• Current Research on Agriculture and Life Sciences
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• v.4
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• pp.55-64
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• 1986

Polyphenol oxidase in japanese pear (Pyrus communis var. mansamkil) was isolated, partially purified and its some properties were investigated. Polyacrylamide disc gel electrophoresis indicated two bands with polyphenol oxidase activity in the extract from acetone dry powder of par flesh. These two polyphenol oxidases (PPO A and PPO B) were purified through acetone precipitation and diethylaminoethyl cellulose column chromatography. PPO A and B were purified 7.8 fold and 8.7 fold by the present procedure, respectively. The Rm values of partially purified PPO A and B were estimated to be 0.58 and 0.68, respectively. The optimum temp, and pH of PPO A activity were $33^{\circ}C$ and pH 7.0, while those of PPO B were $30^{\circ}C$ and pH 4.2, respectively. Two PPO were unstable over the temperature of $60^{\circ}C$. The substrate specificity of pear PPO showed high affinity toward o-diphenolic compounds, especially catechol in PPO A and chlorogenic acid in PPO B, but inactive toward m-diphenol, p-diphenol and monophenols. PPO A showed affinity toward the trihydroxyphenolic compound. $Zn^{{+}{+}}$ activated the PPO A activity but $Fe^{{+}{+}}$ inhibited PPO B activity, while $Fe^{{+}{+}}$ and $Zn^{{+}{+}}$ activated the PPO B activity, while $Fe^{{+}{+}}$ and $Zn^{{+}{+}}$ activated the PPO B activity but $K^+$, $Mg^{{+}{+}}$, $Ca^{{+}{+}}$ and $Hg^{{+}{+}}$ inhibited at 10mM concentration. $Cu^{{+}{+}}$ activated the enzyme action at low concentrations but inhibited at high concentration. Inhibition studies indicated that L-ascorbic acid, L-cysteine and thiourea were most potent. The Km values of PPO A and PPO B for catechol were 20mM and 14.3mM, respectively.

• Microbiology and Biotechnology Letters
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• v.20 no.5
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• pp.519-526
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• 1992

The gene coding for urease of alkalophilic Bacillus pasteurii had been cloned in Escherichia coli previously. The urease protein was purified 63.1-fold by TEAE-cellulose, DEAE-Sephadex A-50, Sephadex G-150 and Sephadex G-200 chromatographies with a 7.3% yield from the sonicated fluid of the E. coli HB1Ol(pBUll) encoding B. pasteurii urease gene. The ureases of E. coli (pBUll) and B. pasteurii possessed as a $K_m$ for urea, 42.1 mM and 40.4 mM, respectively. They hydrolyzed urea with $V_{max}$ of 86.9$\mu$mol/min and 160$\mu$mol/min, respectively. Both ureases were composed with four subunits (Mrs 67,000) and a subunit (Mr 20,000). The molecular weight of both native enzymes was Mr 280,OOO$pm$10,000 determined by gel filtration chromatography and Coomassie blue staining of the subunits. The optimal reaction pH of both ureases were pH 7.5. The ureases were stabled in pH 5.5-10.5. The optimal reaction temperature of both ureases were $60^{\circ}C$, and the ureases were stable for an hour at $50^{\circ}C$, 40min at $60^{\circ}C$ and 10 min at $70^{\circ}C$ The activity of both enzymes were inhibited completely by $Ag^{2+}$, $Hg^{2+}$, $Zn^{2+}$, $Cu^{2+}$, and were inhibited 60% by CoH, 30% by $Fe^{2+}$ and 10% by $Pb^{2+}$. However it was increased by the addition of $Sn^{2+}$, $Mn^{2+}$, $Mg^{2+}$ at concentration of $1{\times}10^{-3}$M. Both ureases were inhibited completely by p-CMB and acetohydroxamic acid. The urease expressed in E. coli (pBU11) was inhibited 70% by SDS. The urease of B. pasteurii was inhibited 40% by hydroxyurea, whereas the recombinant urease of E. coli strain was inhibited 17%. Both enzymes were not inhibited by cyclohexanediaminetetraacetic acid (CDTA) and ethylendiaminetetraacetic acid (EDTA).