Graduate Thesis Or Dissertation
 

Mechanisms of lactose utilization by lactic acid bacteria : enzymic and genetic studies

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  • To learn more about the role β-galactosidase plays in rate of acid production in milk by lactic acid bacteria, several lactobacilli were examined for this enzyme. Cells from different species were grown in lactose broth to induce β-galactosidase activity. The specific activities of whole, solvent treated cells, and of cell-free extracts were compared using the chromogenic substrate o-nitro-phenyl-β-D-galactopyranoside (ONPG). Among those tested, Lactobacillus helveticus possessed the greatest activity and properties of the enzyme in this species were studied. The optimum temperature for the enzyme in a cell-free extract prepared by sonication was 50 C and the optimum pH was about 6.6 when using sodium phosphate buffer (0.05 M) at 50 C; potassium phosphate buffer afforded 30% less activity. Galactose was an inducer of the enzyme but not as effective as lactose. During the isolation of lactose negative mutants from Streptococcus lactis 7962, it was noted that a semi-synthetic medium, containing lactose as the sole carbon source, would not support growth if inoculated with cells previously grown on glucose. Lactose grown cells, however, grew in the medium after a 20 to 24 hour lag period. When glucose or galactose served as the sole carbon source, rapid growth by the glucose or lactose grown cells occurred. Addition of tryptophan to the basal medium permitted the glucose-grown cells to utilize the lactose. Thus tryptophan was stimulatory for growth of S. lactis 7962 on lactose, but was not required for rapid growth on glucose. The mechanism of lactose utilization in two strains of lactic streptococci was examined. Sodium fluoride prevented lactose utilization by whole cells of S. lactis C₂F, but had no effect on S. lactis 7962. Sodium arsenate prevented lactose metabolism in S. lactis 7962 but had only a slight inhibitory response on S. lactis C₂F. Concentrated cell extracts from S. lactis C₂F hydrolyzed ONPG; this hydrolysis was inhibited by sodium fluoride, yet the addition of phosphoenolpyruvate (PEP) in the presence of sodium fluoride, restored maximal activity. Addition of acetyl-P, carbamyl-P, adenosine triphosphate (ATP), guanosine triphosphate (GTP), or uridine triphosphate (UTP) did not stimulate activity. The presence of cofactors did not stimulate nor did sodium fluoride inhibit the hydrolysis of ONPG in cell extracts of S. lactis 7962. The latter organism was shown to hydrolyze lactose into glucose and galactose, whereas S. lactis C₂F was unable to split the disaccharide. A nonmetabolizable analogue of lactose, thiomethyl-β-D-galactoside (TMG), was used to measure the transport process. Uptake of ¹⁴C-TMG was inducible in both organisms. In S. lactis C₂F¹⁴C-TMG rapidly accumulated as a derivative which was negatively charged and which chromatographed as TMG after treatment with phosphatase. The analogue uptake by S. lactis 7962 was defective, yet the accumulated TMG still appeared primarily as a derivative. Galactose was also a better inducer than lactose in S. lactis C₂F and in the several "β-galactosidase-less" lactic streptococci examined. Lactose-negative mutants from S. lactis C₂F all possessed the phenotype lac⁻gal⁻ . Lactose negative mutants from S. lactis 7962 were separated into several classes. Several mutants were unable to transport TMG, although they contained normal levels of β-galactosidase activity, suggesting they possessed the phenotype z⁺y⁻. The instability of β-galactosidase in toluene treated cells or cell-free extracts of over 40 lactic streptococci was explained. These organisms did not hydrolyze lactose, but instead hydrolyzed lactose-P and consequently must possess a different enzyme. The significance of the TMG derivative in 7962, especially since extracts from this strain were shown to hydrolyze lactose, is unclear; it may explain the inability of TMG to induce lactose utilization in this strain.
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