Improving the acidic stability of a methyl parathion hydrolase by changing basic residues to acidic residues

Pichia pastoris is commonly used to express and secrete target proteins, although not all recombinant proteins can be successfully produced. In this study, we used methyl parathion hydrolase (MPH) from Ochrobactrum sp. M231 as a model to study the importance of the N-terminus of the protein for its secretion. While MPH can be efficiently expressed intracellularly in P. pastoris, it is not secreted into the extracellular environment. Three MPH mutants (N66-MPH, D10-MPH, and N9-MPH) were constructed through modification of its N-terminus, and the secretion of each by P. pastoris was improved when compared to wild-type MPH. The level of secreted D10-MPH was increased to 0.21 U/mL, while that of N9-MPH was enhanced to 0.16 U/mL. Although N66-MPH was not enzymatically active, it was secreted efficiently, and was identified by SDS-PAGE. These results demonstrate that the secretion of heterologous proteins in P. pastoris may be improved by modifying their N-terminal structures.

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“…The determination of the enzymatic properties of wild-type and mutant MPH was performed according to methods described previously [33], [34].…”

Section: Methodsmentioning

confidence: 99%

Improving the Secretion of a Methyl Parathion Hydrolase in Pichia pastoris by Modifying Its N-Terminal Sequence

Wang

Huang

et al. 2014

PLoS ONE

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“…Importantly, since the MPH-fold appears to be capable of accommodating a diverse range of substrates ranging from OPs to ␤-lactams and lactones MPH may be a useful agent to be developed into a potent and versatile bioremediator. Toward this aim, site-directed and random mutagenesis, as well as a semi-rational approaches using computer-aided methods and site saturation mutagenesis have all been used to improve the thermal and acid stability of MPH from Ochrobactrum sp M231 [120][121][122]. Several groups have also been successful in increasing the activity of MPH toward less preferred substrates.…”

Section: Methyl Parathion Hydrolase (Mph)mentioning

confidence: 99%

Organophosphate-degrading metallohydrolases: Structure and function of potent catalysts for applications in bioremediation

Schenk

Mateen

et al. 2016

Coordination Chemistry Reviews

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Organophosphate compounds (OPs) have been employed in the agricultural industry as pesticides and insecticides for several decades. Many of the methods used currently for the detoxification of OPs are harmful and possess serious environmental consequences. Therefore, utilizing enzymes for the detection and decontamination of OPs is gaining increasing attention as an efficient and clean bioremediation strategy. Microbial enzymes, such as OP hydrolases, OP acid anhydrolases or methyl parathion hydrolase (MPH), are potent agents for OP decontamination. Their biochemical properties and biotechnological applications are discussed in this review, including a discussion on methods that may be employed to immobilize such enzymes, and essential steps to generate reusable and affordable biocatalytic systems for use in bioremediation and biorestoration.

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“…The pH of the fermentation medium of Pichia pastoris is usually below 5.5, and this may affect the accumulation of secreted proteins in the culture supernatant if they are not stable at low pH. We constructed the MPH mutant K277D, which has improved acidic stability in a previous study [ 27 ]. This mutant was used to evaluate its contribution to protein secretion in a low pH environment.…”

Section: Introductionmentioning

confidence: 99%

Enhanced secretion of a methyl parathion hydrolase in Pichia pastoris using a combinational strategy

Wang

Huang

et al. 2015

Microb Cell Fact

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BackgroundAlthough Pichia pastoris has been successfully used to produce various recombinant heterologous proteins, the efficiency varies. In this study, we used methyl parathion hydrolase (MPH) from Ochrobactrum sp. M231 as an example to study the effect of protein amino acid sequence on secretion from P. pastoris.ResultsThe results indicated that the protein N-terminal sequence, the endoplasmic reticulum (ER) retention signal (KKXX) at the protein C-terminus, and the acidic stability of the protein could affect its secretion from P. pastoris. Mutations designed based on these sequence features markedly improved secretion from P. pastoris. In addition, we found that the secretion properties of a protein can be cumulative when all of the above strategies are combined. The final mutant (CHBD-DQR) designed by combining all of the strategies greatly improved secretion and the secreted MPH activity of CHBD-DQR was enhanced up to 195-fold compared with wild-type MPH without loss of catalytic efficiency.ConclusionsThese results demonstrate that the secretion of heterologous proteins from P. pastoris could be improved by combining changes in multiple protein sequence features.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0315-4) contains supplementary material, which is available to authorized users.

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Improving the acidic stability of a methyl parathion hydrolase by changing basic residues to acidic residues

Cited by 7 publications

References 15 publications

Improving the Secretion of a Methyl Parathion Hydrolase in Pichia pastoris by Modifying Its N-Terminal Sequence

Improving the Secretion of a Methyl Parathion Hydrolase in Pichia pastoris by Modifying Its N-Terminal Sequence

Organophosphate-degrading metallohydrolases: Structure and function of potent catalysts for applications in bioremediation

Enhanced secretion of a methyl parathion hydrolase in Pichia pastoris using a combinational strategy

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