Screening Of Strain Producing Phospholipase A₁Construction Of Recombinant E. Coli And Auto-induction

Phospholipase A₁（PLA₁, EC3.1.1.32）, as one kind of phospholipase, can catalyze thehydrolysis of fatty acids exclusively at the sn-1position of phospholipid. A free fatty acid anda lysophospholipid are the products of a PLA₁reaction. There are many kinds ofcommercialized products of PLA₁, which has been widely used in the oil degumming,pharmaceutical and food processing industries. But the reasearch on PLA₁started late both indomestic and overseas. Up to date, not only very limited information about PLA₁producingmicrobes has been reported, but their yields are rather low as well.Four bacterial strains produced PLA₁were isolated separately from CICIM-CU andCICC. The gene pla, which was cloned by PCR from Serratia liquefacien, was successfulexpressed and secreted by auto-induction in recombinant E. coli. We also obtained theoptimized induction and fermentation conditions that E. coli produce recombination PLA₁. Bythe his-tag of the vector pET-28a, the recombination PLA₁was purified, which was used toinvestigate the enzymatic property and substrate specificity. The main content of this studyincludes the following six parts.（1） Four PLA₁producing stains were isolated respectively from more than600bacteriason the egg-yolk solid medium, which were Xanthomonas retroflexus，Serratia liquefacien，Citrobacter youngae and Shewanella putrefaciens. The PLA₁gene of S. liquefacien wassuccessfully obtained by PCR. After genen sequenced, it showed92.0%identical to PLA₁in the database by the multiple sequence alignment.（2） To research the effect of secretion by different signal peptide in E. coli, tworecombinant plasmids were constructed which were pET20b-dpla and pET28a-pla. Thenthey were transformed into E. coli BL21（DE3） individually to express PLA₁. E. coliBL21（DE3）/pET28a-pla yielded extracellular PLA₁with an activity of248±10U/mL inbatch cultivations of shaken flasks, which was accounted for91.1%of total enzyme activity.We researched two induction patterns of E. coli BL21（DE3）/pET28a-pla producing PLA₁.The result showed biomass and exoenzyme by auto-induction were2.6and3.2folds ofconventional IPTG induction, respectively. It was also researched of the medium andfermentation conditions of E. coli by Single factor and orthogonal test that byauto-induction. When optimized medium contained tryptone10g/L, yeast extract5g/L,glycerine5g/L, seed age6h and incubated for30h at37℃, an extracellular enzymeactivity of512±18U/mL was reached. It had been enhanced by62.3%than initialconditions.（3） It was tested of recombinant E. coli produced PLA₁in7L fermentor level based theoptimization condition on shake flask. By batch fermentation, the extracellular PLA₁was upto619±27U/mL when the recombinant E. coli grew at37℃for20h as followingfenmentation condition: the ventilation of gas as1.5vvm, pH as7.2and DO concentration as30%controlled by agitation speed. However, the extracellular PLA₁was up to922±32U/mL when the recombinant E. coli grew at37℃for28h and it started feeding theinducer lactose after5h inoculating. （4） When the recombinant PLA₁was purified by affinity chromatography, optimal pHand temperature found to be pH6.5and50°C, respectively. The enzyme was stable over a pHrange of5.0to9.0, and at temperatures up to70°C for1hour, with more than60%of itsactivity preserved under these conditions. The activity was inhibited by Li+、Cu²⁺、Mn²⁺andchemical reagent such as EDTA, SDS obviously, and it was increased by Ca²⁺、Ni²⁺and Mg²⁺.Moreover, this PLA₁exhibits broad phospholipid substrate specificity with minimal lipaseactivity. These characteristics will permit much more widespread use of this PLA₁in theprocess of oil degumming.