Detergent for corneum-derived stains, and method for evaluating ability to degrade corneum-derived stains
A cleaning agent and dirt technology, which is applied in the field of keratin and dirt decomposition ability of the composition, and can solve problems such as damage to clothes and the like
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[0204] Hereinafter, the present invention will be described more concretely using examples. However, the technical scope of the present invention is not limited by these examples.
[0205] Table 1 shows the list of primers used in this example.
[0206] [Table 1]
[0207] Primer name sequence serial number BLP_S237signal_F GAAGGAAACACTCGTATGAAAAAAATCTCAAAAGC 9 BLP_S237signal_R AACTAGTTTAATAGATTAGTTCGGTCCAGGATTCAC 10 vector-F TCTATTAAACTAGTTATAGGGTTATTCTAAAGG 11 vector-sig-R ACGAGTGTTCCTTCTGCTGC 12 ΔS237N_fw TGCAGCAATGAAAAAAAATCTCAAAAGCTGGTCTGG 13 ΔS237N_rv TTTTTCATTGCTGCAAGAGCTGCCGGAA 14 Las A_F GCAGCTCTTGCAGCACATGATGATGGCCTG 15 LasA_CR TAGTTTAATAGATTAGTGGTGGTGGTGGTGCAGAGCCAGTCCCGG 16 pHY_just_F TAATCTATTAAACTAGTTATAGGGTTATTCTAAAGG 17 pHY_just_R_NEW TGCTGCAAGAGCTGCCGGAAA 18 LasA_Chis_n_F TAATCTATTAAACTAGTTATAGGGTTATTCTAAAGG 19 LasA_Chis_n_R CAGAGCCAGTCCCGGATTATAC 20 ...
reference example 1
[0208] The preparation of reference example 1 protease
[0209] (1) Preparation of culture supernatant containing BLP
[0210] (1-1) Preparation of expression vector
[0211] A material (BLP / pUC57) in which the BLP gene (SEQ ID NO: 1) was inserted into plasmid pUC57 was prepared using the artificial gene synthesis service of GenScript Corporation. Using BLP / pUC57 as a template, a PCR reaction was carried out using the primer pair BLP_S237signal_F / BLP_S237signal_R (SEQ ID NO. 9 and 10) and PrimeSTAR Max Premix (TAKARA BIO). Using the plasmid pHY-S237 described in Example 7 of WO2006 / 068148A1 as a template, a PCR reaction was performed in the same manner using the primer pair vector-F / vector-sig-R (SEQ ID NO: 11 and 12). DpnI treatment was performed on each PCR product using DpnI (New England Biolabs). Next, an In-Fusion reaction was performed according to the instructions of the In-Fusion HD Cloning Kit (Clontech).
[0212] Use In-Fusion reaction solution to convert ECOS T...
reference example 2
[0231] The mensuration of reference example 2 enzyme activity
[0232] As the substrate, a FRET substrate [hereinafter referred to as FRET-GGGGG] (made to order at PH Japan) with pentaglycine between the fluorescent group Nma and the quencher Lys(Dnp) was used. Here, Nma means 2-(N-methylamino)benzoyl (Nma). In addition, Lys(Dnp) means a group having 2,4-dinitrophenyl (Dnp) in the side chain of lysine (Lys). 190 μL of the enzyme solution (enzyme, 20 mM Tris-HCl (pH 7.5)) obtained in Reference Example 1 (5) was added to a 96-well assay plate (AGC Techno Glass, 3881-096), and FRET-GGGGG solution (1 mM FRET-GGGGG, 100 mM Tris-HCl (pH 7.5)) 10 μL to prepare a reaction solution. The fluorescence intensity of the reaction solution was measured over time at a temperature of 30° C., an excitation wavelength of 340 nm, and a measurement wavelength of 440 nm using infinite M200 (TECAN). Under the same reaction conditions, use 20mM Tris-HCl pH 7.5 solution instead of enzyme solution, ...
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