Study on Free Lipase‐Catalyzed Ethanolysis for Biodiesel Preparation in an Oil/Water Biphasic System

BackgroundWe previously cloned a 1,3-specific lipase gene from the fungus Rhizomucor miehei and expressed it in methylotrophic yeast Pichia pastoris strain GS115. The enzyme produced (termed RML) was able to catalyze methanolysis of soybean oil and showed strong position specificity. However, the enzyme activity and amount of enzyme produced were not adequate for industrial application. Our goal in the present study was to improve the enzyme properties of RML in order to apply it for the conversion of microalgae oil to biofuel.ResultsSeveral new expression plasmids were constructed by adding the propeptide of the target gene, optimizing the signal peptide, and varying the number of target gene copies. Each plasmid was transformed separately into P. pastoris strain X-33. Screening by flask culture showed maximal (21.4-fold increased) enzyme activity for the recombinant strain with two copies of the target gene; the enzyme was termed Lipase GH2. The expressed protein with the propeptide (pRML) was a stable glycosylated protein, because of glycosylation sites in the propeptide. Quantitative real-time RT-PCR analysis revealed two major reasons for the increase in enzyme activity: (1) the modified recombinant expression system gave an increased transcription level of the target gene (rml), and (2) the enzyme was suitable for expression in host cells without causing endoplasmic reticulum (ER) stress. The modified enzyme had improved thermostability and methanol or ethanol tolerance, and was applicable directly as free lipase (fermentation supernatant) in the catalytic esterification and transesterification reaction. After reaction for 24 hours at 30°C, the conversion rate of microalgae oil to biofuel was above 90%.ConclusionsOur experimental results show that signal peptide optimization in the expression plasmid, addition of the gene propeptide, and proper gene dosage significantly increased RML expression level and enhanced the enzymatic properties. The target enzyme was the major component of fermentation supernatant and was stable for over six months at 4°C. The modified free lipase is potentially applicable for industrial-scale conversion of microalgae oil to biodiesel.Electronic supplementary materialThe online version of this article (doi:10.1186/1754-6834-7-111) contains supplementary material, which is available to authorized users.

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“…Sample preparation and detection conditions were as described by Ren et al . [ 54 ]. TLC was performed as in our previous study [ 35 ].…”

Section: Methodsmentioning

confidence: 99%

Improved production of a recombinant Rhizomucor miehei lipase expressed in Pichia pastoris and its application for conversion of microalgae oil to biodiesel

Huang

Xia

Yang

et al. 2014

Biotechnol Biofuels

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“…Among the various lipases, free lipase has become a promising biocatalyst for biodiesel production due to the following merits: the cost of free lipase is tens of times lower compared to immobilized lipase based on the same enzymatic activity; and the risk of using free lipase is greatly reduced; free lipase can catalyze biodiesel production in water-containing system, this is very important for non-edible oils feedstock's conversion and the reuse of free lipase can be achieved through membrane recovery [7][8][9][10][11][12]. In our previous study, it was found that a biodiesel yield of over 90% could be obtained with free lipase NS81006-mediated biodiesel production [10][11][12][13].…”

Section: Introductionmentioning

confidence: 99%

“…However, whether the pathway of direct methanolysis exists in the process still need further exploration. In the current paper, an effective free lipase (NS810006) [7,24] was used as catalyst and two full kinetic models considering both two pathways for biodiesel production were proposed. According to the simulation results, the kinetic mechanism of free lipase NS81006 was finally revealed.…”

Section: Introductionmentioning

confidence: 99%

Kinetic study on free lipase NS81006-catalyzed biodiesel production from soybean oil

Dai

Liu

2015

Journal of Molecular Catalysis B: Enzymatic

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“…The commercial free lipase NS81006 from the genetically modified Aspergillus niger was studied for biodiesel production [160,163]. Fatty acid methyl (FAMEs) and ethyl (FAEEs) esters were produced from soybean oil obtaining yields of 95.1% [160] and 90% [163] respectively. Lipase NS81006 was stable for five batches in the production of FAEEs after simple separation of the water phase [163].…”

Section: Free Lipasesmentioning

confidence: 99%

“…Fatty acid methyl (FAMEs) and ethyl (FAEEs) esters were produced from soybean oil obtaining yields of 95.1% [160] and 90% [163] respectively. Lipase NS81006 was stable for five batches in the production of FAEEs after simple separation of the water phase [163].…”

Section: Free Lipasesmentioning

confidence: 99%

Enzyme-Catalyzed Production of Biodiesel as Alternative to Chemical- Catalyzed Processes: Advantages and Constraints

Sandoval

Casas-Godoy

Bonet-Ragel

et al. 2017

CBE

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Biodiesel represents an interesting alternative to fossil fuels. Traditionally the standard method for biodiesel production from oils is alkaline-catalyzed transesterification. Chemical catalysis can be replaced by enzymatic catalysis using lipases (EC 3.1.1.3, triacylglycerol acyl hydrolases), obtained from plants, animals or microorganisms. These biocatalysts act at milder temperature and normal pressure conditions, resulting in lower energy consumption. Also, undesirable side-reactions do not occur, originating pure products. Refined vegetable oils are the most common feedstocks for biodiesel production, accounting for 70-80% of the overall biodiesel production costs. The search for low-cost feedstocks, i.e. non-edible oils and high acidic waste oils/greases, is an alternative to make biodiesel competitive. Non-regioselective and sn-1,3regioselective lipases can catalyze esterification of free fatty acids and transesterification of triacylglycerols with good yields. The lipases used as catalysts for biodiesel production must present alcohol resistance, thermo-tolerance, high stability and activity. Recently, enzymatic processes for biodiesel production have been implemented at industrial scale. Despite this trend, the conventional chemical process still remains the most popular, mainly due to the high cost of commercial lipases. This review consists of an update of the state of the art of enzymatic biodiesel production, including legislation, feedstocks, lipases used for biodiesel synthesis, the role of acyl acceptors and strategies to avoid lipase inactivation, the mechanisms proposed for biocatalysis and the enzymatic bioreactors used. In addition, the economics of the bioprocess is also presented.

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Study on Free Lipase‐Catalyzed Ethanolysis for Biodiesel Preparation in an Oil/Water Biphasic System

Cited by 41 publications

References 11 publications

Improved production of a recombinant Rhizomucor miehei lipase expressed in Pichia pastoris and its application for conversion of microalgae oil to biodiesel

Improved production of a recombinant Rhizomucor miehei lipase expressed in Pichia pastoris and its application for conversion of microalgae oil to biodiesel

Kinetic study on free lipase NS81006-catalyzed biodiesel production from soybean oil

Enzyme-Catalyzed Production of Biodiesel as Alternative to Chemical- Catalyzed Processes: Advantages and Constraints

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