Dilek Kazan

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

Newly isolated Bacillus clausii GMBAE 42: an alkaline protease producer capable to grow under higly alkaline conditions

Aims:  The isolation and identification of new Bacillus sp. capable of growing under highly alkaline conditions as alkaline protease producers.

Pyrolysis kinetics and thermal characteristics of microalgae Nannochloropsis oculata and Tetraselmis sp.

In this study non-isothermal thermogravimetric analysis were used to investigate pyrolysis behavior and kinetics of microalgae Nannochloropsis oculata (NO) and Tetraselmis sp. (TS). TG/DTG experiments at different heating rates were carried out. Heating rates had slight effect on the decomposition trend, however the maximum temperature and peak of weight loss rate in the DTG curves shifted towards higher temperature with the increase in heating rate. The average activation energy and pre-exponential factor for pyrolysis of NO and TS were estimated by distributed activation energy model. The highest activation energies were observed as 152.20 and 334kJ/mol for NO and TS, respectively, at various conversions. The pre-exponential factors for the corresponding activation energies were observed to be in the order of 10(8)-10(13) and 10(12)-10(25)s(-1) for NO and TS, respectively. Calculated kinetic parameters were used to predict devolatilization curves and results were in well agreement with experimental data.

Halomonas smyrnensis sp. nov., a moderately halophilic, exopolysaccharide-producing bacterium.

Four Gram-negative, moderately halophilic, exopolysaccharide-producing strains, designated AAD6(T), AAD4, AAD17 and AAD21, were isolated from Çamaltı Saltern Area, a wildlife reserve in Sasalı, İzmir province located in the Aegean Region of Turkey. The isolates grew at an optimum NaCl concentration of 10% (w/v). The major cellular fatty acids were C(16:0), C(18:1)ω7c, C(16:1)ω7c and C(12:0) 3OH, respectively and the predominant lipoquinone was ubiquinone Q-9. The G+C content of the genomic DNA of strains AAD6(T), AAD4, AAD17 and AAD21 was 63.0, 63.3, 62.8 and 62.6 mol %, respectively. Comparative 16S rRNA gene sequence studies showed that the isolates belonged to the genus Halomonas. The DNA-DNA hybridization mean values between the representative strain AAD6(T) and the closely related species Halomonas salina DSM 5928(T), Halomonas halophila DSM 4770(T), Halomonas maura DSM 13445(T), Halomonas organivorans DSM 16226(T), Halomonas elongata DSM 2581(T), Halomonas koreensis JCM 12237(T) and Halomonas nitroreducens LMG 24185, were 40.8, 39.6, 24.2, 23.3, 12.6, 14.5 and 12.2%, respectively. Based on these data the strains represent a novel species of the genus Halomonas for which the name Halomonas smyrnensis sp. nov. is proposed. The type strain is AAD6(T) (= DSM 21644(T) = JCM 15723(T)).

Stabilization of Escherichia coli penicillin G acylase against thermal inactivation by cross-linking with dextran dialdehyde polymers

Abstract The thermostabilization of penicillin G acylase (PGA) obtained from a mutant of Escherichia coli ATCC 11105 by cross-linking with dextran dialdehyde molecules, at a molecular mass of 11 500, 37 700 and 71 000 Da, was studied. The thermal inactivation mechanisms of the native and modified PGA were both considered to obey first-order inactivation kinetics during prolonged heat treatment, forming fully active but temperature-sensitive transient states. The highest enhancement to the thermostability of PGA was obtained using dextran-71000-dialdehyde modification, as a␣nearly ninefold increase at temperatures above 50 °C. The modification of PGA by dextran-11500-dialdehyde resulted in a considerable reduction of the Vm and Km parameters of the enzyme. However, other dextran dialdehyde derivatives used for modification did not cause a meaningful change in either Vm and Km. Modification by dextran dialdehyde derivatives did not result in significant change to either the optimal temperature or the activation energy of PGA. All modified PGA preparations showed lower inactivation rate constants but higher half-lives for inactivation than those of the native PGA at all temperatures studied. As indicated by the half-life times and ki values, dextran 71000-dialdehyde was found to be more effective at cross-linking in the thermo-stabilization of PGA than any other agent studied in this work.

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.

Stabilization and functional properties of Escherichia coli penicillin G acylase by covalent conjugation of anionic polysaccharide carboxymethylcellulose

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 Schiffs 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.

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.

Alkaline serine protease from halotolerantBacillus licheniformis BA17

An alkaline protease from halotolerantBacillus licheniformis BA17, isolated from Van Lake in Turkey, was purified 5.4 fold with 58% yield. The molecular weight was 19.7 kDa and the optimum temperature and pH were 60°C and 10, respectively. The half-life of the pure enzyme was 38 h, 93 min, 14 min and 6 min at 40, 50, 60 and 70°C, respectively. BA17 protease is very active at 30°C between pH 8.0 and 10. Enzyme activity increased in the presence of Cu+2, Mg+2, Mn+2 and K+1 ions. Enzyme retained activity with 5% SDS (w/v) and 1% Triton X-100 (v/v). Inhibition with PMSF and EDTA suggested that the enzyme is a serine protease and is a metal-activated enzyme. Based on the N-terminal sequence of the first 13 amino acids,B. licheniformis BA17 alkaline protease did not show identity to any of those from otherBacillus species.