Studies on β-D-galactosidase : purification of β-D-galactosidase by affinity chromatography = β-D-galactosidase 의 affinity chromatography 에 의한 정제 및 microencapsulation 기술에 관한 연구purification of β-D-galactosidase by affinity chromatography

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To purify $\beta$-galactosidase of E. coli by affinity chromatography, various affinity columns using galactosamine, galactosylamine and galactonolactone as ligand were prepared. Galactonolactone adsorbent covalently attached to diaminohexyl-Sepharose 4B was effective to purify $\beta$-galactosidase. This procedure resulted in a 35 folds enrichment of $\beta$-galactosidase activity. The yield of 90\% by the affinity method can be compared to the yield of between 30 and 40\% obtained with conventional techniques. The adsorbent could be used several times without appreciable impairment of binding capacity. The monomeric form of the enzyme was also bound strongly to the substituted agarose, and it was eluted together with the active tetramer. $\beta$-Galactosidase was microencapsulated with the enzyme prepared by affinity chromatography and the microencapsulated enzyme was characterized. The nylon microcapsules, which were obtained by interfacial polymerization of 1,6-diaminohexane and sebacoyl chloride, were generally spherical and have a mean diameter of 80$\mu$ with 45\% of the recovery. The characteristics of the microencapsulated enzyme were similar to those of soluble enzyme. Optimum pH of native enzyme and microcapsule were 7.0 - 7.2 and 7.3 - 7.5, respectively. Optimum temperature for both native and microencapsulated enzyme was 50$^\circ$C and the activation energies were 8.94 Kcal/mole and 9.78 Kcal/mole, respectively. The apparent Km value of microcapsule was about 1.5 times higher than that of native enzyme. In the case of native enzyme, Km for o-nitrophenyl-$\beta$-D-galactopyranoside(ONPG) hydrolysis was $3.33\times10^{-4}$ M and that for lactose hydrolysis was $2.86\times10^{-3}$ M. In microcapsule, Km for ONPG was $5.28\times10^{-4}$ M and that for lactose was $4.25\times10^{-3}$M. The hydrolysis of lactose and milk was carried out by the microencapsulated enzyme eighty \% of lactose in a buffered 5\% lactose solution and 70\% of the lactose in ski...
Byun, Si-Myung변시명
한국과학기술원 : 생물공학과,
Issue Date
62253/325007 / 000761054

학위논문(석사) - 한국과학기술원 : 생물공학과, 1978.2, [ [vii], 72 p. ]


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