A 56.56-kDa extracellular chitinase from Paenibacillus sp. D1 was purified to 52.3-fold by ion exchange chromatography using SP Sepharose. Maximum enzyme activity was recorded at pH 5.0 and 50 °C. MALDI-LC-MS/MS analysis identified the purified enzyme as chitinase with 60% similarity to chitinase Chi55 of Paenibacillus ehimensis. The activation energy (E (a)) for chitin hydrolysis and temperature quotient (Q (10)) at optimum temperature was found to be 19.14 kJ/mol and 1.25, respectively. Determination of kinetic constants k (m), V (max), k (cat), and k (cat)/k (m) and thermodynamic parameters ΔH*, ΔS*, ΔG*, ΔG*(E-S), and ΔG*(E-T) revealed high affinity of the enzyme for chitin. The enzyme exhibited higher stability in presence of commonly used protectant fungicides Captan, Carbendazim, and Mancozeb compared to control as reflected from the t (1/2) values suggesting its applicability in integrated pest management for control of soil-borne fungal phytopathogens. The order of stability of chitinase in presence of fungicides at 80 °C as revealed from t (1/2) values and thermodynamic parameters E (a(d)) (activation energy for irreversible deactivation), ΔH*, ΔG*, and ΔS* was: Captan > Carbendazim > Mancozeb > control. The present study is the first report on thermodynamic and kinetic characterization of chitinase from Paenibacillus sp. D1.

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Aims:  Statistical optimization of medium components for improved chitinase production by Paenibacillus sp. D1. Methods and Results:  Urea, K2HPO4, 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; K2HPO4, 1·17; MgSO4, 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, K2HPO4, 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.

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 3AE4 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.

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