| Summary: | 碩士 === 國立臺灣大學 === 農業化學研究所 === 89 === Abstract
Invertase (EC 3.2.1.26) plays an important role in the carbohydrate metabolism in higher plants. There are one alkaline invertase (IT I), two acid invertases in the soluble fraction and one bound form acid invertase (IT b) in the insoluble fraction of Bambusa edulis suspension cells. Alkaline invertase (IT I) was purified to homogeneity by consecutive use of ammonium sulfate fractionation, DEAE- Sephacel, gel filtration chromatographies and preparative electrophoresis. One of the acid invertases (IT II) was purified by means of ammonium sulfate fractionation, ion-exchange, Con A Sepharose affinity chromatographies and Mono Q ion-exchange chromatography, can be purified to homogeneity. A comparison of molecular mass by SDS-PAGE and gel filtration suggested that the IT I was homotetramer (subunit molecular mass 60 kDa), IT II was a monomer of 68 kDa and IT b was also a monomeric enzyme of 52 kDa.
The pH optima of the IT I、IT II and IT b were 7.0, 4.5 and 5.0, respectively and the optimal temperature was 40℃. Isoelectric focusing analysis indicated that the pI values of IT I and IT II were 4.8 and 7.4, respectively. IT I、IT II and IT b had abilities to hydrolyze sucrose and raffinose but not maltose, therefore, they are β-fructofuranosidase. For IT I, the Km value for sucrose was 10.9 mM. For IT II, the Km values for sucrose and raffinose were 3.7 mM and 8.9 mM, respectively. Glucose and fructose inhibited the enzyme activities. Some proteins such as BSA, Con A, and urease could activate IT II. The metal ions such as Ag+ and Hg2+ strongly inhibited enzyme activities, and so did sulfhydryl group affecting reagents. These indicated that the presence of at least one sulfhydryl group at catalytic site or near the active site of invertases, which did participate the sucrose hydrolysis.
The degradation phenomenon of invertase IT II from B. edulis suspension cells was found during purification and storage. After extended storage at 4℃, the original band (67 kDa) of IT II disappeared. Then, 40 kDa, 36kDa or 31 kDa degraded enzyme fragments were found on SDS-PAGE, and could be detected by the antibody of invertase. Besides, large amount of plant lysozyme was found in the suspension cells. The N-terminal amino acid sequences of the purified enzyme had 90% homology with hen egg white lysozyme. Lysozyme from B. edulis suspension cells seemed to interact with invertase in vitro, and could be separated from invertase on CM Sepharose chromatography. The details of the relationship between them needed to study further.
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