Proteases of the genus <i>Bacillus</i>. II. Alkaline proteases

Keay, Leonard; Moser, Patricia W.; Wildi, Bernard S.

doi:10.1002/bit.260120206

Cited by 67 publications

(29 citation statements)

References 30 publications

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“…Calcium was added to stabilize the enzymes (Keay & Wildi, 1970;Keay, Moser & Wildi, 1970;Moseley & Keay, 1970). EDTA was added to prevent salt precipitation at the fermentation pH.…”

Section: F G H E I N E K E N and R J O'connormentioning

confidence: 99%

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Continuous Culture Studies on the Biosynthesis of Alkaline Protease, Neutral Protease and -Amylase by Bacillus subtilis NRRL-B3411

Heineken

O’Connor

1972

Journal of General Microbiology

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The amounts of three catabolite repressible enzymes, alkaline protease, neutral protease and a-amylase, produced by Bacillus subtilis NRRL-~341 I growing in a chemostat, depended on the growth-limiting substrate. Limiting growth with glucose was advantageous for a-amylase synthesis while nitrogen-limited growth was advantageous for synthesis of the two proteases. Under the conditions used, continuous cultures were unsuitable for large-scale production of the three enzymes since spontaneous mutations to less productive strains occurred in the long term. N T R O D U C T I O NContinuous culture studies were initiated by us for two purposes. The first was to develop a better understanding of the regulatory mechanisms involved in the biosynthesis of alkaline protease, neutral protease and a-amylase by Bacillus subtilis NRRL-~341 I. Such information is fundamental to understanding how to alter these mechanisms in order to provide a desired type of micro-organism. The second was to investigate the possibility of synthesizing these enzymes in a large-scale continuous culture. Decided investment advantages (Ellsworth, Telling & East, 1959) of continuous cultures are discussed in the literature as well as some disadvantages (Butterworth, I 967).A number of publications have appeared in recent years on the synthesis of exoenzymes, many of which are reviewed by Schaeffer (1969). Coleman (1967) reported that amylase and proteases of Bacillus subtilis are synthesized in the stationary phase of growth and appear simultaneously and in parallel fashion. Mandelstam (1967) postulates the concept of a catabolite repressing only the enzymes directly or indirectly producing it. We believe we have data which will help to clarify these concepts even further.

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“…Calcium was added to stabilize the enzymes (Keay & Wildi, 1970;Keay, Moser & Wildi, 1970;Moseley & Keay, 1970). EDTA was added to prevent salt precipitation at the fermentation pH.…”

Section: F G H E I N E K E N and R J O'connormentioning

confidence: 99%

“…Most of the caseinase activity measurements were carried out on a Technicon autoanalyser with reagents as specified for the standard assay (Keay & Wildi, 1970). Extinction was measured at 660 nm after treatment with Folin phenol reagent.…”

Section: F G H E I N E K E N and R J O'connormentioning

confidence: 99%

Continuous Culture Studies on the Biosynthesis of Alkaline Protease, Neutral Protease and -Amylase by Bacillus subtilis NRRL-B3411

Heineken

O’Connor

1972

Journal of General Microbiology

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“…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.…”

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confidence: 99%

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

Durham¹,

Dj²,

Stellwag³

1987

J Bacteriol

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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...

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“…The bacterial suspension was centrifuged and the supernatant was collected for Protease assay (Keay et al, 1970). Protease activity was measured in the presence and absence of diclofenac using a casein substrate; 1 ml of the culture supernatant was mixed with 1 ml 0.05 M phosphate buffer-0.1 M NaOH (pH 7.0) containing 2% casein, and incubated for 10 min at 37°C.…”

Section: Protease Assaymentioning

confidence: 99%

Diclofenac inhibits virulence of Proteus mirabilis isolated from diabetic foot ulcer

Hegazy¹

2016

Afr. J. Microbiol. Res.

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Diclofenac is an analgesic and anti-inflammatory drug, used to relief the secondary complications of diabetes. Wound infections are much more serious particularly in diabetic patients. Proteus mirabilis are Gram negative rods, show a wide range of pathogenesis based on arsenal of diverse virulence factors. Recently, P. mirabilis and other Gram negative rods were isolated from diabetic foot ulcers; moreover, these isolates showed an increase resistance and more aggressive virulence behavior. The promising approaches to overcome such kind of infections include the improvement of patient's immunity and/or challenging the bacterial virulence. Diclofenac is used frequently by diabetic patients, and it showed antimicrobial activity. This study was conducted to screen the effect of diclofenac on the virulence of P. mirabilis isolated from diabetic foot. Interestingly, diclofenac significantly inhibited or decreased the P. mirabilis virulence which indicates its additional beneficial use in diabetic foot patients.

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Proteases of the genus Bacillus. II. Alkaline proteases

Cited by 67 publications

References 30 publications

Continuous Culture Studies on the Biosynthesis of Alkaline Protease, Neutral Protease and -Amylase by Bacillus subtilis NRRL-B3411

Continuous Culture Studies on the Biosynthesis of Alkaline Protease, Neutral Protease and -Amylase by Bacillus subtilis NRRL-B3411

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

Diclofenac inhibits virulence of Proteus mirabilis isolated from diabetic foot ulcer

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