当前位置： SCI文献检索 > PROTEIN EXPRESSION AND PURIFICATION期刊下所有文献 > Production of enzymatically active recombinant full-length barley high pI alpha-glucosidase of glycoside family 31 by high cell-density fermentation of Pichia pastoris and affinity purification.

Production of enzymatically active recombinant full-length barley high pI alpha-glucosidase of glycoside family 31 by high cell-density fermentation of Pichia pastoris and affinity purification.

Abstract：

:Recombinant barley high pI alpha-glucosidase was produced by high cell-density fermentation of Pichia pastoris expressing the cloned full-length gene. The gene was amplified from a genomic clone and exons (coding regions) were assembled by overlap PCR. The resulting cDNA was expressed under control of the alcohol oxidase 1 promoter using methanol induction of P. pastoris fermentation in a Biostat B 5 L reactor. Forty-two milligrams alpha-glucosidase was purified from 3.5 L culture in four steps applying an N-terminal hexa-histidine tag. The apparent molecular mass of the recombinant alpha-glucosidase was 100 kDa compared to 92 kDa of the native barley enzyme. The secreted recombinant enzyme was highly stabile during the 5-day fermentation and had significantly superior specific activity of the enzyme purified previously from barley malt. The kinetic parameters Km, Vmax, and kcat were determined to 1.7 mM, 139 nM x s(-1), and 85 s(-1) using maltose as substrate. This work presents the first production of fully active recombinant alpha-glucosidase of glycoside hydrolase family 31 from higher plants.

journal_name

Protein Expr Purif

journal_title

Protein expression and purification

authors

Naested H,Kramhøft B,Lok F,Bojsen K,Yu S,Svensson B

doi

10.1016/j.pep.2005.10.008

keywords:

subject

Has Abstract

pub_date

2006-03-01 00:00:00

pages

56-63

issue

1

eissn

1046-5928

issn

1096-0279

pii

S1046-5928(05)00353-0

journal_volume

46

pub_type

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