Structural and functional analyses of Pcal_0917, an a-glucosidase from hyperthermophilic archaeon Pyrobaculum calidifontis

Genome analysis of Pyrobaculum calidifontis revealed the presence of a-glucosidase (Pcal_0917) gene. Structural analysis affirmed the presence of signature sequences of Type II alpha-glucosidases in Pcal_0917. We have heterol-ogously expressed the gene and produced recombinant Pcal_0917 in Escherichia coli. Biochemical characteristics of the recombinant enzyme resembled to that of Type I alpha-glucosidases, instead of Type II. Recombinant Pcal_0917 existed in a tetrameric form in solution and displayed highest activity at 95 degrees C and pH 6.0, independent of any metal ions. A short heat-treatment at 90 degrees C resulted in a 35 % increase in enzyme activity. A slight structural shift was observed by CD spectrometry at this temperature. Half-life of the enzyme was >7 h at 90 degrees C. Pcal_0917 exhibited apparent V-max values of 1190 +/- 5 and 3.9 +/- 0.1 U/mg against p-nitrophenyl alpha-D-glucopyranoside and maltose, respectively. To the best of our knowledge, Pcal_0917 displayed the highest ever reported p-nitrophenyl a-D-glucopyranosidase activity among the characterized counterparts. Moreover, Pcal_0917 displayed trans-glycosylation activity in addition to a-glucosidase activity. Furthermore, in combination with alpha-amylase, Pcal_0917 was capable of producing glucose syrup from starch with >40 % glucose content. These properties make Pcal_0917 a potential candidate for starch hydrolyzing industry.

Automated summary

Genome analysis of Pyrobaculum calidifontis identified a gene called a-glucosidase (Pcal_0917), which was confirmed to have Type II alpha-glucosidase characteristics. The gene was heterologously expressed and the resulting recombinant enzyme displayed similar characteristics to Type I alpha-glucosidases. The enzyme existed as a tetramer and showed high activity at high temperature and acidic pH. It also exhibited the highest reported p-nitrophenyl alpha-D-glucopyranosidase activity and demonstrated trans-glycosylation activity. In combination with alpha-amylase, it could produce glucose syrup with a high glucose content, suggesting its potential for the starch hydrolyzing industry.