Cloning and analysis of WF146 protease, a novel thermophilic subtilisin-like protease with four inserted surface loops

Abstract

Cloning and sequencing of the gene encoding WF146 protease, an extracellular subtilisin-like protease from the thermophile Bacillus sp. WF146, revealed that the WF146 protease was translated as a 416-amino acid precursor consisting of a putative 18-amino acid signal peptide, a 10-kDa N-terminal propeptide and a 32-kDa mature protease region. The mature WF146 protease shares a high degree of amino acid sequence identity with two psychrophilic subtilisins, S41 (68.2%) and S39 (65.4%), and a mesophilic subtilisin, SSII (67.1%). Significantly, these closely related proteases adapted to different temperatures all had four inserted surface loops not found in other subtilisins. However, unlike those of S41, S39 and SSII, the inserted loops of the WF146 protease possessed stabilizing features, such as the introduction of Pro residues into the loop regions. Interestingly, the WF146 protease contained five of the seven mutations previously found in a hyperstable variant of subtilisin S41 obtained by directed evolution. The proform of WF146 protease (pro-WF146 protease) was overexpressed in Escherichia coli in an inactive soluble form. After heat treatment, the 42-kDa pro-WF146 protease converted to a 32-kDa active mature form by processing the N-terminal propeptide. The purified mature WF146 protease hydrolyzed casein with an optimum temperature of 85 ‡C, and lost activity with a half-life of 30 min at 80 ‡C in the presence of 10 mM CaCl 2 .

Molecular Basis for Auto- and Hetero-catalytic Maturation of a Thermostable Subtilase from Thermophilic Bacillus sp. WF146

“…WF146, and its precursor is composed of a signal peptide, an N-terminal propeptide, and a mature domain (12,13). The recombinant proform of the wildtype WF146 protease matures effectively at high temperatures (e.g.…”

Improving the Thermostability and Activity of a Thermophilic Subtilase by Incorporating Structural Elements of Its Psychrophilic Counterpart

“…The DNA fragment encoding the proform of the WF146 protease, which comprises the N-terminal propeptide and the catalytic domain, was amplified from the genomic DNA of Bacillus sp. WF146 (25) by PCR using the primer pair PB-F/PX-R (see Table S1 in the supplemental material). Afterwards, the amplified fragment was inserted into pET26b to construct the expression plasmid (pWT) for the proform of the wild-type (WT) WF146 protease.…”

“…The expression of the recombinant proteins was carried out as described previously (25). The cells were then harvested and suspended in buffer A (50 mM Tris-HCl, 10 mM CaCl 2 , 10 mM NaCl, pH 8.0), followed by sonication on ice.…”

“…The WF146 protease is an extracellular subtilase produced by thermophile Bacillus sp. WF146 (25), and its stabilization and maturation mechanisms have been characterized previously (26)(27)(28)(29)(30). We previously engineered a cold-adapted variant of the WF146 protease that displays improved low-temperature activity but shows a decrease in thermostability (31).…”

Identification and characterization of a novel spore-associated subtilase from Thermoactinomyces sp. CDF

“…CDF was extracted according to the method of Orsini & Romano-Spica (2001), except that the cells were disrupted by grinding with a mortar and pestle, rather than microwave treatment. A fragment of the gene (cdf) encoding protease CDF was amplified from the genomic DNA with Pfu DNA polymerase by PCR employing two consensus-degenerate hybrid oligonucleotide primers (CODEHOPs), F and R (Table 1), designed on the basis of two highly conserved amino acid sequences around the catalytic residues His and Ser of subtilases (Wu et al, 2004) ( Supplementary Fig. S1a).…”