High level expression of extracellular secretion of a β-glucosidase gene (PtBglu3) from Paecilomyces thermophila in Pichia pastoris

Protein Expression and Purification
Qiaojuan YanZhengqiang Jiang

Abstract

A novel β-glucosidase gene (designated PtBglu3) from Paecilomyces thermophila was cloned and sequenced. PtBglu3 has an open reading frame of 2,557 bp, encoding 858 amino acids with a calculated molecular mass of 90.9 kDa. The amino acid sequence of the mature polypeptide shared the highest identity (70%) to a glycoside hydrolase (GH) family 3 characterized β-glucosidase from Penicillium purpurogenum. PtBglu3 without the signal peptides was cloned into pPIC9K vector and successfully expressed in Pichia pastoris as an active extracellular β-glucosidase (PtBglu3). High activity of 274.4 U/ml was obtained by high cell-density fermentation, which is by far the highest reported yield for β-glucosidase. The recombinant enzyme was purified to homogeneity with 3.3-fold purification and a recovery of 68.5%. The molecular mass of the enzyme was estimated to be 116 kDa by SDS-PAGE, and 198.2 kDa by gel filtration, indicating that it was a dimer. Optimal activity of the purified enzyme was observed at pH 6.0 and 65 °C, and it was stable up to 60 °C. The enzyme exhibited high specific activity toward pNP-β-D-glucopyranoside, cellooligosaccharides, gentiobiose, amygdalin and salicin, and relatively lower activity against lichenan and laminari...Continue Reading

References

Jun 1, 1996·The Biochemical Journal·B Henrissat, A Bairoch
Dec 19, 2002·Critical Reviews in Biotechnology·Yukti BhatiaV S Bisaria
Oct 6, 2006·Applied Microbiology and Biotechnology·Jiong HongHidehiko Kumagai
Sep 17, 2009·Applied Microbiology and Biotechnology·Kristian B R M KroghLisbeth Olsson
Oct 9, 2009·Antonie van Leeuwenhoek·Miklós TakóTamás Papp
Jan 1, 2010·Applied Microbiology and Biotechnology·Marimuthu JeyaJung-Kul Lee
May 22, 2010·Cellular and Molecular Life Sciences : CMLS·James R Ketudat Cairns, Asim Esen
Aug 24, 2010·Blood Cells, Molecules & Diseases·Nick DekkerJohannes M F G Aerts
Apr 16, 2011·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Hatibe Ertürk KaraYusuf Turan

❮ Previous
Next ❯

Citations

May 23, 2014·Molecular Biotechnology·Sun Mee HongTakahiro Kusakabe
Jul 13, 2018·Microorganisms·Juan-José Escuder-RodríguezMaría-Isabel González-Siso
Dec 3, 2014·Enzyme and Microbial Technology·Yuhong HuangLene Lange

❮ Previous
Next ❯

Related Concepts

Related Feeds

Cardiac Glycosides

Cardiac glycosides are a diverse family of naturally derived compounds that bind to and inhibit na+/k+-atpase. Discover the latest research on cardiac glycosides heres.

Aspergillosis

Aspergillosis is the name given to a wide variety of diseases caused by infection by fungi of the genus Aspergillus. Aspergillosis occurs in chronic or acute forms which are clinically very distinct. Most cases of acute aspergillosis occur in patients with severely compromised immune systems. Chronic colonization or infection can cause complications in people with underlying respiratory illnesses. Discover the latest research on aspergillosis here.

Aspergillosis (ASM)

Aspergillosis is the name given to a wide variety of diseases caused by infection by fungi of the genus Aspergillus. Aspergillosis occurs in chronic or acute forms which are clinically very distinct. Most cases of acute aspergillosis occur in patients with severely compromised immune systems. Chronic colonization or infection can cause complications in people with underlying respiratory illnesses. Discover the latest research on aspergillosis here.