Study On Discovery Of Novel Teichoicase And Analysis Of Exzyme Property

Teichoic acids are important component of Gram-positive bacterial cell wall.It was reported that teichoic acids are related with the growth,the resistance for antibiotics,the adhesion and colonization of host cells for bacteria.And Teichoicase is a kind of phosphodiesterases,which can degrade teichoic acids specifically.So it will give some guidelines for treating diseases caused by bacterial infection with studying Teichoicase from another side of Gram-positive bacteria.Moreover,according to the reports of Wise,Kusse and Myers,we speculated that there were novel Teichoicases in Aspergillus niger and Bacillus.Thus,we find gene An18g03170 in Aspergillus niger with searching functional domain in NCBI and KEGG.And we find gene PRAP（phage-related pre-neck appendage protein）in Bacillus clausii by GP12 sequence homology in the same databases.In this study,An18g03170,PRAP,GP12 were cloned to pET-21a（+）carrier,and the recombinant plasmids were then transferred into E coli BL21（DE3）.Then the recombinant strains were induced to express target proteins.After purification of this three kinds of proteins,we identificated and analysised the properties with them.The results were as follows:（1）The selection of teichoic acids extracted strain and the extraction of teichoic acids: Teichoic acids were extracted by TCA with Staphylococcus aureus and Bacillus subtilis 114.After extraction,we used DEAE Fast Flow to purify teichoic acids,and the determination of teichoic acids were used with phosphomolybdate blue spectrophotometry.With calculation,we got the extraction yield of teichoic acids.According to data,we found that extraction yield of teichoic acids with Staphylococcus aureus,6.13% was higer than extraction yield of teichoic acids with Bacillus subtilis 114,4.23%.In addition,this two samples did not polluted by nucleic acids and proteins.Therefor,we selected Staphylococcus aureus for extraction of teichoic acids.（2）The coloning and expression of An18g03170,PRAP and GP12: An18g03170 was obtained by RT-PCR with total RNA of Aspergillus niger.And the sequence we coloned was different with the sequence on NCBI with 6 amino acids.About thisphenomenon we believed that it was concerned with different strains.Gene PRAP and GP12 were synthesized by GENEWIZ,and the sequences were same with sequences published on NCBI.An18g03170,PRAP,GP12 were cloned to pET-21a（+）carrier,and the recombinant plasmids were then transferred into E coli BL21（DE3）.An18g03170 had significantly higer soluble protein after optimizing the conditions of induction.And PRAP expressed soluble protein in the 90% of the total protein.GP12 always appeared with inclusion body protein.（3）The optimization of GP12: Since GP12 appeared with inclusion body protein,we coloned GP12 into pET-28a（+）after analysis of GP12 reported by Myers.We induced recombinant strain with 1 m M IPTG.Expression was continued for 6 hours at 16℃ and 170 rpm.The protein GP12 was found in the supernatant,although there was still some inclusion body.（4）Purification of purpose protein: Although An18g03170 had quite a few soluble protein,it couldn’t affinity with Ni-NTA Agrose.It was thinked that His label was embedded inside.PRAP was purified very successfully,but its location was higer about 18 KDa than it in theory on SDS-PAGE.We infered that the reason might be that PRAP had too much acid amino acids which led to PRAP could not combine enough SDS.GP12 was also purified successfully after induced with IPTG.（5）The determination of catalysising activity of enzymes: The pure enzymes were measured with bis-pNPP and teichoic acids.The results showed that PRAP and GP12 had catalyzing ability to bis-pNPP.And the optimal temperature of reactions was 37 ℃,the optimal pH was 4.8.Meanwhile,they also had the same thermal stability.However,the K_m/K_cat of PRAP was higher than GP12.In addition,we also determinated degradating activities of PRAP and GP12 with teichoic acids.The results showed that the optimal temperature was 37 ℃,and the optimal pH of PRAP was 6.0.However,the optimal pH of GP12 was 6.6.The mesurments of K_m were proceeded with optimal pH and temperature.The results showed that it was no significantly difference between PRAP and GP12.Compared with broken supernatant which was made with E coli BL21（DE3）containing pET-21a（+）,An18g03170 emerged obvious activity to bis-pNPP,but we had no pure enzyme.（6）The effects of metal ions on the catalyzing of PRAP and GP12 : metal ions were added into reactions to make sure that the final concentrations of them were 5m M.The results showed that Mg²⁺、Ca²⁺ were activator for PRAP and GP12,and Fe²⁺ 、 Mn²⁺ could inhibit the degradation of PRAP and GP12 to teichoic acids.What’s more,Cu²⁺ could promote the degradation of PRAP,while it appeared inhibition on GP12.（7）The effects of target proteins on the growth of Staphylococcus aureus : Crude extract of An18g03170 showed no apparent inhibition on the growth of Saphylococcus aureus,and emerged effects on the growth rate and morphology scarcely.Meanwhile,PRAP and GP12 also showed no effect on this of Staphylococcus aureus.