Cloning Leucine Aminopeptidase Gene From Mucor And Expression In Pichia Pastoris

Protein hydrolysate in food processing usually has bitterness,which is produced by hydrophobic amino acids at the end of polypeptide chain in hydrolysate.Leucine aminopeptidase,which is an exopeptidase,can cut peptide chains or protein N-terminal hydrophobic amino acids,removing bitterness of protein hydrolysate to some extent while promoting the protein hydrolysis.As a main strain to produce fermented bean curd,Actinomucor elegans is rich in peptidase and has good debitterizing effect on soybean protein.Nowadays,a leucine aminopeptidase has been separated and purified from mouldy bran of A.elegans and its enzymatic property has been studied preliminarily,while no research on gene cloning and heterologous expression of leucine aminopeptidase of A.elegans has been reported.In order to clone the leucine aminopeptidase gene from A.elegans,and construct leucine aminopeptidase gene engineering strains,the leucine aminopeptidase gene from Mucor circinelloides was used as template,three pairs of primers were designed to clone the leucine aminopeptidase gene from A.elegans and M.circinelloides and expressed in Pichia pastoris GS115.The expressed products were analyzed to verify whether the cloned gene was leucine aminopeptidase gene or not.First of all,one Alapl fragment was amplified from A.elegans and five fragments from M.circinelloides by using RT-PCR.They were named as Mlapl,Mlap2-1,Mlap2-2,Mlap3-1 and Mlap3-2,respectively.The six fragments were inserted into pPICZaA to construct recombinant expression vectors,respectively.The recombined vectors were named as Alapl-pPICZ?A,Mlapl-pPICZ?A,Mlap2-1-pPICZ?A,Map2-2-pPICZ?A,Mlap3-1-pPICZ?A,Mlap3-2-pPICZ?A,respectively.Linearized by using Sac|,the six recombinant expression vectors and the empty vector were electrotransformed into P.pastoris GS115,respectively.Seven positive recombinants of P.pastoris were verified by PCR amplication and Mut+screening on the MDH and MMH solid medium.The recombinants of P.pastoris were named as Alap1-pPICZ?A-GS115,Mlap1-pPICZ?A-GS115,Mlap2-7-pPICZ?A-GS115,Mlap2-2-pPICZ?A-GS115,Mlap3-1-pPICZ?A-GS115,Mlap3-2-pPICZ?A-GS115 and pPICZaA-GS 115,respectively.All the P.pastoris recombinants were induced with 0.8%methanol at 30 C and 200 rpm for 4 d.Enzymatic activity of leucine aminopeptidase from all the recombinants were analyzed through LAN method,and their protein expression were analyzed by using SDS-PAGE and Western Blot,respectively.Results showed that there was no leucine aminopeptidase activity in all recombinant P.pastoris culture,and the target protein was no detected.Additionally,in order to analyze whether the target gene had been transcribed,the transcription level of leucine aminopeptidase gene from Mlap1-pPICZ?A-GS115 was analyzed by real-time PCR.The result showed that the Mlapl gene was transcribed in the cell and the transcription level of the gene at 36 h was 8.5 times of that at 12 h.To sum up,the one Alapl fragment amplified from Aelegans and five fragments from M.circinelloides had been transcribed in the cell,and the transcription level of them were different under different induced time,but they failed to fulfill secretion expression in P.pastoris GS115.So the six fragments from A.elegans and M.Circinelloides could not be verified as leucine aminopeptidase genes.