Novel alkaline- and heat-stable serine proteases from alkalophilic Bacillus sp. strain GX6638

Abstract

An alkalophilic Bacillus sp., strain GX6638 (ATCC 5327O), was isolated from soil and shown to produce a minimum of three alkaline proteases. The proteases were purified by ion-exchange chromatography and were distinguishable by their isoelectric point, molecular weight, and electrophoretic mobility. Two of the proteases, AS and HS, which exhibited the greatest alkaline and thermal stability, were characterized further. Protease HS had an apparent molecular weight of 36,000 and an isoelectric point of -4.2, whereas protease AS had a molecular weight of 27,500 and an isoelectric point of 5.2. ]Both enzymes had optimal proteolytic activities over a broad pH range (pH 8 to 12) and exhibited temperature optima of 65C. Proteases HS and AS were further distinguished by their proteolytic activities, esterolytic activities, sensitivity to inhibitors, and their alkaline and thermal stability properties. Protease AS was extremely alkali stable, retaining 88% of initial activity it pH 12 over a 24-h incubation period at 25°C; protease HS exhibited similar alkaline stability properties ¢o pH 11. In addition, protease HS had exceptional thermal stability properties. At pH 9.5 (0.1 M CAPS buffer, 5 mM EDTA), the enzyme had a half-life of more than 200 min at 50°C and 25 min at 60°C. At pH above 9.5, protease HS readily lost enzymrtic activity even in the presence of exogenously supplied Ca2+. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60°C. The data presented here clearly indicate that these two alkaline proteases from Bacillus sp. strain GX6638 represent novel proteases that differ fundamentally from the proteases previously described for members of the genus Bacillus.Alkaline proteases secreted by both neutralophilic and alkalophilic bacilli are of interest because they represent a major source of commercially produced proteolytic enzymes (1; 11, 16). These proteases exhibit optimal activity at pHs of 9 to 11 and are inactivated by serine active-site inhibitors, such as phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate. In general, these enzymes have molecular weights ranging from 20,000 to 30,000, are stabilized by Ca2+, and have characteristically high isoelectric points (2,11,12,16,19,23). Alkaline proteases, or subtilisins, secreted by neutralophilic Bacillus spp. are stable from pH 5 to 10 at low temperatures, but are readily inactivated at higher temperatures and alkalinities in the absence of Ca2+ (1, 2). The subtilisins have been divided into two groups based on differences in amino acid composition and immunological and kinetic properties (12). Recent results, however, suggest that these proteases possess identical amino acid residues in 63% of their primary sequences (17). Alkaline proteases from alkalophilic bacteria have been studied in less detail, yet many similarities exist between subtilisins and alkaline proteases from alkalophilic bacilli (11). However, serine proteases from alkalophiles have superior alkaline...

Characterization of detergent stable and feather degrading serine proteases from Bacillus mojavensis A21

“…Ca 2+ has been shown to increase the activity and thermal stability of alkaline proteases at higher temperatures [33][34][35]. The addition of CaCl 2 at 2 and 5 mM resulted in a shift in the apparent optimal temperature from 60 to 65 • C. A similar result was reported by Ghorbel et al [22] for the protease produced by B. cereus BG1.…”

“…For example the half-life was increased by 2.3-fold at 60 • C, 10-fold at 60 • C and 2.5-fold at 50 • C by adding Ca 2+ for alkaline proteases from B. mojavensis, Bacillus spp. GX6638 and B. sphaericus, respectively [26,33,38].…”

Production of Keratinase by Free and Immobilized Cells of Bacillus halodurans Strain PPKS-2: Partial Characterization and Its Application in Feather Degradation and Dehairing of the Goat Skin

“…In general, detergent-compatible enzymes are alkaline thermostable in nature with a high pH optima because the pH of the laundry detergent is generally in the range of 9.0-11.0 and varying thermostability at laundry temperatures (50 to 60°C) [33]. However, the protease from the subtilisin Carlsberg, used in commercial detergents, has a half-life of 2.5 min at 60°C [34]. Besides pH and temperature stability, bleach stability is important because bleach-stable enzymes are not generally available except for a few reports [33,35,36].…”

Thermodynamics and Kinetics of Heat Inactivation of a Novel Keratinase from Chryseobacterium sp. Strain kr6

“…For example, the half-life was increased 2.5 fold at 50°C, and 10-fold at 60°C by adding Ca 2+ to the alkaline proteases from Bacillus sp. GX6638 and B. sphaericus, respectively [25,26]. The addition of CaCl 2 increases the half-life of an alkaline protease of Bacillus mojavensis from 15 min to 57 min, showing the importance of this ion to increase the thermo resistance [11].…”