The specific inhibition of the expression of the lactose operon by D,L-threo-phenylserine inEscherichia coli

Phenylserine, one of the phenylalanine analogues, is incorporated into proteins ofEscherichia coli and replaces the natural amino acid. The incorporation results in the inhibition of the synthesis of both inducible and constitutive β-galactosidase. The rate of the synthesis of β-galactosidase specific m-RNA is only slightly influenced by phenylserine, the steady-state level being decreased by about 40%. The m-RNA formed in the present of the analogue functions normally and its translation after the removal of the inhibitor results in the formation of normal β-galactosidase. The character of the inhibition of the enzyme synthesis by phenylserine is similar to that caused by chloramphenicol. However, phenylserine specifically inhibits only the synthesis of β-galactosidase, whereas other cell proteins are synthesized. No protein immunologically cross-reacting with the antiserum against normal β-galactosidase is formed by inducible ánd constitutiveEscherichia coli strains. The active transport is completely inhibited as the cells induced in the presence of phenylserine do not accumulate¹⁴C-TMG. It follows from the results that phenylserine inhibits both the formation of TMG-specific permease and the synthesis of the active molecule of β-galactosidase inEscherichia coli.     

Synthesis of β-galactosidase inEscherichia coli in the presence of phenylalanine analogues

The effect of phenylalanine analogues (p-F-phenylalanine, phenylserine and furylalanine) is described on the synthesis of inducible β-galactosidase inEscherichia coli ML-30 and phenylalanine requiring mutant ML-48. The incorporation of these analogues into the enzyme molecule results in the formation of a protein sensitive to a different extent to heat, urea and trypsin. The influence of the analogues on the ability to concentrate inducer inside the cells is also described. The different effect of the analogues on the synthesis and stability of the enzyme is discussed.     

Cloning,sequencing, and overexpression in Escherichia coli of a phenylserine dehydratase gene from Ralstonia pickettii PS22

The structural gene coding for phenylserine dehydratase from Ralstonia pickettii PS22 was cloned into Escherichia coli cells, and the nucleotide sequence was identified. The predicted amino acid sequence had high sequence similarity to biodegradative and biosynthetic threonine dehydratases from E. coli and serine dehydratase from human liver. Transformed E. coli cells overproduced phenylserine dehydratase, and the recombinant enzyme was purified to homogeneity with a high yield and characterized.     

Characterization of an inducible phenylserine aldolase from Pseudomonas putida 24-1

An inducible phenylserine aldolase (L-threo-3-phenylserine benzaldehyde-lyase, EC 4.1.2.26), which catalyzes the cleavage of L-3-phenylserine to yield benzaldehyde and glycine, was purified to homogeneity from a crude extract of Pseudomonas putida 24-1 isolated from soil. The enzyme was a hexamer with the apparent subunit molecular mass of 38 kDa and contained 0.7 mol of pyridoxal 5' phosphate per mol of the subunit. The enzyme exhibited absorption maxima at 280 and 420 nm. The maximal activity was obtained at about pH 8.5. The enzyme acted on L-threo-3-phenylserine (Km, 1.3 mM), l-erythro-3-phenylserine (Km, 4.6 mM), l-threonine (Km, 29 mM), and L-allo-threonine (Km, 22 mM). In the reverse reaction, threo- and erythro- forms of L-3-phenylserine were produced from benzaldehyde and glycine. The optimum pH for the reverse reaction was 7.5. The structural gene coding for the phenylserine aldolase from Pseudomonas putida 24-1 was cloned and overexpressed in Escherichia coli cells. The nucleotide sequence of the phenylserine aldolase gene encoded a peptide containing 357 amino acids with a calculated molecular mass of 37.4 kDa. The recombinant enzyme was purified and characterized. Site-directed mutagenesis experiments showed that replacement of K213 with Q resulted in a loss of the enzyme activity, with a disappearance of the absorption maximum at 420 nm. Thus, K213 of the enzyme probably functions as an essential catalytic residue, forming a Schiff base with pyridoxal 5'-phosphate.     

Purification and properties of recombinant β-galactosidase from Bacillus circulans

A gene encoding β-galactosidase from Bacillus circulans which had hydrolysis specificity for the β1-3 linkage was expressed in Escherichia coli. The β-galactosidase was purified from crude cell lysates of E. coli by column chromatographies on Resource Q and Sephacryl S-200 HR. The enzyme released galactose with high selectivity from oligosaccharides which had terminal β1-3 linked galactose residues. However it did not hydrolyse β1-4 linked galactooligosaccharides. Moreover, Galβ1-3GlcNAc, Galβ1-3GalNAc, and their p-nitrophenyl glycosides were regioselectively synthesized in 10–46% yield by the transglycosylation reaction using this enzyme.     

The effect of nucleosides on the induced synthesis of β-galactosidase in non-growing cells ofEscherichia coli

The induced synthesis of β-galactosidase in non-growing cells ofEscherichia coli starving for exogenous carbon and nitrogen sources was stimulated markedly by the addition of any of four nucleosides tested: adenosine, guanosine, cytidine, and uridine. Adenosine was used as a representative of this group of compounds in most experiments. The decrease of ability of the cells to synthesize β-galactosidase, resulting from a prolonged starvation for exogenous carbon and nitrogen, was removed by adenosine. This compound also considerably reduced the inhibitory effect of metabolic poisons on the induced synthesis of β-galactosidase. The blockade of induced β-galactosidase synthesis evoked in aerobically grown cells by anaerobic starvation for exogenous sources of carbon and nitrogen was also significantly reduced by adenosine. The weak transient catabolic repression of induced synthesis of β-galactosidase evoked by glucose in non-growing cells ofEscherichia coli deprived of exogenous carbon and nitrogen sources was prevented by adenosine. The total repression caused by higher glucose concentrations was not influenced by this compound. The results are discussed from the point of view of the role of the energy state ofEscherichia coli cells in the regulation of β-galactosidase synthesis.     

Expression and regulation of the lactose transposon Tn951 in Rhizobium spp.

Abstract The expression of β-galactosidase by the lac transposon Tn 951 , in Escherichia coli , was found to be cAMP-dependent. This finding provided the basis for an investigation of the effect of cAMP on Tn 951 lac expression in Rhizobium , with the ultimate aim of using the Tn 951 system as a specific probe for cAMP mediated catabolite repression. When introduced into Rhizobium , Tn 951 directed the synthesis of β-galactosidase, which was inducible by isopropyl-β- d -thiogalactopyranoside (IPTG). Marked quantitative and qualitative differences in β-galactosidase expression were found between R. meliloti and R. japonicum during the growth cycle, with expression being higher in the former. β-Galactosidase levels were, however, unaffected by exogenous cAMP under catabolite repressing conditions.     

The effect of DL-β-methylaspartic acid on the growth ofEscherichia coli

ВЫла кислота была под готовлена и двух его и зомеры отделить друг от друга. Больше erythro-раст воримой форме стимул ировали рост (itEscherichia титр) в синтетических средн ие на концентрацию 1–5 gmmoles мл. Менее растворимы е фракции, содержащие threo-форма препятствует росту из (itEscherichia коли) на дан ной концентрации. Инг ибирование роста сви детельствует путем п ролонгации лаг фазы; Темпы роста в логари фмической фазы и Макс имальное количество ячеек не были пострад авшим от аналога. Торм озящий последствия, в ызванные threo-форма methylaspartic ки слоты могут быть пода влены Помимо этого из аспарагиновой кисло ты.     

Disulfide bond formation and activation of Escherichia coli β-galactosidase under oxidizing conditions

Escherichia coli β-galactosidase is probably the most widely used reporter enzyme in molecular biology, cell biology, and biotechnology because of the easy detection of its activity. Its large size and tetrameric structure make this bacterial protein an interesting model for crystallographic studies and atomic mapping. In the present study, we investigate a version of Escherichia coli β-galactosidase produced under oxidizing conditions, in the cytoplasm of an Origami strain. Our data prove the activation of this microbial enzyme under oxidizing conditions and clearly show the occurrence of a disulfide bond in the β-galactosidase structure. Additionally, the formation of this disulfide bond is supported by the analysis of a homology model of the protein that indicates that two cysteines located in the vicinity of the catalytic center are sufficiently close for disulfide bond formation.     

Latent β-galactosidase inEscherichia coli

Incubation of washedEscherichia coli cells with crystalline RNase lead to increased β-galactosidase activity. The height of the increase depended on the type of strain and the conditions of cultivation. RNase only raised the level of the β-galactosidase which was bound to the relatively easily sedimenting cellular particles. It had no effect on the activity of β-galactosidase present in soluble form in the supernatant after the disruption of cells or on the activity of purified β-galactosidase in solution. Another basic protein, histone, was found to have a similar effect to that of RNase.     

The effect of oxygen transfer on the activity of encapsulated whole cell β-galactosidase

The effect of oxygen transfer on the production of immobilized whole cell β-galactosidase has been evaluated. The encapsulated whole cell β-galactosidase was prepared by combining cell encapsulation and culture into one-step. Escherichia coli was encapsulated and cultured in the growth and production media to accumulate β-galactosidase in itself. Sunflower seed oil was coimmobilized to increase the oxygen transfer rate through the capsule membrane. The oxygen transfer rate increased 63 percent and the activity of β-galactosidase increased by 10 percent. The activity of encapsulated β-galactosidase obtained in the concentric air lift reactor was 86 percent higher than that in the shaking incubator. In the concentric air lift reactor, the accumulation of encapsulated whole cell β-galactosidase was primarily dependent on the capsule velocity. While the accumulation of specific β-galactosidase in the capsule increased with volumetric oxygen transfer coefficient, the cell biomass accumulated in the capsule decreased.     

Cloning and expression of a β-galactosidase gene of Bacillus circulans

A gene of β-galactosidase from Bacillus circulans ATCC 31382 was cloned and sequenced on the basis of N-terminal and internal peptide sequences isolated from a commercial enzyme preparation, Biolacta(?). Using the cloned gene, recombinant β-galactosidase and its deletion mutants were overexpressed as His-tagged proteins in Escherichia coli cells and the enzymes expressed were characterized.     

Transposon-induced mutants of Listeria monocytogenes incapable of growth at low temperature (4°C)

Abstract When a strain of Escherichia coli , expressing a hybrid protein GalK-β-Gal, is shifted to high osmolarity, the β-galactosidase activity strongly decreases within 20 min of shock. The loss of β-galactosidase activity results from degradation of the hybrid protein under osmotic stress. The results raise the possibility that osmotic stress induces a specific osmodependent protease.     

Mechanism of the glycine cleavage reaction: retention of C-2 hydrogens of glycine on the intermediate attached to H-protein and evidence for the inability of serine hydroxymethyltransferase to catalyze the glycine decarboxylation

Glycine is converted to carbon dioxide and an intermediate attached to a lipoic acid group on H-protein in the P-protein-catalyzed partial reaction of the glycine cleavage reaction [K. Fujiwara and Y. Motokawa (1983) J. Biol. Chem. 258, 8156-8162]. The results presented in this paper indicate that the decarboxylation is not accompanied by the removal of a C-2 hydrogen atom of glycine and instead both C-2 hydrogens are transferred with the alpha carbon atom to the intermediate formed during the decarboxylation of glycine. The purified chicken liver cytosolic and mitochondrial serine hydroxymethyltransferase preparations could not catalyze the decarboxylation of glycine in the presence of either lipoic acid or H-protein. The decarboxylation activity of the serine hydroxymethyltransferase preparation purified from bovine liver by the method similar to that of L. R. Zieske and L. Davis [(1983) J. Biol. Chem. 258, 10355-10359] was completely inhibited by the antibody to P-protein, while the antibody had no effect on the activity of the phenylserine cleavage. Conversely, D-serine inhibited the activity of phenylserine cleavage but the activity of the decarboxylation of glycine was not affected by D-serine. Finally, the two activities were separated by the chromatography on hydroxylapatite. The results clearly demonstrate that serine hydroxymethyltransferase per se cannot catalyze the decarboxylation of glycine.     

Expression of β-galactosidase in Rhizobium japonicum

Abstract The plasmid pGC91.14 was used to introduce via conjugation the Escherichia coli lac operon into fast-growing and slow-growing strains of Rhizobium japonicum . Exconjugants now expressed higher levels of β-galactosidase activity which was still inducible by isopropyl-β- d -thiogalactoside (IPTG). The presence of the lac operon allowed the slow-growing strain 61A76 to grow on lactose as the sole carbon source; the fast-growing strains grew poorly on lactose but growth was not inhibited by lactose as had been reported for Rhizobium meliloti . β-galactosidase could be detected in nodule extracts and bacteroid preparations from soybean plants ( Glycine max L. Merrill) infected with the strain 61A76 (pGC91.14).     

Simultaneous on line monitoring of intracellular β-galactosidase activity and biomass using flow injection analysis inEscherichia coli batch fermentations

Summary A novel method to monitor on-line intracellular β-galactosidase activity and biomass simultaneously, using flow injection analysis (FIA), has been developed. The automatic ultrasonic cell disruption and FIA analysis allow the processing of 10 samples/hour with a wide and variable linear working range of β-galactosidase activity and biomass and a maximum relative standard deviation (RSD) of 1.5%. The system has been optimized by monitoring biomass and intracellular β-galactosidase activity inEscherichia coli batch fermentation.     

Divergent Effects of Chaperone Overexpression and Ethanol Supplementation on Inclusion Body Formation in RecombinantEscherichia coli

The proper folding of aggregation-prone recombinant proteins inEscherichia colican be facilitated by co-overexpressing specific molecular chaperones or by culturing the cells in the presence of ethanol or other agents that upregulate the synthesis of all heat-shock proteins (hsps). We have investigated the effect of combining direct chaperone overproduction with ethanol supplementation on the cytoplasmic folding of two aggregation-prone model proteins, preS2-S′-β-galactosidase and human SPARC. In 25-ml shake flask cultures grown at 30°C, addition of 3% (v/v) ethanol to the growth medium prior to inoculation improved the chaperone-mediated increase in the yields of active preS2-S′-β-galactosidase 1.5- to 2-fold. When cultures overexpressing thednaKJoperon were grown in the presence of ethanol, the levels of enzymatic activity were 5-fold higher relative to control cells and preS2-S′-β-galactosidase aggregation was almost entirely abolished. Combining DnaK–DnaJ overexpression and growth of the cells at temperatures lower than 30°C did not result in a comparable increase in activity. Although the individual effects of ethanol supplementation anddnaKJoverproduction were more limited when the culture volume was raised, a synergistic improvement in preS2-S′-β-galactosidase activity was observed when the two approaches were used in concert. In contrast, ethanol supplementation promoted the aggregation of human SPARC, a protein exhibiting a chaperone dependency similar to that of preS2-S′-β-galactosidase. Our results show that ethanol can exert complex and divergent effects on inclusion body formation and that the beneficial effect of the solvent on recombinant protein folding cannot simply be explained by an increase in the intracellular concentration of molecular chaperones.     

Secreted β-galactosidase from a Flavobacterium sp. isolated from a low-temperature environment

The bacterial strain Flavobacterium sp. 4214 isolated from Greenland was found to express β-galactosidase (EC 3.2.1.23) at temperatures below 25°C. A chromosomal library of Flavobacterium sp. 4214 was constructed in Escherichia coli, and the gene gal4214-1 encoding a β-galactosidase of 1,046 amino acids (114.3 kDa) belonging to glycosyl hydrolase family 2 was isolated. This was the only gene encoding β-galactosidase activity that was identified in the chromosomal library. Expression levels in both Flavobacterium sp. 4214 and in initial recombinant E. coli strains were insufficient for biochemical characterization. However, a combination of T7 promoter expression and introduction of an E. coli host that complemented rare transfer RNA genes yielded 15 mg of β-galactosidase per liter of culture. Gal4214-1-His protein was found to be active in monomeric conformation. The protein was secreted from the cytoplasm, probably through an N-terminal signaling sequence. The Gal4214-1-His protein was found to have optimum activity at a temperature of 42°C, but with short-term stability at temperatures above 25°C.     

Repeated-batch operation of immobilized β-galactosidase inclusion bodies-containing Escherichia coli cell reactor for lactose hydrolysis

In this study, we investigated the performance of an immobilized β-galactosidase inclusion bodies-containing Escherichia coli cell reactor, where the cells were immobilized in alginate beads, which were then used in repeated-batch operations for the hydrolysis of o-nitrophenyl-β-D-galactoside or lactose over the long-term. In particular, in the Tris buffer system, disintegration of the alginate beads was not observed during the operation, which was observed for the phosphate buffer system. The o-nitrophenyl-β-D-galactoside hydrolysis was operated successfully up to about 80 h, and the runs were successfully repeated at least eight times. In addition, hydrolysis of lactose was successfully carried out up to 240 h. Using Western blotting analyses, it was verified that the beta-galactosidase inclusion bodies were sustained in the alginate beads during the repeated-batch operations. Consequently, we experimentally verified that β-galactosidase inclusion bodies-containing Escherichia coli cells could be used in a repeated-batch reactor as a biocatalyst for the hydrolysis of o-nitrophenyl-β-D-galactoside or lactose. It is probable that this approach can be applied to enzymatic synthesis reactions for other biotechnology applications, particularly reactions that require long-term and stable operation.     

Cloning and Expression of a β-Galactosidase Gene of Bacillus circulans

A gene of β-galactosidase from Bacillus circulans ATCC 31382 was cloned and sequenced on the basis of N-terminal and internal peptide sequences isolated from a commercial enzyme preparation, Biolacta^®. Using the cloned gene, recombinant β-galactosidase and its deletion mutants were overexpressed as His-tagged proteins in Escherichia coli cells and the enzymes expressed were characterized.