Abstract：
:The first gene (alpha-gal1) encoding an extracellular alpha-Dgalactosidase from the thermophilic fungus Talaromyces emersonii was cloned and characterized. The alpha-gal1 gene consisted of an open reading frame of 1,792 base pairs interrupted by six introns that encoded a mature protein of 452 amino acids, including a 24 amino acid secretory signal sequence. The translated protein had highest identity with other fungal alpha-galactosidases belonging to glycosyl hydrolase family 27. The alpha-gal1 gene was overexpressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast Pichia pastoris. Recombinant alpha-Gal1 was secreted into the culture medium as a monomeric glycoprotein with a maximal yield of 10.75 mg/l and purified to homogeneity using Hisbinding nickel-agarose affinity chromatography. The purified enzyme was maximally active at 70 degrees C, pH 4.5, and lost no activity over 10 days at 50 degrees C. alpha-Gal1 followed Michaelis-Menten kinetics (Vmax of 240.3 micronM/min/mg, Km of 0.294 mM) and was inhibited competitively by galactose (Km obs of 0.57 mM, Ki of 2.77 mM). The recombinant T. emersonii alpha-galactosidase displayed broad substrate preference, being active on both oligo- and polymeric substrates, yet had strict specificity for the alpha-galactosidic linkage. Owing to its substrate preference and noteworthy stability, alpha-Gal1 is of particular interest for possible biotechnological applications involving the processing of plant materials.
journal_name
J Microbiol Biotechnoljournal_title
Journal of microbiology and biotechnologyauthors
Simila J,Gernig A,Murray P,Fernandes S,Tuohy MGsubject
Has Abstractpub_date
2010-12-01 00:00:00pages
1653-63issue
12eissn
1017-7825issn
1738-8872pii
JMB020-12-07journal_volume
20pub_type
杂志文章abstract：:Cycloisomaltooligosaccharide glucanotransferase (CITase) was isolated from alkaliphilic Paenibacillus daejeonensis via an amino acid homology search for the reported CITase. The recombinant alkaliphilic CITase (PDCITase) from P. daejeonensis was expressed in an Escherichia coli expression system and purified as a sing...
journal_title：Journal of microbiology and biotechnology
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journal_title：Journal of microbiology and biotechnology
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journal_title：Journal of microbiology and biotechnology
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journal_title：Journal of microbiology and biotechnology
pub_type： 杂志文章,评审
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journal_title：Journal of microbiology and biotechnology
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journal_title：Journal of microbiology and biotechnology
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journal_title：Journal of microbiology and biotechnology
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更新日期：2010-02-01 00:00:00
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journal_title：Journal of microbiology and biotechnology
pub_type： 杂志文章,评审
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journal_title：Journal of microbiology and biotechnology
pub_type： 杂志文章
doi：
更新日期：2011-06-01 00:00:00
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journal_title：Journal of microbiology and biotechnology
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journal_title：Journal of microbiology and biotechnology
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更新日期：2009-10-01 00:00:00
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journal_title：Journal of microbiology and biotechnology
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更新日期：2008-09-01 00:00:00
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journal_title：Journal of microbiology and biotechnology
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journal_title：Journal of microbiology and biotechnology
pub_type： 杂志文章
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