Purification of beta-glucosidase from olive (Olea europaea L.) fruit tissue with specifically designed hydrophobic interaction chromatography and characterization of the purified enzyme


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Kara H., Sinan S., Turan Y.

JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES, cilt.879, sa.19, ss.1507-1512, 2011 (SCI-Expanded) identifier identifier identifier

  • Yayın Türü: Makale / Tam Makale
  • Cilt numarası: 879 Sayı: 19
  • Basım Tarihi: 2011
  • Doi Numarası: 10.1016/j.jchromb.2011.03.036
  • Dergi Adı: JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES
  • Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus
  • Sayfa Sayıları: ss.1507-1512
  • Anahtar Kelimeler: beta-Glucosidase, Hydrophobic interaction chromatography, Olive fruit, Purification, BRASSICA-NAPUS, EXPRESSION, BIOSYNTHESIS, MYROSINASE, TOLERANT, PLANTS, RICE, SEED
  • Bursa Uludağ Üniversitesi Adresli: Hayır

Özet

An olive (Olea europaea L) beta-glucosidase was purified to apparent homogeneity by salting out with ammonium sulfate and using specifically designed sepharose-4B-L-tyrosine-1-napthylamine hydrophobic interaction chromatography. The purification was 155 fold with an overall enzyme yield of 54%. The molecular mass of the protein was estimated as ca. 65 kDa. The purified beta-glucosidase was effectively active on p-/o-nitrophenyl-beta-b-glucopyranosides (p-/o-NPG) with K-m values of 2.22 and 14.11 mM and V-max values of 370.4 and 48.5 U/mg, respectively. The enzyme was competitively inhibited by delta-gluconolactone and glucose against p-NPG as substrate. The K-i and IC50 values of delta-gluconolactone were determined as 0.016 mM and 0.23 mM while the enzyme was more tolerant to glucose inhibition with Ki and IC50 values of 6.4 mM and 105.5 mM, respectively, for p-NPG. The effect of various metal ions on the purified beta-glucosidase was investigated. Of the ions tested, only the Fe2+ increased the activity while Cd2+ Pb2+ Cu2+, Ni+, and Ag+ exhibited different levels of inhibitory effects with Ki and IC50 values of 4.29 x 10(-4) and 0.38 x 10(-4), 1.26 x 10(-2) and 5.3 x 10(-3), 2.26 x 10(-4) and 6.1 x 10(-4), 1.04 x 10(-4) and 0.63 x 10(-4), 3.21 x 10(-3) and 3.34 x 10(-3) mM, respectively. (C) 2011 Elsevier B.V. All rights reserved.