Home
Resources
Publications
High-level Expression of Barley beta-D-glucan Exohydrolase HvExoI From a Codon-Optimized cDNA in Pichia Pastoris

High-level Expression of Barley beta-D-glucan Exohydrolase HvExoI From a Codon-Optimized cDNA in Pichia Pastoris

Sukanya Luang, Maria Hrmova, James R Ketudat Cairns

Protein Expr Purif. 2010 Sep;73(1):90-8.

PMID: 20406687

Abstract:

The native beta-d-glucan exohydrolase isoenzyme ExoI from barley seedlings, designated HvExoI, was the first GH3 glycoside hydrolase, for which a crystal structure was determined. A precise understanding of relationships between structure and function in this enzyme has been gained by structural and enzymatic studies. To allow testing of hypotheses gained from these studies, an efficient system for expression of HvExoI in Pichia pastoris was developed using a codon-optimized cDNA. Protein expression at a temperature of 20 degrees C yielded a recombinant enzyme, designated rHvExoI, which had molecular masses of 70-110 kDa due to heavy glycosylation at Asn221, Asn498 and Asn600, the three sites of N-glycosylation in native HvExoI. Most of the N-linked carbohydrate could be removed from rHvExoI, resulting in N-deglycosylated rHvExoI with a substantially decreased molecular mass of 67 kDa. rHvExoI was able to hydrolyse barley (1,3;1,4)-beta-D-glucan, laminarin and lichenans. The catalytic efficiency value k(cat)/K(M) of rHvExoI with barley (1,3;1,4)-beta-D-glucan was similar to that reported for native HvExoI. Further, laminaribiose, cellobiose and gentiobiose were formed through transglycosylation reactions with 4-nitrophenyl beta-D-glucoside and barley (1,3;1,4)-beta-D-glucan. Overall, the biochemical properties of rHvExoI were similar to those reported for native HvExoI, although differences were seen in thermostabilities and hydrolytic rates of certain beta-linked glucosides.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP9041229	β-D-Glucan from barley		9041-22-9	Price

As a specialist in analytical chemical Products, Alfa Chemistry offers consumers a wealth of raw materials and a full range of technologies and services.

QUICK LINKS

HEADQUARTERS

Tel:

Fax:

Email: