Screening,Recombinant Expression And Molecular Modification Of Sn-1,3 Specific Lipase

Enzymatic synthesis of human milk fat substitute is a research hotspot in functional lipids area in recent years, in which the sn-1,3-specific lipases are one of the key raw materials. However, the market provides very limited and expensive sn-1,3-specific lipase preparations, so it’s very necessary to make a breakthrough in the sn-1,3-specific lipase production technology to obtain low-cost, high activity of lipase preparation. This study is aimed to improve the sn-1,3-specific lipases yield and reduce their production costs simutaneously, and the main contents include:The steamers of Chinese dumpling were chosen as the resource for screening of lipase producing strains, and 21 of lipase production microorganisms were obtained. Furthermore, a high yield lipase producing strain Aureobasidium sp. S4 was obtained by further screening. Fermentation medium was optimized and determined by central composite design response surface in flask, and after fermentation under the conditions lipase activity of the strain reached 31.75 U/mL, increased to 3.41 times compared with the original medium. The sn-1,3-specificity of the lipase was identified by TLC experiment.The codon optimized sn-1,3 specific lipase genes of TLL and RML were cloned and transformed into Pichia pastoris expression system for recombinant expression. TLL recombinant strains were fermentated in flask for 168 h and the pNPP hydrolysis activity of lipase in the supernatant of culture medium reached 312.72 U/mL, increasing by 74.29% compared with that of the original TLL gene. However, RML recombinant P. pastoris strains showed very low lipase activity (3.77 U/mL).The recombinant strains GS-pTL, containing lipase TLL with a propeptide, showed higher extracellular lipase activities (434.32 U/mL) in flask fermentation, compared with those without a propeptide in TLL and those with a propeptide linked by Kex2 cleavage site with mature peptide of TLL, which indicated that the 5 amino acid length propeptide of TLL could improve the lipase expression level. Furthermore, the pTL variant recombinant strains with a hydrophobicity-increased TLL propeptide modified by site directed mutation, showed a lipase activity of 483.29 U/mL,11.27% higher than those of the recombiant strain GS-pTL with a original propeptide in TLL. In addition, In the molecular modification of potential Kex2 cleavage site in RML, the result showed no evidence that the site would affect the recombinant expression of RML.Compared among several sn-1,3-specific lipase isozymes co-expression strategies, Kex2-mediated co-expression showed a better result than others according to the extracellular lipase activity comparison. The strains GS--RMk-kTL with Kex2-mediated co-expression of isozymes TLL and RML showed a higher extracellular lipase activity 306.91 U/mL, compared with the independent recombinant expression of RML and TLL in P. pastoris (2.89 and 300.59 U/mL respectively), indicating that co-expression of lipase isoenzymes to some extent could increase the expression level of the enzymes. However, due to the low activity level of RML, the overall effect of isozymes co-expression strategy in improving expression level was limited. The co-expression strategy of fusion protein expression successfully produced the coupled protein TLL and RML, but the chemera showed very lower lipase activity than the corresponding individual TLL activity, due to the influenced structure of TLL and RML in the fusion protein.2A peptide successfully mediated the lipase isozymes co-expression and secretion in P. pastoris, but seriously affected the expression of downstream protein TLL linked with 2A.Through single factor optimization of fermentation condition in flask, pTL recombinant Pichia pastoris strains reached the highest lipase activity 2265.3 U/mL. Using methanol as the sole carbon source, the pTL recombinant strain reached the highest lipase activity of 22561.4 U/mL after inducing 202h in high density fermentation. Co-induction of the strain with methanol and auxiliary carbon source obtained a highest activity of 24522.6 U/mL after 208 h fermentation, which is 10.87% higher than that of the previous induction with methanol only. The macroporous resin D3520 was used to immobilize the recombinant lipase TLL, and the OPO synthesis research of the immobilizied lipase was explored.