Cloning, Expression And Characterization Of Malassezia Globosa Lipase SMG1

Lipase has been widely used in many areas as an excellent enzyme. The swift growth ofmodern industry calls for exploration of novel microbial lipases. Monoglycerols anddiacylglycerols are widely used in cosmetics, food, and detergent industry because of thegood surface activity. While the traditional production method is energy-wasting, using lipasefor the production of monoglycerols and diacylglycerols are economic and environmental. Inmy research, the lipase SMG1from Malassezia globosa showed strictly specific tomonoglycerols and diacylglycerols. Making a research about the characterization of SMG1will have momentous meaning for the industrial production of monoglycerols anddiacylglycerols.In this study, we described the Pichia pastoris expression, the effect of fermentationconditions, purification and characterization of the M.globosa lipase SMG1. The results are asfollows:（1） Artificial synthesis of lipase gene （smg1） from M.globosa and expression inPichia pastoris. Based on the smg1sequence（Genbank ID: XM₀01732152.1）, we obtainedthe full-length smg1sequence using modified two-step approach. And then, the gene wassuccessfully recombinant expressed in Pichia expression system. The fermentation brothshowed lipolytic activity with monoglycerols （29.15U/mL） and diacylglycerols （28.00U/mL）as substrate, but no lipolytic activity was detected with tributyrin and olive oil as substrate.（2）Culture medium and condition optimization in the production of recombinant SMG1. Toenhance the expression level of SMG1in Pichia pastoris, the Response Surface Methodolog（RSM） was used to optimize the fermentation conditions. A4-factor-5-level central compositerotatable design were adopted to evaluate the effects on the lipase production of SMG1. Theoptimal growth condition with3.37%glucose and1.50%of yeast extract, at27.00°C and pH7.69was obtained. The corresponding maximal lipolytic activity of SMG1were33.00U/mL,and the actual lipolytic activity was35.60U/mL. The activity of SMG1was increased by27.60%compared to that in YPD.（3） Purification and characterization the recombinant lipaseSMG1. We obtained the pure lipase SMG1via concentrated by10kDa membran and isolatedby anion-exchange chromatography. Lipase SMG1, which has peak activity at25°C and pH5–6, showed stability over the pH range5–9and at temperatures <40°C. Mg2+and Mn2+enhanced lipase SMG1activity. Lipase SMG1exhibited high levels of tolerance to organic solvents and non-ionic detergents. Lipase SMG1is strictly specific to mono-anddiacylglycerols and showed good activity on glycerol and unsaturated fatty acids, but noactivity toward the mono-or dihydric alcohols or saturated fatty acids. From74.56–82.52%esterification yield was achieved in the esterification of o leic acid and conjugated linoleic acidwith glycerol. No positional selectivity for the production of1,3-diacylglyecerol or1,2-diacylglycerol.