Dataset Information

Lipase from Rhizopus oryzae R1: in-depth characterization, immobilization, and evaluation in biodiesel production.

ABSTRACT:

Background

Rhizopus species is among the most well-known lipase producers, and its enzyme is suitable for use in many industrial applications. Our research focuses on the production of lipase utilizing waste besides evaluating its applications.

Results

An extracellular lipase was partially purified from the culture broth of Rhizopus oryzae R1 isolate to apparent homogeneity using ammonium sulfate precipitation followed by desalting via dialysis. The partially purified enzyme was non-specific lipase and the utmost activity was recorded at pH?6, 40?°C with high stability for 30 min. The constants K_m and V_max, calculated from the Lineweaver-Burk plot, are 0.3?mg/mL and 208.3?U/mL, respectively. Monovalent metal ions such as Na⁺ (1 and 5?mM) and K⁺ (5?mM) were promoters of the lipase to enhance its activity with 110, 105.5, and 106.5%, respectively. Chitosan was used as a perfect support for immobilization via both adsorption and cross-linking in which the latter method attained immobilization efficiency of 99.1% and reusability of 12?cycles. The partially purified enzyme proved its ability in forming methyl oleate (biodiesel) through the esterification of oleic acid and transesterification of olive oil.

Conclusion

The partially purified and immobilized lipase from Rhizopus oryzae R1 approved excellent efficiency, reusability, and a remarkable role in detergents and biodiesel production.

SUBMITTER: Helal SE

PROVIDER: S-EPMC7785608 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Publications

Lipase from Rhizopus oryzae R1: in-depth characterization, immobilization, and evaluation in biodiesel production.

Helal Shimaa E SE Abdelhady Hemmat M HM Abou-Taleb Khadiga A KA Hassan Mervat G MG Amer Mahmoud M MM

Journal, genetic engineering & biotechnology 20210105 1

<h4>Background</h4>Rhizopus species is among the most well-known lipase producers, and its enzyme is suitable for use in many industrial applications. Our research focuses on the production of lipase utilizing waste besides evaluating its applications.<h4>Results</h4>An extracellular lipase was partially purified from the culture broth of Rhizopus oryzae R1 isolate to apparent homogeneity using ammonium sulfate precipitation followed by desalting via dialysis. The partially purified enzyme was n ...[more]

PMID: 33400043

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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

Lipase from Rhizopus oryzae R1: in-depth characterization, immobilization, and evaluation in biodiesel production.

Background

Results

Conclusion

Publications

Lipase from Rhizopus oryzae R1: in-depth characterization, immobilization, and evaluation in biodiesel production.

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