Isolation and characterization of soil chitinolytic bacteria

• Main Author: Ng, Wui Ming
• Format: Thesis
• Language: English; English
• Published: 2007
• Subjects: QR75-99.5 Bacteria
• Online Access: https://eprints.ums.edu.my/id/eprint/9139/1/24%20PAGES.pdf; https://eprints.ums.edu.my/id/eprint/9139/2/FULLTEXT.pdf; https://eprints.ums.edu.my/id/eprint/9139/
• Institution: Universiti Malaysia Sabah

Chitinases (EC 3.2.1.14) hydrolyze the β-1-4-linkages of chitin, the second most abundant biopolymer on earth. Chitinases have high commercial value and their genes have great potential in the development of plant protection scheme against phytopathogenic attacks from fungi and insects. Bacteria are considered as major chitinase producers. Sabah with its mega biodiversity is hypothesized to harbour chitinolytic bacteria with optimal chitinase activity. Isolating local soil chitinolytic bacteria was initiated in which soil from mangrove areas at Sungai Merajah and Abai Bay of Kota Belud, Marudu Bay of Kota Marudu and Sungai Teri of Kimanis were sampled and screened. In addition, desolated normal garden soil enhanced with chitinous materials at Kota Belud was also sampled. Soil suspensions with pH adjusted to pH6.5 was used as inocula, which is the average pH value of soil. Five different solid media supplemented with cycloheximide, an antifungal agent were used for screening. Chitinase Detection Agar was found to be the most suitable screening medium. Selection of bacteria was based on their ability to grow and produce distinct halo on chitin containing medium within the initial five days of incubation. Five isolates designated as BRI 1, BRI 2, BRI 8, BRI 13 and BRI 36 were considered potential as they produce huge halos during the incubation period. Analysis on their partial16S rONA fragments revealed that BRI 1, BRI 2, BRI 13 and BRI 36 belong to the phylum Actinobacteria and are closely related to the genus Streptomyces while BRI 8 belong to the phylum Proteobacteria. However, all the test isolates sporulate indicating that they are actinomycete. Furthermore Gram staining showed that they are Gram positive bacteria, a characteristic of actinomycete. Therefore, a thorough study is proposed for the placing of BRI 8 into its proper taxon. PCR generated amplicon of around 350bp, confirming the presence of family 19 chitinase gene in the genome of all the test isolates. However, amplicon of around 400bp for family 18 chitinase gene was only successfully generated from the genome of BRI 1, BRI 8, BRI 13 and BRI 36. A reverse genetic is proposed to solve the problem encountered for BRI 2. Crude chitinase activity revealed that BRI 1 (8.61 Unit), BRI2 (4.89 Unit), BRI 8 (4.17), BRI 13 (4.82 Unit) and BRI 36 (26.70 Unit) have higher activity compared to the commercially available chitinase from Streptomyces grise us, Sigma C6137 (2.54 Unit). BRI 36 showed the highest crude chitinase activity by 11 folds higher relative to that of Sigma C6137. Cloning of family 18 group A chitinase gene was initiated with BRI 13. However, problems were encountered during the preparation of DNA library. Proposed remedies were discussed. This opens a challenging experimental endeavour in isolating the open reading frame (ORF) of chitinase gene. ORF of chitinase genes can be used for various downstream applications such as in the development of transgenic crops with enhanced resistance towards fungal attacks, development of potent biopesticides and in the development of new strains of soil chitinolytic microbes to control soil fungal attacks on crops.