Constituent Proteins of Globulin Fraction Obtained from Egg White

A globulin fraction which was salted out from egg white by ammonium sulfate was constituted of five kinds of proteins. One of them was macroglobulin and two were G₂ and G₃ of Longsworth et al. [J. Am. Chem. Soc., 62, 2580 (1940)]. Remaining two proteins were assumed to be ovoinhibitors. G₂ and G₃ were separated from each other by CM-cellulose chromatography. The molecular weights of G₂ and G₃ were almost the same, being about 49,000. An euglobulin-like protein which was precipitated during the dialysis of the globulin fraction consisted mainly of macroglobulin and aggregates of some other egg white proteins.     

Metabolism of Serine in Growing Rats and Chicks at Various Dietary Protein Levels

The metabolic fate of the carbon skeleton of l-serine-U-¹⁴C has been investigated, in vivo and in vitro, in growing rats and chicks fed the diets with various protein calories percents (PC%) at 410 kcal of metabolizable energy.

The incorporation of ¹⁴C into body protein at 12 hr after the injection of serine-¹⁴C was about 49% of the injected dose in rats fed the 10 or 15 PC % diet, though the value was reduced in rats fed lower and higher protein diets. The ¹⁴CO₂ production was smaller in rats fed the 10 and 15 PC% diet, and it showed an inverse pattern to that of the ¹⁴C incorporation into body protein. Urinary excretion of ¹⁴C was higher in rats fed 10 and higher PC% diets, whose growth rate and net body protein retention were maximum.

In contrast to the case of rats, the incorporation of ¹⁴C into body protein of chicks at 6 hr after the injection was rather reduced in the 15 PC% group. The proportion of ¹⁴C excreted as uric acid was remarkably increased above the 10 PC% group, and about 19% of the injected dose was recovered in the 50 PC% group.

The catabolic rate of serine in the liver slices of rats and chicks was increased by high protein diets.

These results support the concept that the nutritional significance of metabolism of the carbon skeleton of serine in growing rats and chicks is different from each other, especially at high protein diets.     

On the Mechanism for the Formation of Indole Alkaloids in Penicillium concavo-rugulosum

Experiments on the biosynthesis and microbiological conversion of indole alkaloids in Pénicillium concavo-rugulosum were carried out with the growing and resting mycelia, respectively, of a selected strain of the same mold. The former experiments were performed by the use of dl-tryptophan-3-¹⁴C or dl-mevalonic acid-2-¹⁴C-lactone as a precursor, while the latter experiments by the use of rugulovasine A–³H, dihydrorugulovasine A–³H, 4-[γ, γ-dimethylallyl]-tryptophan-³H, chanoclavine-[I]–³H or the other tritilated ergoline alkaloids. The results of these experiments suggested that in the Penicillium mold employed there exist the following biosynthetic route: tryptophan + mevalonic acid→4-[γ, γ-dimethylallyl]-tryptophan→rugulovasine A→dihydrorugulovasine A→dihydrorugulovasine A-lactam.     

Bacterial species selective toxicity of two isomeric α/β-peptides: Role of membrane lipids

We have studied how membrane interactions of two synthetic cationic antimicrobial peptides with alternating α- and β-amino acid residues (“α/β-peptides”) impact toxicity to different prokaryotes. Electron microscopic examination of thin sections of Escherichia coli and of Bacillus subtilis exposed to these two α/β-peptides reveals different structural changes in the membranes of these bacteria. These two peptides also have very different effects on the morphology of liposomes composed of phosphatidylethanolamine and phosphatidylglycerol in a 2:1 molar ratio. Freeze fracture electron microscopy indicates that with this lipid mixture, α/β-peptide I induces the formation of a sponge phase. ³¹P NMR and X-ray diffraction are consistent with this conclusion. In contrast, with α/β-peptide II and this same lipid mixture, a lamellar phase is maintained, but with a drastically reduced d-spacing. α/β-Peptide II is more lytic to liposomes composed of these lipids than is I. These findings are consistent with the greater toxicity of α/β-peptide II, relative to α/β-peptide I, to E. coli, a bacterium having a high content of phosphatidylethanolamine. In contrast, both α/β-peptides display similar toxicity toward B. subtilis, in accord with the greater anionic lipid composition in its membrane. This work shows that variations in the selectivity of these peptidic antimicrobial peptides toward different strains of bacteria can be partly determined by the lipid composition of the bacterial cell membrane.     

OPTIMIZATION OF FERMENTATION MEDIUM FOR ACETOIN PRODUCTION BY Bacillus subtilis SF4-3 USING STATISTICAL METHODS

To improve the acetoin-producing ability of Bacillus subtilis SF4-3, isolated from “natto,” a Japanese traditional food, the fermentation medium was optimized in shake-flask fermentation by statistically designed methods. Based on results of the single-factor experiment, orthogonal experiment, and Plackett–Burman design, yeast extract, corn steep liquor, and urea were identified as showing significant influence on the acetoin production. Subsequently, the optimum combination of the three factors was investigated by the Box–Behnken design (BBD) of response surface methodology (RSM) in order to further enhance the acetoin production. The maximum acetoin yield of 45.4 g/L was predicted when the concentrations of yeast extract, corn steep liquor, and urea were 8.5 g/L, 14.6 g/L, and 3.8 g/L, respectively. The results were further confirmed in triplicate experiments using the optimized medium (glucose 160 g/L, yeast extract 8.5 g/L, corn steep liquor 14.6 g/L, urea 3.8 g/L, manganese sulfate 0.05 g/L, ferrous sulfate 0.05 g/L), and an acetoin yield of 46.2 g/L was obtained in the validation experiment, which was in agreement with the prediction. After the optimization of medium components, an increase of 36.28% in acetoin production was achieved in comparison to that at the initial medium levels.     

Rhizobacterial-mediated induction of defense enzymes to enhance the resistance of turmeric (Curcuma longa L) to Pythium aphanidermatum causing rhizome rot

Rhizobacteria isolated from the rhizosphere soil were evaluated for their ability to control rhizome rot in turmeric (Curcuma longa L). These isolates were characterised as Pseudomonas fluorescens and Bacillus subtilis. Under in vitro condition, two isolates, namely P. chlororaphis (PcPA23) and B. subtilis (BsCBE4), showed maximum inhibition of mycelial growth of Pythium aphanidermatum, were found effective in reducing rhizome rot of turmeric both under greenhouse and field conditions and increased the plant growth and rhizome yield. Both the isolates were further tested for its ability to induce production of defense-related enzymes and chemicals in plants. Increased activities of phenylalanine ammonia lyase, peroxidase, polyphenol oxidase, chitinase and β-1,3-glucanase were observed in PcPA23 and BsCBE4 pre-treated turmeric plants challenged with P. aphanidermatum. Moreover, higher accumulation of phenolics was noticed in plants pre-treated with PcPA23 and BsCBE4 challenged with P. aphanidermatum. Thus, the present study shows that in addition to direct antagonism and plant growth promotion, induction of defense-related enzymes involved in the phenyl propanoid pathway collectively contributed to enhance resistance against invasion of Pythium in turmeric.     

Enhancing the efficiency of the bioagent Bacillus subtilis JF419701 against soil-borne phytopathogens by increasing the productivity of fungal cell wall degrading enzymes

This study was devoted to increasing the production of fungal cell wall degrading enzymes by Bacillus subtilis JF419701 to enhance its efficiency in the biological control process. In dual culture, B. subtilis JF419701 showed the highest antagonistic effect of the 256 bacterial strains tested against six soil-borne pathogens, Alternaria alternata, Exserohilum rostratum, Fusarium oxysporum, Macrophomina phaseolina, Pythium ultimum and Rhizoctonia solani. The production potentiality of the enzymes α-1,3-glucanase, β-1,3-glucanase, chitinase and protease by B. subtilis JF419701 was studied in vitro. Results proved that the maximum production of enzymes by this bacterium was achieved after a two-day incubation period at a slightly alkaline pH (8). The addition of colloidal chitin or S-glucan to the growth media enhanced the production of all the enzymes except protease, which was stimulated by casein. This study therefore recommends that to obtain an efficient and strong bioagent culture of B. subtilis JF419701, it is necessary to grow this micro-organism on a specific medium containing either chitin or its derivatives at pH 8 for two days.     

In vitro antifungal activity of chitinolytic enzymes produced by bio-agents against root rot pathogenic fungi

A study was carried out using simple laboratory techniques to examine the influence of the antagonistic isolates of Trichoderma harzianum, T. viride, Bacillius subtilis and Pseudomons flourescence and their culture filtrates on selected soil-borne root rot pathogens Rhizoctonia solani and Fusarium solani. Testing procedures were standardised using two different methods. The experiments were based on the principle of dual culture and agar diffusion techniques. The experiment involved the recording of the percentage of reduction in growth and inhibition zones formed by various filtrates of antagonistic culture growth. The results showed that the antagonists tested had the ability to reduce the linear growth of fungal pathogens. Also, the cultures filtrates of antagonists had antifungal activities by forming inhibition zones. Culture filtrates have shown a strong clear inhibition zone which increases in diameter as the incubation period of antagonists increases. This observation was related to the increase in the activity of chitinolytic enzymes as secondary metabolic compounds produced in growth media by prolonging the period of incubation. The study has proved that such enzymes can be effectively used for suppression of soil-borne pathogens and that it can evolve as a potential biocide.     

Association of IL-12, IL-18 variants and serum IL-18 with bladder cancer susceptibility in North Indian population

IL-12 and IL-18 are immunomodulatory cytokines that play important roles in host immune response against cancers. Variation in DNA sequence in gene promoter may lead to altered IL-18 production and/or activity, and hence can modulate an individual's susceptibility to BC. To test this hypothesis, we investigated the relationship of IL-18 gene promoter −137 G/C and −607C/A polymorphisms and IL12 (− 16974) A/C with the risk of BC in North Indian population. Polymorphisms in IL-18 and IL-12 genes were analyzed in 200 BC patients and 200 age, ethnicity and sex-matched controls, using restriction fragment length polymorphism-polymerase chain reaction (PCR-RFLP) and amplification refractory mutation specific-polymerase chain reaction (ARMS) method. The concentrations of IL-18 in serum were determined by ELISA. Significant association was observed with IL18 (− 137) G/C heterozygous genotype (GC) with 1.96 folds risk of BC as well at C allele carrier and variant C allele having 2 fold and 1.6 fold risk for BC respectively. IL18 (− 607) C/A, heterozygous CA genotype also showed a high risk (OR = 1.59) for BC. While IL12 (− 16974) A/C heterozygote genotype and C allele carrier demonstrated reduced risk of BC. Hetero genotype of IL18 (− 137) G/C was associated with risk of recurrence (HR = 2.35) in superficial BC patients receiving BCG treatment thus showing least survival. The distributions of IL-18 gene haplotypes were not significantly different between patients and controls. Serum IL-18 levels were significantly higher in BC patients than in the healthy subjects (p = 0.025). Serum IL-18 levels was also significantly associated with IL18 (− 137) G/C in heterozygous genotype (GC) (p = 0.048). Our results suggest that IL-18 gene polymorphism contributes to bladder cancer risk whereas IL-12 is protective. A relation between IL18 (− 137) G/C in heterozygous genotype with elevated IL-18 serum level and bladder cancer risk has been registered in the present study.     

Modular pathway engineering of key carbon‐precursor supply‐pathways for improved N‐acetylneuraminic acid production in Bacillus subtilis

N‐acetylneuraminic acid (NeuAc) is widely used as a nutraceutical for facilitating infant brain development, maintaining brain health, and enhancing immunity. Currently, NeuAc is mainly produced by extraction from egg yolk and milk, or via chemical synthesis. However, its low concentration in natural resources and its non‐ecofriendly chemical synthesis result in insufficient NeuAc production and environmental pollution, respectively. In this study, improved NeuAc production was attained via modular pathway engineering of the supply pathways of two key precursors—N‐acetylglucosamine (GlcNAc) and phosphoenolpyruvate (PEP)—and by balancing NeuAc biosynthesis and cell growth in engineered Bacillus subtilis. Specifically, we used a previously constructed GlcNAc‐producing B. subtilis as the initial host for NeuAc biosynthesis. First, we constructed a de novo NeuAc biosynthetic pathway utilizing glucose by coexpressing glucosamine‐6‐phosphate acetyl‐transferase (GNA1), N‐acetylglucosamine 2‐epimerase (AGE), and N‐acetylneuraminic acid synthase (NeuB), resulting in 0.33 g/l NeuAc production. Next, to balance the supply of the two key precursors for NeuAc biosynthesis, modular pathway engineering was performed. The optimal strategy for balancing the GlcNAc module and PEP supply module involved the use of an engineered, unique glucose and malate coutilization pathway in B. subtilis, supplied with both glucose (for the GlcNAc moiety) and malate (for the PEP moiety) at high strength. This led to 1.65 g/L NeuAc production, representing a 5.0‐fold improvement over the existing methods. Furthermore, to enhance the NeuAc yield on cell, glucose and malate coutilization pathways were engineered to balance NeuAc biosynthesis and cell growth via the blocking of glycolysis, the introduction of the Entner–Doudoroff pathway, and the overexpression of the malic enzyme YtsJ. NeuAc titer reached 2.18 g/L, with 0.38 g/g dry cell weight NeuAc yield on cell, which represented a 1.32‐fold and 2.64‐fold improvement over the existing methods, respectively. The strategy of modular pathway engineering of key carbon precursor supply pathways via engineering of the unique glucose‐malate coutilization pathway in B. subtilis should be generically applicable for engineering of B. subtilis for the production of other important biomolecules. Our study also provides a good starting point for further metabolic engineering to achieve industrial production of NeuAc by a Generally Regarded As Safe bacterial strain.