Extracellular Production, Characterization, and Engineering of a Polyextremotolerant Subtilisin-Like Protease From Feather-Degrading Thermoactinomyces vulgaris Strain CDF

Here, the gene encoding a subtilisin-like protease (protease Als) was cloned from Thermoactinomyces vulgaris strain CDF and expressed in Escherichia coli. The recombinant enzyme was released into the culture medium of E. coli as a mature form (mAls). Purified mAls displayed optimal activity at 60–70°C and pH 10.0 using azo-casein as the substrate, and showed a half-life of 13.8 h at 70°C. Moreover, the activity of thermostable mAls was comparable to or higher than those of mesophilic subtilisin Carlsberg and proteinase K at low temperatures (10–30°C). Protease Als was also stable in several organic solvents and showed high compatibility with commercial laundry detergents. Notably, mAls exhibited approximately 100% of its activity at 3 M NaCl, and showed enhanced thermostability with the increase of NaCl concentration up to 3 M. Protease Als possesses an excess of solvent-accessible acidic amino acid residues, which may account for the high halotolerance of the enzyme. Compared with homologous protease C2 from the same strain, protease Als exhibits substantially lower activity toward insoluble keratin substrates but efficiently hydrolyzes soluble keratin released from chicken feathers. Additionally, direct substitution of the substrate-binding site of protease Als with that of protease C2 improves its activity against insoluble keratin substrates. By virtue of its polyextremotolerant attribute and kerationolytic capacity, protease Als may find broad applications in various industries such as laundry detergents, food processing, non-aqueous biocatalysis, and feather processing.

PARTIAL CHARACTERIZATION AND CLONING OF PROTEASE FROM BACILLUS

Objective: The present research focused on amplification of protease gene from Bacillus strain which was then assessed for maximal enzyme activity.Methods: A putative Bacillus strain was isolated from soil, inoculated into protease production media, and optimized with appropriate pH and temperature conditions for maximal enzyme activity. Genomic DNA was isolated from the strain and amplified the fragment by polymerase chain reaction (PCR) using gene-specific primers for protease. The fragment is then ligated into a T/A cloning vector and transformed into calcium chloride-treated competent Escherichia coli DH5α cells. The plasmids were then isolated and confirmed the presence of the gene.Results: A specific amplification of 1.1 kb was observed following PCR. The amplified product includes the coding sequence and a signal peptide sequence of the protease gene. After cloning with T/A cloning vector pTZ57R/T and transformed into E. coli DH5α competent cells, the recombinant plasmid was selected using blue-white selection. Plasmid DNA isolated from the recombinant strains and confirmed the presence of a gene of interest using PCR and quantified by an assay for maximal protease activity. The optimum pH was found to be 10.1 and giving an activity of 21.566 international unit (IU)/ml, and the optimum temperature was found to be on 60°C giving an activity of 38.708 IU/ml.Conclusion: Amplification of protease gene by PCR isolated from Bacillus strain and optimization of pH and temperature conditions for the assessment of subtilisin Carlsberg produced by it. Subtilisin which is protein engineered can be used in commercial products such as stain cutter, dishwashing detergents, cosmetics and food processing, and contact lens cleaner.

Crystal structure of the M14R mutant of oryctin in complex with trypsin

Oryctin is a 66-amino-acid protein purified from the larval haemolymph of the coconut rhinoceros beetle Oryctes rhinoceros, which shows no sequence similarity to any other protein known. We determined the solution NMR structure of oryctin, and found that oryctin had a similar backbone fold to the turkey ovomucoid domain 3, OMTKY3, a Kazal-type serine protease inhibitor [1]. Based on the structural similarity, we tested the serine protease inhibitory activity of oryctin, and found that oryctin does inhibit some serine proteases, such as α-chymotrypsin, endopeptidase K, subtilisin Carlsberg, and leukocyte elastase [1]. However, oryctin cannot inhibit trypsin at all. In this study, we have introduced point mutations to the putative inhibition loop of oryctin to obtain oryctin mutants that can inhibit trypsin. Then, we have solved the crystal structure of such an oryctin mutant, M14R-oryctin with a Ki value of 3.410.8 nM, in complex with trypsin to reveal how it binds to and inhibits trypsin. As predicted, the putative inhibition loop lay on the substrate binding cleft of trypsin. Particularly, the side chain of R14 fit into the S1 pocket of trypsin by forming hydrogen/ionic bonds with D191, S192 and G216 at the bottom of the S1 pocket and G195, D196, S197 and S212 at its entrance. In addition, R65 located in the C-terminal α-helix of M14R-oryctin formed hydrogen bonds with S40 and F44 of trypsin. The latter interaction, which is unique to oryctin, enhances its binding affinity to trypsin.