Digestive enzyme of cell walls of lactic acid bacterium

Labiase™ Bacterial Cell Lytic Enzymes


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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.

Specifications

Appearance: Lyophilized powder (containing lactose)
Package Size: 500mg
Activity: ≥10 units/g (β-N-Acetyl-D-glucosaminidase)
Other activities contained: Lysozyme≥2×104 units/g
Optimum pH and temperature: See Fig. 1,2
pH and thermal stability: See Fig. 1,2

Fig.1 pH-stability and activity
Stability:18 hours treatment at 25°C
Activity:37°C

Fig.2 Thermal-stability and activity
Stability:10 minutes treatment
Activity:pH4.0

ASSAY

ASSAY FOR ß-N-ACETYL-D-GLUCOSAMINIDASE ACTIVITY:

Unit Definition
One unit of enzyme catalyzes the release of 1µmole of p-nitrophenol from p-nitrophenyl N-acetyl-ß-D-glucosaminide per minute at 37°C, pH4.0.

Method
Reaction mixture

Substrate 5mM p-nitrophenyl N-acetyl-ß-D-glucosaminide in 0.2M
  Citrate-phosphate buffer, pH 4.0 0.95ml
Enzyme suitably diluted enzyme 0.05ml
  Total volume    1.00ml

 

Procedure
After incubation for 10 minutes at 37°C, terminate by 2ml of 1M Na2CO3. Read A405.

Calculation

Enzyme unit = A405 × 3 × 103
17.7 × 103 × 10

 

The molar extinction coefficient of free p-nitrophenol under these conditions is 17,700.

 

ASSAY FOR LYTIC ACTIVITY TOWARD LACTIC ACID BACTERIUM:

 

Method
Reaction mixture

 

Buffer 50mM Citrate -sodium citrate buffer, pH 4.0 1ml
Substrate Lactic acid bacterium suspension(A660≈5.0) 1ml
Enzyme 500µg-protein/ml solution 1ml
Distilled water 2ml
  Total volume    5ml

 

Procedure
Before adding the enzyme solution preincubation is carried out for 5 minutes at 37°C.After incubation for 2 hours at 37°C with gentle shaking, A660 of the mixture is determined. As a reference, 1 ml of distilled water is used instead of enzyme solution.

Calculation

Percentage decrease in A660 = (d0-dt)-(D0-Dt) × 100
d0

d(0 or t):A660 of reaction mixture after 0 or 2 hours
D(0 or t):A660 of reference mixture after 0 or 2 hours

Result
See Table 1.
The extent of lysis of lactic acid bacterium cells by Labiase varies with lactic acid bacterium strain, growth stage of lactic acid bacterium, or cultural condition.

Table 1.Lysis of lactic acid bacterium cells by Labiase.

Strains Lysis(%)
Leuconostoc mesenteroides IFO 3832 92.8
Leuconostoc lactis IFO 12455 99.6
Streptococcus faecalis IFO 3971 96.5
Lactobacillus acidophilus IFO 13951 97.9
Lactobacillus plantarum IAM 1041 96.8
Lactobacillus casei subsp.casei IFO 3533 59.4
Lactobacillus casei subsp.ramnosus IAM 1118 91.8
Lactobacillus brevis IFO 3345 95.6
Lactobacillus fermentum IFO 3071 70.4
Lactobacillus sp.(buchneri) IFO 3961 89.4
Lactobacillus fructivorans IFO 13954 96.9
Lactobacillus delbrueckii subsp.bulgaricus JCM 1002 97.7
Lactobacillus delbrueckii subsp.delbrueckii JCM 1012 92.0
Lactobacillus helvetics JCM 1120 98.7
Lactobacillus helveticus JCM 1005 84.5
Lactobacillus kefir 5818 93.0
Lactobacillus sanfrancisco JCM 5818 82.1
Bifidobacterium bifidum JCM 1255 62.9
Streptococcus salivaris subsp.thermophilus IFO 13957 94.0
Streptococcus mutans IFO 5705 73.9

ico_caution_s.pngPlease note that the above data is an example. The values vary widely depending on the condition.

Storage

Lyophilized preparation is stable for at least 1 year when stored at 4°C.

 

Labiase technical information

 Antibacterial effects of Labiase

 DNase activity of Labiase

 

FAQ

Q.What is the recommended concentration for reconstitution of this enzyme?
A. We recommend you use in 0.5% concentration of the enzyme. However please prepare and an appropriate change the concentration according to your culturing condition or kinds of strain.B

Q. What is the recommended concentration for reconstitution of this enzyme?
A. We recommend you use in 0.5% concentration of the enzyme. However please prepare and an appropriate change the concentration according to your culturing condition or kinds of strain.

Product List

Product Name Cat# Quantity Price

LabiaseTM Bacterial Cell Lytic Enzymes DataSheet ,

OZK-OZ-30EX

500 MG ¥25,000
$334

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.



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