Comparison of chito-oligosaccharide production from three different colloidal chitosans using the endochitonsanolytic system of Bacillus thuringiensis.

Bacillus thuringiensis is a nonhuman pathogen bacterium that is used as a fungal and insect biocontrol agent. Because of its environmental interaction, it possesses several extracellular enzymes that are able to degrade chitin and chitosan, two of the most important polymers because of their application in numerous fields. However, in recent years, it has been observed that oligosaccharides from the enzymatic degradation of chitosan have important benefits for human health. Comparison and exploration of the production of chito-oligosaccharides from different sources of chitosan will improve the process parameters and expand the biotechnology based in these molecules. This study shows the production of chito-oligosaccharides from three different sources of colloidal chitosan and conducts a qualitative-quantitative comparison between them, using the extracellular enzyme of B. thuringiensis. We found that in the three substrates, it is possible to get a mixture of chito-oligosaccharides from dimer to hexamer in a concentration range from 0.72 to 8.09 mg · g-1 of original substrate. The best substrate to obtain these molecules was commercial chitosan as it has the highest production yields.

Characterization of a novel Bacillus thuringiensis phenotype possessing multiple appendages attached to a parasporal body.

Bacillus thuringiensis is a bacterium best known for its production of crystal-like bodies comprised of one or more Cry-proteins, which can be toxic to insects, nematodes or cancer cells. Although strains of B. thuringiensis have occasionally been observed with filamentous appendages attached to their spores, appendages in association with their parasporal bodies are extremely rare. Herein we report the characterization of Bt1-88, a bacterial strain isolated from the Caribbean that produces a spore-crystal complex containing six long appendages, each comprised of numerous thinner filaments approximately 10 nm in diameter and 2.5 µm in length. Each of the multi-filament appendages was attached to a single, small parasporal body located at one end of the bacterial spore. Biochemical tests, 16S rDNA gene sequencing, and the identification of two Cry proteins by partial protein sequencing (putatively Cry1A and Cry2A), unambiguously identified Bt1-88 as a strain of B. thuringiensis. Bt1-88 represents the second reported strain of B. thuringiensis possessing a parasporal body/appendage phenotype characterized by one or more long appendages, comprised of numerous filaments in association with a parasporal body. This finding suggests that Bt1-88 is a member of a new phenotypic class of B. thuringiensis, in which the parasporal body may perform a novel structural role through its association with multi-filament appendages.

Molecular cloning and purification of an endochitinase from Serratia marcescens (Nima).

An endochitinase gene from the Serratia marcescens Nima strain (chiA Nima) was cloned, sequenced, and expressed in Escherichia coli DH5alphaF', and the recombinant protein (ChiA Nima) was purified by hydrophobic interaction chromatography. chiA Nima contains an open reading frame (ORF) that encodes an endochitinase with a deduced molecular weight and an isoelectric point of 61 kDa and 6.84, respectively. A sequence at the 5'-end was identified as a signal peptide, recognized by Gram-negative bacteria transport mechanism. Comparison of ChiA Nima with other chitinases revealed a modular structure formed by the catalytic domain and a putative chitin-binding domain. The purified chitinase was able to hydrolyze both trimeric and tetrameric fluorogenic substrates, but not a chitobiose analog substrate. ChiA Nima showed high enzymatic activity within a broad pH range (pH 4.0-10.0), with a peak activity at pH 5.5. The optimal temperature for enzymatic activity was detected at 55 degrees C.