Purification and Biochemical Characterization of Phytase Enzyme from Lactobacillus brevis Isolated from Fresh Kashar Cheese

Year 2024, Volume: 13 Issue: 1, 123 - 127, 26.03.2024

Neslihan Dikbaş , Şeyma Alım , Sümeyra Uçar , Sevda Uçar , Ayşe Gül Kasapoğlu , Esma Yigider

https://doi.org/10.46810/tdfd.1440314

Abstract

In the last 20 years, phytase enzyme has attracted the attention of scientists in the fields of environmental protection, nutrition and biotechnology. Myo-inositol hexaphosphate phosphohydrolase (phytase), which is a type of phosphatase enzyme, catalyzes the hydrolysis of phytate into less phosphorylated inorganic phosphates and phytate. Phytases of microbial origin are widely used in biotechnological applications (paper industry, feed industry, food industry and soil improvement).
In the present study, phytase enzyme was partially purified from Lactobacillus brevis NM-34 strain isolated from fresh kashar cheese and the pH and temperature values at which the enzyme showed optimum activity were determined. L. brevis NM-34 showed a phytase activity of 243.80 U/mL as a result of ammonium sulfate precipitation. In the ammonium sulfate range (40-60%), where the highest phytase activity was observed, the protein concentration was measured as 0.989 mg/mL. Km and Vmax values of phytase enzyme were determined as 0.0146 mM and 1.6 µmol/min, respectively. The pH and temperature values at which the partially purified phytase showed optimum activity were found to be pH 5 and 50 °C, respectively. Based on the findings from our research, the enzyme purified from this bacterium was found to have unique properties that make it suitable for use in industrial applications.

Keywords

Lactobacillus brevis, Phytase, Enzyme purification

Taze Kaşar Peynirinden İzole Edilen Lactobacillus brevis'ten Fitaz Enziminin Kısmi Saflaştırılması ve Biyokimyasal Karakterizasyonu

Abstract

Son 20 yılda fitaz enzimi çevre koruma, beslenme ve biyoteknoloji alanlarında bilim insanlarının dikkatini çekmiştir. Fosfataz enzim grubunda yer alan miyo-inositol heksafosfat fosfohidrolaz (fitaz), fitatın daha az fosforile edilmiş inorganik fosfatlara ve fitata hidrolizini katalizler. Mikrobiyal kaynaklı fitazlar biyoteknolojik uygulamalarda (kâğıt endüstrisinde, yem sanayinde, gıda endüstrisinde ve toprak iyileştirmede) yaygın bir şekilde kullanılmaktadır. Mevcut çalışmada, taze kaşar peynirinden izole edilen Lactobacillus brevis NM-34 suşundan fitaz enzimi kısmi olarak saflaştırıldı ve enzimin optimum aktivite gösterdiği pH ve sıcaklık değerleri belirlendi. L. brevis NM-34 amonyum sülfat çöktürmesi sonucunda 243.80 U/mL’lik bir fitaz aktivitesi sergiledi. En yüksek fitaz aktivitesi görülen amonyum sülfat aralığında (%40-60) protein konsantrasyonu 0.989 mg/mL olarak ölçüldü. Fitaz enziminin Km ve Vmax değerleri sırasıyla 0.0146 mM ve 1.6 µmol/min olarak belirlendi. Kısmi olarak saflaştırılan fitazın optimum aktivite gösterdiği pH ve sıcaklık değerleri sırasıyla pH 5.0 ve 50 °C olarak bulundu. Araştırmamızdan elde edilen bulgulara dayanarak, bu bakteriden saflaştırılan enzimin endüstriyel uygulamalarda kullanılmasını uygun kılan kendine özgü niteliklere sahip olduğu tespit edilmiştir.

Keywords

Lactobacillus brevis, Fitaz, Enzim saflaştırması

References

• Mora-Villalobos JA, Montero-Zamora J, Barboza N, Rojas-Garbanzo C, Usaga J, Redondo-Solano M, et al. Multi-product lactic acid bacteria fermentations: A review. Ferment. 2020; 6(1):23.
• Wang Y, Wu J, Lv M, Shao Z, Hungwe M, Wang J, et al. Metabolism characteristics of lactic acid bacteria and the expanding applications in food industry. Front Bioeng Biotechnol.2021;9:612285.
• Mbandlwa P. LAB as probiotics and silage inoculants for enteric methane [dissertation]. University College Cork; 2022.
• Pirouzian HR, Pourjafar H, Probiotic Lactic Acid Bacteria 46. Handbook of Food Bioactive Ingredients;1473.
• Onoruoiza MA, Ayodele AM, David-Momoh TE, A Review on the Effects of Functional Food on Humans and Microorganisms. Int J Probiotics and Dietetics.2024; 4(1):01-14.
• Ayivi RD, Gyawali R, Krastanov A, Aljaloud SO, Worku M, Tahergorabi R, et al. Lactic acid bacteria: Food safety and human health applications. Dairy.2020;1(3):202-232.
• Mathur H, Beresford TP, Cotter PD, Health benefits of lactic acid bacteria (LAB) fermentates. Nutrients.2020; 12(6):1679.
• Dikbaş N, Uçar S, Alım Ş, Purification of phytase enzyme from Lactobacillus brevis and biochemical properties. Biol.2023;1-9.
• Handa V, Sharma D, Kaur A, Arya SK, Biotechnological applications of microbial phytase and phytic acid in food and feed industries. Biocatal Agric Biotechnol. 2020; 25:101600.
• Sharma N, Angural S, Rana M, Puri N, Kondepudi KK, Gupta N, Phytase producing lactic acid bacteria: Cell factories for enhancing micronutrient bioavailability of phytate rich foods. Trends Food Sci Technol.2020; 96:1-12.
• Karagöz FP, Demir Y, Kotan MŞ, Dursun A, Beydemir Ş, Dikbaş N, Purification of the phytase enzyme from Lactobacillus plantarum: The effect on pansy growth and macro–micro element content. Biotechnol Appl Biochem.2021; 68(5):1067-1075.
• Han R, Chen JY, He SX, Liu CJ, Dai ZH, Liu X, et al. Phytate and arsenic enhance each other’s uptake in As-hyperaccumulator Pteris vittata: root exudation of phytate and phytase, and plant uptake of phytate-P. Environ Sci Tech.2022; 57(1):190-200.
• Rizwanuddin S, Kumar V, Naik B, Singh P, Mishra S, Rustagi S, et al. Microbial phytase: Their sources, production, and role in the enhancement of nutritional aspects of food and feed additives. J Agric Food Res.2023;100559.
• Pragya Sharma KK, Kumar S, Manisha Singh D, Kumar V, Singh B, (2023). Enhanced production and immobilization of phytase from Aspergillus oryzae: a safe and ideal food supplement for improving nutrition. Lett Appl Microbiol. 2023;76(2): ovac077.
• Dikbaş N, Parlakova Karagöz F, Uçar S, Demir Y, Ornamental cabbage (Brassica oleracea var. acephala) responses to phytase enzyme purified from Lactobacillus coryniformis application. Biotechnol Appl Biochem.2023.
• Joudaki H, Aria N, Moravej R, Rezaei Yazdi M, Emami-Karvani Z, Hamblin, MR, Microbial Phytases: Properties and Applications in the Food Industry. Curr Microbiol. 2023; 80(12):374.
• Priyodip P, Prakash PY, Balaji S, Phytases of probiotic bacteria: characteristics and beneficial aspects. Indian J Microbiol. 2017; 57:148-154.
• Demir Y, Şenol Kotan M, Dikbaş N, Beydemir Ş, Phytase from Weissella halotolerans: purification, partial characterisation and the effect of some metals. Int J Food Prop. 2017;20(2):2127-2137.
• Bradford MM, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72(1-2):248-254.
• Caglayan C, Taslimi P, Türk C, Gulcin İ, Kandemir FM, Demir Y, et al. Inhibition effects of some pesticides and heavy metals on carbonic anhydrase enzyme activity purified from horse mackerel (Trachurus trachurus) gill tissues. Environ Sci Pollut Res. 2020; 27:10607-10616.
• Sanbuga E, Nadaroglu H, Dikbas N, Senol M, Cetin B, Purification, characterization of phytase enzyme from Lactobacillus plantarum bacteria and determination of its kinetic properties. Afr J Biotechnol. 2014;13(23).
• Demir Y, Dikbaş N, Beydemir Ş, Purification and biochemical characterization of phytase enzyme from Lactobacillus coryniformis (MH121153). Mol Biotechnol. 2018; 60:783-790.
• Sharma R, Kumar P, Kaushal V, Das R, Navani NK, A novel protein tyrosine phosphatase like phytase from Lactobacillus fermentum NKN51: cloning, characterization and application in mineral release for food technology applications. Bioresour Technol. 2018; 249:1000-1008.
• Bhagat D, Slathia PS, Raina N, Sharma P, Production of phytase from Lactobacillus paracasei strain and its probiotic profile. Indian J Exp Biol. 2019; 57:839–851
• Ahire JJ, Jakkamsetty C, Kashikar MS, Lakshmi SG, Madempudi RS, In vitro evaluation of probiotic properties of Lactobacillus plantarum UBLP40 isolated from traditional indigenous fermented food. Probiotics Antimicrob Proteins. 2021;13(5):1413-1424.
• Sandez Penidez SH, Velasco Manini MA, Gerez CL, Rollán GC, Partial characterization and purification of phytase from Lactobacillus plantarum CRL1964 isolated from pseudocereals. J Basic Microbiol. 2020;60(9):787-798.
• Georgiev D, Dobrev G, Shilev S, Purification and properties of a phytase from Candida melibiosica 2491. Emir J Food Agric.2018;927-934.
• Abdolshahi A, Yancheshmeh BS, Arabameri M, Marvdashti LM, Phytase from Bacillus sp. Strain LA12: isolation, purification and characterization. J Microbiol Biotechnol Food Sci. 2021;10(4):572-576.
• Sumengen M, Dincer S, Kaya A, Production and characterization of phytase from Lactobacillus plantarum. Food Biotechnol.2013;27(2):105-118.
• Arif A, Nawaz M, Rabbani M, Iqbal S, Mustafa A, Yousuf MR, et al. Screening, characterization and physicochemical optimization of phosphorus solubilization activity of potential probiotic Lactobacillus spp. Pak Vet J. 2018;38(3):316-320.