Purification Of Cryptococcus Laurentii B40 Lipase And Construction Of Mutant Penicillium Expansum Lipase

Cryptococcus laurentii B40 is an yeast which can secret extracellular lipase, the cuture condition for Cryptococcus laurentii B40 was optimized according to the cell growth status, enzyme secretion and if it is suitable for purification.The lipase was purified by ammonium sulphate precipitation, DEAE Sephadex A-25 and Superdex 75 chromatography and the molecular weight of the enzyme was estimated to be 21 kDa. The optimal temperature and pH of the enzyme are about 43℃and 10.7 respectively. A double-mutant lipase from Penicillium expansum was obtained by site-directed mutagenesis at R182K using the random mutant ep8 as a template.The activity of the double mutant is higher than that of the wild type lipase PEL.1. Purification and characterization of extracellular lipases produced by Cryptococcus laurentii B40Several culture was designed and used to find one which is suitable for cell growth, lipase secret and purification, the ingredients of the media are as below: corn meal 0.5%, bean cake 2%, (NH₄)₂SO₄ 0.1%, Na₂HPO₄12H₂O 0.2% , MgSO₄7H20 0.25%, CaCl₂ 0.01% , pH 7.2.Cryptococcus laurentii B40 secrets extracellular lipase to medium when it grows at 26.5℃for 28 hour.A lipase was purified from the supernatant of medium by the method of ammonium sulphate precipitation,DEAE Sephadex A-25 and Superdex 75 chromatography.The molecular weight of the enzyme was estimated to be 21 kDa by SDS-PAGE.The lipase shows activities in a pH range of 9.2-11.2 and the optimum pH of the enzyme is 10.7.The optimum temperature of enzyme is about 43℃and it is stable for at least 45 min at 55℃.The lipase will lost its activity quickly when it was incubated at the temperature above 55℃.It's activity was inhibited by many heavy metal ions,especially Zn²⁺,Hg²⁺,Cu³⁺,Al³⁺. But some detergents, such as 0.5mM sodium desoxycholate and 0.05% Trition X-100 promoted the lipase activity. While SDS and EDTA at the concentration of 0.05% stongly reduced the lipase activity.The enzyme shows higher activity on the short chain(butyrate) than the long chain(bean oil). The N-terminal sequencing was performed but we did not get the result. The mainly reason is most likely the N-terminal is blocked,the further sequencing will be done by tryed some deblocking methods.2. PEL-ep8-R182K Double-Mutagenesis of Penicillium expansum lipaseIn order to improve the activity of the Penicillium expansum Lipase (PEL), the lipase encoding gene was mutated by site-directed mutagenesis. A recombinant vector pAO815-ep8-R182K which contain double mutant lipase was constructed by overlap extension PCR using the cDNA of a random-mutant lipase ep8 (a single Site mutant) as the template and two special primers were used to generate another mutation site R182K.The recombinant vector was transformed into Pichia pastoris GSll5 by electroporation and the recombinant mutant GS-pAO815-ep8-R182K can secret double mutant lipase PEL-ep8-R182K-GS to the medium when it was induced by methanol. The yield of the double mutant was 781.81 U/mL, which is 106.8% of that of the wild type lipase PEL-GS(732 U/mL).The optimum temperature of the double-mutant lipase is same with the wild type lipase PEL-GS and random-mutant lipase PEL-ep8-GS.And its thermostability (38.71℃) was similar with the wild type (38.81℃) and 2.25℃lower than that of the random mutant (40.96℃).