Purification, Characterization, and cDNA Cloning of a Prominent β-Glucosidase from the Gut of the Xylophagous Cockroach Panesthia angustipennis spadica

Abstract

In this study, a β-glucosidase (PaBG1b) with high specific activity was purified from gut extracts of the wood-feeding cockroach Panesthia angustipennis spadica using Superdex 75 gel filtration chromatography and High-Trap phenyl hydrophobic chromatography. The protein was purified 14-fold to a single band identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with an apparent molecular mass of 56.7 kDa. The specific activity of the purified enzyme was 708 μmol/min/mg protein using cellobiose as substrate. To the best of our knowledge, this is the highest specific activity reported among β-glucosidases to date. The purified PaBG1b showed optimal activity at pH 5.0 and retained more than 65 % of the activity between pH 4.0 and 6.5. The activity was stable up to 50 °C for 30 min. Kinetic studies on cellobiose revealed that the K _m was 5.3 mM, and the V _max was 1,020 μmol/min/mg. The internal amino acid sequence of PaBG1b was analyzed, and two continuous sequences (a total of 39 amino acids) of the C-terminal region were elucidated. Based on these amino acid sequences, a full-length cDNA (1,552 bp) encoding 502 amino acids was isolated. The encoded protein showed high similarity to β-glucosidases from glycoside hydrolase family 1. Thus, the current study demonstrated the potential of PaBG1b for application in enzymatic biomass-conversion as a donor gene for heterologous recombination of cellulase-producing agents (fungi or bacteria) or an additive enzyme for cellulase products based on the high-performance of PaBG1b as a digestive enzyme in cockroaches.

Keywords: cellobiase; glycoside hydrolase family 1; wood-feeding cockroach; β-glucosidase.

Fig. 1.

Adults and midsize nymphs of the wood-feeding cockroach Panesthia angustipennis spadica. A male adult (middle) lost its right antenna. Nymphs (right) exhibited medium sizes. First instar nymphs are born oviparously at around 5 mm in body length, and full-grown adults are around 30 mm in length.

Fig. 2.

Elution profile and distribution of β-glucosidase activity from the Superdex-75 gel-filtration column. Activities (lateral bars) are described as percentages of the peak value. The protein curve (broken line) indicates the relative absorbance (280 nm). Recovery is indicated by the horizontal bar.

Fig. 3.

Elution profiles of protein and β-glucosidase activity from the HiPrep Phenyl hydrophobic chromatography column. The inset shows SDS-PAGE of the eluted fractions (A-P) negatively stained with Oriole. The activity curve is presented as relative values (%) based on the highest activity in the fractions.

Fig. 4.

SDS-PAGE of purified β-glucosidase stained with Coomassie Brilliant Blue. S, Purified β-glucosidase; M, Precision Plus Protein Standards (Bio-Rad). The numbered molecular weight markers were used for estimation of the molecular weights of proteins in the sample.

Fig. 5.

Lineweaver-Burk plot of the β-glucosidase purified from the wood-feeding cockroach Panesthia angustipennis spadica. The concentration of the purified enzyme used for these plots was 0.012 mg/mL, and the regression line was given as: 1 / (β-glucosidase activity [U/mL]) = −0.21 × 1 / (cellobiose concentration [mM]) + 0.04.

Fig. 6.

cDNA and putative amino acid sequences of the β-glucosidase of the wood-feeding cockroach Panesthia angustipennis spadica. The bold characters in the amino acid sequence indicate consentaneity with the internal amino acid sequences. Double and single underlines with arrow heads indicate positions of the degenerate primers. Potential N-glycosylation sites are designated by “+” on the right side of the corresponding amino acids.