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. 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 11.0 and 60 degrees C. CONCLUSIONS: The results suggest that isolated strain GMBAE 42 is a new type of B. clausii capable of growing at pH 10.0 and produce extracellular alkaline protease very active at pH 11.0. 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.

On the excited-state energy transfer between tryptophan residues in proteins: the case of penicillin acylase.

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.

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

The thermostabilization of penicillin G acylase (PGA) obtained from a mutant of Escherichia coli ATCC 11,105 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 degrees 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.

Stabilization of Escherichia 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 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 heart 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 Vm, K(m), the activation energy, and the Kcat values of enzyme were markedly decreased because of the mixed inhibition by PEG compounds. The type of inhibition was found to be hyperbolic uncompetitive.

Thermal inactivation kinetics of penicillin G acylase obtained from a mutant derivative of Escherichia coli ATCC 11105.

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.

Kinetic investigation of penicillin G acylase from a mutant strain of Escherichia coli ATCC 11105 immobilized on oxirane-acrylic beads.

Highly purified penicillin G acylase from a mutant derivative of Escherichia coli ATCC 11105 was immobilized on oxirane-acrylic beads by covalent binding via oxirane groups. The highest specific activity, (322 U g-1 dry matrix at 40 degrees C and at pH 8.0) was obtained by using an enzyme solution having 13.8 U mg-1 specific activity and 72.73 mg total protein. The efficiency of immobilization was 95.8%. Kinetic parameters of immobilized penicillin G acylase were determined at the same pH and temperature by a preparation having 8.1 mg bound protein. The Km value and substrate inhibition constant of the enzyme were found to be 11.36 mmol dm-3 and 680 mmol dm-3 penicillin G respectively. Phenylacetic acid and 6-aminopenicillanic acid were estimated as the competitive and non-competitive inhibitors of the enzyme and their inhibition constants were found to be 90 mmol dm-3 phenylacetic acid and 76.1 mmol dm-3 for 6-aminopenicillanic acid. The activation energy of the hydrolytic reaction was calculated to be 7.75 kcal mol-1. The immobilized enzyme showed highest activity at pH 8.0 and at 55 degrees C. The enzyme was stable when incubated at 4 degrees C for one day at a pH range of 5.0 to 9.0. Thermal stability (over 30 min) was observed up to 40 degrees C but decreased at higher temperatures and was almost absent at 60 degrees C. A 95% conversion rate was observed at 28 degrees C and at 40 degrees C with 60 and 30 min operation times respectively. Operational stability of the enzyme was improved further with dithiothreitol treatment. Activity loss was around 5% following 20 cycles of repeated use of the enzyme at 40 degrees C. No significant loss of activity was observed at 28 degrees C when the enzyme was used for 20 cycles. 6-Aminopenicillanic acid in the reaction mixture was observed to be stable during conversion reactions which were carried out at both temperatures.