Purification, Characterization, and Heterologous Expression of a Thermostable β-1,3-1,4-Glucanase from Bacillus altitudinis YC-9

Abstract

Purification, characterization, gene cloning, and heterologous expression in Escherichia coli of a thermostable β-1,3-1,4-glucanase from Bacillus altitudinis YC-9 have been investigated in this paper. The donor strain B. altitudinis YC-9 was isolated from spring silt. The native enzyme was purified by ammonium sulfate precipitation, diethylaminoethyl-cellulose anion exchange chromatography, and Sephadex G-100 gel filtration. The purified β-1,3-1,4-glucanase was observed to be stable at 60 °C and retain more than 90% activity when incubated for 2 h at 60 °C and remain about 75% and 44% activity after incubating at 70 °C and 80 °C for 10 min, respectively. Acidity and temperature optimal for this enzyme was pH 6 and 65 °C. The open reading frame of the enzyme gene was measured to be 732 bp encoding 243 amino acids, with a predicted molecular weight of 27.47 kDa. The gene sequence of β-1,3-1,4-glucanase showed a homology of 98% with that of Bacillus licheniformis. After being expressed in E. coli BL21, active recombinant enzyme was detected both in the supernatants of the culture and the cell lysate, with the activity of 102.7 and 216.7 U/mL, respectively. The supernatants of the culture were used to purify the recombinant enzyme. The purified recombinant enzyme was characterized to show almost the same properties to the wild enzyme, except that the specific activity of the recombinant enzyme reached 5392.7 U/mg, which was higher than those ever reported β-1,3-1,4-glucanase from Bacillus strains. The thermal stability and high activity make this enzyme broad prospect for industry application. This is the first report on β-1,3-1,4-glucanase produced by B. altitudinis.

Directed evolution of a β‐1,3‐1,4‐glucanase from Bacillus subtilis MA139 for improving thermal stability and other characteristics

“…All of the K m values were lower than the wild-type enzyme, so it could be inferred that these mutant enzymes had higher substrate affinity. These mutant enzymes had good application prospects in the studies of b-1,3-1,4-glucanase, especially of enzyme 7-32 which had better thermal stability and activity simultaneously than enzymes produced in previous studies [42,43]. After analysis of their amino acid sequence, the obtained mutant enzyme 7-32 was shown to have one amino-acid substitution T113S.…”

Engineering of a thermostable β‐1,3‐1,4‐glucanase from Bacillus altitudinisYC‐9 to improve its catalytic efficiency

“…21 In previous work in our laboratory, a -1,3-1,4-glucanase gene from Bacillus altitudinis YC-9 was isolated, characterized and expressed in Escherichia coli. 22 The results showed that the enzyme had good thermostability and great application potential. In this study, we designed a use of directed evolution technology to improve the catalytic efficiency of -1,3-1,4-glucanase from Bacillus altitudinis YC-9.…”

“…In previous work in our laboratory, a β‐1,3‐1,4‐glucanase gene from Bacillus altitudinis YC‐9 was isolated, characterized and expressed in Escherichia coli . The results showed that the enzyme had good thermostability and great application potential.…”

Characterization of a GH family 8 β-1,3-1,4-glucanase with distinctive broad substrate specificity from Paenibacillus sp. X4

“…Several bacterial b-1,3-1,4-glucanase genes have been isolated from many Bacillus species such as B. subtilis (Qiao et al 2009;Jung et al, 2010;Furtado et al 2011), B. licheniformis (Teng et al 2006), B. halodurans (Akita et al 2005), B. circulans (Bueno et al 1990b), B. altitudinis (Mao et al 2013), and a Bacillus sp. (Kim et al 2013).…”