Pal ADWITTIYA, Arun Kumar AVINASH, Rajagopal BADRI, Dadhe KAJAL, Shivakumar SRIVIDYA, Ponnamma VOMSI, Prabhu ASHWINI

Mutagenesis of Bacillus thuringiensis IAM 12077 for increasing poly (-$beta$-) hydroxybutyrate (PHB) production

PHB biriktiren gram pozitif bir bakteri Bangalore’nin yerel bahçe topraklarından izole edildi. Morfolojik ve fizyolojik özellikleri ve 16S rDNa nükleotit dizi analizine (yaklaşık 15 kb) göre Bacillus thuringiensis IAM 12077 olarak tanımlandı. Bu çalışmada Bacillus thuringiensis IAM 12077’nin varsayılan mutant suşlarının PHB üretim kapasiteleri çalışıldı. 48 saat nutrient brothda büyüdüğünde % 10-15 kuru hücre ağırlığında PHB üretme yeteneğine sahip Bacillus thuringiensis IAM 12077 olarak tanımlanan bir Bacillus sp. rasgele mutagenlere [fiziksel (UV) ve kimyasal (akridin oranj)] tabi tutuldu. Total UV mutant taramasında, iki suş PHB veriminde bir azalma gösterirken, 19 varsayılan mutant suş PHB üretiminde ebeynlerin üzerinde artış göstermiştir. Bu mutantların, (B8) ebeveyn suşların üzerinde PHB üretiminde bir artış ile (% 24,68; 1,54 kat) PHB birikimi ümit verici görülmektedir. Kimyasal mutajenlerde PHB üretiminde varasayılan mutantların yalnızca iki tanesinde (B3, C2) bir artış gözlenirken, 6 tanesinde bir azalma, 3 tanesinde önemsiz değişiklikler görülmüştür. Birikimdeki artış UV mutant B8 suşu ile görülen artış ile (B3 ve C2 için sırasıyla % 19,69; 1,43 kat ve % 22,22; 1,62 kat) karşılaştırılabilirken, verimlerde aynı anda bir artış (sırasıyla 0,43g/l; 1,13 kat ve 0,6g/l; 1,57 kat) bulunamamıştır.

Poli (-$beta$-) hidroksibutirat üretiminin artışı için Bacillus thuringiensis IAM 12077'nin mutagenezi

A gram-positive bacterium that accumulated PHB was isolated from local garden soil in Bangalore. Based on its morphological and physiological properties, and nucleotide sequence (about 1.5 kb) of its 16S rDNA it was identified as Bacillus thuringiensis IAM 12077. In this study the PHB-producing capacity of putative mutant strains of Bacillus thuringiensis IAM 12077 was evaluated. A Bacillus sp. identified as a Bacillus thuringiensis IAM 12077 strain capable of producing 10%-15% dry cell weight (DCW) PHB when grown in nutrient broth for 48 h was subjected to random mutagenesis [physical (UV) and chemical (acridine orange)]. Among the UV-mutants screened, 19 putative mutants produced more PHB than the parental, while 2 strains produced less. Of these mutants, B8 exhibited promising PHB accumulation (24.68%; 1.54-fold) with more PHB production (1.3 g/l; 5.4-fold) than the parental strain. Chemical mutagenesis yielded putative mutants, of which 6 had a decrease, 3 had no change, and 2 had an increase (B3, C2) in PHB production. While the increase in accumulation (19.69%, 1.43-fold and 22.22%, 1.62-fold, respectively, for B3 and C2) was comparable to the increase shown by the UV mutant (B8), the yields did not concomitantly increase (0.43g/l, 1.13- fold and 0.6g/l, 1.57-fold, respectively).

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1. Anderson AJ, Haywood GW, Dawes EA. Biosynthesis and composition of bacterial polyhydroxyalkanoates. International Journal of Biological Macromolecules. 12:102-105, 1990.
2. Byrom D. Polymer synthesis by microorganisms: technology & economics. Trends Biotechnol. 5: 246-250, 1987.
3. Fiechter A. Plastics from bacteria & for bacteria: Poly (betahydroxyalkanoates) as natural, biocompatible & biodegradable polyesters. Springer-Verlag, New York, 77-93, 1990.
4. Lee SY. Plastic bacteria? Progress and prospects for polyhydroxyalkanonate production in bacteria. Trends in Biotechnology. 14: 431-438, 1996.
5. Belma A, Zehra Nur Y, Yavuz B. Determination of PHB growth quantities of certain Bacillus sp. isolated from soil. Turkish Electronic J Biotechnol Special issue, 24-30, 2002.
6. Hikmet K, Belma A, Zehra NK, et al. Production of PHB & differentiation of putative Bacillusmutant strains by SDS-PAGE of total cell protein. African J Biotechnol 2: 47-149, 2003.
7. Hori K, Kaneko M, Tanji Y et al. Construction of self-disruptive Bacillus megaterium in response to substrate exhaustion for PHB production. Appl Microbiol & Biotechnol. 59: 211-216, 2002.
8. Kofronova O, Ptackova L, Chaloupka J. Poly (3- hydroxybutyrate) granules in Bacillus megaterium. Folia Microbiologiya. 39: 166-167, 1994.
9. Lach DA, Sharma VK, Vary PS. Isolation & characterization of unique division of mutant of Bacillus megaterium. J Gen Microbiol. 136: 545-553, 1990.
10. Holt JG, Krieg NR, Sneath PHA et al. Bergey’s manual of determinative bacteriology, 1993, vol. 9, p. 559-564.
11. Law JH, Slepecky RA. Assay of Poly β-hydroxybutyric acid. J Bacteriol 82: 33-36, 1961.
12. Dave H, Ramakrishna C, Desai JD. Production of PHB by petrochemical activated sludge and Bacillus sp. IPCB-403. Ind J Exp Biol 34: 216-219, 1996.
13. Finlay RH, White DC. Polymeric beta-hydroxyalkanoates from environmental samples and Bacillus megaterium. Appl Environ Microbiol 45: 71-78, 1983.
14. Chen G, Konig K, Lafferty RM. Occurrence of poly-D (-)-3- hydroxyalkanoates in the genus Bacillus. FEMS Microb Lett 84: 173-176, 1991.