Purification, Enzymatic Characterization, and Nucleotide Sequence of a High-Isoelectric-Point α-Glucosidase from Barley Malt

Author:

Frandsen Torben Peter1,Lok Finn2,Mirgorodskaya Ekaterina3,Roepstorff Peter3,Svensson Birte1

Affiliation:

1. Department of Chemistry, Carlsberg Laboratory (T.P.F., B.S.), and

2. Department of Physiology (F.L.), Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK–2500 Copenhagen Valby, Denmark; and

3. Department of Molecular Biology, University of Southern Denmark, Odense University, Campusvej 55, DK–5230 Odense M, Denmark (E.M., P.R.)

Abstract

Abstract High-isoelectric-point (pI) α-glucosidase was purified 7,300-fold from an extract of barley (Hordeum vulgare) malt by ammonium sulfate fractionation, ion-exchange, and butyl-Sepharose chromatography. The enzyme had high activity toward maltose (k  cat = 25 s−1), with an optimum at pH 4.5, and catalyzed the hydrolysis by a retaining mechanism, as shown by nuclear magnetic resonance. Acarbose was a strong inhibitor (K  i = 1.5 μm). Molecular recognition revealed that all OH-groups in the non-reducing ring and OH-3 in the reducing ring of maltose formed important hydrogen bonds to the enzyme in the transition state complex. Mass spectrometry of tryptic fragments assigned the 92-kD protein to a barley cDNA (GenBank accession no. U22450) that appears to encode an α-glucosidase. A corresponding sequence (HvAgl97; GenBank accession no. AF118226) was isolated from a genomic phage library using a cDNA fragment from a barley cDNA library. HvAgl97 encodes a putative 96.6-kD protein of 879 amino acids with 93.8% identity to the protein deduced from U22450. The sequence contains two active site motifs of glycoside hydrolase family 31. Three introns of 86 to 4,286 bp interrupt the coding region. The four exons vary from 218 to 1,529 bp. Gene expression analysis showed that transcription reached a maximum 48 h after the start of germination.

Publisher

Oxford University Press (OUP)

Subject

Plant Science,Genetics,Physiology

同舟云学术

1.学者识别学者识别

2.学术分析学术分析

3.人才评估人才评估

"同舟云学术"是以全球学者为主线,采集、加工和组织学术论文而形成的新型学术文献查询和分析系统,可以对全球学者进行文献检索和人才价值评估。用户可以通过关注某些学科领域的顶尖人物而持续追踪该领域的学科进展和研究前沿。经过近期的数据扩容,当前同舟云学术共收录了国内外主流学术期刊6万余种,收集的期刊论文及会议论文总量共计约1.5亿篇,并以每天添加12000余篇中外论文的速度递增。我们也可以为用户提供个性化、定制化的学者数据。欢迎来电咨询!咨询电话:010-8811{复制后删除}0370

www.globalauthorid.com

TOP

Copyright © 2019-2024 北京同舟云网络信息技术有限公司
京公网安备11010802033243号  京ICP备18003416号-3