Effect of chitin sources on production of chitinase and chitosanase byStreptomyces griseus HUT 6037

The advantage of usingStreptomyces griseus HUT 6037 in the production of chitinase or chitosanase is that the organism is capable of hydrolyzing amorphous or crystal-line chitin and chitosan according to the type of the substrate used. We investigated the effects of the enzyme induction time and chitin sources, CM-chitosan and deacetylated chitosan (degree of deacetylation 75–99%), on production of chitosanase. We found that this strain accumulated chitosanase when cells were grown in the culture medium containing chitosanaceous substrates instead of chitinaceous substrates. The highest chitosanase activity was obtained at 4 days of cultivation with 99% deacetylated chitosan. Soluble chitosan (53% deacetylated chitosan) was found to induce chitinase as well as chitosanase. The specific activities of chitinase and chitosanase were 0.91 and 1.33 U/mg protein at 3 and 5 days, respectively. From the study of the enzymatic digestibility of various degrees of deacetylated chitosan, it was found that (GlcN)₃, (GlcN)₄ and (GlcN)₅ were produced during the enzymatic hydrolysis reaction. The results of this study suggested that the sugar composition of (GlcN)₃ was homogeneous and those of (GlcN)₄ and (GlcN)₅ were heterogeneous.     

Structural studies of a two-domain chitinase from Streptomyces griseus HUT6037

Chitinase C (ChiC) from Streptomyces griseus HUT6037 was the first glycoside hydrolase family 19 chitinase that was found in an organism other than higher plants. An N-terminal chitin-binding domain and a C-terminal catalytic domain connected by a linker peptide constitute ChiC. We determined the crystal structure of full-length ChiC, which is the only representative of the two-domain chitinases in the family. The catalytic domain has an alpha-helix-rich fold with a deep cleft containing a catalytic site, and lacks three loops on the domain surface compared with the catalytic domain of plant chitinases. The chitin-binding domain is an all-beta protein with two tryptophan residues (Trp59 and Trp60) aligned on the surface. We suggest the binding mechanism of tri-N-acetylchitotriose onto the chitin-binding domain on the basis of molecular dynamics (MD) simulations. In this mechanism, the ligand molecule binds well on the surface-exposed binding site through two stacking interactions and two hydrogen bonds and only Trp59 and Trp60 are involved in the binding. Furthermore, the flexibility of the Trp60 side-chain, which may be involved in adjusting the binding surface to fit the surface of crystalline chitin by the rotation of chi2 angle, is shown.     

Effects of several metals on spore,biomass, and geosmin production byStreptomyces tendae andPenicillium expansum

Cultures ofStreptomyces tendae andPenicillium expansum grown on Actinomyces and Czapek's media, respectively, were exposed to 5 mg L^–1 of manganese, magnesium, iron, cobalt, nickel, copper and zinc, supplied as sulfate salts. Only copper markedly increased geosmin (1, 10-dimethyl-9-decalol), biomass, and spore production. inductively coupled plasma-atomic emission spectrometric analysis ofS. tendae andP. expansum cells did not indicate an accumulation of copper. Both 1 and 5 mg L^–1 copper, as copper sulfate, increased total geosmin production in cultures ofS. tendae on several media, but decreased production on others, suggesting that substrate composition affects responses to copper.     

The effect of feedback regulation and in situ product removal on the conversion of sugar to cycloheximide by Streptomyces griseus

An addition of cycloheximide to cycloheximide-producing Streptomyces griseus cultures resulted in reductions in the production rate and in the conversion of sugar into cycloheximide. In situ cycloheximide adsorption was observed to enhance: total cycloheximide titers; productivities; and the conversion of sugar to cycloheximide. During the secondary metabolite-producing phase, sugar consumption was observed to be linearly dependent on cycloheximide productivity. From this analysis a true product yield and maintenance coefficient were estimated to be 0.08 g cycloheximide/g glucose and 0.028 g glucose/g cell-h, respectively. The sixfold difference between this true product yield and a theoretical value obtained from knowledge of the biosynthetic pathway is discussed. Since the maintenance sugar requirement for cycloheximide production is large, stimulation of biosynthesis through in situ adsorption significantly increases the overall efficiency of sugar conversion to this secondary metabolite.     

The degrees of polymerization and N-acetylation of chitosan determine its ability to elicit callose formation in suspension cells and protoplasts of Catharanthus roseus

Partially and fully deacetylated chitosan fragments and oligomers were compared for their potency to elicit formation of the 1.3--glucan callose in suspension-cultured cells and protoplasts of Catharanthus roseus (line 385). Chitosan oligomers induced little callose formation, while callose synthesis increased with the degree of polymerization of chitosan up to several thousand corresponding to a molecular mass near 10⁶ Da. At a comparable degree of polymerization, partially N-acetylated chitosan fragments were less effective. Colloidal chitin and chitin oligomers induced only trace callose synthesis in protoplasts. These results indicate that the primary interaction involved the amino groups of chitosan and numerous negative charges at the surface of the plasma membrane with spacing in the nanometer range and occurring regularly over micrometer stretches. Charged phospholipid head-groups may fulfill these requirements. The resulting alteration of membrane fluidity may lead to the changes in ion transport known to be associated with the induction of callose formation.Abbreviations DP degree of polymerization - FDA fluorescein diacetate - PE pachyman equivalents     

Conditions for the production of jadomycin B byStreptomyces venezuelae ISP5230: Effects of heat shock,ethanol treatment and phage infection

Summary The novel benzoxazolophenanthridine antibiotic, jadomycin B, is produced byStreptomyces venezuelae ISP5230 following a 42 °C heat shock or exposure to ethanol. To further characterize these unusual culture conditions, studies were carried out using different media, varying nutrient concentrations, initial pH, and time of application of heat or ethanol stress. Highest titers of jadomycin B accumulated 48 h afterS. venezuelae ISP5230 was inoculated into ad-galactose-l-isoleucine production medium (pH 7.5) which was supplemented with ethanol (6%, v/v) between 6 and 13 h. Cultures supplemented with ethanol later than 17 h post inoculation into the production medium produced little or no jadomycin B. Among other heat-shock inducing treatments examined, infection with phage SV1 was associated with increased jadomycin B production. Although jadomycin B titers showed little change with variations in the concentration of phosphate in the production medium, the nature of the nitrogen source was found to be important. Different colored pigments, presumed to be jadomycin B analogs, were formed when other amino acids replacedl-isoleucine in the medium as the sole nitrogen source. Increased jadomycin B titers accompanied increasedl-isoleucine andd-galactose concentrations in the production medium.     

Study on the production of chitin and chitosan from shrimp shell by using Bacillus subtilis fermentation

Fermentation of shrimp shell in jaggery broth using Bacillus subtilis for the production of chitin and chitosan was investigated. It was found that B. subtilis produced sufficient quantities of acid to remove the minerals from the shell and to prevent spoilage organisms. The protease enzyme in Bacillus species was responsible for the deprotenisation of the shell. The pH, proteolytic activity, extent of demineralization and deprotenisation were studied during fermentation. About 84% of the protein and 72% of the minerals were removed from the shrimp shell after fermentation. Mild acid and alkali treatments were given to produce characteristic chitin and their concentrations were standardized. Chitin was converted to chitosan by N-deacetylation and the properties of chitin and chitosan were studied. FTIR spectral analysis of chitin and chitosan prepared by the process was carried out and compared with spectra of commercially available samples.     

Molecular cloning and characterization of a pea chitinase gene expressed in response to wounding,fungal infection and the elicitor chitosan

The fungicidal class I endochitinases (E.C.3.3.1.14, chitinase) are associated with the biochemical defense of plants against potential pathogens. We isolated and sequenced a genomic clone, DAH53, corresponding to a class I basic endochitinase gene in pea, Chil. The predicted amino acid sequence of this chitinase contains a hydrophobic C-terminal domain similar to the vacuole targeting sequences of class I chitinases isolated from other plants. The pea genome contains one gene corresponding to the chitinase DAH53 probe. Chitinase RNA accumulation was observed in pea pods within 2 to 4 h after inoculation with the incompatible fungal strain Fusarium solani f. sp. phaseoli, the compatible strain F. solani f.sp. pisi, or the elicitor chitosan. The RNA accumulation was high in the basal region (lower stem and root) of both fungus challenged and wounded pea seedlings. The sustained high levels of chitinase mRNA expression may contribute to later stages of pea's non-host resistance.     

Altered expression of an ankyrin-repeat protein results in leaf abnormalities,necrotic lesions,and the elaboration of a systemic signal

Summary The PR-like proteins, class I -1,3-glucanase (GLU I) and chitinase (CHN I), are induced as part of a stereotypic response that can provide protection against viral, bacterial, and fungal pathogens. We have identified two Nicotiana plumbaginifolia ankyrin-repeat proteins, designated lucanohydrolase inding roteins (GBP) 1 and 2, that bind GLU I and CHN I both in vitro and when expressed in yeast cells. Sense as well as antisense transformants of tobacco carrying the GBP1 gene elaborated graft-transmissible acropetally moving signals that induced the downward curling of young leaves. This phenotype was associated with reduced starch, sucrose, and fructose accumulation; the formation of necrotic lesions; and, the induction of markers for the hypersensitive response. GBP1/2 are members of a conserved lant-specific yrin- repeat (PANK) family that includes proteins implicated in carbohydrate allocation, reactive oxygen metabolism, hypersensitive cell death, rapid elicitor responses, virus pathogenesis, and auxin signaling. The similarity in phenotype of PANK transformants and transformants altered in carbohydrate metabolism leads us to propose that PANK family members are multifunctional proteins involved in linking plant defense responses and carbohydrate metabolism.