Optimization of medium constituents for improved chitinase production by Paenibacillus sp. D1 using statistical approach.

AIMS: Statistical optimization of medium components for improved chitinase production by Paenibacillus sp. D1. METHODS AND RESULTS: Urea, K(2)HPO(4), chitin and yeast extract were identified as significant components influencing chitinase production by Paenibacillus sp. D1 using Plackett-Burman method. Response surface methodology (central composite design) was applied for further optimization. The concentrations of medium components for improved chitinase production were as follows (g l(-1)): urea, 0.33; K(2)HPO(4), 1.17; MgSO(4), 0.3; yeast extract, 0.65 and chitin, 3.75. This statistical optimization approach led to the production of 93.2 +/- 0.58 U ml(-1) of chitinase. CONCLUSIONS: The important factors controlling the production of chitinase by Paenibacillus sp. D1 were identified as urea, K(2)HPO(4), chitin and yeast extract. Statistical approach was found to be very effective in optimizing the medium components in manageable number of experimental runs with overall 2.56-fold increase in chitinase production. SIGNIFICANCE AND IMPACT OF THE STUDY: The present investigation provides a report on statistical optimization of medium components for improved chitinase production by Paenibacillus sp. D1. Paenibacillus species are gram-positive, spore-forming bacteria with several PGPR and biocontrol potentials. However, only few reports concerning mycolytic enzyme production especially chitinases are available. Chitinase produced by Paenibacillus sp. D1 represents new source for biotechnological and agricultural use.

Pesticide tolerance of Paenibacillus sp. D1 and its chitinase.

Excessive use of pesticides in agriculture has led to several problems pertaining to loss of soil fertility and environmental degradation. Biological control agents offer the best alternative to reduce use of toxic pesticides. Paenibacillus sp. D1 isolated from the effluent treatment plant of a seafood processing industry exhibited broad spectrum tolerance towards a number of pesticides at concentrations higher than recommended for field applications. The isolate showed enhanced growth and chitinase production in the presence of some protectant fungicides. None of the tested demethylase inhibitor (DMI) fungicides inhibited growth and chitinase production except triadimefon. The isolate was also tolerant to most commonly used insecticides belonging to the organophosphate, carbamate and cyclodiene organochloride classes. Chitinase of Paenibacillus sp. D1 was found to be more tolerant than the organism itself and was highly stable in the presence of pesticides at the temperature under field conditions in Gujarat, India, i.e. 40 degrees C. This was suggestive of its potential in integrated pest management (IPM) to significantly reduce the use of harmful chemicals. To our knowledge this is the first extensive study on pesticide tolerance of the Paenibacillus species and its chitinase.

Formulation of medium constituents by multiresponse analysis of central composite design to enhance chitinase production in Pantoea dispersa.

In the present study, a high chitinase producing strain Pantoea dispersa was isolated from the sea dumps at Bhavnagar, India. Chitin, urea, CaCl2 and MgSO4 x 7H2O were variables used in central composite design for chitinase production. Chitinase, biomass and pH were the responses used in different models to evaluate individually fit ones. Quadratic model was found to be fit for chitinase response whereas in the case of biomass and pH, linear model was found to be fit without the effect of others. Chitinase production was optimized with respect to other responses such as biomass and pH in multiresponse analysis of response surface design by using desirability approach. In multiresponse analysis, following medium formulation (g/l), chitin, 15; urea, 0.32; CaCl2, 0.10 and MgSO4 x 7H2O, 0.08 was found to predict optimum chitinase production of 482.77 units/ml with overall highest desirability of 0.854 as compared to other formulations. The selection of model was done on the basis of high Adjusted R-squared value and lowered p-value for each model in individual analysis of each response. In multiresponse experiment, it was found that for response chitinase quadratic model and for responses pH and biomass linear models were well fit. Through desirability analysis, it was found that in the chitinase production, pH was essential as compared to biomass in P. dispersa. Endochitinase and chitobiase actvities were also studied.

Isolation and identification of marine chitinolytic bacteria and their potential in antifungal biocontrol.

Chitinolytic marine bacterial strains (30) were isolated from the sea dumps at Bhavnagar, India. They were screened as chitinase producers on the basis of zone of clearance on chitin agar plates incorporated with calcofluor white M2R for the better resolution. Out of these, three strains namely, Pseudomonas sp., Pantoea dispersa and Enterobacter amnigenus showed high chitinase production. They were also found to produce proteases and therefore have a good potential for use as antifungal biocontrol agents for the control of fungal plant pathogens. These strains could degrade and utilize the mycelia of Macrophomina phaseoliena (Tassi) Goidanich and Fusarium sp. In vitro, these strains could inhibit the growth of Fusarium sp. and M. phaseolina. The culture filtrate inhibiting hyphal elongation was observed microscopically.

Biological control of Fusarium wilt of pigeonpea Cajanus cajan (L.) Millsp with chitinolytic Alcaligenes xylosoxydans.

Alcaligenes xylosoxydans protected pigeonpea from Fusarium wilt in a pot experiment and field trials. When seeds of pigeonpea (C. cajan) were treated with A. xylosoxydans and sown in soil infested with Fusarium, the incidence of wilt was reduced by 43.5% and resulted in 58% higher grain yield. The antifungal activity of A. xylosoxydans was based on chitinase production and was comparable in efficacy to commercial antifungal agents such as benlate, monitor WP, thiram and bavistin.

Relations of enzymes inAspergillus repens grown under sodium chloride stress.

Aspergillus repens, a salt-pan isolate, was halotolerant. When grown for 72 h (log phase) and 144 h (beginning of stationary phase) in a medium containing 2M sodium chloride, the activities of invertase, malate dehydrogenase (MDH), glucose-6-phosphate dehydrogenase (G6PDH), and glutamate dehydrogenase (GDH) were found to have increased. Control cultures grown in a medium devoid of 2M NaCl failed to show such changes. The activities of MDH, G6PDH, and GDH increased with rising concentrations of Na(+) (as NaCl) when added up to 100MM in vitro. At higher concentrations they decreased. Changes in kinetic constants, Km and Vmax of these enzymes, as well as their de novo synthesis, were found to be some of the responses to NaCl stress-mediated changes.

Polyol concentrations in Aspergillus repens grown under salt stress.

Na(+), K(+) and the ratio of Na(+)/K(+) were higher in cells of the halotolerant Aspergillus repens grown with 2 M NaCl than without NaCl. The osmolytes, proline, glycerol, betaine and glutamate, did not affect the Na(+)/K(+) ratio, nor the polyol content of cells under any conditions. The concentrations of polyols, consisting of glycerol, arabitol, erythritol and mannitol, changed markedly during growth, indicating that they have a crucial role in osmotic adaptation.

Biochemical changes in mango after infection with Rhizoctonia bataticola.

Rhizoctonia bataticola is responsible for the spoilage of mango fruits (Mangifera india) during post-harvest preservation and storage. Culture of R. bataticola exhibited significant pectinase and cellulase activity. In Rhizoctonia-infected fruits an increase of protease and cellulase activity, and a decrease in certain enzymes of carbohydrate metabolism, were observed in comparison to healthy fruits.

Photoregulation of some enzymes from Neurospora crassa.

Light-grown cultures of Neurospora crassa showed photoregulation of a number of enzymes. Proteases and cytosolic malate dehydrogenase showed an increase in activity. There was a decrease in the activity of mitochondrial malate dehydrogenase, isocitrate dehydrogenase and cytosolic glucose-6P-dehydrogenase, isocitrate dehydrogenase and isocitrate lyase.

The novel method for isolating chitinolytic bacteria and its application in screening for hyperchitinase producing mutant of Alcaligenes xylosoxydans.

AIMS: To develop a novel, rapid and effective screening method for chitinase producing bacteria. METHODS AND RESULTS: A simple and rapid technique for screening of potential chitinolytic bacteria has been developed using the chitin binding dye calcofluor white M2R in chitin agar. Microorganisms possessing high chitinolytic potential gave a clear zone under ultraviolet light after 24-48 h of incubation. This method was successfully applied for isolating the hyperchitinase mutant of Alcaligenes xylosoxydans. The mutant Alc. xylosoxydans EMS 33 was found to produce 3.4 times more chitinase than the wild type. CONCLUSIONS: In this study, the screening method for chitinase producing bacteria has been developed and it was applied to screen chitinase-overproducing mutant of Alc. xylosoxydans. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel screening method for chitinase producer is more sensitive, rapid, user-friendly and reliable, which can also be used for screening of recombinants having chitinase gene.

Purification and characterization of chitinase from Alcaligenes xylosoxydans.

Extracellular chitinase from Alcaligenes xylosoxydans was purified to electrophoretic homogeneity using affinity and gel filtration chromatography. The molecular mass of chitinase was estimated to be 45 kDa and 44 kDa by SDS-PAGE and gel-filtration, respectively. The enzyme was optimally active at 50 degrees C (over 30 min) and pH 5. Activity staining after PAGE showed a single band. The Km for chitin was 3 g l-1. Cu2+ and Na+ at 5 mM inhibited chitinase activity to 25% while Ca2+, Mg2+ and Ba2+ had no effect at the same concentration. The purified enzyme degraded mycelia of Aspergillus niger.

Aminopeptidase-N from the Helicoverpa armigera (Hubner) brush border membrane vesicles as a receptor of Bacillus thuringiensis crylac delta-endotoxin.

Brush border membrane vesicles (BBMVs) were prepared from the 2nd instar larvae of Helicoverpa armigera. Binding of the activated Cry1Ac of Bacillus thuringiensis (Bt) toxin was shown by immunoblot. A 120-kDa protein was identified as a receptor for the Cry1Ac type delta-endotoxin. The aminopeptidase-N activity of BBMVs was measured as the hydrolysis of L-leucine p-nitroanilide. The specific activity was 35 units/mg protein. The BBMV preparation also showed low level of alkaline phosphatase activity. Zn++ chelating agents 2,2'-dipyridyl and 1,10-phenanthroline inhibited aminopeptidase activity at 10 mM concentration, indicating the presence of zinc-dependent aminopeptidase in the brush border of H. armigera. The aminopeptidase activity was increased with increasing concentration of delta-endotoxin. The purified 120-kDa binding protein was N-terminally sequenced. The first 10-amino-acid sequence showed 60-77% similarity with human cysteine-rich secretory protein-1 precursor, inhibin alpha chain precursor. Salmonella flagellar hook protein and yeast carboxypeptidase S.