Cloning And Expression And Separation And Purification Of Leucine Aminopeptidase From Bacillus Cereus CZ

With the development of economic, people’s material and cultural needs are also growing. Especially, demand for food from the full transtition to quality requirements, which enhance the demand for safe and effective food additive. Protein by hydrolysis to free the small peptides and amino acid fragment to thermal stability, solubility and nutritional properties have been significantly improved, which is an important raw material in today’s food processing market. Therefore, the aminopeptidase research will play an important effect to the entire food processing industry and people’s quality life.This paper aims to take advantage of two different expression vectors were transformed into three different expression hosts, explore the most suitable leucine aminopeptidase heterologous expression vectors and host. In this paper, we compare the Bacillus cereus CZ 16 SrRNA and design primers to get leucine aminopeptidase full-length gene CZ-lap. The recombinant cloning vector pMD18-T-LAP and two recombinant expression vectors pEASY-E2-LAP and pET22b-LAP are successful constructed. Successful get five recombinant expression strain pEASY-E2-LAP- BL21(pEASY-B), pEASY-E2-LAP-OrigamiTM(pEASY-O), pEASY-E2-LAP- Rosetta(pEASY-R) and pET22b-LAP- BL21(pET22b-B), pET22b-LAPOrigamiTM(pET22b-O), and which pEASY-O 、pET22b-B and pET22b-O three strains expressing soluble expression and activity is detected. Because aminopeptidase is a metal enzyme, so the activation of metal ions is studied, that research results showed that 0.5 mM of Ni2+ activation pEASY-O strain of the best, up 14.5 times.Conducted a preliminary study of the expression strain optimized conditions, the results showed that the cell OD joined between 1.0-1.2 IPTG induced expression, induced 4 h at 28℃ have the best expression, activity reached 22.40 U/mL. Finally, in order to explore several leucine aminopeptidase of Bacillus cereus contained, that isolated and purified was carried out. Using ammonium sulfate precipitation, Sephadex G50 gel chromatography preliminary purification, respectively, and using HiTrapCapto DEAE anion exchange chromatography and Phenyl Fast Flow was moderate purified, and using Phenyl HP water purification chromatography fine. SDS-PAGE protein gel are clearly seen to reduce the total protein bands after each step. Final leucine aminopeptidase purified reaches 93.56 times.