Purification and Characterization of Serine Proteinase 2 from Bacillus intermedius 3-19

Abstract

A proteinase secreted in the late stationary phase was isolated from the culture fluid of Bacillus intermedius 3-19 by ion-exchange chromatography on CM-cellulose followed by FPLC on a Mono S column. The enzyme was completely inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. The maximum proteolytic activity against the synthetic chromogenic substrate Z-Ala-Ala-Leu-pNA was observed at pH 9.0. The molecular weight of the enzyme is 28 kD and its isoelectric point is 9.2. We have also determined pH- and thermostability and Km and kcat of this proteinase. The enzyme has been classified as a thiol-dependent serine proteinase. N-Terminal amino acid sequence (10 residues) and amino acid composition of the protein were also determined. By the mode of hydrolysis of peptide bonds in the oxidized B-chain of insulin, this enzyme is similar to the thiol-dependent serine proteinase 1 from B. intermedius 3-19 secreted during vegetative growth.

Subtilisin-like proteinase secreted by the Bacillus pumilus KMM 62 strain at different growth stages

“…5). The detected activity pH optima for B. pumilus proteinases were close to those of B. intermedius [16] and B. amylolique faciens [17] proteinases. Interestingly, the pH opti mum for the classical subtilisins such as BPN' and Carlsberg is shifted to the alkali range and measures 10.5, but BPN' is stable in the range of pH 7-8 [18].…”

An extremely thermotolerant, alkaliphilic subtilisin-like protease from hyperthermophilic Bacillus sp. MLA64

“…The relative activities at pH 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0 were about 77, 84, 93, 88, 80 and 75%, respectively. The optimum pH for this protease was found to be around 9.5 which is greater than subtilisin from G. stearothermophilus and Bacillus intermedius, pH 9, Alkalimonas collagenimarina, pH 8.5, B. licheniformis, pH 8, and B. subtilis, pH 7.5 [32][33][34][35], but lower than those reported for Bacillus sp. DJ-4, pH 10, Rhizopus microspores, pH 10.5, and Bacillus clausii GMBAE42, pH 11.3 [36][37][38].…”

“…Other subtilisin-like proteases from Bacillus species are stable in a narrow range of pH. For example, B. subtilis, B.intermedius and B.clausii are stable in pH from 5.5 to 9, 6.3 to 9.5 and 9 to 12, respectively [32,34,38].…”

Purification of a subtilisin-like serine proteinase from recombinantBacillus subtilis during different phases of growth

“…The Gram-positive spore-forming bacterium Bacillus intermedius secretes subtilisin-like proteinase into the culture medium (Balaban et al, 2004). The enzyme appears in culture broth when the culture shows an overall slow-down of the growth kinetics, with maximum levels of the enzyme activity appearing at the 24th and 48th hour of growth (Balaban et al, 2004).…”

“…The enzyme appears in culture broth when the culture shows an overall slow-down of the growth kinetics, with maximum levels of the enzyme activity appearing at the 24th and 48th hour of growth (Balaban et al, 2004). Two fractions of enzyme produced by the bacteria in the early and late stationary phases were products of the single gene coding for B. intermedius extracellular serine subtilisin-like proteinase (AprBi), which was cloned and its complete nucleotide sequence was determined (GeneBank accession number AY754946) (Sharipova et al, 2008).…”