Leather modifier
A leather modifier and leather treatment technology, applied in small raw hide/big raw hide/leather/fur treatment, small raw hide/big raw hide/leather skin/fur chemical treatment, animal husbandry, etc.
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Embodiment 1
[0111] Embodiment 1: the preparation of BLP and LgBLP
[0112] (1-1) Preparation of transformants
[0113] As the host, Bacillus subtilis strain 168 (Bacillus subtilis Marburg No. 168 strain: Nature, 390, 1997, p. 249) was used. The plasmids pHY-BLP2 and pHY-LgBLP described in Example 1 of Patent Document 4 above were introduced in the following manner, respectively. Bacillus subtilis strain 168 was inoculated in 1 mL of LB medium, and cultured with shaking at 30° C. and 200 rpm overnight. 10 μL of this culture solution was inoculated into 1 mL of new LB medium, and cultured at 37° C. and 200 rpm for 3 hours. The culture solution was centrifuged to collect pellets. SMMP (0.5M sucrose, 20mM disodium maleate, 20mM magnesium chloride hexahydrate, 35% (w / v) Antibiotic medium No. 3) (Difco)) 500 μL, incubate at 37°C for 1 hour. Next, the pellet was collected by centrifugation and suspended in 400 µL of SMMP. 33 μL of the suspension and 20 ng of each plasmid were mixed, and 10...
Embodiment 2
[0122] Example 2: Determination of degradative activity of elastin and collagen
[0123] As proteases, BLP, LgBLP, Savinase (SIGMA, P3111) and purified elastase (hereinafter PPE) derived from porcine pancreas (Worthington Biochemical, ESFF) were used. Bacillus-derived subtilisin containing Savinase is a general softening enzyme (Tanning Chemistry: The Science of Leather).
[0124] As the substrate, elastin (SIGMA, E1625) derived from bovine taurus ligament and collagen (SIGMA, C9879) derived from bovine Achilles tendon were used. Each enzyme was added to 1 mL of 50 mM Tris-HCl (pH 7.5) containing 20 mg of the substrate so that the final concentration would be 1 mg / L. After reacting at 30° C. for 1 hour, centrifugation was performed at 4° C. and 15,000 rpm for 5 minutes, and the supernatant was recovered. The peptides in the supernatant were quantified using TaKaRa BCA Protein Assay Kit (TaKaRa BCA Protein Assay Kit, TaKaRa). The unadded sample was used as a blank, and the a...
Embodiment 3
[0126] Embodiment 3: the cowhide degradation test of BLP
[0127] (3-1) Preparation of cowhide for softening
[0128] The cowhide that has been shaved, depilated, and lime dipped is cut into 1.5cm squares for use. 12 skin pieces were placed in one centrifuge tube, immersed in 40 mL of an aqueous ammonium chloride solution containing 3% (w / w) of the skin weight of ammonium chloride, and incubated at room temperature for 60 minutes to perform deliming. The deashed skin pieces were washed once with 50 mM Tris-HCl (pH 7.5), and then used for the following tests.
[0129] (3-2) Protease treatment of cowhide
[0130] Two skin pieces prepared in (3-1) were placed in a 5 mL screw vial (27 mm×55 mm) containing 50 mM Tris-HCl (pH 7.5). A protease (Savinase, PPE, BLP) was added to the screw vial so that the final concentration became 50 mg / L to start the reaction. After culturing at 30° C. and 150 spm for 4 hours, each skin piece was transferred into 10 mL of 0.1 M sulfuric acid to s...
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