General Properties of Endo-N-acetylmuramidase of Bacillus subtilis YT-25

The enzymatic behaviour, amino acid composition and some physical properties of a new endo-N-acetylmuramidase (B-enzyme) of Bacillus subtilis YT–25 were determined and compared with hen’s egg white lysozyme. The molecular weight was estimated to be about 13000 by the sedimentation equilibrium method. The isoelectric point was pH 9.8. The amino acid composition indicates that the enzyme is rich in basic amino acids, especially lysin. Maximal activity on the lysis of cell walls of M. lysodeikticus occurred at pH 6.2. The enzyme was stable at pH 3.5 ~ 6.0. The specific activity for the lysis of cell walls of M. lysodeikticus was less than fourth part of that of hen’s egg white lysozyme. Digest of cell walls of M. lysodeikticus with B-enzyme consisted greater numbers of high molecular products than digest with egg white lysozyme. Substrate specificity of B-enzyme seemed to be different from that of egg white lysozyme.     

Studies on Isomerization of Sugars by Bacteria

An enzyme, which catalyzes the isomerization of d-glucose to d-fructose, has been found in a newly isolated bacterium which tentatively identified as Pacacolobacterum aerogenoides. The enzyme converts not only d-glucose but also d-mannose to d-fructose, and NAD and Mg⁺⁺ are required as cofactor for this isomerization. The properties of this enzyme were summarized as follows: (1) As a cofactor for the isomerization by this enzyme, NAD was absolutely necessary, whereas NADP, FMN and FAD were not. (2) The optimum pH was found to be at 7.5 and optinum temperature was at about 40°C. (3) The enzyme activity was markedly reduced by EDTA treatment and the reduced activity by EDTA was restored by the addition of Mg⁺⁺, Mn⁺⁺ or Co⁺⁺. (4) The enzyme activity was strongly inhibited by monoiodoacetate, p-chloromercuribenzoate, and Cu⁺⁺, however, the activity was recovered by adding cysteine or glutathione.     

Formation of Glucose Isomerase by Streptomyces sp.

Sophoradin (I) [2′,4,4′-trihydroxy-3,3′,5-tris(3-methyl-2-butenyl)chalcone] which had been isolated from “Guang-Dou-Gen” (the root of Sophora subprostrata Chun et T. Chen) was synthesized through Claisen rearrangement. The reaction of p-hydroxybenzaldehyde and 3-chloro-3-methyl-1-butyne (III) gave 4-(1,1-dimethylpropargyloxy)benzaldehyde (VIII), which was catalytically hydrogenated over Lindlar catalyst to afford 4-(1,1-dimethylallyloxy)benzaldehyde (IX). IX was converted to 4-hydroxy-3-(3-methyl-2-butenyl)benzaldehyde (X) by Claisen rearrangement. The reaction of X and III gave 3-(3-methyl-2-butenyl)-4-(1,1-dimethylpropargyloxy)benzaldehyde (XI). Condensation of 2-hydroxy-4-(1,1-dimethylpropargyloxy)acetophenone (IV) and XI in alkaline solution gave a chalcone (XIII), which was catalytically hydrogenated over Lindlar catalyst to give 2′-hydroxy-4,4′-bis(1,-dimethylallyloxy)-3-(3-methyl-2-butenyl)chalcone (XIV). XIV was converted to I by Claisen rearrangement.     

Comparison of Extracellular Polysaccharide Produced by Pullularia pullulans with Polysaccharides in the Cells and Cell Wall

The purified swep hydrolase appeared homogeneous on SDS disc electrophoresis, and the molecular weights of the enzyme and the subunit were 280,000 and 70,000, respectively. The isoelectric point of this enzyme was pH 4.8.     

Structural Uniformity of Pullulan Produced by Several Strains of Pullularia pullulans

Extracellular polysaccharides produced by 3 strains of Pullularia pullulans were fractionated by treating with cetyl trimethyl ammonium hydroxide into soluble and insoluble fractions, and the structure of the former fraction, i.e., pullulan, was studied. The yield and the ratio of 2 fractions varied widely according to the strains. But the structure of pullulan was found to be uniform irrespective of the strains used. All 3 samples of pullulan gave only glucose on complete acid hydrolysis, and 93~95% maltotriose and 5~7% maltotetraose after isoamylase (pullulanase) action. The ratio of α-1,4- to α-1,6-glucosidic linkages calculated from periodate oxidation data coincided very well with the value expected from the ratio of maltotriose to maltotetraose units. An evidence for the complete absence of branch structure in pullulan was presented from the results of hydrolysis by pullulan 4-glucanohydrolase.     

The enhancing effect of fucoidan derived from Undaria pinnatifida on immunoglobulin production by mouse spleen lymphocytes

In this study, we revealed that a Mekabu (Udaria pinnantifida) extract enhanced immunoglobulin (Ig) production of mouse spleen lymphocytes. Furthermore, it was suggested that water-soluble and high molecular weight ingredients in the Mekabu extract have significant enhancing effect on Ig production. Therefore, fucoidan was estimated as the active component.     

SIZ1 deficiency causes reduced stomatal aperture and enhanced drought tolerance via controlling salicylic acid‐induced accumulation of reactive oxygen species in Arabidopsis

Transpiration and gas exchange occur through stomata. Thus, the control of stomatal aperture is important for the efficiency and regulation of water use, and for the response to drought. Here, we demonstrate that SIZ1‐mediated endogenous salicylic acid (SA) accumulation plays an important role in stomatal closure and drought tolerance. siz1 reduced stomatal apertures. The reduced stomatal apertures of siz1 were inhibited by the application of peroxidase inhibitors, salicylhydroxamic acid and azide, which inhibits SA‐dependent reactive oxygen species (ROS) production, but not by an NADPH oxidase inhibitor, diphenyl iodonium chloride, which inhibits ABA‐dependent ROS production. Furthermore, the introduction of nahG into siz1, which reduces SA accumulation, restored stomatal opening. Stomatal closure is generally induced by water deficit. The siz1 mutation caused drought tolerance, whereas nahG siz1 suppressed the tolerant phenotype. Drought stresses also induced expression of SA‐responsive genes, such as PR1 and PR2. Furthermore, other SA‐accumulating mutants, cpr5 and acd6, exhibited stomatal closure and drought tolerance, and nahG suppressed the phenotype of cpr5 and acd6, as did siz1 and nahG siz1. Together, these results suggest that SIZ1 negatively affects stomatal closure and drought tolerance through the accumulation of SA.     

A novel tag-free probe for targeting molecules interacting with a flavonoid catabolite

3,4-Dihydroxyphenylacetic acid (DOPAC) is one of the colonic microflora-produced catabolites of quercetin 4′-glucoside (Q4′G). Although the interaction of DOPAC with cellular proteins might be involved in its biological activity, the actual proteins have not yet been identified. In this study, we developed a novel tag-free DOPAC probe to label the targeted proteins by the copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) and verified its efficacy. Various labeled proteins were detected by the DOPAC probe with the azide labeled biotin and a horseradish peroxidase (HRP)-streptavidin complex. Furthermore, a pulldown assay identified Keap1 and aryl hydrocarbon receptor (AhR) as the target proteins for the phase 2 enzyme up-regulation.