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Alkaline proteases are one of the most important group of enzymes that are indispensable in a number of different industrial sectors. In this work, the effect of copper ions (Cu2+) was investigated for improving the thermostability and hydrolytic performance of Bacillus clausii GMBE 42 alkaline protease at different temperatures (45–65°C). Maximal residual activity was observed in the presence of 5 mM CuCl2. The enzyme was thermoinactivated according to first-order kinetics. A stabilization effect caused by copper ions was the result of a decrease in both autolysis and thermoinactivation rates. Thermodynamic analysis of the thermoinactivation process showed that Ea,i, ΔGi, and ΔHi values of the enzyme were higher in the presence of copper ions, but there was no measurable change in ΔSi values. These results show the thermostabilizing potential of copper ions on the enzyme. Lower Km values and higher kcat and kcat/Km values were obtained in the presence of copper ions, which is an indication of the nonessential activation of the enzyme by copper ions. Thermodynamic analysis of casein hydrolysis showed that in presence of copper ions Ea, ΔG≠, ΔH≠, , and values of enzyme were lower, but there was no change in ΔS≠ values. This is so far the first study that investigates the effect of cations on the basic catalytic and thermodynamic properties of an alkaline serine protease, which may be used to remove protein wastes from various industries such as food and leather processing.

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 stabilization of Escherichia coli penicillin G acylase (PGA) conjugated with carboxymethylcellulose (CMC) against temperature and pH was studied. The 2,3-dialdehyde derivative of CMC obtained by periodate oxidation was covalently conjugated to PGA via Schiff's base formation. The inactivation mechanism of both native and CMC-conjugated PGA appeared to obey first order inactivation kinetics during prolonged incubations at 40–60 °C and in the pH range 4–9. Inactivation rate constants of conjugated enzyme were always lower, and half-life times were always higher than that of native PGA. The activation free energy of inactivation (ΔGi values) of CMC-conjugated enzyme were found to be always higher than that of native PGA at all temperatures and pH values studied as another indicator of enzyme stabilization. Highest stability of CMC-conjugated enzyme was observed as nearly four-fold at 40 °C and pH 8.0. No changes were observed on the temperature and pH profiles of PGA after CMC conjugation. Lower Km and higher kcat values of PGA obtained after CMC conjugation indicates the improved effect of conjugation on the substrate affinity and catalytic performance of the enzyme.

The problem, whether excited-state energy transfer occurs between Trp residues in a multi-tryptophan proteins and if it does, what kind of changes it induces in different parameters of protein fluorescence, is currently under active investigation. In our previous paper [Biophys. Chem. 72 (1998) 265], the energy transfer was found and studied in detail for Na,K-ATPase. It was shown that this transfer influences all parameters of fluorescence emission, which is detected at site-selective conditions (red-edge of excitation, blue and red edges of emission). Present experiments were performed on unusually tryptophan-rich protein, bacterial penicillin acylase (28 Trp per dimer of 82 kDa) and were aimed to extend these observations. They demonstrate substantial heterogeneity in the environments of tryptophan residues within the protein structure. This suggests, that in the present case, if the energy transfer exists, it should be directed from short-wavelength-emitting to long-wavelength-emitting tryptophan residues and thus could be easily observed by a number of time-resolved and steady-state fluorescence techniques. Unexpectedly, no signature of inter-tryptophan energy transfer was found.

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.

The effects of polyhydric compounds (PHC) on the stabilization of penicillin G acylase (PGA) obtained from a mutant of Escherichia coli ATCC 11105 against pH were studied. Glucose and sucrose in I M concentration and polyethylene glycols (PEG) at a molecular weight of 400, 4000 and 15 000 in 250, 150 and 50 mM concentrations, respectively were used as PNC. The inactivation mechanisms of native and PHC containing PGA are considered to obey first order inactivation kinetics during prolonged incubation time interval of enzyme solutions at different pH values in 40 degrees C. PHC containing PGA preparations showed lower inactivation rate constants than native PGA preparations at the pH values studied. Lowest inactivation rates were obtained at pH 7.0. Inactivation rates were higher when the enzyme solutions were incubated at pH values above 7.0. Among the PHC compounds used, the sucrose, PEG 400 and PEG 4000 showed the highest enhancement to the pH stability of PGA. The addition of PHC into PGA. solution did not cause any change in the pH optimum of enzyme in penicillin G (Pen G) hydrolysis reaction. Copyright (C) 1996 Elsevier Science Ltd

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.

The effects of the 15 polyol compounds on the thermostability of penicillin G acylase (PGA) from a mutant of Escherichia coli ATCC 11105 were investigated. PGA solutions containing glucose, sucrose, mannose, adonitol, sorbitol or xylitol at 1 m concentration showed an initial activity rate (IAR) higher than 3. The IAR values of these six sugars at 50°C increased with their concentrations in PGA solution. The investigations on half-life times and stabilization factor (SF) values of PGA at 45 and 50°C, in the presence of these compounds, showed a tendency for the half-life and SF of the enzyme, at 50°C, to increase with the OH/C ratios of polyol compounds. Sucrose and glucose were the only two compounds having higher SF values at 50°C than at 45°C. Sucrose showed almost twice the half-life of glucose and was selected as the best stabiliser of PGA activity against thermal inactivation. PGA showed almost the state of pH and temperature activity profiles in both presence and absence of sucrose. Km and Vm values decreased with the increasing sucrose concentrations, with a greater decrease in Km values.

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The hydrolysis of cephalosporin G to 7-amino-3-deacetoxy cephalosporanic acid (7-ADCA) by free and immobilized penicillin G acylase (PGA) was studied in a mutant of Escherichia coli ATCC 11105. V m and K m values of soluble enzyme (specific activity: 25·0 U mg −1 , protein concentration: 0·4 mg ml −1 ) were 15·92 U ml −1 min −1 and 2·94 m m cephalosporin G (Cep G) respectively at pH 8·0 and 40°C. Optimal pH and temperature values of Cep G hydrolysis by the soluble enzyme were 8·0 and 55°C. The K m value of immobilized enzyme obtained by covalent attachment on oxirane acrylic beads (specific activity: 120 U g −1 dry matrix at pH 8·0 and 40°C, bound protein: 12·8 mg) was 3·32±0·06 m M Cep G. The immobilized enzyme showed highest activity at pH 8·0 and at 50°C. A 95% conversion rate to 7-ADCA was observed in 40 min at 40°C and pH 8·0 with 1·0% (w/v) Cep G. Excess Cep G, PAA and 7-ADCA were uncompetitive, competitive and non-competitive inhibitors respectively for both soluble and immobilized PGA. The activation energy of Cep G hydrolysis was calculated to be 7·83 kcal mol −1 for soluble PGA and 5·25 kcal mol −1 for immobilized PGA.

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.

The effects of five polyethylene glycol (PEG) compounds of different molecular weight on the thermal stability of penicillin G acylase (PGA) obtained from a mutant of Escherichia coli ATCC 11105 have been investigated. The molecular weights of PEG compounds were 400, 4000, 6000, 10,000, and 15,000. The thermal inactivation mechanisms of both native and PEG-containing PGA were considered to obey first order inactivation kinetics during prolonged heat treatments. Optimal concentrations of PEGs at molecular weights of 400, 4000, 6000, 10,000, and 15,000 were found to be 250, 150, 150, 100, and 50 mM, respectively. The greatest enhancement of thermostability was observed with PEG 4000 and PEG 6000, as a nearly 20-fold increase above 50 degrees C. PGA showed almost the same temperature activity profile and optimal temperature values both in the presence and absence of PEG. The addition of PEGs did not cause any change in the optimal temperature value of PGA, but the parameters V-m, K-m, the activation energy, and the k(cat) values of enzyme were markedly decreased because of the mixed inhibition by PEG compounds. The type of inhibition was found to be hyperbolic uncompetitive.

The methods and Stabilisation methods for thermal inactivation of enzymes are classified as 1. Screening of micro-organisms for enzymes with enhanced intrinsic stability, 2. Addition of stabilising agents, 3. Chemical modification of enzyme molecules, 4. Immobilisation of enzyme molecules. The criteria for the stabilization of biocatalytic proteins against irreversible inactivation are described with numerous applications

The effect of pHs between 2·0 and 10·0 on the inactivation kinetics of penicillin G acylase (PGA) obtained from a mutant of Escherichia coli ATCC 11105 and the stabilization of enzyme against pH by chemical cross-linking with dimethyladipimidate (DMA) were studied. The inactivation mechanisms of native and DMA cross-linked PGA both appeared to obey first-order inactivation kinetics during prolonged incubation of enzyme solutions at 40°C and at different pH values. The lowest inactivation rate constants were obtained with both native and DMA cross-linked PGA at pH 7·0. Inactivation rate constants decreased with an increase in pH from 2·0 to 7·0 and then increased again at pH values above 7·0. Chemical cross-linking of PGA by DMA resulted in the stabilization of enzyme against pH. Highest enhancement of pH stabilization (nearly four-fold) was obtained at pH 7·0 and 8·0.

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