Abstract
A glycoside hydrolase family 32 invertase from Bacillus sp. HJ14 was expressed in Escherichia coli. The purified recombinant enzyme (rInvHJ14) showed typical biochemical properties of low-temperature-active and alkaline enzymes: (i) rInvHJ14 was active and stable in the range of pH 7.0–9.5 with an apparent pH optimum of 8.0; (ii) rInvHJ14 was most active but not stable at 30–32.5 °C, with 19.7, 48.2 and 82.1% of its maximum activity when assayed at 0, 10 and 20 °C, respectively, and the Ea, ΔG* (30 °C), Km (30 °C) and kcat (30 °C) values for hydrolysis of sucrose by rInvHJ14 was 47.6 kJ mol−1, 57.6 kJ mol−1, 62.9 mM and 746.2 s−1, respectively. The enzyme also showed strong sucrose tolerance. rInvHJ14 preserved approximately 50% of its highest activity in the presence of 2045.0 mM sucrose. Furthermore, potential factors for low-temperature-active and alkaline adaptations of rInvHJ14 were presumed. Compared with more thermostable homologs, rInvHJ14 has a higher frequency of glycine residues and a longer loop but a lower frequency of proline residues (especially in a loop) in the catalytic domain. The catalytic pockets of acid invertases were almost negatively charged while that of alkaline rInvHJ14 was mostly positively charged.
Highlights
Invertases have been isolated from a variety of sources, including bacteria[17], yeast[12], fungi[10], higher plants[18] and animals[19]
A BLASTP search against NCBI protein database showed that InvHJ14 was most similar to a number of putative invertases translated from genome sequences
Compared with rInvHJ14, most invertases show properties of mesophilic or thermophilic enzymes as their optimal activities are at temperatures of equal to or higher than 45 °C, they are thermostable at 50 °C or higher than 50 °C, and they show no activity or less than 10% relative activity at 10 °C2,3,7,8,12,18,24,28–35
Summary
Invertases have been isolated from a variety of sources, including bacteria[17], yeast[12], fungi[10], higher plants[18] and animals[19]. Acid invertases have been the most extensively studied and isolated from various sources while much less is known about alkaline invertases which have been reported only from plants and cyanobacteria[2,4,5,20,21,22,23,24]. A new GH 32 invertase, designated InvHJ14, was discovered from Bacillus sp. The recombinant InvHJ14 (rInvHJ14) was tolerant to sucrose and showed typical properties of low-temperature-active and alkaline enzymes. The potential molecular adaptations to low-temperature and alkaline environments of the enzyme were presumed
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