Dilek KAZAN, Aziz Akın DENİZCİ, Kamil Serkan UZYOL, Berna AKBULUT SARIYAR

Thermostable α-amylase from moderately halophilic Halomonas sp. AAD21

Isılkararlı α-amilaz üreticisi ılımlı halofilik Halomonas sp. AAD21, İzmir’de bulunan Çamaltı Tuzlası’ndan izole edilmiştir. Enzim üretimini arttırmak amacı ile NaCl, karbon ve azot kaynakları optimize edilmiştir. En yüksek enzim aktivitesi azot kaynağı olarak pepton, karbon kaynağı olarak nişastanın kullanıldığı besi yerinde, % 20 NaCl konsantrasyonunda elde edilmiştir. İzole edilen mikroorganizmanın patates kabuklarını da karbon kaynağı olarak kullandığı belirlenmiştir. Karbon ve azot kaynaklarının konsantrasyonu istatistiksel yaklaşımla optimize edilmiş ve α-amilaz aktivitesi 4,07 U $mL^ {-1}$ dak–1 den 26,25 U $mL^ {-1}$ dak–1 değerine yükselmiştir. En yüksek α-amilaz üretimi büyümenin 48.nci saatinde % 20 NaCl, % 4,12 nişasta, % 1 pepton, % 0,2 KCl, % 2 MgSO4·7H2O, ve % 0,03 trisodyum sitrat pentahidrat içeren besi yerinde elde edilmiştir. α-Amilazın optimum sıcaklık ve pH değeri sırası ile 50 °C ve pH 7 olarak belirlenmiştir. Ayrıca enzimin yüksek sıcaklıklarda kararlı olduğu da bulunmuştur. Ham enzim 50 °C ve 60 °C sıcaklıklarda iki saat inkübasyonda aktivitesinin tamamını korumaktadır. 90 °C ise 120 dakika inkübasyon sonunda ise aktivitesinin %70’i korunmuştur.

Ilımlı halofilik Halomonas sp. AAD21’in ısılkararlı α-amilazı

The moderately halophilic Halomonas sp. strain AAD21, which produces extracellular thermostable α-amylase, was isolated from the Çamaltı Saltern area located in İzmir, Turkey. NaCl, carbon, and nitrogen sources in the growth medium were optimized to enhance α-amylase yield. The highest enzyme yield was measured in the presence of 20% NaCl with peptone as the nitrogen and starch as the carbon sources in the fermentation broth. This microorganism was also found to utilize waste potato peel as a carbon source for α-amylase production. Concentrations of carbon and nitrogen sources were optimized using a statistical approach, and α-amylase activity increased from 4.07 U $mL^ {-1}$ min–1 to 26.25 U $mL^ {-1}$ min–1. Maximum α-amylase production was achieved at the end of 48 h of growth in the presence of 20% NaCl, 4.12% starch, 1.0% peptone, 0.2% KCl, 2% MgSO4·7H2O, and 0.03% trisodium citrate pentahydrate. The optimum pH and temperature of the α-amylase were found to be 7.0 and 50 °C, respectively. The α-amylase synthesized by Halomonas sp. AAD21 was also thermostable. Crude α-amylase did not lose its original activity after 2 h of incubation at 50 °C and 60 °C, and it retained 70% of its original activity after 120 min of incubation at 90 °C.

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1. Ventosa A, Nieto JJ, Oren A. Biology of moderately halophilic aerobic bacteria. Microbiol Mol Biol Rev 62: 504-544, 1998.
2. DasSarma S, Kennedy SP, Berquist B et al. Genomic perspective on the photobiology of Halobacterium species NRC-1, a phototropic, phototactic and UV-tolerant haloarcheon. Photosynth Res 70: 3-17, 2001.
3. Gomes J, Steiner W. The biocatalytic potential of extremophiles and extremozymes. Food Technol Biotechnol 42: 223-235, 2004.
4. Prakash B, Vidyasagar M, Madhukumar MS et al. Production, purification and characterization of two extremely halotolerant, thermostable, and alkali-stable α-amylases from Chromohalobacter sp. TVSP 101. Process Biochem,44: 210- 215, 2009.
5. Gupta R, Gigras P, Mohapatra H et al. Microbial α-amylases: a biotechnological perspective. Process Biochem 38: 1599-1616, 2003.
6. Kiran KK, Chandra TS. Production of surfactant and detergent-stable, halophilic, and alkalitolerant alpha-amylase by a moderately halophilic Bacillus sp. strain TSCVKK. Appl Microbiol Biotechnol 77: 1023-1031, 2008.
7. Bozic N, Ruiz J, Lopez-Santin J et al. Production and properties of the highly efficient raw starch digesting α-amylase from a Bacillus licheniformis ATCC 9945a. Biochem Eng J 53: 203-209, 2011.
8. Wang SL, Liang YC, Liang TW. Purification and characterization of a novel alkali-stable α-amylase from Chryseobacterium taeanense TKU001, and application in antioxidant and prebiotic. Process Biochem 46: 745-750, 2011.
9. Onishi H, Hidaka O. Purification and some properties of amylase produced by a moderately halophilic Acinetobacter sp. Can J Microbiol 24: 1017-1023, 1978.
10. Onishi H, Sonada K. Purification and some properties of an extracellular amylase from moderate halophilic Micrococcus halobius. Appl Environ Microbiol 38: 616-620, 1979.
11. Kobayashi T, Kamekura M, Kanlayakrit W et al. Production, purifi cation and characterization of an amylase from the moderate halophile Micrococcus varians subspecies halophilus. Microbios 46: 165-177, 1986.
12. Onishi H. Halophilic amylase from a moderately halophilic Micrococcus. J Bacteriol 109: 570-574, 1972.
13. Khire JM. Production of moderately halophilic amylase by newly isolated Micrococcus sp. 4 from a salt-pen. Lett Appl Microbiol 19: 210-212, 1994.
14. Coronado M, Vargas C, Hofemeister J et al. Production and biochemical characterization of an α-amylase from the moderate halophile Halomonas meridiana. FEMS Microbial Lett 183: 67-71, 2000.
15. Deutch CE. Characterization of a salt tolerant extracellular α-amylase from Bacillus dipsosauri. Lett Appl Microbiol 35: 78-84, 2002.
16. Amoozegar MA, Malekzadeh F, Malik KA. Production of amylase by newly isolated moderate halophile Halobacillus sp. strain MA-2. J Microbiol Methods 52: 353-359, 2003.
17. Mukherjee AK, Borah M, Rai SK. To study the influence of different components of fermentable substrates on induction of extracellular α-amylase synthesis by Bacillus subtilis DM- 03 in solid-state fermentation and exploration of feasibility for inclusion of α-amylase in laundry detergent formulations. Biochem Eng J 43: 149-156, 2009.
18. Rao RS, Kumar CG, Prakasham RS et al. The Taguchi methodology as a statistical tool for biotechnological applications: a critical appraisal. Biotechnol J 3: 510-523, 2008.
19. Grant WD, Kamekura M, McGenity TJ et al. Class III. Halobacteria, Order I. Halobacteriales. In: Garrity GM. ed. Bergey’s Manual of Systematic Bacteriology. Vol 1: The Archaea and the Deeply Branching and Phototrophic Bacteria. Springer; 2001: pp. 294-334.
20. Gerhardt P, Murray RGE, Costilow RN et al. Manual of Methods for General Bacteriology. American Society for Microbiology. Washington, D.C.; 1981.
21. Prescott LM, Harley JP, Klein DA. Microbiology. Wm. C. Brown. Dubuque, IA, USA; 1993.
22. Gonzalez C, Gutierrez C, Ramirez C. Halobacterium vallismortis sp. nov. An amylolytic and carbohydratemetabolizing, extremely halophilic bacterium. Can J Microbiol 24: 710-715, 1978.
23. Brenner DJ, Krieg NR, Staley JT et al. eds. Bergey’s Manual of Determinative Bacteriology, Vol. 2. Williams and Wilkins. Baltimore; 1986.
24. Altschul SF, Madden, TL, Schaffer AA et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389-3402, 1997.
25. Tamura K, Dudley J, Nei M et al. MEGA 4: Molecular Evolutionary Genetics Analysis (MEGA) soft ware version 4.0. Mol Biol Evol 24: 1596-1599, 2007.
26. Thompson JD, Gibson TJ, Plewniak F et al. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876-4882, 1997.
27. Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequence. J Mol Evol 16: 111-120, 1980.
28. Kimura M. The Neutral Theory of Molecular Evolution. Cambridge University. Cambridge; 1983.
29. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791, 1985.
30. Mehrotra S, Pandey PK, Gaur R et al. The production of alkaline protease by a Bacillus species isolate. Bioresour Technol 67: 201-203, 1999.
31. Bernfeld P. Amylases, alpha and beta. Methods Enzymol 1: 149-158, 1955.
32. Dobrev GT, Pishtiyski IG, Stanchev VS et al. Optimization of nutrient medium containing agricultural wastes for xylanase production by Aspergillus niger B03 using optimal composite experimental design. Bioresour Technol 98: 2671-2678, 2007.
33. Wejse PL, Ingvorsen K, Mortensen KK. Xylanase production by a novel halophilic bacterium increased 20-fold by response surface methodology. Enzyme Microb Technol 32: 721-727, 2003.
34. García MT, Mellado E, Ostos JC et al. Halomonas organivorans sp. nov., a moderate halophile able to degrade aromatic compounds. Int J Syst Evol Microbiol 54: 1723-1728, 2004.
35. Mata A, Canovas JM, Quesada E et al. A detailed phenotypic characterisation of the type strains of Halomonas species. System Appl Microbiol 25: 360-375, 2002.
36. Arahal DR, Vreeland RH, Litchfield CD et al. Recommended minimal standards for describing new taxa of the family Halomonadaceae. Int J Syst Evol Microbiol 57: 2436-2446, 2007.
37. Shukla J, Kar R. Potato peel as a solid state substrate for thermostable α-amylase production by thermophilic Bacillus isolates. World J Microbiol Biotechnol 22: 417-422, 2006.
38. Patel S, Jain N, Madamwar D. Production of α-amylase from Halobacterium halobium. World J Microbiol Biotechnol 9: 25- 28, 1993.
39. Sivaramakrishnan S, Gangadharan D, Nampoothiri KM et al. α-Amylases from microbial sources - an overview on recent developments. Food Technol Biotechnol 44: 173-184, 2006.