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Generation of a Functionally Distinct Rhizopus oryzae Lipase through Protein Folding Memory.

ABSTRACT: Rhizopus oryzae lipase (ROL) has a propeptide at its N-terminus that functions as an intramolecular chaperone and facilitates the folding of mature ROL (mROL). In this study, we successfully generated a functionally distinct imprinted mROL (mROLimp) through protein folding memory using a mutated propeptide. The mutated propeptide left its structural memory on mROL and produced mROLimp that exhibited different substrate specificities compared with mROLWT (prepared from the wild type propeptide), although the amino acid sequences of both mROLs were the same. mROLimp showed a preference for substrates with medium chain-length acyl groups and, noticeably, recognized a peptidase-specific substrate. In addition, ROLimp was more stable than mROLWT. These results strongly suggest that proteins with identical amino acid sequences can fold into different conformations and that mutations in intramolecular chaperones can dynamically induce changes in enzymatic activity.

SUBMITTER: Satomura A

PROVIDER: S-EPMC4430139 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Generation of a Functionally Distinct Rhizopus oryzae Lipase through Protein Folding Memory.

Satomura Atsushi A Kuroda Kouichi K Ueda Mitsuyoshi M

PloS one 20150513 5

Rhizopus oryzae lipase (ROL) has a propeptide at its N-terminus that functions as an intramolecular chaperone and facilitates the folding of mature ROL (mROL). In this study, we successfully generated a functionally distinct imprinted mROL (mROLimp) through protein folding memory using a mutated propeptide. The mutated propeptide left its structural memory on mROL and produced mROLimp that exhibited different substrate specificities compared with mROLWT (prepared from the wild type propeptide), ...[more]

PMID: 25970342

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Project description:BackgroundProduction of biodiesel from non-edible oils is receiving increasing attention. Tung oil, called "China wood oil" is one kind of promising non-edible biodiesel oil in China. To our knowledge, tung oil has not been used to produce biodiesel by enzymatic method. The enzymatic production of biodiesel has been investigated extensively by using Rhizopus oryzae lipase as catalyst. However, the high cost of R. oryzae lipase remains a barrier for its industrial applications. Through different heterologous expression strategies and fermentation techniques, the highest expression level of the lipase from R. oryzae reached 1334 U/mL in Pichia pastoris, which is still not optimistic for industry applications.ResultsThe prosequence of lipases from Rhizopus sp. is very important for the folding and secretion of an active lipase. A chimeric lipase from R. oryzae was constructed by replacing the prosequence with that from the R. chinensis lipase and expressed in P. pastoris. The maximum activity of the chimera reached 4050 U/mL, which was 11 fold higher than that of the parent. The properties of the chimera were studied. The immobilized chimera was used successfully for biodiesel production from tung oil, which achieved higher FAME yield compared with the free chimeric lipase, non-chimeric lipase and mature lipase. By response surface methodology, three variables, water content, methanol to tung oil molar ratio and enzyme dosage were proved to be crucial parameters for biosynthesis of FAME and the FAME yield reached 91.9±2.5% at the optimized conditions by adding 5.66 wt.% of the initial water based on oil weight, 3.88 of methanol to tung oil molar ratio and 13.24 wt.% of enzyme concentration based on oil weight at 40°C.ConclusionsThis is the first report on improving the expression level of the lipase from R. oryzae by replacing prosequences. The immobilized chimera was used successfully for biodiesel production from tung oil. Using tung oil as non-edible raw material and a chimeric lipase from R. oryzae as an economic catalyst make this study a promising one for biodiesel applications.

Project description:BACKGROUND: The analysis of transcriptional levels of the genes involved in protein synthesis and secretion is a key factor to understand the host organism's responses to recombinant protein production, as well as their interaction with the cultivation conditions. Novel techniques such as the sandwich hybridization allow monitoring quantitatively the dynamic changes of specific RNAs. In this study, the transcriptional levels of some genes related to the unfolded protein response (UPR) and central metabolism of Pichia pastoris were analysed during batch and fed-batch cultivations using an X-33-derived strain expressing a Rhizopus oryzae lipase under control of the formaldehyde dehydrogenase promoter (FLD1), namely the alcohol oxidase gene AOX1, the formaldehyde dehydrogenase FLD1, the protein disulfide isomerase PDI, the KAR2 gene coding for the BiP chaperone, the 26S rRNA and the R. oryzae lipase gene ROL. RESULTS: The transcriptional levels of the selected set of genes were first analysed in P. pastoris cells growing in shake flask cultures containing different carbon and nitrogen sources combinations, glycerol + ammonium, methanol + methylamine and sorbitol + methylamine. The transcriptional levels of the AOX1 and FLD1 genes were coherent with the known regulatory mechanism of C1 substrates in P. pastoris, whereas ROL induction lead to the up-regulation of KAR2 and PDI transcriptional levels, thus suggesting that ROL overexpression triggers the UPR. This was further confirmed in fed-batch cultivations performed at different growth rates. Transcriptional levels of the analysed set of genes were generally higher at higher growth rates. Nevertheless, when ROL was overexpressed in a strain having the UPR constitutively activated, significantly lower relative induction levels of these marker genes were detected. CONCLUSION: The bead-based sandwich hybridization assay has shown its potential as a reliable instrument for quantification of specific mRNA species in P. pastoris cells grown in fed-batch cultures. As a proof-of-principle, the influence of the carbon and nitrogen sources, the specific growth rate, as well as the ROL overexpression on the transcriptional levels of a reduced set of bioprocess-relevant genes has been quantitatively studied, revealing that ROL overexpression and secretion seems to trigger the UPR in P. pastoris, resulting in a physiological bottleneck for the production process.

Dataset Information

Generation of a Functionally Distinct Rhizopus oryzae Lipase through Protein Folding Memory.

Publications

Generation of a Functionally Distinct Rhizopus oryzae Lipase through Protein Folding Memory.

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