Characterization of a Thermally Stable β-galactosidase Produced by Thermophilic Anoxybacillus sp. AH1

Yıl 2021, , 130 - 136, 25.06.2021

Ömer Acer , Fatma Matpan Bekler

https://doi.org/10.46810/tdfd.835632

Cited By: 2

Öz

Thermostable β-galactosidases from thermophilic bacteria have attracted increasing interest to have various advantages in industrial and biotechnological applications. In this study, a highly thermally stable β-galactosidase produced by Anoxybacillus sp. AH1was purified and characterized. The highest enzyme production was achieved after the bacterium was incubated for 24 hours. The enzyme was purified by precipitation with ammonium sulphate dialysis, gel filtration chromatography using Sephadex G-75. After the purification steps, β-galactosidase was found to be purified 10.2-fold and a yield of 13.9%. The molecular mass of the galactosidase was estimated to be 75 kDa by SDS-PAGE. The purified enzyme was highly stable and retained at 71% of the original activity at 60 °C and 53% at 70 oC within 120 minutes. The Km and Vmax values of purified β-galactosidase were calculated as 1.249 mM and 0.5 μmol minutes-1, respectively. Ca2+, Zn2+, and Mg2+ significantly activated β-galactosidase activity, whereas enzyme activity was inhibited significantly by Cu+2 as well as by the metal ion chelators1,10-phenanthroline (phen) and ethylenediaminetetraacetic acid (EDTA). The Purified β-galactosidase activity was increased by PMSF (phenylmethylsulfonyl fluoride), PCMB (p-chloromercuribenzoic acid), DTT (dithiothreitol), and β-ME (β-mercaptoethanol) at 2 mM, but inhibited completely by NEM (N-ethylmaleimide) at 1 mM.

Anahtar Kelimeler

enzyme activity inhibition, thermostable B-galactosidase, enzyme purification, Anoxybacillus sp. AH1

Thermofilik Anoxybacillus sp. AH1'den Üretilen Termostabil β-galaktosidazın Karakterizasyonu

Öz

Termofilik bakterilerden elde edilen termostabil β-galaktosidazlar, endüstriyel ve biyoteknolojik uygulamalarda çeşitli avantajlara sahip oldukları için ilgi çekmektedir. Bu çalışmada, Anoxybacillus sp. AH1'den üretilen, oldukça termostabil olan β-galaktosidaz, saflaştırıldı ve karakterize edildi. En yüksek enzim üretimi, bakterinin 24 saat inkübe edilmesinden sonra elde edildi. Enzim, amonyum sülfat çöktürmesi, diyaliz ve jel filtrasyon kromatografisi (Sephadex G-75) kullanılırak saflaştırıldı. Saflaştırma aşamalarından sonra, β-galaktosidazın % 13,9 verimle 10,2 kata kadar saflaştırıldığı tespit edildi. β-galaktosidazın moleküler kütlesi, SDS-PAGE ile 75 kDa olarak tahmin edildi. Saflaştırılmış enzimin oldukça stabil olduğu ve 120 dakika sonunda 60 ° C'de orijinal aktivitenin% 71'ini, 70 ° C'de ise % 53'ünü koruduğu tespit edildi. Saflaştırılmış β-galaktosidazın Km ve Vmax değerleri sırasıyla 1,249 mM ve 0,5 μmol dakika-1 olarak hesaplandı. Ca2+, Zn2+ ve Mg2+ β-galaktosidaz aktivitesini önemli ölçüde aktive ederken, Cu2+ ve metal iyon şelatörleri, 1,10-phenanthroline (phen) ve ethylenediaminetetraacetic acid (EDTA) enzim aktivitesini önemli ölçüde inhibe etmiştir. Saflaştırılmış β-galaktosidaz aktivitesi 2 mM PMSF (phenylmethylsulfonyl fluoride), PCMB (p-chloromercuribenzoic acid), DTT (dithiothreitol), ve β-ME (β-mercaptoethanol) ile artar iken, 1 mM NEM (N-ethylmaleimide) ile tamamen inhibe edildiği belirlendi.

Anahtar Kelimeler

enzim aktivitesi inhibisyonu, termostabil b-galaltosidaz, enzim saflaştırma, Anoxybacillus sp. AH1.

Kaynakça

• 1. Rani V, Sharma P, Dev K. Characterization of thermally stable β-galactosidase from Anoxybacillus flavithermus and Bacillus licheniformis isolated from Tattapani Hotspring of North-Western Himalayas, India. Int. J. Curr. Microbiol. 2019; 8 (1): 2517-2542.
• 2. Banerjee G, Ray A, Das SK, Kumar R, Ray AK. Chemical extraction and optimization of intracellular β-galactosidase production from the bacterium Arthrobacter oxydans using Box-Behnken design of response surface methodology. Acta Alimentaria. 2016;45 (1):93-103.
• 3. Natarajan J, Christobell C, Kumar DM, Balakumaran MD, Kumar MR, Kalaichelvan, PT. Isolation and characterization of β-galactosidase Producing Bacillus sp. from dairy effluent. World Appl. Sci. J. 2012;17 (11):1466-1474.
• 4. Gül-Güven R, Kaplan A, Guven K, Matpan-Bekler F, Dogru M. Effects of various inhibitors on β-galactosidase purified from the thermoacidophilic Alicyclobacillus acidocaldarius subsp. rittmannii isolated from Antarctica. Biotechnol. Bioprocess Eng. 2011;16 (1):114-119.
• 5. Liu Z, Zhao C, Deng Y, Huang Y, Liu B, 2015. Characterization of a thermostable recombinant β-galactosidase from a thermophilic anaerobic bacterial consortium YTY-70. Biotechnol. Biotechnol. Equip. 2015;29 (3):547-554.
• 6. Chanalia P, Gandhi D, Attri P, Dhanda S. Purification and characterization of β-galactosidase from probiotic Pediococcus acidilactici and its use in milk lactose hydrolysis and galactooligosaccharide synthesis. Bioorg. Chem.2018; 77:176-189.
• 7. Gul-Guven R, Guven K, Poli A, Nicolaus B. Purification and some properties of a β-galactosidase from the thermoacidophilic Alicyclobacillus acidocaldarius subsp. rittmannii isolated from Antarctica. Enzyme Microb. Technol. 2007; 40 (6): 1570-1577.
• 8. Chen W, Chen H, Xia Y, Yang J, Zhao J, Tian F, et al. Immobilization of recombinant thermostable β-galactosidase from Bacillus stearothermophilus for lactose hydrolysis in milk. J. Dairy Sci. 2009; 92 (2): 491–498.
• 9. Princely S, Basha NS, Kirubakaran JJ, Dhanaraju, MD. Biochemical characterization, partial purification, and production of an intracellular beta-galactosidase from Streptococcus thermophilus grown in whey. Eur. J. Exp. Biol. 2013; 3 (2): 242-251.
• 10. Kong F, Wang Y, Cao S, Gao R, Xie G Cloning, purification and characterization of a thermostable β-galactosidase from Thermotoga naphthophila RUK-10. Process Bioche. 204; 49 (5): 775–82.
• 11. Acer Ö, Pirinççioğlu H, Bekler FM, Gül-Güven R, Güven K. 2015. Anoxybacillus sp. AH1, an α-amylase-producing thermophilic bacterium isolated from Dargeçit Hot Spring. Biologia. 2015; 70 (7): 853-862. 12. Matpan-Bekler F, Acer Ö, Güven, K. Production and purification of novel thermostable alkaline protease from Anoxybacillus sp. KP1. Cell. Mol. Biol. 2015; 61 (4): 113-120.
• 13. Karaoglu H, Yanmis D, Sal FA, Celik A, Canakci S, Belduz AO. 2013. Biochemical characterization of a novel glucose isomerase from Anoxybacillus gonensis G2T displays a high level of activity and thermal stability. J. Mol. Catal. 2013;97: 215–24.
• 14. Ay F, Karaoglu H, Inan K, Canakci S, Belduz AO, 2011. Cloning, purification, and characterization of a thermostable carboxy- lesterase from Anoxybacillus sp. PDF1. Protein Expr. Purif. 2011;80 (1): 74–9.
• 15. Chiş L, Hriscu M, Bica A, Toşa M, Nagy G, Róna G, et al. 2013. Molecular cloning and characterization of a thermostable esterase/lipase produced by a novel Anoxybacillus flavithermus strain. J. Gen. Appl. Microbiol. 2013;59 (2): 119-134.
• 16. Bakir ZB, Metin K. Purification and characterization of an alkali-thermostable lipase from thermophilic Anoxybacillus flavithermus HBB 134. J Microbiol Biotechnol, 2016;26 (6): 1087-97.
• 17. Matpan Bekler F, Yalaz S, Acer O, Guven K. Purification of thermostable β-galactosidase from Anoxybacillus sp. KP1 and estimation of the combined effect of some chemicals on enzyme activity using semiparametric errors in variables model. Fresenius Environ Bull. 2017;26: 2251-2259.
• 18. Matpan-Bekler F, Yalaz S, Güven RG, Acer O, Güven K 2018. Characterization of thermostable β-galactosidase from Anoxybacillus ayderensis and optimal design for enzyme inhibition using semiparametric EIV models. TOJSAT. 2018;8 (2): 32-37.
• 19. Lowry OH, Rosebrough NJ, Farr AL. Protein measurement with the folin phenol reagent. The Journal of Biological Chemistry. 1951;193 (1): 265–275.
• 20. Laemmli U. Cleavage of structural proteins during the assembly of the head of Bacteriphage T4. Nature. 1970;277: 680-685.
• 21. Osiriphun S, Jaturapiree P. Isolation and Characterization of β-galactosidase from the Thermophile B1.2. AJOFAI. 2009;04: 135-143.
• 22. Vidya B, Palaniswamy M, Angayarkanni J, Nawaz KA, Thandeeswaran M, Chaithanya, KK, et al. Purification and characterization of β-galactosidase from newly isolated Aspergillus terreus (KUBCF1306) and evaluating its efficacy on breast cancer cell line (MCF-7). Bioorg. Chem. 2020; 94:103442.
• 23. Liu Y, Wu Z, Zeng X, Weng P, Zhang X., Wang, C. A novel cold-adapted phospho-beta-galactosidase from Bacillus velezensis and its potential application for lactose hydrolysis in milk. Int. J. Biol. Macromol. 2021;166: 760-770.
• 24. Huang J, Zhu S, Zhao L, Chen L, Du M, Zhang C, et al. A novel β- galactosidase from Klebsiella oxytoca ZJUH1705 for efficient production of galacto-oligosaccharides from lactose. Appl. Microbiol. Biotechnol. 2020;104: 6161-6172.
• 25. Di Lauro B, Strazzulli A, Perugino G, Cara FL, Bedini E, Corsaro MM, et al. Isolation and characterization of a new family 42 β-galactosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius: Identification of the active site residues. Biochim Biophys Acta Proteins Proteom. 2008;1784 (2): 292–301.
• 26. Murphy J, Ryan MP, Walsh G. Purification and characterization of a novel β galactosidase from the thermoacidophile Alicyclobacillus vulcanalis. Appl. Biochem. Biotechnol. 2020;191(3):1190-1206.
• 27. Ohtsu N, Motoshima H, Goto K, Tsukasaki F, Matsuzawa H.. Thermostable β-galactosidase from an extreme thermophile, Thermus sp. A4: Enzyme purification and characterization, and gene cloning and sequencing. Biosci. Biotechnol. Biochem. 1998;62 (8): 1539–45.
• 28. Ustok FI, Tari C, Harsa S. Biochemical and thermal properties of β-galactosidase enzymes produced by artisanal yoghurt cultures. Food Chem. 2010; 119 (3): 1114–20.