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A xylose-stimulated xylanase-xylose binding protein chimera created by random nonhomologous recombination.

ABSTRACT: BACKGROUND:Saccharification of lignocellulosic material by xylanases and other glycoside hydrolases is generally conducted at high concentrations of the final reaction products, which frequently inhibit the enzymes used in the saccharification process. Using a random nonhomologous recombination strategy, we have fused the GH11 xylanase from Bacillus subtilis (XynA) with the xylose binding protein from Escherichia coli (XBP) to produce an enzyme that is allosterically stimulated by xylose. RESULTS:The pT7T3GFP_XBP plasmid containing the XBP coding sequence was randomly linearized with DNase I, and ligated with the XynA coding sequence to create a random XynA-XBP insertion library, which was used to transform E. coli strain JW3538-1 lacking the XBP gene. Screening for active XBP was based on the expression of GFP from the pT7T3GFP_XBP plasmid under the control of a xylose inducible promoter. In the presence of xylose, cells harboring a functional XBP domain in the fusion protein (XBP+) showed increased GFP fluorescence and were selected using FACS. The XBP+ cells were further screened for xylanase activity by halo formation around xylanase producing colonies (XynA+) on LB-agar-xylan media after staining with Congo red. The xylanase activity ratio with xylose/without xylose in supernatants from the XBP+/XynA+ clones was measured against remazol brilliant blue xylan. A clone showing an activity ratio higher than 1.3 was selected where the XynA was inserted after the asparagine 271 in the XBP, and this chimera was denominated as XynA-XBP271. The XynA-XBP271 was more stable than XynA at 55 °C, and in the presence of xylose the catalytic efficiency was ~3-fold greater than the parental xylanase. Molecular dynamics simulations predicted the formation of an extended protein-protein interface with coupled movements between the XynA and XBP domains. In the XynA-XBP271 with xylose bound to the XBP domain, the mobility of a ?-loop in the XynA domain results in an increased access to the active site, and may explain the observed allosteric activation. CONCLUSIONS:The approach presented here provides an important advance for the engineering enzymes that are stimulated by the final product.

SUBMITTER: Ribeiro LF

PROVIDER: S-EPMC4896006 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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A xylose-stimulated xylanase-xylose binding protein chimera created by random nonhomologous recombination.

Ribeiro Lucas Ferreira LF Tullman Jennifer J Nicholes Nathan N Silva Sérgio Ruschi Bergamachi SR Vieira Davi Serradella DS Ostermeier Marc M Ward Richard John RJ

Biotechnology for biofuels 20160606

<h4>Background</h4>Saccharification of lignocellulosic material by xylanases and other glycoside hydrolases is generally conducted at high concentrations of the final reaction products, which frequently inhibit the enzymes used in the saccharification process. Using a random nonhomologous recombination strategy, we have fused the GH11 xylanase from Bacillus subtilis (XynA) with the xylose binding protein from Escherichia coli (XBP) to produce an enzyme that is allosterically stimulated by xylose ...[more]

PMID: 27274356

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Project description: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 k cat 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).

Project description:Four unrelated families with the same unbalanced translocation der(4)t(4;11)(p16.2;p15.4) were identified. Both of the breakpoint regions in 4p16.2 and 11p15.4 were narrowed to large ~359-kb and ~215-kb low-copy repeat (LCR) clusters, respectively, by aCGH and SNP array analyses. DNA sequencing enabled mapping the breakpoints of one translocation to 24-bp within interchromosomal paralogous LCRs of ~130-kb in length and 94.7% DNA sequence identity located in olfactory receptor gene clusters, indicating nonallelic homologous recombination (NAHR) as the mechanism for translocation. To investigate the potential involvement of interchromosomal LCRs in recurrent chromosomal translocation formation, we performed computational genome-wide analyses and identified 5292 interchromosomal LCR substrate pairs, > 5-kb in size and sharing > 94% sequence identity that can potentially mediate chromosomal translocations. Additional proof for interchromosomal NAHR mediated translocation formation was provided by sequencing the breakpoints of another recurrent translocation, der(8)t(8;12)(p23.1;p13.31). The NAHR sites were mapped within 55-bp in ~7.8-kb paralogous subunits of 95.3% sequence identity located in the ~579-kb (chr8) and ~287-kb (chr12) LCR clusters. We demonstrate that NAHR mediates recurrent constitutional translocations throughout the human genome and provide a computationally determined genome-wide “recurrent translocation map”. Affymetrix Genome-Wide SNP Array 6.0 arrays (Affymetrix, Santa Clara, California) were employed to define the breakpoints on chromosomes 4, 8, 11, and 12. Analysis was performed according to the Genome-Wide Human SNP Nsp/Sty Assay Kit 5.0/6.0 protocol provided by the supplier. The arrays were scanned using a GeneChip® Scanner 3000 7G (Affymetrix, Inc.) and results were analyzed using Genotyping Console version 2.1 software. patient 1: Version 5 BAC array contained 853 BAC/PAC clones designed to cover genomic regions of 75 known genomic disorders, all 41 subtelomeric regions, and 43 pericentromeric regions. For each patient sample, two experiments were performed with reversal of the dye labels for the control and test samples. patient 2: Version 5.1 BAC array contained 853 BAC/PAC clones designed to cover genomic regions of 75 known genomic disorders, all 41 subtelomeric regions, and 43 pericentromeric regions. For each patient sample, two experiments were performed with reversal of the dye labels for the control and test samples. patient 3: The BAC emulated Version 6.1 OLIGO array was comprised of approximately 42,460 oligonucleotides representing 1400 BAC clones, covering ~150 genomic disorders, all 41 subtelomeric regions up to 12 Mb and 43 pericentromeric regions with backbone coverage of every chromosome at the 650-band level of cytogenetic resolution (http://www.bcm.edu/geneticlabs/cma/tables.html). The 42.46K oligonucleotides (oligos) were selected from initial testing of 105,000 oligos derived from the Agilent eArray library with strict selection criteria and removal of repetitive sequences to ensure optimal performance with greater dynamic range. This targeted 42.46 K OLIGO array (V6 OLIGO) corresponds to genomic regions covered by the V6 BAC arrays and was manufactured in a 4 x 44K format with an average of 28-30 oligos per region previously covered by a single BAC clone. patient 5: The BAC emulated Version 6.3 OLIGO array was comprised of approximately 43,580 oligonucleotides representing 1400 BAC clones, covering ~150 genomic disorders, all 41 subtelomeric regions up to 12 Mb and 43 pericentromeric regions with backbone coverage of every chromosome at the 650-band level of cytogenetic resolution (http://www.bcm.edu/geneticlabs/cma/tables.html). This targeted OLIGO array corresponds to genomic regions covered by the V6 BAC arrays and was manufactured in a 4 x 44K format with an average of 28-30 oligos per region previously covered by a single BAC clone. patient 6: The BAC emulated Version 6.4 OLIGO array was comprised of approximately 43,700 oligonucleotides representing 1400 BAC clones, covering ~150 genomic disorders, all 41 subtelomeric regions up to 12 Mb and 43 pericentromeric regions with backbone coverage of every chromosome at the 650-band level of cytogenetic resolution (http://www.bcm.edu/geneticlabs/cma/tables.html). This targeted OLIGO array corresponds to genomic regions covered by the V6 BAC arrays and was manufactured in a 4 x 44K format with an average of 28-30 oligos per region previously covered by a single BAC clone. Patient 4 was a known t(4;11) translocation patient form previous report and only ran on Affymetrix array.

Dataset Information

A xylose-stimulated xylanase-xylose binding protein chimera created by random nonhomologous recombination.

Publications

A xylose-stimulated xylanase-xylose binding protein chimera created by random nonhomologous recombination.

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