Properties of γ-aminobutyraldehyde dehydrogenase from Escherichia coli

γ-Aminobutyraldehyde dehydrogenase from Escherichia coli K-12 has been purified and characterized from cell mutants able to grow in putrescine as the sole carbon and nitrogen source. The enzyme has an M_r of 195 000±10 000 in its dimeric form with an M_r of 95 000±1000 for each subunit, a pH optimum at 5.4 in sodium citrate buffer, and does not require bivalent cations for its activity. K_m values are 31.3±6.8 μM and 53.8±7.4 μM for Δ-1-pyrroline and NAD⁺, respectively. An inhibitory capacity for NADH is also shown using the purified enzyme.     

High-level expression of soluble human β-defensin-2 in Escherichia coli

Human β-defensin-2 (hBD2) is a short cationic peptide with a broad antimicrobial spectrum. The coding sequence of hBD2 was cloned into pET-32a (+) to construct a fusion expression plasmid, pET32–hBD2, which was transformed into E. coli BL21 (DE3) for expression. The cultivation parameters of the expression vector harboring strain were optimized to produce the fusion protein in soluble form efficiently and to avoid the formation of insoluble inclusion bodies. The optimal conditions were determined as following: cultivation at 28 °C in MBL medium, induction at middle stage of exponential growth with 0.8 mM IPTG, and post-induction expression for 8 h. Under the above conditions, a high percentage of the target fusion protein (≥92.3%) was expressed in soluble form and the volumetric productivity of soluble fusion protein reached 1.3 g/l. The culture process was successfully scaled up in a 10 l bench-top fermentor.     

Enzymatic production of γ-L-glutamyltaurine through the transpeptidation reaction of γ-glutamyltranspeptidase from Escherichia coli K-12

γ-L-Glutamyltaurine is a naturally occurring peptide and known to have several physiological functions in mammals. This paper describes a new method for the enzymatic production of γ-L-glutamyltaurine from L-glutamine and taurine through the transpeptidation reaction of γ-glutamyltranspeptidase (EC 2.3.2.2) of Escherichia coli K-12. The optimum conditions for the production of γ-L-glutamyltaurine were 200 mM L-glutamine, 200 mM taurine and 0.2 U/ml γ-glutamyltranspeptidase, pH 10, and 1-h incubation at 37°C. Forty-five mM γ-L-glutamyltaurine was obtained, the yield being 22.5%. γ-L-Glutamyltaurine was purified on Dowex 1 × 8 and C₁₈ columns, and identified by means of NMR and a polarimeter.     

Optimization of β-galactosidase extraction from Kluyveromyces marxianus

The influence of several parameters, such as temperature, pH, and concentration of buffer and solvent, on the release of β-galactosidase from Kluyveromyces marxianus cells was studied. In optimal conditions (37°C, pH 9.5–10.5) greater than 90% of the intracellular β-galactosidase activity was released into 0.1-0.5 phosphate buffer after 1.5-2.0 h treatment with 1% chloroform. The described method is simple, effective, relatively fast, and selective.     

Synthesis of the biologically active β-subunit of human nerve growth factor in Escherichia coli

The gene(NGFB) encoding the β subunit of mature human nerve growth factor (hNGFB) was subcloned into the pJLA503 expression vector under the control of bacteriophage promoters p_R and p_L, and expressed in Escherichia coli. The recombinant protein represented approximately 3% of the total cellular protein. Biologically active hNGFB was solubilized (0.2% total NGFB) and purified by cation-exchange chromatography and it yielded two bands on polyacrylamide-gel electrophoresis under nonreducing conditions, corresponding to the monomeric (14 kDa) and homodimeric (26.5 kDa) forms of the molecule. Both hNGFB forms were immunopositive on Western blots with rabbit anti-NGFB antibodies; however, following additional purification, only the species corresponding to the hNGFB homodimer was biologically active on cultured chicken dorsal root ganglion neurons. These results demonstrate the feasibility of synthesizing the biologically active form of hNGFB in E. coli.     

Expression of a mouse metallothionein-Escherichia coli β-galactosidase fusion gene (MT-βgal) in early mouse embryos

We have microinjected DNA containing the inducible mouse metallothionein-I (MT-I) promoter, coupled to the structural gene for Escherichia coli β-galactosidase (lacZ), into the pronuclei of one-cell mouse embryos. A qualitative histochemical assay, with 5-bromo-4-chloro-3-indolylβ- -galactopyranoside (X-Gal) as a substrate, was used to detect expression of lacZ at several preimplantation stages. We observed staining indicative of exogenous β-galactosidase activity in 5–17% of DNA-injected embryos assayed at preimplantation stages after 16–24 h treatment with ZnSO₄. Thus, lacZ can be used as an indicator gene for promoter function during early mouse embryogenesis, and the incorporation of the MT-I promoter into fusion genes can be a useful means of controlling the expression of exogenous genes in preimplantation mouse embryos.     

Immobilization of thermostable β-glucosidase from Sulfolobus shibatae by cross-linking with transglutaminase

Thermostable β-glucosidase from Sulfolobus shibatae was immobilized on silica gel modified or not modified with 3-aminopropyl-triethoxysilane using transglutaminase as a cross-linking factor. Obtained preparations had specific activity of 3883 U/g of the support, when measured at 70 °C using o-nitrophenyl β-d-galactopyranoside (GalβoNp) as substrate. The highest immobilization yield of the enzyme was achieved at pH 5.0 in reaction media. The most active preparations of immobilized β-glucosidase were obtained at a transglutaminase concentration of 40 mg/ml at 50 °C. The immobilization was almost completely terminated after 100 min of the reaction and prolonged time of this process did not cause considerable changes of the activity of the preparations. The immobilization did not influence considerably on optimum pH and temperature of GalβoNp hydrolysis catalyzed by the investigated enzyme (98 °C, pH 5.5). The broad substrate specifity and properties of the thermostable β-glucosidase from S. shibatae immobilized on silica-gel indicate its suitability for hydrolysis of lactose during whey processing.     

The catalytic potency of β-glucosidase from Pyrococcus furiosus in the direct glucosylation reaction

Enzymes from extremophiles operate at conditions that are different from their ‘normal’ counterparts, and are therefore a useful extension of the enzyme toolbox. In this paper, the direct glucosylation reaction mediated by a hyperthermophilic β-glucosidase from Pyrocuccus furiosus was investigated. Hexanol was successfully coupled to glucose with this enzyme. A preliminary study was conducted to improve the product yield. A maximum product concentration of 12.9 g.l⁻¹ was attainable by increasing the glucose concentration to the maximum solubility of 2000 g.(kg buffer solution)⁻¹ at the reaction temperature. The highest glucose based yield of 2.64% was achieved with a glucose concentration of 900 g.(kg buffer solution)⁻¹ at a reaction temperature of 65°C and a pH of 6.0. Performing the reaction at higher pH and temperature led to lower product concentrations. This was caused by deactivation of the enzyme accompanied by browning of the reaction mixture. A pH of 4.4 did have a negative effect on both the storage and the operational stability of the enzyme.     

Interaction of an Escherichia coli mutator gene with a deoxyribonucleotide effector

Summary Strains of Escherichia coli carrying mutD5 display very high mutation rates (about 10⁴-fold above wild-type) when grown in rich medium, but relatively low mutation rates (about 10- to 50-fold above wild-type) in minimal medium. Thymidine, deoxycytidine, and deoxyuridine are all capable of stimulating mutation when added to minimal medium. Studies with mutants blocked in various steps of thymidine metabolism implicate a phosphorylated thymidine effector which mediates the mutagenic action of the added deoxyribonucleotides. In addition, an unidentified compound or compounds other than thymidine present in rich medium (L-broth) can also increase the mutation rate.     

A radioactive assay for the physiological activity of the tryptophan synthetase α subunit in crude extracts of Escherichia coli

A modified assay has been devised for the physiological reaction, indole-3-glycerol phosphate to Trp, of the enzyme tryptophan synthetase. The assay may be applied to crude bacterial extracts, and is based on the measurement of incorporation of radioactivity from [³H]Ser into Trp. Comparison with previous colorimetric assays indicates an improvement in sensitivity of about 30-fold, and advantages in terms of sample economy and simplified manipulation.     

The purification and immobilization of Penicillium notatum β-galactosidase

Penicillium notatum No. 1 as a producer of β-galactosidase was cultivated in a 5–1 fermenter. Various methods of protein isolation and concentration from the culture fluid were optimized. Then the conditions of β-galactosidase purification using an affinity chromatographic technique were established. The purified enzyme was immobilized on a controlled porous glass (CPG). The optimum temperature and pH values of the native and immobilized forms of β-galactosidase were determined as 50°C and 30–50°C as well as pH 3 and pH 3–5, respectively.     

The cloning of the rorB gene of Escherichia coli

Summary A segment of the Escherichia coli genome which complements the ionising radiation sensitivity of the rorB mutation was cloned into pBR322. This DNA segment also complements the mitomycin C sensitivity of the rorB mutation. The gene was subcloned until defined in a fragment of 1.05 kb. Only one gene product, a protein of approximately 16.5 kDa, was found on maxicell analysis of the various subclones. Iso-electric focusing of this gene product suggests it may function in a complex.     

Hydrolysis of lactose in whey permeate by immobilized β-galactosidase from Kluyveromyces fragilis

Neutral β-galactosidase from Kluyveromyces fragilis was immobilized on silanized porous glass modified by glutaraldehyde binding, with retention of more than 90% of its activity. Marked shifts in optimum pH (from 7.0 to 6.0) and temperature (from 35°C to 50°C) of the solid-phase enzyme were observed together with high catalytic activity and reasonable stability at wider pH and temperature ranges than those of the free enzyme. Highly efficient lactose saccharification (86–90%) in whey permeate was achieved both in a batch process and in a recycling packed-bed bioreactor.     

Expression of a foreign eukaryotic gene in Saccharomyces cerevisiae: β-galactosidase from Kluyveromyces lactis

Three recombinant DNA vectors carrying the β-galactosidase structural gene, LAC4, from the yeast Kluyveromyces lactis were constructed and transformed into Saccharomyces cerevisiae. All transformants expressed the β-galactosidase activity of LAC4. However, the level of enzyme activity varied, being highest in cells transformed with vectors which are maintained as multicopy plasmids and lowest in cells transformed with a vector which integrates into chromosomes. Enzyme levels probably reflect gene dosage. LAC4 is very stable when integrated into a chromosome, but unstable when carried on a plasmid. Therefore, stability is a property of the recombinant vector rather than of LAC4, LAC4-coded β-galactosidase synthesized in either S. cerevisiae or in K. lactis is the same as judged by two-dimensional polyacrylamide gel electrophoresis. However, S. cerevisiae transformed with