Abstract
BackgroundThe 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.ResultsA 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.ConclusionsA 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.
Highlights
The biodiesel production can be carried out by transesterification using either chemical or enzymatic process
Cold-active/adapted lipases have been found to be attractive in biodiesel production over other lipases mainly in terms of energy saving, since the biodiesel synthesis by most other lipases was performed at elevated temperatures [2, 4,5,6]
Several mesophilic lipaseencoding genes have been successfully expressed in P. pastoris [20, 21], but only a few cold-adapted lipase genes have been expressed in P. pastoris [4]
Summary
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. Lipases with high catalytic efficiency and good stability play a key role in biodiesel production process. The yields of cold-active/ adapted lipases by wild-type strains are very low, and hardly meet the requirements for large-scale production. These can be overcome by high-level heterologous expression of lipase genes from various microorganisms in suitable hosts. Some cold-active/ adapted lipases have been gene cloned, expressed, and characterized, such as the lipases from Geomyces sp. The yields of cold-adapted lipases still remain low [22,23,24], and the highest yield of 2760 U mL−1 was observed from CALIP1 which was derived from a metagenomics library [25]
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