1. Statistical Optimization of the Production of NaCl-Tolerant Proteases by a Moderate Halophile, Virgibacillus sp. SK37
Sornchai Sinsuwan, Anuvat Jangchud, Sureelak Rodtong, Sittirak Roytrakul, Jirawat Yongsawatdigul Food Technol Biotechnol. 2015 Jun;53(2):136-145.
The objectives of this study are to optimize the conditions for providing high yield of NaCl-tolerant extracellular protease from Virgibacillus sp. SK37 based on a fish-based medium and to investigate the effects of the key factors (mass per volume ratios of dried anchovy, yeast extract and NaCl, and initial pH of the medium) on the secretion pattern of proteases. Based on the predicted response model, the optimized medium contained 1.81% of dried anchovy, 0.33% of yeast extract and 1.25% of NaCl at pH=7.8. Under these conditions, a 5.3-fold increase in protease production was achieved, compared with the broth containing only 1.2% of dried anchovy (5% of NaCl at pH=7). The cubic regression adequately described the protease production. Protease activity was determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on the synthetic substrate (Suc-Ala-Ala-Pro-Phe-AMC). Proteases of molecular masses of 19, 34, 35 and 44 kDa were secreted in the presence of NaCl, whereas those of 22 and 42 kDa were the main proteases detected in the absence of NaCl. In addition, no secreted proteases were detected when initial pH of the medium was pH=6. The peptide mass fingerprint of the medium cultured with 10% NaCl showed a higher abundance of peptides with lower mass of 500-1000 m/z compared with the medium containing 0% NaCl, indicating the higher proteolytic activity of the high-salt medium. The Virgibacillus sp. SK37 proteases showed a marked preference towards Lys, Arg and Tyr in the presence of NaCl and towards Lys and Arg in the absence of NaCl.
2. NaCl-activated extracellular proteinase from Virgibacillus sp. SK37 isolated from fish sauce fermentation
S Sinsuwan, S Rodtong, J Yongsawatdigul J Food Sci. 2007 Jun;72(5):C264-9. doi: 10.1111/j.1750-3841.2007.00375.x.
Virgibacillus sp. SK37 exhibited high extracellular proteolytic activity in skim milk broth containing 10% NaCl. Optimum conditions of the crude proteinase were at pH 8.0 and 65 degrees C. The proteinase was strongly inhibited by phenylmethanesulfonyl fluoride (PMSF) and preferably hydrolyzed Suc-Ala-Ala-Pro-Phe-AMC, suggesting the serine proteinase with a subtilisin-like characteristic. Proteolytic activity increased with NaCl concentration up to 20%. Ca(2+) activated the enzyme activity but reduced enzyme stability at 65 degrees C. Several proteinases with dominant molecular mass (MW) of 81, 67, 63, 50, 38, and 18 kDa were detected on native-polyacrylamide gel electrophoresis (native-PAGE) activity staining in the absence and presence of 25% NaCl. These results demonstrated that Virgibacillus sp. SK37 produced salt-activated extracellular proteinases. Virgibacillus sp. SK37 could be a promising strain for starter culture development used in fish sauce fermentation.
3. Converting trypsin to chymotrypsin: ground-state binding does not determine substrate specificity
L Hedstrom, S Farr-Jones, C A Kettner, W J Rutter Biochemistry. 1994 Jul 26;33(29):8764-9. doi: 10.1021/bi00195a018.
Rat trypsin II has been converted to a protease with chymotrypsin-like substrate specificity [Hedstrom, L., et al. (1994) Biochemistry (preceding paper in this issue)]. The key alteration in this conversion is the exchange of two surface loops for the analogous loops of chymotrypsin. k(inact)/Ki for the inactivation of chymotrypsin, trypsin, a trypsin mutant with poor activity (D189S), and the chymotrypsin-like mutants Tr-->Ch[S1+L1+L2] and Tr-->Ch[S1+L1+L2+Y172W] by Suc-Ala-Ala-Pro-Phe-chloromethylketone correlates with kcat/Km for hydrolysis of Suc-Ala-Ala-Pro-Phe-AMC. k(inact)'s for the inactivation of Tr-->Ch[S1+L1+L2] and Tr-->Ch[S1+L1+L2+Y172W] are comparable to that of chymotrypsin, while Ki's were much higher. Ki for the inhibition of these enzymes by the transition-state analog MeOSuc-Ala-Ala-Pro-boro-Phe also correlates with kcat/Km for hydrolysis of Suc-Ala-Ala-Pro-Phe-AMC. These results suggest that the surface loops stabilize the transition state for hydrolysis of chymotrypsin substrates by improving the orientation of bound substrates relative to the catalytic residues. Lastly, trypsin and chymotrypsin have comparable affinities for proflavin, while the Kd for the Tr-->Ch[S1+L1+L2+Y172W]-proflavin complex is 10-fold higher. No proflavin binding could be observed for either D189S or Tr-->Ch-[S1+L1+L2], which suggests that the S1 binding pockets of these two mutant enzymes are deformed. This work confirms that enzyme specificity is expressed in the chemical steps of the reaction rather than in substrate binding.
