Cloning, expression, and characteristic analysis of the novel β-galactosidase from silkworm, Bombyx mori.

Yongzhu Yi,Xingjian Liu,Haozhi Song,Haining Wang,Jialei Li,Yinü Li,Huan Zhang,Zhifang Zhang,Zhipeng Zong

doi:10.1002/dvg.23446

Abstract

β-Galactosidase is a critical exoglycosidase involved in the hydrolysis of lactose, the modification and degradation of glycoprotein in vivo. In this study, the β-galactosidase gene of silkworm (BmGal), whose cDNA comprises 11 exons and contains an intact ORF of 1,821 bp, was cloned. The protein sequence of BmGal showed high similarity with other known insect β-galactosidases. No activity of the BmGal expressed in Escherichia coli or Pichia pastoris was detected while it was successfully expressed with high enzyme activity in baculovirus expression system in silkworm, and the electrophoresis result revealed that the BmGal showed activity in oligomer mode. Enzyme activity assay showed that its optimum pH was 8.4 and its optimum temperature was 40 °C. What is more, we found that iron ions can stimulate the activity of the enzyme while cobalt, nickel, or lead ions can inhibit its activity significantly. Besides, the temporal-spatial transcription pattern of the BmGal mRNA level was analyzed, which showed that BmGal was transcribed at the highest level in the fifth larval instar but relatively low level in the pupal and adult stage, and the highest transcriptional level of BmGal was found in testis among all the tissues concerned.

Highlights

Introduction β-galactosidase (β-D-galactosidase, galactosidase, EC 3.2.1.23), called lactase, is a kind of glycoside hydrolase (GH), which catalyses the hydrolysis of β-D-galactopyranosides or transgalactosylation
Most research of β-galactosidase is limited to that encoded by the Escherichia coli lacZ gene,which is widely used as a reporter molecule in molecular biology in a wide variety of animals
Escherichia coli strains Top 10, BL21(DE3) and pET-28a, Pichia receptor strains GS115 and Pichiapastoris expression vector pPIC9-γ, baculovirus transfer vector pVL1393, Bmbacpak-6 virus containing the β-galactosidase gene of Escherichia coli, Bombyx mori cell line, parental virus BmBacmid made from Bombyx mori nucleopolyhedrovirus genome which lacks the ORF1629 gene [16, 17], Bm-N cell and silkworm variety JY-1 were all kept in our own laboratory

Summary

Introduction

Introduction β-galactosidase (β-D-galactosidase, galactosidase, EC 3.2.1.23), called lactase, is a kind of glycoside hydrolase (GH), which catalyses the hydrolysis of β-D-galactopyranosides or transgalactosylation. Most research of β-galactosidase is limited to that encoded by the Escherichia coli lacZ gene,which is widely used as a reporter molecule in molecular biology in a wide variety of animals. This enzyme is composed of four homologous monomers, each of which contains 1023 amino residue with a molecular weight of 116483 Da [4]. Studies on β-galactosidase in insects were limited and only a few paper reported in this area, mainly in Drosophila melanogaster. It was reported that Drosophila βgalactosidase showed enzymatic activity in the physiological conditions of living animals when they were ectopically expressed in a desired specific spatial and temporal pattern [10]. The temporal-spatial expression pattern of the BmGal was analyzed

Methods

Results

Conclusion