RESUMO
Bacillus thuringiensis is a Gram-positive soil bacterium that is known to be a bacterial biopesticide that produces insecticidal proteins called crystal proteins (Cry). In the insecticidal process, chitinases are suggested to perforate the peritrophic membrane barrier to facilitate the invasion of the Cry proteins into epithelial membranes. A chitinase gene from B. thuringiensis was successfully expressed in a soluble form in Escherichia coli, and the gene product was purified and characterized. The purified recombinant enzyme, BthChi74, hydrolyzed an artificial substrate, 4-nitrophenyl N,N'-diacetyl-ß-D-chitobioside [4NP-(GlcNAc)2], and the natural substrates, colloidal chitin and crystalline α-chitin, but it did not hydrolyze cellulose. BthChi74 exhibited catalytic activity under a weakly acidic to neutral pH range at 50 °C, and it was stable over a wide pH range for 24 h. Differential scanning fluorimetry (DSF) indicated a protein melting temperature (T m) of 63.6 °C. Kinetic analysis revealed k cat and K M values of 1.5 s-1 and 159 µM, respectively, with 4NP-(GlcNAc)2 as a substrate. BthChi74 produced (GlcNAc)2 and GlcNAc from colloidal chitin and α-chitin as substrates, but the activity toward the latter was lower than that toward the former. BthChi74 could bind similarly to chitin beads, crystalline α-chitin, and cellulose through a unique family 2 carbohydrate-binding module (CBM2). The structure-function relationships of BthChi74 are discussed in relation to other chitinases, such as Listeria chitinase, which possesses a family 5 carbohydrate-binding module (CBM5).
RESUMO
The present study investigated the simultaneous oxidation of pyruvate and amino acids during H2-evolving growth of the hyperthermophilic archaeon Thermococcus kodakarensis. The comparison of mass balance between a cytosolic hydrogenase (HYH)-deficient strain (the ΔhyhBGSL strain) and the parent strain indicated that NADPH generated via H2 uptake by HYH was consumed by reductive amination of 2-oxoglutarate catalyzed by glutamate dehydrogenase. Further examinations were done to elucidate functions of three enzymes potentially involved in pyruvate oxidation: pyruvate formate-lyase (PFL), pyruvate:ferredoxin oxidoreductase (POR), and 2-oxoisovalerate:ferredoxin oxidoreductase (VOR) under the HYH-deficient background in T. kodakarensis. No significant change was observed by deletion of pflDA, suggesting that PFL had no critical role in pyruvate oxidation. The growth properties and mass balances of ΔporDAB and ΔvorDAB strains indicated that POR and VOR specifically functioned in oxidation of pyruvate and branched-chain amino acids, respectively, and the lack of POR or VOR was compensated for by promoting the oxidation of another substrate driven by the remaining oxidoreductase. The H2 yields from the consumed pyruvate and amino acids were increased from 31% by the parent strain to 67% and 82% by the deletion of hyhBGSL and double deletion of hyhBGSL and vorDAB, respectively. Significant discrepancies in the mass balances were observed in excess formation of acetate and NH3, suggesting the presence of unknown metabolisms in T. kodakarensis grown in the rich medium containing pyruvate.
