Altan Erarslan

Structure and Protein Separation Efficiency of Poly(N-isopropylacrylamide) Gels: Effect of Synthesis Conditions

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

Bacillus marmarensis sp. nov., an alkaliphilic, protease-producing bacterium isolated from mushroom compost

A Gram-stain-positive, obligately alkaliphilic bacterium designated strain GMBE 72(T) 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 A1gamma peptidoglycan based on meso-diaminopimelic acid. The strain grew at pH 8.0-12.5. The major cellular fatty acid was anteiso-C(15 : 0). The genomic DNA G+C content was 40.2 mol%. Phylogenetic analyses based on 16S rRNA gene sequencing showed that strain GMBE 72(T) belonged to the genus Bacillus and exhibited 98.2 % sequence similarity to Bacillus pseudofirmus DSM 8715(T). DNA-DNA reassociation was 56 % between GMBE 72(T) and B. pseudofirmus DSM 8715(T). According to our polyphasic characterization, strain GMBE 72(T) represents a novel species of the genus Bacillus, for which the name Bacillus marmarensis sp. nov. is proposed. The type strain is GMBE 72(T) (=DSM 21297(T) =JCM 15719(T)).

Stabilization ofEscherichia coli penicillin G acylase by polyethylene glycols against thermal inactivation

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 ofEscherichia 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°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 parametersVm,Km, the activation energy, and thekcat 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 effect of polyol compounds on the thermostability of penicillin G acylase from a mutant of Escherichia coli ATCC 11105

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.

Stabilization of penicillin G acylase against pH by chemical cross-linking

Abstract 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.

Thermostabilization of penicillin G acylase obtained from a mutant of Escherichia coli ATCC 11105 by bisimidoesters as homobifunctional cross-linking agents

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 hydrolysis of cephalosporin G by free and immobilized penicillin G acylsse from a mutant of Escherichia coli ATCC 11105

Abstract 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.

Cross-linked stabilization of Escherichia coli penicillin G acylase against pH by dextran-dialdehyde polymers

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 modified PGA obeyed first order kinetics. The lowest inactivation rate constants for native and dextran-11500-dialdehyde modified PGA were 9.0310 and 1.5310 min respectively at pH 7.0. The highest pH stabilization (nearly ten-fold) was obtained at pH 7.0.

Studies on Alkaline Serine Protease Produced by Bacillus clausii GMBE 22

Abstract 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°C and 12.0, respectively. The enzyme showed highest specificity to N-Suc-Ala-Ala-Pro-Phe-pNA. The Km and kcat 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°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.

Effect of dextran polymers on the stability of soluble Escherichia coli penicillin G acylase

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.