Recombinant Expression And Characterization Of Four Glycosidases

Glycosidase?EC 3.2.1?is a class of enzyme that hydrolyze of glycosidic bonds in various glycosyl compounds and plays an important role in the study of glycobiology.Enterococcus faecalis and Enterococcus faecium are the major intestinal floras in mammals.There are abundant glycoside metabolic enzymes in their body,which are potential biological sources for glycosidase preparation.Based on the genome datas of E.faecalis and E.faecium in the NCBI database,primers were designed to amplify endo-beta-N-acetylglucosaminidase?ENGase/Endo,EC 3.2.1.96?genes endoEf,endoE and endo-alpha-N-acetylgalactosamine?Endo-GalNAcase/Ogly,EC.3.2.1.97?gene oglyEf from Enterococcus faecalis,as well as autolysin gene autoEfm from Enterococcus faecium.These genes were constructed into expression vectors using seamless cloning technology.The enzymatic properties and catalytic properties were analyzed on the basis of recombinant expression and purification.The results showed:?1?The coding sequences of endoEf gene without signal peptide was 822 bp,which encoded a protein consisted of 273 amino acids.The predicted molecular weight of EndoEf was 30.12 kDa and the isoelectric point was 5.41.EndoEf shared 70%similarity with the indicated ENGase from Streptomyces pleurotus,and belonged to glycoside hydrolase family18 which contained conservative motifs DXDXE.The prokaryotic expression vector pET-28a-endoEf was constructed,and the recombinant protein EndoEf was efficiently expressed in Escherichia coli in souble form,and the target protein was successfully purified from bacteria solution by one step Co²⁺affinity chromatography with a yield of 202.1 mg per liter.The specific activity of EndoEf was 1.0×10⁴ U/mg using denatured RNaseB as substrate.EndoEf could hydrolyze both natural or denatured RNaseB and Ova,but could not hydrolyze IgG and Trf which had complex sugar chains.MALDI-TOF-MS further confirmed the hydrolysis on N-linked sugar chains in RNaseB.The optimum temperature and pH range of EndoEf were 40?and 5.0-7.0.EndoEf had salt-tolerance up to 1 mol/L NaCl,and remained full activity under either condition of 100 mmol/L DTT,2%SDS and Triton X-100.Site-directed mutagenesis studies showed that glutamic acid at position 129 was essential for catalytic reactions.Activation of Sepharose CL 6B with epichlorohydrin and coupled with EndoEf,and the coupling rate was 78.95%.The immobilized EndoEf could hydrolyze RNase B and Ova,and still had hydrolytic activity after storaged at 4?for three months.?2?endoE and oglyEf genes without signal peptide were 2508 bp and 3891 bp in length,which encoded 835 and 1296 amino acids respectively.The predicted Mw and pI of EndoE were 94.06 kDa and 4.96,that of OglyEf were 147.11 kDa and 5.22.EndoE included a GH18 domain in N-terminus and a GH20 domain in C-terminus,while OglyEf had a conservative GH101 domain.endoE and oglyEf genes were ligated to expression vetor pET-28a respectively,and both were successfully expressed in Escherichia coli.EndoE and OglyEf were both purified by combination of Co²⁺affinity chromatography and anion exchange chromatography.EndoE could not only digest natural or denatured RNaseB and Ova,but also hydrolyze natural or denatured IgG.The specific activity of EndoE was measured as 8.33×10³ U/mg using denatured RNaseB as substrate.EndoE could play its maximum hydrolytic activity in neither temperature range of 20-50?nor pH range of4.0-6.0,and was not affected by DTT,NaCl,and Triton X-100,but its activity was strongly inhibited by the anionic surfactant SDS.The specific activity of OglyEf was 2.2×10⁶ U/mg using Gal?-1,3GalNAc-?-PNP as substrate,and the optimum temperature and pH were 50?and 5.0 respectively.OglyEf has a certain tolerance to SDS and Triton X-100,and the activity of OglyEf was significantly activated by calcium ion.The Km and Vmax of OglyEf were 95.2?mol/L and 153.8 mmol/mg·min respectively.?3?The coding sequences of autoEfm gene without signal peptide was 1194 bp,which encoded a protein consisted of 397 amino acids.The predicted Mw of this protein was 44.00kDa and the pI was 4.82.AutoEfm had a conserved GH73 domain and shared a similarity of58%with the known autolysin from Listeria monocytogenes.The recombinant expression vector pET-28a-autoEfm and the corresponding N-terminal truncation,C-terminal truncation anddouble-truncatedtruncationexpressionvectorspET-28a-autoEfm?N,pET-28a-autoEfm?C and pET-28a-autoEfm?N+C were constructed.The above expression vectors were transformed into Escherichia coli respectively,and all achieved expression.While AutoEfm?N still expressed in a form of inclusion body after optimization of the expression conditions.Lysozyme activity analysis showed that AutoEfm and AutoEfm?C had no activity,while AutoEfm?N+C showed obvious lysozyme activity and the specific activity was 8.2×10⁴ U/mg,which could hydrolyze the cell wall of Micrococcus lysodeikticus.These results implied that there may be an inhibition domain at AutoEfm?s N-terminus.Enzymatic properties analysis showed that the optimum temperature and pH of AutoEfm?N+C were 37?and 5.0.In addition,the activity of AutoEfm?N+C was significantly inhibited by divalent iron ion.