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Cloning, sequences, and characterization of two chitinase genes from the Antarctic Arthrobacter sp. strain TAD20: isolation and partial characterization of the enzymes.

ABSTRACT: Arthrobacter sp. strain TAD20, a chitinolytic gram-positive organism, was isolated from the sea bottom along the Antarctic ice shell. Arthrobacter sp. strain TAD20 secretes two major chitinases, ChiA and ChiB (ArChiA and ArChiB), in response to chitin induction. A single chromosomal DNA fragment containing the genes coding for both chitinases was cloned in Escherichia coli. DNA sequencing analysis of this fragment revealed two contiguous open reading frames coding for the precursors of ArChiA (881 amino acids [aa]) and ArChiB (578 aa). ArChiA and ArChiB are modular enzymes consisting of a glycosyl-hydrolase family 18 catalytic domain as well as two and one chitin-binding domains, respectively. The catalytic domain of ArChiA exhibits 55% identity with a chitodextrinase from Vibrio furnissii. The ArChiB catalytic domain exhibits 33% identity with chitinase A of Bacillus circulans. The ArChiA chitin-binding domains are homologous to the chitin-binding domain of ArChiB. ArChiA and ArChiB were purified to homogeneity from the native Arthrobacter strain and partially characterized. Thermal unfolding of ArChiA, ArChiB, and chitinase A of Serratia marcescens was studied using differential scanning calorimetry. ArChiA and ArChiB, compared to their mesophilic counterpart, exhibited increased heat lability, similar to other cold-adapted enzymes.

SUBMITTER: Lonhienne T

PROVIDER: S-EPMC95064 | biostudies-literature | 2001 Mar

REPOSITORIES: biostudies-literature

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Cloning, sequences, and characterization of two chitinase genes from the Antarctic Arthrobacter sp. strain TAD20: isolation and partial characterization of the enzymes.

Lonhienne T T Mavromatis K K Vorgias C E CE Buchon L L Gerday C C Bouriotis V V

Journal of bacteriology 20010301 5

Arthrobacter sp. strain TAD20, a chitinolytic gram-positive organism, was isolated from the sea bottom along the Antarctic ice shell. Arthrobacter sp. strain TAD20 secretes two major chitinases, ChiA and ChiB (ArChiA and ArChiB), in response to chitin induction. A single chromosomal DNA fragment containing the genes coding for both chitinases was cloned in Escherichia coli. DNA sequencing analysis of this fragment revealed two contiguous open reading frames coding for the precursors of ArChiA (8 ...[more]

PMID: 11160110

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Project description:BackgroundChitin is an important biopolymer next to cellulose, extracted in the present study. The exoskeleton of marine bycatch brachyuran crabs, namely Calappa lophos, Dromia dehaani, Dorippe facchino and also from stomatopod Squilla spp. were used to extract chitin through fermentation methods by employing two bacterial strains such as Pseudomonas aeruginosa, Serratia marcescens. The yield of chitin was 44.24%, 37.45%, 11.56% and 27.24% in C. lophos, D. dehaani, D. facchino and Squilla spp. respectively. FT-IR spectra of the produced chitin exhibit peaks which is more or less coherent to that of standard chitin which is further analysed by Scanning Electron Microscope. The quality of produced chitin was assessed through moisture, protein, ash and lipid content analysis ensured that chitin obtained from trash crustaceans are on par with that of standard chitin.ResultsA total of 10 samples were collected from different areas of Jiangsu China for screening of chitinase-producing bacteria. Based on the clearance zone, two of the best samples were chosen for further study. 16S rRNA sequence analysis showed that this strain belongs to genus Myxococcus and species Myxococcus fulvus. Phylogenetic analysis was performed and it shows strain UM01 is a novel bacterial strain. UM01 isolate shows maximum chitinase production at 35 °C and 8 pH. Among all, these colloidal chitins were found to be the best for chitinase production. Three chitinase-producing genes were identified and sequenced by using degenerative plasmid. UMCda gene (chitin disaccharide deacetylase) was cloned into E. coli DH5a by using PET-28a vector, and antagonistic activity was examined against T. reesei.ConclusionTo our knowledge, this is the earliest study report to gene cloning and identification of the chitinase gene in Myxococcus fulvus. Chitinase plays a key role in decomposition and utilization of chitin as a raw material. This research indicates that Myxococcus fulvus UM01 strain is a novel myxobacteria strain and can produce large amounts of chitinase within a short time. The UMCda gene cloned into E. coli DH5a showed a promising effect as antifungal activity. In overall findings, the specific strain UM01 has endowed properties of bioconversation of waste chitin and other biological applications.

Dataset Information

Cloning, sequences, and characterization of two chitinase genes from the Antarctic Arthrobacter sp. strain TAD20: isolation and partial characterization of the enzymes.

Publications

Cloning, sequences, and characterization of two chitinase genes from the Antarctic Arthrobacter sp. strain TAD20: isolation and partial characterization of the enzymes.

Similar Datasets

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