Studies on glucose isomerase from lactobacillus brevis
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Glucose isomerase (E.C. 126.96.36.199) was extracted from Lactobacillus brevis N.CD.O 474 grown in xylose, containing medium with a yield of cells (dry weight) of 2.3 - 3.3gll of medium and 300-310 glucose isomerase units. Several methods for releasing the intracellular enzyme were investigated and the specific activity recovery was highest with the heat autolysis method. The crude extract preparation was further purified by nucleic acid precipitation with MnCl2, protein fractionation by ammonium sulphate and dialysis followed by chromatography on CM-cellulose, DEAE-cellulose and gel filtration on Sephadex G-200. A final purification of 24 fold was achieved with about 25% activity recovery in 4 purification steps as follows: enzyme extraction by heat autolyis, MnCl2 treatment (nucleic acid precipitation), ammonium sulphate (2-3.6M2pH 7.0) protein precipitation and CM-cellulose chromatography. A mol. wt. of approximately 120,000 was calculated for the purified enzyme by gel filtration (Sephadex G-200) which dissociated in small subunits with mol wt. of 54,000-42,600 calculated by electrophoresis on 5% polyacrylamide - 3% SDS-8M urea. The purified enzyme was immobilised with a PEI-derivative of nylon (polyethyleneimine) and the kinetic properties of both free and immobilized enzyme were investigated. Apparent Km values for the free purified enzyme were 7.4 x 10<super>-3</super>M (D-xylose); 2.8M (D-glucose); 1.9M (D-fructose). The corresponding apparent V values were 0.45; 0.015 and 0.022 mumoles min-1. mug enzyme-1 respectively. Investigations were also carried out into several other possibilities of assaying glucose isomerase activity. Parameters for the coupled reaction assay system using sorbitol dehydrogenase -NADH were optimised.
Thesis, PhD Doctor of Philosophy
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