Polypeptides having protease activity and polynucleotides encoding same
A protease activity, polynucleotide technology for use in hydrolases, microorganism-based methods, biochemical equipment and methods, etc., to solve problems such as complex stains and soil composition
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example 1
[0318] Example 1: Expression and purification:
[0319] Isolation, genome sequencing and identification of the coding gene from Bacillus sp. (SEQ ID NO 1).
[0320] Bacterial strains from Bacillus spp. were isolated from environmental samples and the species identified by sequencing of the 16S ribosomal subunit gene are listed in Table 1 .
[0321] Table 1 :
[0322]
[0323] Chromosomal DNA from bacterial strains was isolated by using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). 2ug of chromosomal DNA was sent to FASTERISSA, Switzerland for genome sequencing. Genomes were sequenced by Illumina sequencing. The resulting genomic sequence was analyzed and the S8 protease was identified by searching for comparison with protease TY-145 (SEQ ID NO: 4) using the BLAST program. The DNA sequence of the identified gene encoding the polypeptide of the present invention is included in the Sequence Listing as SEQ ID NO:1.
example 2
[0330] Example 2 Purification of proteases from Bacillus species
[0331] The broth was centrifuged (26000 x g, 20 min) and the supernatant was carefully decanted from the precipitate. The supernatant was filtered through a Nalgene 0.2 μm filter unit to remove remaining Bacillus host cells. The 0.2μm filtrate was mixed with 3.0M (NH 4 ) 2 SO 4 Mix and apply the mixture to a Phenyl-Sepharose FF (high sub) column (from GE Healthcare) in 100 mM H 3 BO 3 , 10mM MES / NaOH, 2mM CaCl 2 , 1.5M (NH 4 ) 2 SO 4 (pH 6.0) equilibrium. After washing the column with equilibration buffer, the protease was washed with 100 mM H 3 BO 3 , 10mMMES, 2mM CaCl 2 (pH 6.0) for stepwise elution. The eluted peak (containing protease activity) was collected and applied to 3 BO 3 , 10mM MES, 2mM CaCl 2 (pH 6.0) on a bacitracin sepharose column (from Upfront chromatography). After washing the column well with equilibration buffer, the protease was washed with 100 mM H2O with 25% (v / v) 2-prop...
example 3
[0338] Example 3 Construction of protease variants by site-directed mutagenesis
[0339] Site-directed variants were constructed from the Bacillus protease (SEQ ID NO: 3) comprising specific substitutions according to the invention. These variants were made by traditional DNA fragment cloning using PCR together with properly designed mutagenic oligonucleotides (which introduced the desired mutation in the resulting sequence) (Sambrook et al., Molecular Cloning: A Laboratory Manual [Molecular Cloning : Laboratory Manual], 2nd ed., Cold Spring Harbor, 1989).
[0340] Mutagenized oligonucleotides are synthesized corresponding to the DNA sequence flanking the desired mutation site or sites, separated by DNA base pairs defining insertions / deletions / substitutions. In this way, the variants listed in Table 2 below were constructed and produced.
[0341] To test the protease variants of the invention, mutant DNA comprising the variants of the invention was transformed into a compete...
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