Graduate Thesis Or Dissertation
 

Purification and properties of Streptococcus lactis β-galactosidase

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  • Experiments were carried out to purify and characterize the galactosidase of the Streptococcus lactis 7962. Purification was accomplished using standard procedures; however the lability of the enzyme to numerous treatments limited the techniques that could be used and the amount of active enzyme recovered. Gel filtration revealed that the enzyme existed in two forms differing in size by a factor of two (molecular weights of 5 x 10⁵ and 10⁶); the smaller of the two forms was purified and characterized in detail. Chemical characteristics of the two forms were the same: pH optimum, 7.0; temperature optimum, 37 C; and Michaelis constant, 10⁻³ M ONPG. Ammonium sulfate exerted a stabilizing effect on the enzyme and caused an association of the smaller unit to form a larger molecular weight aggregate. Because of the size and the chemical properties of the two forms a subunit (monomer) - native enzyme (dimer) relationship was established. The observations that para-chloromecuribenzoate and ethylenediamine tetraacetic acid inactivated the enzyme and that ammonium sulfate prevented this inactivation suggested that essential sulfhydryl and metal ion groups became masked in the enzyme structure during salt stabilization. Comparison of the amount of change seen in the U.V. spectra of salt-stabilized and nonstabilized enzyme indicated that ammonium sulfate stabilized the structure of the enzyme. The salt also protected the enzyme from the inactivating effect of urea, but to a much lower extent than was seen with the sulfhydryl antagonist or metal chelator. Chemical analysis of the purified enzyme, when compared with ß- galactosidase from bacteria of the enteric group, showed similarities and differences in the amino acid composition. For example, alanine, aspartic acid, glutamic acid, glycine, isoleucine, phenylalanine, serine, threonine and valine were present in the same amounts. Incubation of the purified enzyme at 27 C caused a similar association (quaternization) as was observed in the presence of ammonium sulfate. This indicated that hydrophobic bonding is involved in holding the dimer form together as has been suggested for other polymeric proteins. The same relative amount of the two enzyme forms (monomer and dimer) observed following quaternization was also found in enzyme extracted from log phase culture; this suggested that an equilibrium between the two forms existed. When enzyme was obtained from bacteria in the late logarithimic phase of growth (12 hours) a disproportionately large amount of monomer was observed. This indicated that lactose was essential for association to occur in vivo since this carbohydrate was probably depleated at that time. No evidence for dissociation of the dimer form of enzyme was seen, however, prolonged incubation at either 27 C or 5 C caused an increase in the amount of monomer form of enzyme present. This effect apparently was caused by the activation of an inactive form of enzyme, possibly monomer, as has been suggested in other proteins.
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