Development of a new affinity chromatography method for purification of horseradish peroxidase enzyme

Gerni S., ÖZDEMİR H.

Biotechnology and Applied Biochemistry, cilt.71, sa.1, ss.202-212, 2024 (SCI-Expanded) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 71 Sayı: 1
  • Basım Tarihi: 2024
  • Doi Numarası: 10.1002/bab.2532
  • Dergi Adı: Biotechnology and Applied Biochemistry
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Academic Search Premier, Applied Science & Technology Source, BIOSIS, Biotechnology Research Abstracts, CAB Abstracts, Chemical Abstracts Core, Compendex, Computer & Applied Sciences, EMBASE, Environment Index, Food Science & Technology Abstracts, INSPEC, MEDLINE, Veterinary Science Database
  • Sayfa Sayıları: ss.202-212
  • Anahtar Kelimeler: affinity chromatography, enzyme inhibition, enzyme purification, horseradish peroxidase, hydroxamic acid
  • Atatürk Üniversitesi Adresli: Evet

Özet

In this study, benzohydroxamic acid molecules were synthesized from methyl 4-amino-2-methoxy, methyl 4-amino-3-nitro, methyl 4-amino-3-methyl, and methyl 4-amino-3-chloro benzoate molecules, and the horseradish peroxidase (HRP) enzyme was purified in one step using the affinity chromatography technique for the first time. The IC50 and Ki values for the 4-amino 3-methyl benzohydroxamic acid molecule were 0.136 and 0.132 ± 0.054 μM, respectively, while the IC50 and Ki values for the 4-amino-3-nitro benzohydroxamic acid molecule were 56.00 and 51.90 ± 9.90 μM, respectively. It was found that the IC50 and Ki values for the 4-amino-3-chloro benzohydroxamic acid molecule were 218.33 and 175.67 ± 43.78 μM, respectively, whereas the IC50 and Ki values for the 4-amino-2-methoxy benzohydroxamic acid molecule were 306.00 and 218.00 ± 68.80 μM, respectively. The HRP enzyme was synthesized from 4-amino-2-methoxy hydroxamic acid column with a 35.97% yield 601.13 times, 4-amino-3-nitro hydroxamic acid column, with a 14.00% yield 404.11 times, 4-amino-3-methyl hydroxamic acid column with an 8.70% yield 394.88 times, and 4-amino-3-chloro hydroxamic acid column with a 4.48% yield 284.85 times. Thus, the HRP enzyme was purified in a single step with hydroxamic acids, and its molecular weight was found to be 44 kDa. The optimum pH was 8.0, the optimum temperature was 15°C, and the optimum ionic strength was 0.4 M for the purified HRP enzyme.