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Proteases are largely employed in biocatalysis. In order to increase the number of their applications it is useful to shed light on the reasons that cause a non-optimal activity of these enzymes when used in inactivating experimental conditions (e.g., in the presence of co-solvent to favor substrate dissolution). To this end the effect of different mono alcohols on the activity and the conformation of alkaline protease from Bacillus clausii GMBAE 42 was investigated. We found that the enzyme in the presence of 20–25% of methanol, ethanol, 1-propanol or 2-propanol halves its activity. At the concentration of 10%, all the alcohols caused a slightly more intense far-UV (CD) circular dichroism signal of the protease at around 208 and 220 nm with respect to the protein in only buffer, which suggests an increase of helicity in the secondary structure of the protein. Monitoring the shift of the fluorescence emission of the protease with respect to that of the standard N -acetyl- l -tryptophan-ethyl ester, we suggest that with all the alcohols tested the decrease of activity might be due to the loss of tertiary structure (even though at a lower extents in methanol and ethanol compared to 1-propanol and 2-propanol).

An alkali tolerant Bacillus strain having extracellular serine alkaline protease activity was newly isolated from compost and identified as Bacillus clausii GMBE 22. An alkaline protease (AP22) was 4.66-fold purified in 51.5% yield from Bacillus clausii GMBE 22 by ethanol precipitation and DEAE-cellulose anion exchange chromatography. The purified enzyme was identified as serine protease by LC-ESI-MS analysis. Its complete inhibition by phenylmethanesulfonylfluoride (PMSF) also justified that it is a serine alkaline protease. The molecular weight of the enzyme is 25.4 kDa. Optimal temperature and pH values are 60 degrees C and 12.0, respectively. The enzyme showed highest specificity to N-Suc-Ala-Ala-Pro-Phe-pNA. The K(m) and k(cat) values for hydrolysis of this substrate are 0.347 mM and 1141 min(-1) respectively. The enzyme was affected by surface active agents to varying extents. The enzyme is stable for 2 h at 30 degrees C and pH 10.5. AP22 is also stable for 5 days over the pH range 9.0-11.0 at room temperature. AP22 has good pH stability compared with the alkaline proteases belonging to other strains of Bacillus clausii reported in the literature.

Thermal inactivation kinetics of native and glutaraldehyde cross-linked forms of penicillin G acylase obtained from a mutant derivative of Escherichia coli ATCC 11105 were studied. Apparent activation energies for thermal inactivation of both native and cross-linked forms of enzyme were calculated to be [57.71 +/- 8.46] and [67.11 +/- 13.83] kcal mol-1 respectively. This slight increase in activation energy suggested that glutaraldehyde cross-linking did not markedly protect against thermal activation. Cross-linked enzyme did, however, have a significantly improved half-life at temperatures between 40 degrees C and 50 degrees C.

A mutant strain with high penicillin G acylase activity was derived from Escherichia coli ATCC 11105 by chemical mutagenesis, using N-methyl-N′-nitro-N-nitrosoguanidine. Penicillin acylase was extracted from the mutant strain and highly purified by DEAE-cellulose and hydroxyapatite column chromatography followed by preliminary precipitation steps. Vm and Km values of the enzyme (specific activity: 24·81 U mg−1, protein concentration: 0.56 mg cm−3) were found to be 22.73 U cm−3 mm−1 and 3.18 mmold m−3 penicillin G, respectively. The enzyme was shown to be uncompetitively inhibited by excess of substrate. The inhibition by phenylacetic acid was found to be competitive, and 6-aminopenicillanic acid to be noncompetitive. Inhibition constants for excess of penicillin G, phenylacetic acid and 6-aminopenicillanic acid were estimated as 74.20, 18.74 and 15.00 mmol dm−3 respectively, at pH 8.0 and 40°C. The activation energy of the enzymatic hydrolysis reaction of penicillin G was found to be 10.78 kcal mol−1. Optimal pH and temperature values of the enzyme were determined as 8.0 and 60°C. Complete loss of the enzyme stability occurred when the enzyme was incubated at pH 10.0 for 2 days or at 50°C for 2 h.

An extracellular serine alkaline protease of Bacillus clausii GMBAE 42 was produced in protein-rich medium in shake-flask cultures for 3 days at pH 10.5 and 37 degrees C. Highest alkaline protease activity was observed in the late stationary phase of cell cultivation. The enzyme was purified 16-fold from culture filtrate by DEAE-cellulose chromatography followed by (NH(4))(2)SO(4) precipitation, with a yield of 58%. SDS-PAGE analysis revealed the molecular weight of the enzyme to be 26.50 kDa. The optimum temperature for enzyme activity was 60 degrees C; however, it is shifted to 70 degrees C after addition of 5 mM Ca(2+) ions. The enzyme was stable between 30 and 40 degrees C for 2 h at pH 10.5; only 14% activity loss was observed at 50 degrees C. The optimal pH of the enzyme was 11.3. The enzyme was also stable in the pH 9.0--12.2 range for 24 h at 30 degrees C; however, activity losses of 38% and 76% were observed at pH values of 12.7 and 13.0, respectively. The activation energy of Hammarsten casein hydrolysis by the purified enzyme was 10.59 kcal mol(-1) (44.30 kJ mol(-1)). The enzyme was stable in the presence of the 1% (w/v) Tween-20, Tween-40,Tween-60, Tween-80, and 0.2% (w/v) SDS for 1 h at 30 degrees C and pH 10.5. Only 10% activity loss was observed with 1% sodium perborate under the same conditions. The enzyme was not inhibited by iodoacetate, ethylacetimidate, phenylglyoxal, iodoacetimidate, n-ethylmaleimidate, n-bromosuccinimide, diethylpyrocarbonate or n-ethyl-5-phenyl-iso-xazolium-3'-sulfonate. Its complete inhibition by phenylmethanesulfonylfluoride and relatively high k (cat) value for N-Suc-Ala-Ala-Pro-Phe-pNA hydrolysis indicates that the enzyme is a chymotrypsin-like serine protease. K (m) and k (cat) values were estimated at 0.655 microM N-Suc-Ala-Ala-Pro-Phe-pNA and 4.21 x 10(3) min(-1), respectively.

The stabilisation of Escherichia coli penicillin G acylase (PGA) by dextran polymers (of molecular weight 11.5, 37.7 and 71kDa) was studied. The inactivation of both the native and dextran-containing enzyme preparations obeyed first-order kinetics at the temperature and pH values studied. The optimal concentrations of dextran polymers of molecular weight 11.5, 37. 7 and 71 kDa stabilising PGA against inactivation were 50, 20 and 7.5 mmol dm -3 respectively. Dextran 11500 (11.5kDa) gave 100-fold protection of PGA against thermal inactivation of enzyme above 50 °C. The kinetic constants of the enzyme were slightly altered, but temperature and pH profiles were not altered by the dextrans.

Crosslinked poly(N-isopropylacrylamide) (PNIPA) gels with different crosslink densities in the form of rods and beads were prepared by free-radical crosslink- ing copolymerization. Solution and inverse suspension polymerization techniques were used for the gel synthesis. The gels were utilized to concentrate dilute aqueous solutions of penicillin G acylase (PGA), bovine serum albumin (BSA), and 6-aminopenicillanic acid (6-APA). The discontinuous volume transition at 347C observed in the gel swelling was used as the basis of concentrating dilute aqueous protein solutions. PNIPA gels formed below 187C were homogeneous, whereas those formed at higher temperatures exhibited heterogeneous structures. The water absorption capacity of PNIPA gels in the form of beads was much higher, and their rate of swelling was much faster than the rod-shaped PNIPA gels. It was also found that the polymerization techniques used significantly affect the properties of PNIPA gels. The separation efficiency decreased when the protein molecules PGA or BSA in the external solution were replaced with small-molecular-weight compounds, such as 6-APA. The protein separation efficiency by the gel beads increased to 100% after coating the bead surfaces with BSA. q 1998

The inactivation kinetics of Escherichia coli penicillin G acylase (PGA), and cross-linked stabilization of the enzyme by dextran-dialdehyde derivatives of molecular weights of 11500, 37000 and 71000, were similar from pH 2 to pH 10. Inactivation of the native and modiÞed PGA obeyed Þrst order kinetics. The lowest inactivation rate constants for native and dextran-11500-dialdehyde modiÞed PGA were 9.0 3 10 Ð4 and 1.5 3 10 Ð4 min Ð1 respectively at pH 7.0. The highest pH stabilization (nearly ten-fold) was obtained at pH 7.0.

In this study, our investigations showed that the increasing concentrations of all examined mono alcohols caused a decrease in the V m, k cat and k cat/K m values of Bacillus clausii GMBE 42 serine alkaline protease for casein hydrolysis. However, the K m value of the enzyme remained almost the same, which was an indicator of non-competitive inhibition. Whereas inhibition by methanol was partial non-competitive, inhibition by the rest of the alcohols tested was simple non-competitive. The inhibition constants (K I) were in the range of 1.32–3.10 M, and the order of the inhibitory effect was 1-propanol>2-propanol>methanol>ethanol. The ΔG ≠ and ΔG ≠ E − T values of the enzyme increased at increasing concentrations of all alcohols examined, but the ΔG ≠ ES value of the enzyme remained almost the same. The constant K m and ΔG ≠ ES values in the presence and absence of mono alcohols indicated the existence of different binding sites for mono alcohols and casein on enzyme the molecule. The k cat of the enzyme decreased linearly by increasing log P and decreasing dielectric constant (D) values, but the ΔG ≠ and ΔG ≠ E − T values of the enzyme increased by increasing log P and decreasing D values of the reaction medium containing mono alcohols.

We investigated the effects of three different bisimidoesters as homobifunctional cross-linking agents on the thermostabilization of penicillin G acylase (PGA) obtained from a mutant of Escherichia coli ATCC 11105. Cross-linkers were dimethyladipimidate (DMA), dimethylsuberimidate (DMS), and dimethyl-3,3′-dithiobispropionimidate (DTBP). The thermal inactivation mechanisms of the native and cross-linked PGA were both considered to obey first-order inactivation kinetics during prolonged heat treatment, forming fully active, susceptible transient states. The efficacy of the cross-linkers on the thermostabilization of PGA was estimated to be DMA > DMS > DTBP. Optimal concentrations of DMA, DMS, and DTBP for cross-linking of PGA were found to be 0.5, 0.4, and 0.3% (w/v), respectively. The greatest enhancement of the thermostabilities was observed during DMA cross-linking, as a nearly 15-fold increase at temperatures above 50°C. Cross-linking by DMA did not cause much change in the parameters Vm, Km, and the optimal temperature values of PGA, but the activation energy of the enzyme was slightly decreased and kcat value slightly increased after cross-linking.

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The competitive inhibition of pH on the kinetics of penicillin G (Pen G) hydrolysis by penicillin G acylase (PGA) obtained from a mutant derivative of Escherichia coli ATCC 11105 was investigated. The effect of pH on the initial reaction rate was interpreted according to a mechanism discussed by Tippon and Dixon. Molecular dissociation constants pK E B and pK ES B of PGA were calculated to be 9·783 and 10·851 respectively.

A Gram-stain-positive, obligately alkaliphilic bacterium designated strain GMBE 72T was isolated from mushroom compost from Yalova, located in the Marmara region of Turkey. Cells were aerobic, straight rods and they formed subterminal to terminal ellipsoidal endospores. The isolate was catalase-positive, oxidase-negative and motile and contained a type A1γ peptidoglycan based on meso-diaminopimelic acid. The strain grew at pH 8.0–12.5. The major cellular fatty acid was anteiso-C15 : 0. The genomic DNA G+C content was 40.2 mol%. Phylogenetic analyses based on 16S rRNA gene sequencing showed that strain GMBE 72T belonged to the genus Bacillus and exhibited 98.2 % sequence similarity to Bacillus pseudofirmus DSM 8715T. DNA–DNA reassociation was 56 % between GMBE 72T and B. pseudofirmus DSM 8715T. According to our polyphasic characterization, strain GMBE 72T represents a novel species of the genus Bacillus, for which the name Bacillus marmarensis sp. nov. is proposed. The type strain is GMBE 72T (=DSM 21297T =JCM 15719T).

A . A . D E N I Z C I , D . K A Z A N , E . C . A . A B E L N A N D A . E R A R S L A N . 2003. Aims: The isolation and identification of new Bacillus sp. capable of growing under highly alkaline conditions as alkaline protease producers. Methods and Results: A Bacillus strain capable of growing under highly alkaline conditions was isolated from compost. The strain is a Gram-positive, spore-forming, motile, aerobic, catalase- and oxidase-positive, alkaliphilic bacterium and designated as GMBAE 42. Good growth of the strain was observed at pH 10. The strain was identified as Bacillus clausii according to the physiological properties, cellular fatty acid composition, G + C content of genomic DNA and 16S rRNA gene sequence analyses. The result of 16S rRNA sequence analyses placed this bacterium in a cluster with B. clausii. The G + C content of the genomic DNA of the isolate GMBAE 42 was found to be 49 mol%. The crude extracellular alkaline protease produced by the isolate showed maximal activity at pH 11AE0 and 60� C. Conclusions: The results suggest that isolated strain GMBAE 42 is a new type of B. clausii capable of growing at pH 10AE0 and produce extracellular alkaline protease very active at pH 11AE0. Significance and Impact of the Study: Isolated strain could be used in commercial alkaline protease production and its enzyme can be considered as a candidate as an additive for commercial detergents.

The chemical modification of penicillin G acylase (PGA) obtained from a mutant of Escherichia coli ATCC 11105 was studied in order to identify the catalytically essential amino acid residues of the enzyme. The modification of PGA by serine specific phenylmethylsulphonylfluoride (PMSF) and tryptophan specific N-bromosuccinimide (NBS) resulted in the complete inactivation of the enzyme. Modification by arginine specific phenylglyoxal and lysine specific ethylacetimidate caused the partial inactivation of the enzyme and partial protection of enzyme activity was observed when modifications were carried out in the presence of the strong competitive inhibitor penicillin G sulphoxide (PenGSO). Enzyme activity was not affected by the modification of cysteine specific ethylmaleiimide and histidine specific reagents tosyl-L-lysine-chloromethyl ketone (TLCK) and tosyl-L-phenylalanine-chloromethyl ketone (TPCK). The inactivation of PGA by amino acid specific modifying reagents obeyed first-order kinetics. The kinetic constants (V-m, K-m and k(cat) values) and pH-activity profiles of native and modified PGA preparations were also investigated. The results of these investigations showed that the active site of the enzyme contained a catalytically essential serine residue. It may be argued that the modification of arginine residues induces the conformational change of the entire enzyme molecule. These residues are located at a site other than the active site of the enzyme and are not essential for catalysis. The modification of lysine residues induces conformational changes in the active site of the enzyme but these residues can contribute neither catalysis nor substrate binding. (C) 2001 Elsevier Science Ltd. All rights reserved.