Abstract
Thirty bacterial isolates were obtained from different sources and sites at Jeddah, Saudi Arabia, on chitin agar medium; 9 of the 30 isolates were cultured in liquid medium containing chitin as sole carbon and nitrogen sources. Isolate SM21, which was isolated from shrimp shells, showed the best growth and chitinase production in liquid medium. According to its morphological, physiological and biochemical characteristics, SM21 belongs to the genus Streptomyces and was identified as Streptomyces anulatus SM21. Identification was confirmed using 16S rDNA analysis. The chitinase enzyme was precipitated with 80% NH4SO4 and purified using DEAE-cellulose ion exchange chromatography followed by Sephadex G-100 gel filtration. The molecular weight determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis was 28 kDa. Genetic improvement using the protoplast fusion technique was carried out between the identified Streptomyces isolate and Streptomyces coelicolor SM1. These two species, which have different resistance profiles to streptomycin and tetracycline (400 μg/ml and 10 μg/ml, respectively), were used in an intraspecific protoplast fusion using PEG 6000. The percentage of real protoplasts that could regenerate successfully was 71% for S. coelicolor SM1 and 80% for S. anulatus SM21. Out of three recombinant fusants obtained, one (named Fu3) showed higher chitinase production compared to both parents (5 fold increase).
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Agwa A, Aly MM, Bonaly R (2000) Isolation and characterization of two Streptomyces species produced non polyenic antifungal agents. J Union Arab Biol 7B:62–84
Anné J, Van Melleart L, Eyssen H (1990) Optimal conditions for efficient transformation of Streptomyces venezuelae protoplasts. Appl Microbiol Biotechnol 32:431–435
Apichaisataienchote B, Altenbuchner J, Buchenauer H (2005) Isolation and identification of Streptomyces fradiae SU-1 from Thailand and protoplast transformation with the chitinase B gene from OPC-131. Curr Microbiol 51:116–121
Baltz RH, Matsushima P (1981) Protoplast fusion in Streptomyces: conditions for efficient genetic recombination and cell regeneration. J Gen Microbiol 127:137–146
Bhattacharya D, Nagpure A, Gupta RK (2007) Bacterial chitinases: properties and potential. Crit Rev Biotechnol 27:21–28
Bradford MM (1976) A rapid and sensitive for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Butte W (1983) Rapid method for the determination of fatty acid profiles from fats and oils using trimethylsulphonium hydroxide for transesterification. J Chromatogr 261:142–145
Dahiya N, Tewari R, Hoondal GH (2006) Biotechnological aspects of chitinolytic enzymes: a review. Appl Microbiol Biotechnol 71:773–782
El-Sayed ESA, Ezzat SM, Ghaly MF, Mansour M, El-Bohey MA (2000) Purification and characterization of two chitinases from Streptomyces albovinaceus S-22. World J Microbiol Biotechnol 16:87–89
Faramarzi MA, Fazeli M, Tabatabaei Yazdi M, Adrangi S, Jami Al-Ahmadi K, Tasharrofi N, Aziz Mohseni F (2009) Optimization of cultural conditions for production of chitinase by a soil isolate of Massilia timonae. Biotechnology 8:93–99
Fenice M, Leuba JL, Federici F (1998) Chitinolytic enzymes activity of Penicillium janthinellum P9 in bench top bioreactor. J Ferment Bioeng 86:620–623
Gooday GW (1990) Physiology of microbial degradation of chitin and chitosan. Biodegradation 1:177–190
Hasegawa T, Takizawa M, Tanida S (1983) A rapid analysis for chemical grouping of aerobic actinomycetes. J Gen Appl Microbiol 29:319–322
Hoischen C, Gura K, Luge C, Gumpert J (1997) Lipid and fatty acid composition of cytoplasmic membranes from Streptomyces hygroscopicus and Its stable protoplast-type L form. J Bacteriol 179(11):3430–3436
Huang JH, Chen CJ, Su YC (1996) Production of chitinolytic enzymes from a novel species of Aeromonas. J Ind Microbiol Biotechnol 17:89–95
Keyhani NO, Li XB, Roseman S (2000) Chitin catabolism in the marine bacterium Vibrio furnissii. Identification and molecular cloning of a chitoporin. J Biol Chem 275:33068–33088
Khamna S, Yokota A, Peberdy JF, Lumyong S (2010) Indole-3-acetic acid production by Streptomyces sp. isolated from some Thai medicinal plant rhizosphere. Green Chem 12:35–38
Kim KJ, Yang YJ, Kim JG (2003) Purification and characterization of chitinase from Streptomyces sp. M-20. J Biochem Mol Biol 36(2):185–189
Kishore GK, Pande S, Podile AR (2005) Biological control of late leaf spot of peanut (Arachis hypogaea) with chinitolytic bacteria. Phytopathology 95:1157–1165
Konagaya Y, Tsuchiya C, Sugita H (2006) Purification and characterization of chitinases from Clostridium sp. E16 isolated from the intestinal tract of the South American sea lion (Otraia flavescens). Lett Appl Microbiol 43(2):187–193
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Madigan MT, Martinko JM (2006) Industrial microbiology. In: Madigan MT, Martinko JM (eds) Biology of microorganisms, 11th edn. Pearson Prentice Hall, Upper Saddle River, NJ
Mahadevan B, Crawford DL (1997) Properties of the chitinase of the antifungal biocontrol agent Streptomyces lydicus WYEC 108. Enzyme Microb Technol 20:489–493
Matsushima P, Baltz RH (1986) Protoplast fusion. In: Demain AL, Solomon NA (eds) Manual of industrial microbiology and biotechnology. American Society of Microbiology, Washington DC, pp 170–183
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–429
Narayana KJP, Vijayalakshmi M (2009) Chitinase production by Streptomyces sp. ANU 6277. Braz J Microbiol 40(10):725–733
Nawani NN, Kapadnis BP (2004) Production dynamics and characterization of chitinolytic system of Streptomyces sp. NK 1057, a well equipped chitin degrader. World J Microbiol Biotechnol 20:487–494
Nawani NN, Kapadinis BP, Das AD, Rao AS, Mahajan SK (2002) Purification and characterization of a thermophilic and acidophilic chitinase from Micromonospora sp. V2. J Appl Microbiol 93(6):965–975
Nazari R, Akbarzadeh A, Norouzian D, Farahmand B, Vaez J, Sadegil A, Hormozi F, Kiani-rad M, Zarbakhsh B (2005) Applying intra-specific protoplast fusion in Streptomyces griseoflavus to increase the production of Desferrioxamine B. Curr Sci 88(11):1815–1820
Ochi K (1982) Protoplast fusion permits high- frequency transfer of a Streptomyces determinant which mediates actinomycin synthesis. J Bacteriol 150(2):592–597
Pinto ADS, Barreto CC, Schrank A, Ulho ACJ, Vainstein MH (1997) Purification and characterization of an extracellular chitinase from the entomopathogen Metarhizium anisopliae. Can J Microbiol 43:322–327
Shirling EB, Gottlieb D (1966) Methods for characterization of Streptomyces species. Int J Syst Bacteriol 16:313–340
Tanabe T, Kawase T, Watanabe T, Uchida Y, Mitsutomi M (2000) Purification and characterization of a 49 kDa chitinase from Streptomyces griseus HUT 6037. J Biosci Bioeng 89:27–32
Tork S, Aly MM, Nawar L (2010) Biochemical and molecular characterization of a new local keratinase producing Pseudomomanas sp., MS21. Asian J Biotechnol 2(1):1–13
Trachuk LA, Revina LP, Shemyakina TM, Chestukhina GG, Stepanov VM (1996) Chitinases of Bacillus lichenitinolytic B-6839: isolation and properties. Can J Microbiol 42:307–315
Ulhoa CJ, Peberdy JF (1991) Regulation of chitinase synthesis in Trichoderma harzianum. J Gen Microbiol 14:2163–2169
Vetrivel KS, Dharmalingam K (2000) Isolation of a chitinase overproducing mutant of Streptomyces peucetius defective in daunorubicin biosynthesis. Can J Microbiol 46(10):956–960
Weisberg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S Ribosomal DNA amplification for phylogenetic study. J Bacteriol 173(2):697–703
Williams ST, Sharpe ME, Molt JC (1989) Bergey’s manual of systematic bacteriology, 4th edn. Williams and Wilkins, Baltimore, pp 2300–2648
Acknowledgment
This study was supported by Deanship of Scientific Research (Grant No. 17-022/ 429), King Abdul-Aziz University, Saudi Arabia.
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Aly, M.M., Tork, S., Al-Garni, S.M. et al. Chitinolytic enzyme production and genetic improvement of a new isolate belonging to Streptomyces anulatus . Ann Microbiol 61, 453–461 (2011). https://doi.org/10.1007/s13213-010-0158-5
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DOI: https://doi.org/10.1007/s13213-010-0158-5