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Purification, characterization and substrate specificity of thermostable α-galactosidase from Bacillus stearothermophilus (NCIM-5146)

Paper ID Volume ID Publish Year Pages File Format Full-Text
36339 45129 2006 7 PDF Available
Title
Purification, characterization and substrate specificity of thermostable α-galactosidase from Bacillus stearothermophilus (NCIM-5146)
Abstract

An extracellular thermostable α-galactosidase from Bacillus stearothermophilus (NCIM-5146) has been purified to homogeneity by chromatographic step, using Phenyl Sepharose CL-4B column. The specific activity of the enzyme was increased approximately 389-fold, from 1.03 U/mg protein to 400 U/mg protein. The molecular mass of the purified enzyme as determined by SDS-PAGE and gel filtration was 79.9 and 165.9 kDa, respectively, suggesting dimeric nature. The purified α-galactosidase is a non-glycosylated protein with a pI of 4.9. The pH and temperature optima for the purified enzyme are 6.5–7.0 and 65 °C, respectively. The α-galactosidase is stable over a broad pH range (3–9) and its half-life of inactivation (t1/2) at 70 °C is 30 min. The partial N-terminal sequence of α-galactosidase showed remarkable homology (80% similarity) with earlier reported α-galactosidase from B. stearothermophilus NUB 3621. The secondary structure of the enzyme determined by circular dichroism (CD) spectroscopy exhibited α/β class of protein and showed temperature induced conformational forms below and above the transition temperature. The purified enzyme showed biphasic Arrhenius plot with break point at 55 °C for pNPG and 50 °C for melibiose, raffinose and stachyose. The enzyme hydrolyzes α-1-3, α-1-4, and α-1-6 galactosidic linkages and not the β-galactosidic linkages. Synthetic substrates pNPG and oNPG had lower Km and higher Kcat as compare to natural substrates, melibiose, raffinose, and stachyose.

Keywords
CD, circular dichroism; α-d-gal-(1-3)-d-gal, 3-O-α-d-galactopyranosyl-d-galactobiose; α-d-gal-(1-4)-d-gal, 4-O-α-d-galacopyranosyl-d-galactopyranose; pNP, p-nitrophenyl; mNP, m-nitrophenyl; TLC, thin layer chromatography; BSA, bovine serum albumin; PCMB,
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Purification, characterization and substrate specificity of thermostable α-galactosidase from Bacillus stearothermophilus (NCIM-5146)
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Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 41, Issue 6, June 2006, Pages 1311–1317
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us