Recombinant expression, purification and characterization of Bombyx mori (Lepidoptera: Bombycidae) pyridoxal kinase

Title:

Recombinant expression, purification and characterization of Bombyx mori (Lepidoptera: Bombycidae) pyridoxal kinase

Creator:

Huang, Shuo-Hao, Ma, Wang, Zhang, Ping-Ping, Zhang, Jian-Yun, Xie, Yan-Feng, and Huang, Long-Quan

Identifier:

https://cdk.lib.cas.cz/client/handle/uuid:ec96b65e-a5a7-4267-84ea-ae99bc546216
uuid:ec96b65e-a5a7-4267-84ea-ae99bc546216

Subject:

Zoologie, entomologie, Bombyx mori, pyridoxal kinase, recombinant expression, purification, characterization, 2, and 59

Type:

article, články, model:article, and TEXT

Description:

Pyridoxal kinase (PLK; EC 2.7.1.35) is a key enzyme in the metabolism of vitamin B6 (VB6) in Bombyx mori. A fusion expressional vector pET-22b-BPLK-His was constructed using a sub-cloning technique, the recombinant B. mori PLK was then expressed in Escherichia coli, purified and characterized. Bioinformatics were used to deduce the protein structure and genomic organization of this enzyme. Using Ni Sepharose affinity column chromatography, the recombinant protein was purified to very high degree (approximately 90%). The recombinant PLK exhibits a high specific enzymatic activity (1800 nmol/min/mg of protein). The maximum catalytic activity of this enzyme was recorded over a narrow pH range (5.5-6.0) and Zn2+ is the most effective cation for catalysis under saturating substrate concentrations. When only triethanolamine is present as the cation, K+ is an activator of PLK. A double reciprocal plot of initial velocity suggests that the enzyme catalyses the reaction by means of a sequential catalytic mechanism. Under optimal conditions, the Km value for the substrates of ATP and pyridoxal are 57.9 ± 5.1 and 44.1 ± 3.9 µM. B. mori's genome contains a single copy of the PLK gene, which is 7.73 kb long and contains five exons and four introns, and is located on the eighth chromosome. The PLK may be a dimer with two identical subunits under native conditions, and it is hypothesized that each monomer contains eight α-helices (α1-8), nine β-strands (β1-9) and two segments of 310 helices. and Shuo-Hao HUANG, Wang MA, Ping-Ping ZHANG, Jian-Yun ZHANG, Yan-Feng XIE, Long-Quan HUANG.

Language:

English

Rights:

http://creativecommons.org/publicdomain/mark/1.0/
policy:public

Source:

European Journal of Entomology | 2011 Volume:108 | Number:1

Harvested from:

CDK

Metadata only:

false