title: 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.
authors.affiliation: Bioresource Utilization Laboratory, College of Engineering, China Agricultural University, Beijing, 100083 China.
authors.forename: Qiaojuan
authors.initials: Q
authors.lastname: Yan
authors.affiliation: College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center for Food Nutrition and Human Health, China Agricultural University, Beijing, 100083 China.
authors.forename: Xiaojie
authors.initials: X
authors.lastname: Duan
authors.affiliation: College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center for Food Nutrition and Human Health, China Agricultural University, Beijing, 100083 China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Liu
authors.affiliation: College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center for Food Nutrition and Human Health, China Agricultural University, Beijing, 100083 China.
authors.forename: Zhengqiang
authors.initials: Z
authors.lastname: Jiang
authors.affiliation: College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center for Food Nutrition and Human Health, China Agricultural University, Beijing, 100083 China.
authors.forename: Shaoqing
authors.initials: S
authors.lastname: Yang
keywords: Biodiesel synthesis; Cold-adapted; Gene cloning; Lipase; Pichia pastoris; Rhizomucor endophyticus