Gdsl lipase, genetically-engineered bacteria and application thereof

Abstract

The invention relates to a GDSL lipase, genetically-engineered bacteria and an application thereof. The GDSL lipase is derived from Streptomyces diastaticus CS1801 and its amino acid sequence is as shown in SEQ ID NO.2. After construction of a genetically-engineered bacterium strain, a GDSL lipase is generated through fermentation. Through this enzyme, vitamin A and palmitic acid are converted to produce vitamin A palmitate. The content of the vitamin A palmitate obtained from the conversion is 16.35 mg/L at most. The conversion efficiency is 81.75% at most. This lipase provides a new path to synthesize vitamin A palmitate by the enzymatic method and has an important application prospect.

1 . A GDSL lipase, wherein its amino acid sequence is as shown in SEQ ID NO.2. 
     
     
         2 . A gene encoding the GDSL lipase as in  claim 1 . 
     
     
         3 . The gene according to  claim 2 , wherein its nucleotide sequence is as shown in SEQ ID NO.1 
     
     
         4 . A recombinant vector containing the gene as in  claim 3 . 
     
     
         5 . The recombinant vector according to  claim 4 , wherein its expression vector is pET 32a(+). 
     
     
         6 . An engineered bacterium containing the recombinant vector as in  claim 5 . 
     
     
         7 . The engineered bacterium according to  claim 6 , wherein the host cell is  E. coli  BL21(DE3). 
     
     
         8 . An application of the engineered bacterium as in  claim 6  in the production of vitamin A palmitate, including:
 (1) inoculating the engineered bacterium to an LB medium for seed culture; 
 (2) transferring the seed solution to a fermentation medium for fermentation culture, and then adding an inducer to induce expression of enzymes; 
 (3) centrifuging the fermentation broth to obtain a supernate, and obtaining enzyme powder through precipitation by ammonium sulfate and lyophilization; and 
 (4) adding the enzyme powder to an organic phase system containing vitamin A and palmitic acid to produce vitamin A palmitate. 
 
     
     
         9 . The application according to  claim 8 , wherein the fermentation medium comprises:
 tryptone 10 g/L, yeast powder 5 g/L, NaCl 10 g/L, MgSO 4 .7H 2 O 1 g/L, KH 2 PO 4  0.5 g/L, K 2 HPO 4  0.5 g/L, olive oil emulsion 12 mL/L, and distilled water added till volume 1 L.   
     
     
         10 . The application according to  claim 9 , wherein the olive oil emulsion is prepared by the following method: mixing olive oil emulsifier PVA with olive oil at a volume ratio of 3:1 and emulsifying the mixture by ultrasound.