Peptidoglycan (PG) hydrolases or autolysins are a group of enzymes which degrade bacterial cell wall at specific sites. Staphylococcus aureus produces two major PG hydrolases: major autolysin (Atl) and Aaa, a autolysin/adhesin protein. Amidase are surface-associated proteins that have both enzymatic and adhesive functions. The atl gene product is a bifunctional protein that has an N-terminal N-acetyl L-alanine amidase (Ami) domain, three internal repeat domains (R1, 2, 3) and a C terminal endo-ß-N-acetylglucosaminidase (GL) domain which undergo proteolytic processing to generate the two extracellular lytic enzymes (62 kDa Ami-R1, 2 and 51 kDa R3-GL) found in the culture broth of S. aureus. In the mature protein repeats R1 and R2 are located at the C-terminal portion of the amidase (Ami-R1, 2) and repeat R3 is located at N-terminal portion of the glucosaminidase (R3-GL) The AltA protein only with an amidase domain and the two repeat sequences R1 and R2 is 62 kDa. Another autolysin/ adhesin present on the cell surface of S.aureus (Aaa) is a 35 kDa protein containing two direct LysM (lysine motif) repeats at the N-terminal and catalytic domain in the C-terminus. The glucosaminidase domain has one repeat region (R3). All the proteins were cloned in the pBAD/Myc-His Vector with arabinose promoter in M13 strain of E. coli. We grew the transformed bacteria carrying the glucosaminidase gene encoding the His-tagged protein in LB medium containing ampicillin; expression was then induced with different concentrations of arabinose.
Cells were harvested by centrifugation, and lysed by freeze thaw method with the addition of lysozyme. Proteins were purified under denaturing conditions with 8M urea and analyzed by 10 % SDS PAGE. Isolation of insoluble and soluble fractions of recombinant protein from the bacterial cell lysate and their analysis confirmed the presence of both the recombinant proteins in the insoluble fraction possibly as inclusion bodies. Large scale purification of the recombinant proteins was done using nickel (Ni-NTA) affinity chromatography. Maximum protein elution was found in 20mM imidazole buffer. Refolding
was optimized in refolding buffer containing 0.5mM reduced glutathione: 0.5mM oxidised glutathione (1:1) in 50mM TrisHCl pH-8.0 followed by dialysis for 24hrs in 50mM TrisHCl pH-7.4 with 3 times intermittent change of buffer. After refolding the bioactivity of both the protein were assayed. After refolding the activity was tested on different E. coli (Gram-ve), Bacillus clausii and S. aureus. It was most active against Bacillus clausii . The activity of recombinant Amidase was more than that of glucosaminidase. Thus we could successfully expressed, purified and refolded the glucosaminidase protein from E. coli and compared its activity with recombinant Amidase., raising the possibility of use the enzymes as anti-microbial agent.
