Synthesis of γ-Glutamyl Peptides Catalyzed by Transamidase from Bacillus natto

Crude ammonium sulfate fraction of a cell free extract from Bacillus natto contained an enzyme (or enzymes) which catalyzed the transamidation reaction specific for glutamine. Both l- and d-isomers of glutamine were active as substrate. On incubation of l- or d-glutamine with the enzyme preparation, two peptides consisting of glutamic acid and glutamine were formed. The main component of the peptides was readily isolated by ion-exchange chromatography and identified as γ-glutamylglutamine by paper chromatography and by paper electrophoresis using authentic peptides. The optical configuration of the amino acid residues in the dipeptide was determined by digestion of the acid hydrolyzate with l-glutamic acid decarboxylase, and the result showed that the dipeptide obtained from l-glutamine was a l-l isomer, while the dipeptide from d-glutamine was a d-d isomer.     

Formation of Phage-Induced γ-Polyglutamic Acid Depolymerase in Lysogenic Strain of Bacillus natto

The influence of tea catechins on the absorption of starch or sucrose was investigated in vivo. Tea catechins were administered orally to rats before soluble starch or sucrose administration. Saccharide-dosed rats were killed and the blood and the contents of the intestine were collected at intervals over two hours. Catechins of certain concentrations suppressed the increase of plasma glucose levels, thus concurrently suppressing insulin activity. Increased activity of intestinal α-amylase by starch dosing was inhibited markedly in the catechin-administered rats. Sucrase on the brush border membrane was also inhibited by prior catechin administration. From these results it was assumed that orally administered catechins will inhibit intestinal α-amylase or sucrase, thereby deterring the digestion of certain amounts of starch or sucrose and eventually reducing the plasma glucose levels.     

Hydrolysis of starches by the action of an α-amylase from Bacillus subtilis

A moderately thermophilic Bacillus subtilis strain, isolated from fresh sheep’s milk, produced extracellular thermostable α-amylase. Maximum amylase production was obtained at 40 °C in a medium containing low starch concentrations. The enzyme displayed maximal activity at 135 °C and pH 6.5 and its thermostability was enhanced in the presence of either calcium or starch. This thermostable α-amylase was used for the hydrolysis of various starches. An ammonium sulphate crude enzyme preparation as well as the cell-free supernatant efficiently degraded the starches tested. The use of the clear supernatant as enzyme source is highly advantageous mainly because it decreases the cost of the hydrolysis. Upon increase of reaction temperature to 70 °C, all substrates exhibited higher hydrolysis rates. Potato starch hydrolysis resulted in a higher yield of reducing sugars in comparison to the other starches at all temperatures tested. Soluble and rice starch took, respectively, the second and third position regarding reducing sugars liberation, while the α-amylase studied showed slightly lower affinity for corn starch and oat starch.     

Structure of Bacillus subtilis Bacteriophage φ25 and φ25 Deoxyribonucleic Acid

The structure of Bacillus subtilis bacteriophage phi25 and phi25 deoxyribonucleic acid (DNA) were studied by electron microscopy. The head of phi25 is a regular polyhedron measuring 75 nm in diameter. The uncontracted tail of phi25 is 130 nm in length and includes a large, complex tail plate. Phage phi25 DNA is double-stranded and has a molecular weight of approximately 100 million as determined by electron microscopic length measurements and analytical band sedimentation in CsCl. The complementary strands of phi25 DNA contain numerous random interruptions. Chemical analysis of phi25 DNA demonstrated that 5-hydroxymethyluracil replaces thymine and that the DNA has a mole per cent (guanine plus cytosine) of 42.     

Investigation on enzymatic degradation of γ-polyglutamic acid from Bacillus subtilis NX-2

The preparation of γ-polyglutamic acid (γ-PGA) from Bacillus subtilis NX-2 has been previously investigated, and its depolymerization during the batch culture was studied in this paper. The results suggested that the γ-PGA depolymerase was present and active extracellularly in the culture. The ywtD gene from B. subtilis NX-2, encoding the γ-PGA depolymerase was cloned and expressed in Escherichia coli. The YwtD protein was purified by metal-chelating affinity chromatography. YwtD was proved to be an endo-hydrolase enzyme and exhibited a remarkable activity in γ-PGA degradation at a wide range of temperature (30–40 °C) and pH (5.0–8.0). On an optimal condition of 30 °C and pH 5.0, an efficient γ-PGA enzymatic degradation was achieved. The molecular weight of γ-PGA could be reduced within the range of 1000–20 kDa and the polydispersity also decreased as a function of depolymerization time. Therefore, a controllable degradation of γ-PGA could be available by enzymatic depolymerization.     

Glutamic Acid Independent Production of Poly(γ-glutamic acid) by Bacillus subtilis TAM-4

A bacterium that produced a large amount of poly(γ-glutamic acid) (PGA) when it was grown aerobically in a culture medium containing ammonium salt and sugar as sources of nitrogen and carbon, respectively, was isolated from soil. The bacterium, strain TAM-4, was classified as Bacillus subtilis. The maximum PGA production (22.1 mg/ml) was obtained when it was grown in a medium containing 1.8% ammonium chloride and 7.5% fructose at 30°C for 96 h with shaking. Some properties of the PGA obtained at different times of cultivation were investigated by gel permeation chromatography, SDS–PAGE, and measurement of viscosity, and calculation of the d/l ratio of glutamic acid constituting PGA. The results suggested that PGA was elongated with no changes in the diastereoisomer ratio in the molecule.     

Characterization of Infectious Deoxyribonucleic Acid from Temperate Bacillus subtilis Bacteriophage φ 105

Phenol-extracted, infectious deoxyribonucleic acid (DNA) species from phi105 phage particles, from phi105 lysogenic bacteria, and from induced phi105 lysogenic bacteria were sedimented in sucrose gradients. Infectious DNA from phi105 particles sedimented like the bulk of mature phage DNA in neutral sucrose. Infectivity of prophage DNA was associated with fast-sedimenting material of heterogenous size. Infectious vegetative phage DNA sedimented somewhat faster than mature phage DNA; it was rapidly converted to a poorly infectious form during the infection.     

Improving the acidic stability of a β-mannanase from Bacillus subtilis by site-directed mutagenesis

β-Mannanase can randomly hydrolyze the (1→4)-β-d-mannosidic linkages in mannans, galactomannans and glucomannans, yielding manno-oligosaccharides. In this study, the β-mannanase (MAN) from Bacillus subtilis B10-02 was overexpressed successfully in B. subtilis 168 as a hexa-histidine tagged, secreted protein. The recombinant enzyme BsMAN6H was not stable under acidic conditions, which restricts its use in food and feed industry. We aimed to improve the acid stability of BsMAN6H by changing several surface-exposed amino acid residues to acidic or neutral ones. Among the mutations, the His54Asp resulted in a shift in the optimal pH from 6.5 to 5.5. Accordingly, the acid stability was improved by a factor of a negative potential on the structure surface around the mutated site. Furthermore, the H54D variant showed the enzyme activity up to 3207.82 U/mL in bioreactors using the cheap Kojac powder as substrate. As a result, a bacterial β-mannanase was produced efficiently with increased acid stability, improving its applicability in the animal feed industry.     

Purification and Properties of Two Isozymes of γ-Glutamyltranspeptidase from Bacillus subtilis TAM-4

Two isozymes of γ-glutamyltranspeptidase, GGT-A and GGT-B, were purified to electrophoretic homogeneity from a culture broth of Bacillus subtilis TAM-4, which produces poly(γ-glutamic acid) (PGA) de novo. GGT-A was composed of three subunits with molecular weights of 23,000 (I), 39,000 (II), and 40,000 (III). GGT-B was composed of two subunits with molecular weights of 22,000 (I) and 39,000 (II). The N-terminal amino acid sequences of GGT-A subunit I and GGT-B subunit I were very similar. GGT-A subunit II and GGT-B subunit II had an identical N-terminal amino acid sequence. That of GGT-A subunit III showed no similarity to the other subunits. Both GGTs had similar enzymatic properties (optimum pH and temperature: pH 8.8 and 55°C) but showed a significantly different thermal stability at 55°C. Both GGT-A and -B used d-γ-glutamyl-p-nitroanilide as well as the l-isomer as the γ-glutamyl donor and used various amino acids and peptides as the acceptor. It was also found that the PGA produced by the strain was hydrolyzed to glutamic acid by its own GGTs.     

The DNA sequence of γ-glutamyltranspeptidase gene of Bacillus subtilis (natto) plasmid pUH1

Summary The -glutamyltranspeptidase (-GTP) gene of Bacillus subtilis (natto) plasmid designated pUH1, which is responsible for polyglutamate production, has been cloned and the nucleotide sequence determined. The sequence contains a single open-reading frame stretching for 1260 bp with a relative molecular mass of 49356. Putative -35 and -10 sequences, TTCAAA and TATTAT, were observed as the consensus sequence for the promoter recognized by the ⁴³ RNA polymerase of B. subtilis, and the ribosome binding site, the sequence of which was AACGAG, was complementary to the binding sequence of B. subtilis 16S rRNA except for one base. The amino acid sequence of the gene with the segment of putative protein C403 of staphylococcal plasmid pE194 indicates homology, whereas that with Escherichia coli and mammalian -GTPs does not show any similarity at all.     

Highly efficient gene expression of a fibrinolytic enzyme (subtilisin DFE) in Bacillus subtilis mediated by the promoter of α-amylase gene from Bacillus amyloliquefaciens

Subtilisin DFE is a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4. The promoter and signal peptide-coding sequence of alpha-amylase gene from B. amyloliquefaciens was cloned and fused to the sequence coding for pro-peptide and mature peptide of subtilisin DFE. This hybrid gene was inserted into the Escherichia coli/Bacillus subtilis shuttle plasmid vector, pSUGV4. Recombinant subtilisin DFE gene was successfully expressed in B. subtilis WB600 with a fibrinolytic activity of 200 urokinase units ml(-1).     

Abortive Infection of Sporulating Bacillus subtilis 168 by φ2 Bacteriophage

Bacteriophage phi2 is unable to replicate in Bacillus subtilis 168. Although some phage deoxyribonucleic acid (DNA) synthesis can occur, the DNA made is not biologically active and sedimentation analysis reveals that it is smaller in size than that of mature DNA or DNA isolated from phi2-infected permissive hosts. Messenger ribonucleic acid hybridizable with phi2 DNA is also synthesized in phi2-infected cells of 168. Mutants of 168 which are permissive hosts for phi2 have been isolated. These mutants are defective in sporulation and possess the phenotype of "early sporulation mutants." The majority map in two locations, one near the lys locus opposite the trp locus (spoA locus) and the other tightly linked to a phe locus.     

The γ-Aminobutyrate Permease GabP Serves as the Third Proline Transporter of Bacillus subtilis

PutP and OpuE serve as proline transporters when this imino acid is used by Bacillus subtilis as a nutrient or as an osmostress protectant, respectively. The simultaneous inactivation of the PutP and OpuE systems still allows the utilization of proline as a nutrient. This growth phenotype pointed to the presence of a third proline transport system in B. subtilis. We took advantage of the sensitivity of a putP opuE double mutant to the toxic proline analog 3,4-dehydro-dl-proline (DHP) to identify this additional proline uptake system. DHP-resistant mutants were selected and found to be defective in the use of proline as a nutrient. Whole-genome resequencing of one of these strains provided the lead that the inactivation of the γ-aminobutyrate (GABA) transporter GabP was responsible for these phenotypes. DNA sequencing of the gabP gene in 14 additionally analyzed DHP-resistant strains confirmed this finding. Consistently, each of the DHP-resistant mutants was defective not only in the use of proline as a nutrient but also in the use of GABA as a nitrogen source. The same phenotype resulted from the targeted deletion of the gabP gene in a putP opuE mutant strain. Hence, the GabP carrier not only serves as an uptake system for GABA but also functions as the third proline transporter of B. subtilis. Uptake studies with radiolabeled GABA and proline confirmed this conclusion and provided information on the kinetic parameters of the GabP carrier for both of these substrates.     

Monodehydro-2,2′-iminodi-2(2′)-deoxy-l-ascorbic Acid,a Radical Product from the Reaction of Dehydro-l-ascorbic Acid with an α-Amino Acid

One of the radical species produced by the reaction of dehydro-l-ascorbic acid with an α-amino acid gave a very characteristic hyperfine structure in its electron spin resonance spectrum. The same spectrum was also obtained when l-scorbamic acid was oxidized with some oxidants, indicating the formation of the radical via the oxidation of l-scorbamic acid. From the results of deuterium exchange experiments, simulation spectra and the reduction of 2,2′-nitrilodi-2(2′)-deoxy-l-ascorbic acid monoammonium salt, the radical was concluded to be monodehydro-2,2′-iminodi-2(2′)-deoxy-l-ascorbic acid. Possible formation mechanism of the radical was also discussed.     

Enhancement of γ-aminobutyric acid production in Chungkukjang by applying a Bacillus subtilis strain expressing glutamate decarboxylase from Lactobacillus brevis

For a foreign glutamate decarboxylase (GAD) to be expressed in Bacillus host system, a recombinant DNA (pLip/LbGAD) was constructed by ligating an LbGAD gene from Lactobacillus brevis OPK-3 into Escherichia coli–Bacillus shuttle vector, pLip. The pLip/LbGAD construct was then transformed into Bacillus subtilis. The culture of the transformed Bacillus strain with the pLip/LbGAD construct had higher GAD activity and γ-aminobutyric acid (GABA) concentration than those of untransformed Bacillus counterpart. In addition, Chungkukjang, a traditional Korean fermented soybean product prepared by the transformed Bacillus subtilis, contained a significantly higher level of GABA than conventional ones. Thus, by introducing a foreign GAD gene, Bacillus strains have been genetically engineered to produce high levels of GAD and GABA.     

Selective Replication of Bacteriophage φ29 Deoxyribonucleic Acid in 6-(p-Hydroxyphenylazo)-Uracil-Treated Bacillus subtilis

Phage phi29 deoxyribonucleic acid (DNA) replicated under conditions where semiconservative DNA production in Bacillus subtilis host cells was blocked with 6-(p-hydroxyphenylazo)-uracil (HPUra). The time of initiation of phi29 DNA replication was not affected by HPUra, and normal quantities of viable phage were produced in the presence of the inhibitor. Studies with conditional lethal mutants of phage phi29 demonstrated the usefulness of HPUra for detection of viral-specific DNA production.     

Biosynthesis and Hydrolysis of Poly(γ-glutamic acid) from Bacillus subtilis IF03335

Poly(γ-glutamic acid) (PGA) production in Bacillus subtilis IF03335 was studied. When citric acid as a carbon source was added to a glutamic acid medium containing L-glutamic acid and ammonium sulfate, a large amount of pure PGA was produced. On the other hand, when glucose was added to the glutamic acid medium, a by-product was produced, which seemed to be a polysaccharide. Moreover, the mode of hydrolysis was investigated with PGA in aqueous solutions at 80, 100, and 120°C by monitoring the time-dependent changes in the molecular weights. Hydrolytic degradation of PGA was found to proceed through a random chain scission.