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.
|Appearance:||Lyophilized powder (containing lactose)|
|Activity:||10 units/g (β-N-Acetyl-D-glucosaminidase)|
|Other activities contained:||Lysozyme2×104 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
Fig.2 Thermal-stability and activity
Stability:10 minutes treatment
ASSAY FOR -N-ACETYL-D-GLUCOSAMINIDASE ACTIVITY:
One unit of enzyme catalyzes the release of 1mole of p-nitrophenol from p-nitrophenyl N-acetyl-D-glucosaminide per minute at 37°C, pH4.0.
|Substrate||5mM p-nitrophenyl N-acetyl-D-glucosaminide in 0.2M|
|Citrate-phosphate buffer, pH 4.0||0.95ml|
|Enzyme||suitably diluted enzyme||0.05ml|
After incubation for 10 minutes at 37°C, terminate by 2ml of 1M Na2CO3. Read A405.
|Enzyme unit =||A405 × 3||x 103|
|17.7 x 103 x 10|
The molar extinction coefficient of free p-nitrophenol under these conditions is 17,700.
ASSAY FOR LYTIC ACTIVITY TOWARD LACTIC ACID BACTERIUM:
|Buffer||50mM Citrate -sodium citrate buffer, pH 4.0||1ml|
|Substrate||Lactic acid bacterium suspension(A6605.0)||1ml|
|Total volume||5 ml|
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.
|Percentage decrease in A660 =||(d0-dt)-(D0-Dt)||x 100|
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
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.
|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|
Please note that the above data is an example. The values vary widely depending on the condition.
Lyophilized preparation is stable for at least 1 year when stored at 4°C.
Labiase technical information
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.。
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.
Labiase Bacterial Cell Lytic Enzymes
To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.