Abstract
BackgroundProduct inhibition can reduce catalytic performance of enzymes used for biofuel production. Different mechanisms can cause this inhibition and, in most cases, the use of classical enzymology approach is not sufficient to overcome this problem. Here we have used a semi-rational protein fusion strategy to create a product-stimulated enzyme.ResultsA semi-rational protein fusion strategy was used to create a protein fusion library where the Bacillus subtilis GH11 xylanase A (XynA) was inserted at 144 surface positions of the Escherichia coli xylose binding protein (XBP). Two XynA insertions at XBP positions 209 ([209]XBP-Xyn-XBP) and 262 ([262]XBP-Xyn-XBP) showed a 20% increased xylanolytic activity in the presence of xylose, conditions where native XynA is inhibited. Random linkers of 1-4 Gly/Ala residues were inserted at the XynA N- and C-termini in the [209]XBP and [262]XBP, and the chimeras 2091A and 2621B were isolated, showing a twofold increased xylanolytic activity in the presence of xylose and kcat values of 200 and 240 s−1 in the 2091A and 2621B, respectively, as compared to 70 s−1 in the native XynA. The xylose affinity of the XBP was unchanged in the chimeras, showing that the ~3- to 3.5-fold stimulation of catalytic efficiency by xylose was the result of allosteric coupling between the XBP and XynA domains. Molecular dynamics simulations of the chimeras suggested conformation alterations in the XynA on xylose binding to the XBP resulted in exposure of the catalytic cavity and increased mobility of catalytic site residues as compared to the native XynA.ConclusionsThese results are the first report of engineered glycosyl hydrolase showing allosteric product stimulation and suggest that the strategy may be more widely employed to overcome enzyme product inhibition and to improve catalytic performance.Graphical abstractProtein fusion of a GH11 xylanase (in red) and a xylose binding protein (XBP, in blue) results in a xylanase-XBP chimera that presents allosteric activation of the xylanase activity by xylose (shown as a space-filled molecule bound to the xylanase-XBP chimera)Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0293-0) contains supplementary material, which is available to authorized users.
Highlights
Product inhibition can reduce catalytic performance of enzymes used for biofuel production
Semi‐rational insertion library construction and screening for xylose‐stimulation Initially, 144 positions in xylose binding protein (XBP) were defined as targets for insertion of XynA
It is observed that the insertions were distributed throughout the sequence of XBP (Fig. 1), and the selected clones with the highest activation effect showed an increase in XynA catalytic activity of approximately 20% in the presence of xylose (Table 1)
Summary
Product inhibition can reduce catalytic performance of enzymes used for biofuel production. Regulated enzymes present spatially distinct locations for regulation and catalysis and frequently present oligomeric states in which tertiary and quaternary structure changes transmitted across protein–protein interfaces can mediate the communication between allosteric effector binding and the modulation of catalytic activity. These attributes can be exploited by protein engineering strategies that aim to introduce fine modulation of catalytic activity without modification of the active site. A powerful method for constructing allosteric proteins is through random domain insertion, which is potentially a general strategy for introducing coupling between fused domains [8,9,10]
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