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Expression and characterization of a novel 1,3-regioselective cold-adapted lipase from Rhizomucor endophyticus suitable for biodiesel synthesis.

ABSTRACT: BACKGROUND:The biodiesel production can be carried out by transesterification using either chemical or enzymatic process. The enzymatic transesterification is more promising as it offers an environmental friendly option compared to the chemical process, where the lipases with high catalytic efficiency and good stability play a key role. Hence, it is of great value to identify novel lipases which are suitable for biodiesel production. RESULTS:A lipase gene (ReLipA) from Rhizomucor endophyticus was cloned and heterologously expressed in Pichia pastoris. ReLipA shared the highest identity of 61 % with the lipases from Rhizopus delemar, Rhizopus oryzae, and Saccharomyces cerevisiae. The recombinant lipase (ReLipA) was secreted as an active protein with the highest activity of 1961 U mL(-1) in a 5-L fermentor by high cell-density fermentation. ReLipA was purified to homogeneity with a recovery yield of 75.7 %. The purified enzyme was most active at pH 6.0 and 40 °C, respectively, and it was stable up to 55 °C. ReLipA displayed 75 % of its maximal activity at 0 °C, indicating that it is a cold-adapted lipase. It exhibited broad substrate specificity toward various p-nitrophenyl esters and triglycerides. ReLipA hydrolyzed triolein to release mainly 1,2-diolein without the formation of 1,3-diolein, suggesting that it is a sn-1,3 regiospecific lipase. Furthermore, ReLipA synthesized different types of oleates by esterification using oleic acid and short chain alcohols (e.g., methanol, ethanol, and butanol) as the substrates with the highest conversion yield of 82.2 %. Therefore, the cold-adapted lipase may be a good biocatalyst in ester synthesis in biodiesel industry. CONCLUSIONS:A novel cold-adapted lipase was identified and characterized. The high yield and excellent properties may confer the enzyme with great potential for biodiesel production in bioenergy industry. This is the first report on a cold-adapted lipase from Rhizomucor species.

SUBMITTER: Yan Q

PROVIDER: S-EPMC4831154 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Expression and characterization of a novel 1,3-regioselective cold-adapted lipase from Rhizomucor endophyticus suitable for biodiesel synthesis.

Yan Qiaojuan Q Duan Xiaojie X Liu Yu Y Jiang Zhengqiang Z Yang Shaoqing S

Biotechnology for biofuels 20160414

<h4>Background</h4>The biodiesel production can be carried out by transesterification using either chemical or enzymatic process. The enzymatic transesterification is more promising as it offers an environmental friendly option compared to the chemical process, where the lipases with high catalytic efficiency and good stability play a key role. Hence, it is of great value to identify novel lipases which are suitable for biodiesel production.<h4>Results</h4>A lipase gene (ReLipA) from Rhizomucor ...[more]

PMID: 27081399

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Project description: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.

Dataset Information

Expression and characterization of a novel 1,3-regioselective cold-adapted lipase from Rhizomucor endophyticus suitable for biodiesel synthesis.

Publications

Expression and characterization of a novel 1,3-regioselective cold-adapted lipase from Rhizomucor endophyticus suitable for biodiesel synthesis.

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