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    Purification, crystallization and cloning of tributyrin esterase from Lactococcus : a thesis presented in partial filfillment of the requirements for the degree of Master of Technology in Biotechnology in the Institute of Molecular Biosciences at Massey University, New Zealand
    (Massey University, 1998) Zheng, Jiong
    Tributyrin esterase is an enzyme that has been isolated and purified from lactococcal starter strain by research staff at the New Zealand Dairy Research Institute. It has been shown to play an important role in production and control of flavour development during cheese ripening, but little is known about its biochemical characteristics. New studies on tributyrin esterase have been initiated, with the aim of carrying out a three dimensional structure determination to completely understand the molecular basis and the nature of its in vivo activity. This thesis is divided into three main parts. In the first part, the purification of tributyrin esterase from a genetically modified strain Lc. lactis subsp. cremoris B1079 is described. The procedure investigated for optimization of the protocol and a partial study of factors affecting tributyrin esterase activity are described. In the second part, crystallization trials for tributyrin esterase are described. Several crystals were obtained, with the best ordered crystals being grown from 2.6M ammonium sulfate, these have been shown to diffract to 3.0 Å, and belong to the space group C222 with cell dimensions a=76Å, b=178Å c=179Å. In the third part, the lipase gene was ligated into 4.75kbp expression vector proEX, which contains a his-tag sequence upstream of the multiple cloning site. The ligation reaction mixture was transformed into competent E.coli DH5α cells. This should allow the expression of tributyrin esterase in E.coli and eventually provide a great yield of protein and make purification easier.
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    The refolding of recombinant human liver methylmalonyl-CoA mutase from inclusion bodies produced in Escherichia coli : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University
    (Massey University, 1998) Hayes, Michelle Marie
    Human methylmalonyl-CoA mutase (hMCM) is an adenosylcobalamin-dependent enzyme that catalyses the structural rearrangement of (R)-methylmalonyl-CoA to succinyl-CoA as pan of the catabolism of the branched chain amino acids valine, leucine and isoleucine, odd chain fatty acids and intermediates of cholesterol metabolism. Reactions that require adenosylcobalamin (AdoCbl) have been intensively studied, and the first step in the catalysis is widely agreed to involve homolytic cleavage of the unusual carbon-cobalt bond in the cofactor. A reliable source of recombinant hMCM would be useful in defining more fully the mechanistic pathway of AdoCbl-dependent enzymes. Recombinant hMCM overexpressed in E. coli forms insoluble aggregates of inactive protein known as inclusion bodies. hMCM inclusion bodies were purified, solubilised and then several different in vitro refolding techniques were tested in attempts to produce active recombinant hMCM from purified solubilised inclusion body material. These methods included refolding by rapid dilution, refolding by dialysis, detergent-assisted refolding, refolding by gel filtration chromatography and chaperonin-assisted refolding. Chaperonin-assisted refolding necessitated the purification of recombinant E. coli chaperonins GroES and GroEL from the E. coli strain DH1/pGroESL. Refolding by rapid dilution of the GdmHCl-solubilised inclusion bodies into a refolding buffer was judged to be the simplest and most effective method, however the refolding process was extremely inefficient. Refolding by rapid dilution was scaled up to 2 litres to produce as much active hMCM as possible. The refolded protein was concentrated by batch adsorption to and stepwise elution from hydroxyapatite, and further purified using a synthesised 5'adenosylcobalamin- agarose 'affinity' chromatography column. The final refolded hMCM preparation contained a single ~29 kDa contaminant protein, tentatively identified as E. coli branched-chain amino acid aminotransferase (EC 2.6.1.42), present in approximately equal amounts to the hMCM, and had a specific activity of ~3.11 units/mg.