Labiase Bacterial Cell Lytic Enzymes

Cosmo Bio LTD

Catalog No: OZK-OZ-30EX

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Background
Labiase, produced by a submerged culture of Streptomyces fulvissimus TU-6, is a new enzyme preparation that lyses effectively cell walls of numerous lactic acid bacterium. This preparation is used as a tool for studies of cell walls structure of lactic acid bacterium and the preparation of plasmid DNA, intracellular enzyme and protoplast from lactic acid bacterium and so on.

DNase activity of Labiase
A previous questionnaire survey showed that labiase was most commonly used for the preparation of genomic and plasmid DNA from lactic acid bacteria. Thus, the DNA degradation activity of labiase was examined. (Methods)
1. A 0.5% labiase solution was prepared using McIlvaine buffer (pH 4.0).
2. To 50 μl of the 0.5% labiase solution, 1 μg of DNA was added as a substrate.
3. Five reaction solutions were prepared. Of these, two each were heated at 30oC for 1 or 4 hours, and the remaining one was not heated as a blank (0 hours). 4. The above five samples were treated with phenol and chloroform twice, followed by ethanol precipitation and washing.
5. The precipitate was dried up and dissolved in 45 µl of TE. To the solution, 5 µl of 10x loading buffer was added for 0.8% agarose gel electrophoresis. Lot Nos. JJAD1 and AAAC2 were tested.

Antibacterial effects of Labiase
(1) Bacteriolytic Spectrum of Labiase
The bacteriolytic spectrum of labiase was examined by measuring the bacteriolytic activity against bacterial strains. Turbidity reduction rates in bacterial suspensions were determined as the bacteriolytic activities of labiase.
Bacterial suspensions in a logarithmic growth phase were prepared with McIlvaine buffer (pH 4.0) at turbidity of 1.0 (OD660) and at a final labiase concentration of 0.25% to measure turbidity after 2-hour reaction.
The turbidity of a bacterial suspension prepared in the same manner without labiase was also measured as a control. Dissolution rates were determined using the formula below
[Formula]
                                 (d0-dt)-(D0-Dt)
Dissolution rate (%) = ------------------- ×100
                                         d0

d(0,t): Turbidity of reaction solution at 0 and t hours
D(0,t): Turbidity of the control at 0 and t hours

labiase efficiently lysed gram-negative bacteria (e.g., Escherichia coli and Pseudomonas spp.), as well as gram-positive bacteria (e.g., lactic acid and hiochi bacteria), suggesting a broad bacteriolytic spectrum. Of note, it lysed hiochi bacteria (i.e., causative bacteria of sake spoilage), Staphylococcus aureus IFO12732 (i.e., causative bacteria of food poisoning), and Streptococcus mutans JCM5705 (i.e., causative bacteria of dental caries), suggesting the possible applications of labiase to the prevention of hiochi, food poisoning, and dental caries.

(2) Bacteriolytic activity of Labiase in the presence of alcohol
To examine the bacteriolytic activities of labiase in the presence of alcohol, the dissolution rates of lactic acid and hiochi bacteria were compared in the same reaction system as that in Test (1) with or without 15% ethanol. Results are shown in Table 2.

As shown in Table 2, the bacteriolytic activities of labiase were comparable in cases with or without 15% ethanol. Specifically, the bacteriolytic activities of labiase were sufficiently high even when labiase was directly contacted with alcoholic beverages (e.g., sake and beer) of an alcohol concentration of 15%.

(3) Growth inhibition test of Staphylococcus aureus using Labiase (S. aureus IFO12732)

A filter-sterilized aqueous solution of labiase (or sterilized distilled water as a control) was added at a final concentration of 0.25% to an autoclaved general bacterial medium, Brain Heart Infusion Broth "Nissui" (Nissui Pharmaceutical Co., Ltd.). These media were inoculated with a loopful of Staphylococcus aureus (IFO12732), a causative bacterium of food poisoning, using a platinum loop, followed by shaking culture at 37oC for 24 hours to compare the time course of turbidity (OD660). As a control, egg white lysozyme (Sigma) was added at a final concentration of 0.25%. Results are shown in Figure 1.

For example, if there had been bacteriostatic effects until absorbance changed, the effects were noted for about 2 and 6 hours with egg white lysozyme and labiase added at 0.25% to the medium, respectively, as compared with an additive-free medium (Figure 1). Thus, the growth of causative bacteria of food poisoning can be suppressed by adding labiase to food. The effects of labiase were more potent than those of egg-white lysozyme used as a preservative.

(4) Growth inhibition test of hiochi bacteria using labiase

A filter-sterilized aqueous solution of labiase (or sterilized distilled water as a control) was added at a final concentration of 0.25, 0.125, or 0.025% to an autoclaved SI medium (1% yeast extract, 0.5% polypeptone, 2.5% glucose, 1% sodium acetate, 0.01% magnesium sulfate, 0.00025% manganese sulfate, 0.00025% ferrous sulfate, 0.005% mevalonic acid, and 10% ethanol, pH5.1). These media were inoculated with a loopful of hiochi bacteria (S24) using a platinum loop, followed by static culture at 30oC for 120 hours to compare the time course of turbidity (OD660). As a control, egg white lysozyme (Sigma) was added at a final concentration of 0.25%. Results are shown in Figure 2.

As shown in Figure 2, no growth of hiochi bacteria S24 was observed in labiase-added medium as compared with additive-free medium, demonstrating the marked growth inhibitory effects of labiase on hiochi bacteria. On the other hand, little growth inhibitory effect on hiochi bacteria was observed for egg white lysozyme. A similar test was conducted with other hiochi bacterial strains. Then, similar results were obtained for all the strains. The present study was conducted in the presence of 10% ethanol, demonstrating the growth inhibitory effects of labiase even in the presence of at least 10% ethanol. Thus, labiase can prevent the contamination of alcoholic beverages (e.g., sake and beer) with hiochi and lactic acid bacteria.

(5) Growth inhibition test of S. mutans JCM5705 using Labiase
A filter-sterilized aqueous solution of labiase (or sterilized distilled water as a control) was added at a final concentration of 0.25, 0.125, or 0.25% to an autoclaved general bacterial medium, Brain Heart Infusion Broth "Nissui" (Nissui Pharmaceutical Co., Ltd.). These media were inoculated with a loopful of S. mutans JCM5705, a causative bacterium of dental caries, using a platinum loop, followed by static culture at 37oC for 24 hours to compare the time course of turbidity (OD660). As a control, egg white lysozyme (Sigma) was added at a final concentration of 0.25%. Results are shown in Figure 3.

As shown in Figure 3, no growth of S. mutans JCM5705 was observed in labiase-added medium as compared with additive-free medium, demonstrating the marked growth inhibitory effects of labiase on S. mutans. On the other hand, no growth inhibitory effect on S. mutans was observed for egg white lysozyme. Thus, labiase can suppress the growth of S. mutans, a causative bacterium of dental caries, and can be used as an anticaries agent when mixed in toothpaste, powdered dentifrice, or mouthwash.

(6) Heat susceptibility of Labiase

Using the same method as in Tests (3), (4), and (5), the growth inhibitory effects of labiase on bacteria were examined with or without heat treatment in boiling water for 15 minutes before the addition of labiase into medium at a final concentration of 0.25%. Results are shown in Figures 4, 5, and 6. The present study was conducted to examine whether the growth inhibitory effects were definitely caused by the bacteriolytic activity of labiase or the activities of other bacterial components, by heat-inactivating the bacteriolytic activity of labiase (verified). (284 characters

As shown in Figure 4, the growth inhibitory effects on Staphylococcus aureus remained for about 3 hours even after enzyme inactivation by heat treatment, demonstrating the effects of growth inhibitory compounds, besides the bacteriolytic activity of labiase.

As shown in Figure 5, the growth of hiochi bacteria (S24) was completely inhibited for at least 120 hours with or without heat treatment, demonstrating effects, besides the bacteriolytic activity of labiase.

As shown in Figure 6, the growth of S. mutans was incompletely inhibited by heating, suggesting the effects of unknown growth inhibitory compounds, besides the bacteriolytic activity of labiase. In conclusion, the growth inhibitory effects of labiase were the combination of the effects of unknown compounds in labiase and the bacteriolytic activity of cell wall lytic enzyme, although the inhibitory effects slightly varied with strains. This could be easily predicted from the facts that S. fulvissimus, a labiase-producing bacterium, belongs to actinomycetes and, therefore, is likely to produce an antibacterial substance and that the enzyme is a crude enzyme preparation. The antibacterial effects of labiase were highly heat-resistant.

Citations for Labiase Bacterial Cell Lytic Enzymes – 4 Found

Sakamoto et al. 2019. Dialister hominis sp. nov., isolated from human faeces. Int J Syst Evol Microbiol. :.   , Journal

Sakamoto et al. 2019. Alistipes communis sp. nov., Alistipes dispar sp. nov. and Alistipes onderdonkii subsp. vulgaris subsp. nov., isolated from human faeces, and creation of Alistipes onderdonkii subsp. onderdonkii subsp. nov. Int J Syst Evol Microbiol. :.   , Journal

Sakamoto et al. 2019. Comparative genomics of Parolsenella catena and Libanicoccus massiliensis: Reclassification of Libanicoccus massiliensis as Parolsenella massiliensis comb. nov. Int J Syst Evol Microbiol. 69(4):1123-29.   , Journal

Okai et al. 2019. Rapid identification and quantification of Lactobacillus rhamnosus-targeting real-time PCR using a TaqMan probe. Jpn J Infect Dis. :.   , Journal