Study On The Function And Characterization Of Glutamate-Specific Endopeptidase From Bacillus Licheniformis

Glutamate-specific endopeptidase (GSE) is a kind of serine protease, which have strongsepcificity for peptide bonds formed by carboxyl of Glu residues，and GSEs have a broadapplication in the field of protein sequence and structure analysis, peptide synthesis and therecovery of bioactive peptide. The specifity of GSEs is not thoroughly understood until nowbecause the crystal structure of most GSEs have not been resolved. Compared with Glutamatespecific endopeptidase from Staphylococcus aureus V8(V8-GSE) which have been widelyused in many industries, Glutamate-specific endopeptidase from Bacillus lichefomis (GSE-BL)exhibits higher catalytic efficiency and better specificity towards Glu residues. Hence, it is ofgreat significance for widening the application of GSEs and understanding the mechanism ofaction of GSEs to investigate the structure and function of GSE-BL.Recombinant GSE-BL was expressed in E.coli as inative inclusion bodies, mature activeGSE-BL was obtained with a yield of140.5mg/L after dialysis refolding, and trypsintreatment. The results of Western blot, enzymatic activity assaying and amino acid sequencingdemonstrated that GSE-BL intermediate was formed by self-cleavage and the maturation ofGSE-BL was stepwise. Mature GSE-BL could also be obtained without introducingexogenous trypsin when Lys at-1position in GSE-BL precursor was replaced by Glu, thusproviding a new strategy for the preparation of mature GSE-BL with enzymatic activity, theactivity of resultant mature GSE-BL towards Z-Phe-Leu-Glu-pNA also decreased by27.4%compared with native mature GSE-BL with His-tag, demonstrating that Lys-1Glu mutationhad slight negative effect on correct refolding of GSE-BL. Mature GSE-BL withoutpropeptide was majorly expressed as soluble form in E.coli, the purified product of whichshowed little activity towards Z-Phe-Leu-Glu-pNA;27kD intermediate was expressed asinclusion bodies, corresponding product after trypsin treatment had little activity towardsZ-Phe-Leu-Glu-pNA, demonstrating the presence of propeptide was indispensable for thecorrect refolding of GSE-BL to form active conformation.The investigation on characteristic of GSE-BL implied us that GSE-BL had an optimalreaction pH of8.5, an optimal reaction temperature of37℃，and the Km of GSE-BL towardsZ-Phe-Leu-Glu-pNA was1.495±0.034mM, the Vmaxof which was50.237μmol/mg.minThe presence of2mM calcium ion could significantly enhance the thermostability ofGSE-BL at50℃, and EDTA could partially inhibite the activity of GSE-BL. The catalytic efficiency of GSE-BL towards Z-Phe-Leu-Glu-pNA was925fold as that of GSE-BL towardsZ-Asp-pNA.A computer model of GSE-BL based on glutamate-specific endopeptidase from Bacillusintermidius (GSE-BI) was built. And we predicted that His47, Asp96and Ser167constitutestable catalytic triad of GSE-BL by the proton transport, site-directed mutagenesis wasperformed to prove this prediction. The results of site-directed mutagenesis also demonstratedthat Val2, Cys32, Arg89and His190was essential for the formation of catalytic triad andspecificity of GSE-BL. Val2was involved in the formation of substrate binding pocketing,and it is important for the correct folding of GSE-BL. Cys32binded withZ-Phe-Leu-Glu-pNA and was involved in the formation of disulfide bond. Arg89providedpositive charge for substrate binding pocket, facilitating the entrance of substrate; His190binded with the γ-carboxyl of Glu residue, compensating for the negative charge of Gluresidue. Cys181Ala mutation decreased the enzymatic activity and thermostability of GSE-BLtowards Z-Phe-Leu-Glu-pNA. The catalytic efficiency of GSE-BL towardsZ-Phe-Leu-Glu-pNA was enhanced50%by Phe57Ala mutation. The Asp51Arg, Glu101Alaand Glu104Arg mutation resulted in the destabilization of GSE-BL catalytic triad becauseof unbalanced charge distribution.In this study, GSE-BL was applied to the removal of affinity tag of recombinant proteinand the maturation of bioactive peptide.Vitreoscilla hemoglobin is a kind of hemoglobin that can enhance oxygen uptake rate.VHb-His6was expressed in E.coli as soluble form. His-Tag of VHb-His6could be removedwith a ratio of91.6%by GSE-BL treatment for15min, thus obtaining tagless VHb with thesame amino acid sequence as native VHb. The yield of tagless VHb was31.8mg/L. TaglessVHb showed higer affinity for carbonic oxide than VHb-His6. The Vmaxof tagless VHbtowards hydrogen peroxide was1169μmol/g.min, while the Vmaxof VHb-His6towardshydrogen peroxide was781μmol/g.min. And the welan gum yield could be increased16.2%by the addition of0.1mM tagless VHb to fermentation liquid of Alcaligenes ATCC31555,while the addition of0.1mM VHb-His6only increased welan gum by4.9%.Human glucagon can stimulate the liver glycogenolysis, thus enhancing blood sugar levels.Human glucagon fused with N-His6tag was expressed in Pichia pastoris as secreted forms insupernatant. His-Tag of recombinant human glucagon could be removed with a ratio of91.6%by GSE-BL treatment for1h, and the molecular weight of tagless human glucagon was thesame as the standard synthesized human glucagon. The results of HPLC illustrated thatpurified human glucagon had the same retetion time with the standard synthesized human glucagon. The yield of tagless glucagons was50.5mg/L.Human beta-defensin-2(HBD-2) is a kind of cysteine-rich and cationic antimicrobialpeptide, and Glu residue was introduced as recognition site before mature HBD-2, andHis-Tag was fused at N-terminus to facilitate purification. Mature HBD-2was obtain fromHBD-2precursor by GSE-BL treatment for1h, and the yield of which was23.2mg/L.Mature HBD-2exhibited higher antimicrobial activity towards Pseudomonas aeruginosa thanstaphylococcus aureas, and higer cytotoxity of mature HBD-2for HCT-8was observed thancytoxity of mature HBD-2for dendritic cells from mice marrow.