Eur. J. Entomol. 108 (1): 25-34, 2011 | 10.14411/eje.2011.003

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

Shuo-Hao HUANG1,2, Wang MA1, Ping-Ping ZHANG3, Jian-Yun ZHANG3, Yan-Feng XIE1, Long-Quan HUANG*,1
1 Key Laboratory of Tea Biochemistry & Biotechnology of Ministry of Education and Ministry of Agriculture, Anhui Agricultural University, Hefei 230036, People's Republic of China
2 Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan
3 College of Life Science, Anhui Agricultural University, Hefei 230036, People's Republic of China

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.

Keywords: Bombyx mori, pyridoxal kinase, recombinant expression, purification, characterization

Received: February 10, 2010; Accepted: July 7, 2010; Published: January 3, 2011

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