Identification of a Recombinant Inulin Fructotransferase (Difructose Dianhydride III Forming) from Arthrobacter sp. 161MFSha2.1 with High Specific Activity and Remarkable Thermostability

Abstract

Difructose dianhydride III (DFA III) is a functional carbohydrate produced from inulin by inulin fructotransferase (IFTase, EC 4.2.2.18). In this work, an IFTase gene from Arthrobacter sp. 161MFSha2.1 was cloned and expressed in Escherachia coli. The recombinant enzyme was purified by metal affinity chromatography. It showed significant inulin hydrolysis activity, and the produced main product from inulin was determined as DFA III by nuclear magnetic resonance analysis. The molecular mass of the purified protein was calculated to be 43 and 125 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively, suggesting the native enzyme might be a homotrimer. The recombinant enzyme showed maximal activity as 2391 units/mg at pH 6.5 and 55 °C. It displayed the highest thermostability among previously reported IFTases (DFA III forming) and was stable up to 80 °C for 4 h of incubation. The smallest substrate was determined as nystose. The conversion ratio of inulin to DFA III reached 81% when 100 g/L inulin was catalyzed by 80 nM recombinant enzyme for 20 min at pH 6.5 and 55 °C. All of these data indicated that the IFTase (DFA III forming) from Arthrobacter sp. 161MFSha2.1 had great potential for industrial DFA III production.

Probing the Role of Two Critical Residues in Inulin Fructotransferase (DFA III-Producing) Thermostability from Arthrobacter sp. 161MFSha2.1

“…As seen in Figure , the individual mutant protein bands on the SDS-PAGE gel were approximately 42 kDa. This agreed well with the molecular mass of wild-type As IFTase in our previous work . Wild-type As IFTase was also purified, and the SDS-PAGE result was similar to our previous work (data not shown).…”

“…However, in Bs IFTase, linkages between Thr-308, Ser-309, and Ala-333 (corresponding to Ser-308, Ser-309, and Ser-333 in As IFTase, respectively) were absent (Figure B). Therefore, in comparison with As IFTase, the lower thermostability (103 min half-life at 60 °C for Bs IFTase and 240 min half-life at 80 °C for As IFTase) may be partly ascribed to this absence. As shown in Figure S2, all of the IFTases (DFA III-forming) with GenBank accession had 82% sequence identity.…”

“…Induction, expression, and purification of the As IFTase mutants were carried out similarly to that of As FTase in our previous work …”

“…In this work, we constructed a model of IFTase from Arthrobacter sp. 161MFSha2.1 ( As IFTase) based on the Bs IFTase structure ( As IFTase, which is a homotrimer as Bs IFTase has been fully characterized in our previous investigation). In this model, two serine residues, Ser-309 and Ser-333, showed potential critical roles in stabilizing the structure.…”

Identification of a Novel Di-D-Fructofuranose 1,2’:2,3’ Dianhydride (DFA III) Hydrolysis Enzyme from Arthrobacter aurescens SK8.001

“…5–12 represented IFTases (DFA III-forming) from Arthrobacter sp. 161MFSha2.1 (WP_018778058) [ 49 ], A . aurescens SK 8.001 (ADJ19283.1) [ 31 ], Arthrobacter sp.…”