carboxypeptidase B for P(G6). Moreover, the molecular function of the gene under the control of P(G1) was determined to encode a high-affinity glucose transporter and named GTH1.ConclusionsA set of novel regulated promoters, enabling induction without methanol, was successfully identified by using DNA microarrays and shown to be suitable for high level expression of recombinant proteins in glucose-based protein production processes.