Subtilase variants and compositions comprising same
A technology of subtilase and composition, applied in the directions of detergent composition, enzyme, peptidase, etc., can solve the problems of loss of washing performance, inactivation of enzyme, difficulty in completely removing stains, etc.
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[0202] Preparation of variants
[0203] The present invention also relates to methods for obtaining subtilase variants as described herein.
[0204] One embodiment relates to a method for obtaining a subtilase variant comprising:
[0205] (a) providing a host cell comprising a polynucleotide encoding a variant of a parent protease comprising the mutations X9R+X19L+X62D compared to SEQ ID NO: 1, wherein the position numbers correspond to SEQ ID NO: 1 The position of the polypeptide of ID NO: 2, wherein the variant has protease activity, and the sequence identity of the variant to SEQ ID NO: 1 is at least 80% but less than 100%, and with the proviso that the variant does not contain a histidine residue in position 14;
[0206] (b) cultivating the host cell under conditions suitable for expressing the variant; and
[0207] (c) recovering the variant.
[0208] In a particular embodiment, the method for obtaining a subtilase variant comprises:
[0209] (a) providing a host cel...
example
[0414] Materials and Methods
[0415] Suc-AAPF-pNA activity assay
[0416] Proteolytic activity can be determined by a method using Suc-AAPF-PNA as a substrate. Suc-AAPF-PNA is an abbreviation for N-succinyl-alanine-alanine-proline-phenylalanine-p-nitroaniline, and is a blocking peptide that can be cleaved by endoproteases. After cleavage, free PNA molecules are released, which have a yellow color and can therefore be measured by visible spectrophotometry at a wavelength of 405 nm. The Suc-AAPF-PNA substrate was manufactured by Bachem (Catalog # L1400, dissolved in DMSO).
[0417] Protease samples to be analyzed were diluted in residual activity buffer (100 mM Tris pH 8.6). The assay was performed by transferring 30 μl of diluted enzyme samples to a 96-well microtiter plate and adding 70 μl of substrate working solution (0.72 mg / ml in 100 mM Tris pH 8.6). The solution was mixed at room temperature and the absorbance was measured every 20 seconds for 5 minutes at OD 405 n...
example 1
[0419] Example 1: Preparation and purification of polypeptides
[0420] Mutagenesis and introduction of the expression cassette into Bacillus subtilis are performed by standard methods known in the art. All DNA manipulations were performed by PCR (eg, as described in Sambrook et al., 2001, supra) using standard methods known to those skilled in the art.
[0421] The recombinant Bacillus subtilis construct encoding the subtilase polypeptide was inoculated into a complex medium (TBgly) for culture, and cultured at 37° C. for 24 h for antibiotic selection. Inoculate the overnight culture at a ratio of 1:100 into a rich medium (PS-1: 100 g / L sucrose (Danisco Cat. No. 109-0429), 40 g / L soybean hulls (soy flour), 10 g / L Na 2 HPO 4 .12H 2 O (Merck Cat. No. 106579), 0.1 ml / L Dowfax63N10 (Dow) in shake flasks. Shake flask cultures were performed at 30°C with shaking at 270 rpm for 4 days.
[0422] Purification of the culture supernatant was performed as follows: the culture broth ...
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