Catalysis Letters https://doi.org/10.1007/s10562-018-2431-3

Inhibiting the Catalytic Activity of Family GH11 Xylanases by Recombinant Rice Xylanase-Inhibiting Protein Ya‑hui Dang1 · Ming‑qi Liu1 · Qian Wang2 Received: 26 March 2018 / Accepted: 20 May 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract ricexip, a xylanase-inhibiting protein (XIP)-type gene, was expressed in Escherichia coli BL21 (DE3). The recombinant protein, reEriceXIP significantly inhibited catalytic activity of several xylanases. Optimal inhibitory activity of reEriceXIP on xylanase (TfxA_CD214 and reBaxA454) occurred at 40 °C for 30 min. reEriceXIP decreased fluorescence intensity of xylanase and changed its secondary structure. reEriceXIP decreased global concentration of hydrolytes released from beechwood xylan. Graphical Abstract

Keywords  Rice xylanase-inhibiting protein (riceXIP) · Inhibitory activity · Fluorescence spectrum · Hydrolysis Abbreviations PCR Polymerase chain reaction SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis XIs Xylanase inhibitors

Electronic supplementary material  The online version of this article (https​://doi.org/10.1007/s1056​2-018-2431-3) contains supplementary material, which is available to authorized users. * Ming‑qi Liu [email protected] 1



National and Local United Engineering Lab of Quality Controlling Technology and Instrumentation for Marine Food, College of Life Science, China Jiliang University, Hangzhou 310018, China



College of Biological and Environmental Science, Zhejiang Wanli University, Ningbo 315100, China

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CD Circular dichroism HPLC-ELSD High performance liquid chromatography with evaporative light scattering detection IPTG Isopropyl-β-d-thiogalactopyranoside BaxA  Bacillus amyloliquefaciens xylanase A (mature peptide) reBaxA454 A BaxA mutant (S100G) reBaxA199 A BaxA mutant (T33I) TfxA_CD Catalytic domain of Thermomonospora fusca TF xylanase A TfxA_CD214 A TfxA_CD mutant (D19V-S64C-I114T) TfxA_CD162 A TfxA_CD mutant (D19V-S35G-S64C-P107S) TfxA_CD309 A TfxA_CD mutant (D19V-S64C-H159Y) TfxA_CD310 A TfxA_CD mutant (D19V-S64C) TfxA_CD311 A TfxA_CD mutant (D19V-S64C-T111A)

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TfxA_CD526 A TfxA_CD mutant (F14L-D19V-S64CT144I-T185M), all of those mutants were from the DNA shuffling library constructed in our previous study using TfxA_CD and BaxA as parents AnxA  Aspergillus niger xylanase A TLx  Thermomonospora lanuginosus xylanase TRx  Trichoderma reesei xylanase

TF vector. The inhibitory activities of recombinant riceXIP (reEriceXIP) on different family GH11 xylanases were investigated. The interaction between reEriceXIP and xylanases was studied by fluorescence spectroscopy and CD spectroscopy. The effect of reEriceXIP on hydrolysates of beechwood xylan by xylanase was studied through highperformance liquid chromatography with evaporative light scattering detection (HPLC-ELSD).

1 Introduction

2 Materials and Methods

Xylanases (EC 3.2.1.8) belonging to the glycoside hydrolase (GH) family catalyze the hydrolysis of internal β-1,4 bonds of xylan and belong to glycoside hydrolase (GH) [1]. Xylanase inhibitors (XIs) are classified into thaumatinlike xylanase inhibitor (TLXI), Triticum aestivum endoxylanase inhibitor (TAXI), and xylanase inhibiting protein (XIP) inhibitors [2]. The XIP inhibitors was initially found from wheat [3]. To date, four XIP-type XIs, riceXIP, RIXI, OsXIP, and OsHI-XIP, have been identified in rice crops [4–7]; these XIs are involved in plant defense against herbivores or pathogens by inhibiting activity of xylanases produced by phytopathogenic microorganisms [8–10]. For examples, RIXI plays a role in plant defense and affects the activity of family GH11 Aspergillus niger xylanase (AnxA) [11]. OsHI-XIP also plays a role in resistance among rice herbivores and inhibits activity of Thermomyces lanuginosus xylanase and T. longibrachiatum xylanase [4]. Zhan et al. suggested that OsXIP involved in defense responses in rice and inhibits activity of T. lanuginosus xylanase (TLx), and Trichoderma reesei xylanase (TRx) [12]. riceXIP is a non-chitinolytic homolog of chitinases in rice plants, which is active against AnxA and participates in plant defense against herbivores too [2, 5]. The molecular inhibition mechanism of the XIP-I inhibitor against family GH10 A. nidulans xylanase and family GH11 Penicillium funiculosum xylanase can be explained on the basis of the solved 3-D structure, which illustrated that xylanase towards an essential role of the “thumb” hairpin loop binds to the XIP-I inhibitor from wheat [6, 13, 14]. However, its properties and the interaction between xylanases and riceXIP have yet to be determined. The interaction mechanism of two compounds can be investigated through fluorescence spectroscopy in combination with circular dichroism (CD) spectroscopy. Fluorescence spectroscopy of xylanase interaction with XIs was investigated according to the Stern–Volmer equation. CD spectroscopy was used to analyze the structural changes of xylanase in the presence of XIs [15, 16]. In the present study, the ricexip gene was cloned and expressed in Escherichia coli BL21 (DE3) using the pCold

The rice genomes of Oryza sativa L. spp. Japonica cv. Nipponbare and A. niger xylanase A (AnxA) were preserved in the Central Laboratory of Food Science Department, China JiLiang University. Recombinant Bacillus amyloliquefaciens xylanase A mutant (reBaxA454 and reBaxA199), and recombinant Thermomonospora fusca TF xylanase A mutant (TfxA_CD214, TfxA_CD162, TfxA_CD309, TfxA_CD310, TfxA_CD311 and TfxA_CD526) were from the DNA shuffling library constructed in our previous study [17]. Beechwood xylan, T. lanuginosus xylanase (TLx), and Trichoderma reesei xylanase (TRx) were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. (Shanghai, China). Xylanases used in the study were summarized in Supplemental file Table 1. The pCold TF DNA cold-shock expression vector and restriction endonucleases were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Primers were synthesized at Shanghai Sunny Biotechnology Co., Ltd (Shanghai, China). Protein marker and antibodies were purchased from Sangon Biotech (Shanghai, China). d-Xylose was from Merck (Darmstadt, German). Standard xylooligosaccharides (XOs: xylobiose, X2; xylotriose, X3; xylotetraose, X4; xylopentaose, X5; and xylohexaose, X6) were obtained from Megazyme (Wicklow, Ireland).

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2.1 Cloning and Recombinant Expression of ricexip Gene Full-length ricexip was amplified from rice genome of Oryza sativa L. spp. Japonica cv. Nipponbare with specific primers: E1: 5′-CCA​GAA​TTC​ATG​GGT​CTC​GTA​CACGC-3′ and E2: 5′-GCTC​TCT​AGA​ACC​CTC​ACC​AGT​GTA​GTT​ -3′, which contain EcoRI and XbaI restriction sites (underlined), respectively. ‘touchdown’ PCR was conducted under the following conditions: initial denaturation at 94 °C for 4 min; 30 consecutive cycles of denaturation at 94 °C for 30 s, annealing at 64 °C for 30 s with a 0.1 °C decline per cycle and extension at 72 °C for 1 min; and a final extension at 72 °C for 10 min. The resulting PCR product was then recovered and sequenced.

Inhibiting the Catalytic Activity of Family GH11 Xylanases by Recombinant Rice…

The recovered ricexip was doubly digested by EcoRI and XbaI and then inserted into pCold TF vector. The pColdricexip plasmid was transformed into E. coli BL21 (DE3) [18]. The positive clones were isolated on Luria–Bertani (LB) agar plates containing ampicillin (100 µg/mL) and then confirmed through PCR [19]. The positive transformant ColdXIP-5 was selected for scale-up expression. ColdXIP-5 (1.0 mL) was cultured in 50 mL of LB medium (100 µg/ mL ampicillin) at 37 °C and 150 rpm. When the bacterial density reached O ­ D600 = 0.4 (3 h), the baffled shake flask was settled at 15 °C for 30 min. To induce recombinant protein production, isopropyl-β-d-thiogalactopyranoside (IPTG) was added into the medium at a final concentration of 200 µg/ mL, and then cells were cultured at 15 °C for 24 h [20]. The recombinant xylanase inhibitor produced by ColdXIP-5 was named as reEriceXIP and was used in subsequent studies.

2.2 Purification, SDS‑PAGE, and Western Blot Analysis of reEriceXIP After induction by IPTG at 15 °C for 24 h, the fermentation supernatant of ColdXIP-5 was collected. The cell pellets were ultrasonicated for 30 min. The ultrasonicated supernatant was mixed with high-affinity N ­ i2+-charged resin and incubated for 1 h by gently inverting the supernatant in an ice bath to bind the protein to the resin. The slurry was transferred to a column, which was then washed with eight bed volumes of wash buffer (50 mM N ­ aH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) and five bed volumes of elution buffer (50 mM ­NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). Elutes were collected tube by tube (1 mL per tube) for subsequent analysis [20]. Protein concentration was measured using Bradford method with bovine serum albumin as the standard [21]. The samples from the ColdXIP-5 were subjected to SDSPAGE, with 5% stacking gel and 12% separating gel [22]. Proteins were dyed in with Coomassie blue R-250. For Western blot analysis, the proteins were transferred onto a polyvinylidene difluoride membrane, which was then incubated with anti-His monoclonal antibody. Immunoreactive protein was visualized using horseradish peroxidase-labeled goat anti-mouse IgG as the secondary antibody on a chromogenic substrate [23].

2.3 Inhibitory Activity Assay The inhibitory activity of reEriceXIP was assayed as follows. reEriceXIP (0.3 mg) preincubated with 11 types of xylanases (TfxA_CD214, TfxA_CD309, TfxA_CD310, TfxA_CD526, TfxA_CD162, TfxA_CD311, reBaxA454, reBaxA199, AnxA, TLx, and TRx, 0.3 U) for 30  min at 30 °C, respectively. Sample group: 11 glass tubes of pre-warmed (50 °C) beechwood xylan (0.5%, w/v, 500

µL) were each added with 1 type of the 11 reEriceXIPxylanase mixtures. The tubes were then placed in a water bath at 50 °C for 5 min. Blank contrast group: beechwood xylan (0.25%, 500 µL) was added into 11 glass tubes and placed in a water bath at 50 °C for 5 min. Xylanase contrast group: 11 types of xylanase (0.3 U) and pre-warmed beechwood xylan (50 °C, 0.5%, 500 µL) were simultaneously placed into glass tubes and then subjected to a water bath at 50 °C for 5 min. The reactions of the three groups were terminated by adding 3,5-dinitrosalicylic acid (1.0 mL) [24, 25]. The corresponding xylanase (0.3 U) and reEriceXIP (0.3 mg) were added into the blank contrast group and reEriceXIP (0.3 mg) was added into the xylanase contrast group. All tubes were boiled for 5 min and then were added 2.0 mL water. The blank contrast group was used as the control and the absorbance (ABS) at 540 nm of the sample group and the xylanase contrast group was measured. The xylanase inhibitory rate was displayed as a percentage of inhibitory activity of reEriceXIP on xylanase [25]. One unit of xylanase activity was defined as the amount of the enzyme that catalyzed the formation of 1.0 µmol of reducing sugar (d-xylose) from beechwood xylan per minute under its optimal conditions. Furthermore, xylanases (TfxA_CD214 and reBaxA454) that showed the highest sensitivity to the inhibitor were selected for further experiments. For each assay in this study, triplicate experiments were conducted, and the mean value was obtained.

2.4 Effects of Dose, Time, and Temperature on Inhibitory Activity of reEriceXIP Xylanases (0.3 U) incubated with reEriceXIP at concentrations of 0, 0.057, 0.114, 0.171, 0.228, and 0.285 mg at 30 °C for 30 min, the residual activities of TfxA_CD214 and reBaxA454 were determined, respectively. The relatively inhibitory rates of reEriceXIP on xylanase were then calculated, and the highest inhibiting activity was taken as 100%. reEriceXIP was incubated with TfxA_CD214 and reBaxA454 (0.3 U) at 30 °C for 10, 20, 30, 40, 50, and 60 min, respectively. The residual activity of xylanase was tested, and the inhibitory rates were calculated. The optimum inhibiting time is the reaction time when reEriceXIP obtains the highest inhibiting rate. reEriceXIP incubated with TfxA_CD214 and reBaxA454 at 20–70 °C for 30 min, respectively. Then the inhibitory activities were assayed. The temperature with the highest inhibitory rate was considered the optimal temperature of reEriceXIP.

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2.5 Fluorescence Spectroscopy of Interaction Between Xylanases and reEriceXIP The interactions between xylanases and reEriceXIP were investigated with fluorescence spectroscopy at 30 °C. Samples with a series of concentration of diluted purified reEriceXIP and diluted purified and fix-concentration xylanase (A < 0.05) were stored at 30 °C in the dark for 0.5 h [26]. Fluorescence was then determined in a 1 cm path-length quartz cell using an F-4600 fluorescence spectrophotometer (Hiachi High-Tech Science, Japan). Fluorescence emission spectra of the samples upon excitation at 280 nm were recorded from 300 to 450 nm. The fluorescence quenching data were analyzed according to Stern–Volmer equation, and the binding constant obtained is illustrated as follows: (1) where ­F0 is the fluorescence emission intensity of xylanases (TfxA_CD214 and reBaxA454), F is the fluorescence

F0 /F = 1 + Kq τ0 [Q] = 1 + KSV [Q]

Fig. 1  Nucleotide sequence of ricexip and the deduced amino acid sequences. The signal peptide is underlined. The stop codon is indicated with an asterisk. The potential xylanase binding domains with family GH11 xylanase are in gray

emission intensity of xylanases with reEriceXIP at Q concentration, ­Kq is the quenching rate constant of the molecule; τ0 is the average lifetime (τ0 = 10−8 s) of the molecule, and ­KSV is the Stern–Volmer dynamic quenching constant. For the static quenching, the equation below can be used to obtain the binding constant KA and binding sites n: ( ) lg F0 − F /F = lg KA + n lg [Q] (2) where ­F0 is the fluorescence intensity of xylanases, F is the fluorescence emission intensity of xylanases with reEriceXIP at Q concentration, ­KA is the association constant, and n is the number of binding sites.

2.6 CD Spectroscopy Analysis TfxA_CD214 and reBaxA454 were mixed with reEriceXIP (mass ratio: 1:1) at 30 °C for 30 min, and analyzed by CD spectroscopy, respectively. CD analysis of reEriceXIP on TfxA_CD214 and reBaxA454 was conducted with a Jasco

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Inhibiting the Catalytic Activity of Family GH11 Xylanases by Recombinant Rice…

J-815 CD spectrometer. CD spectra of TfxA_CD214 and reBaxA454 from 190 to 240 nm at 25 °C with and without reEriceXIP were acquired using a 0.2 cm path-length cuvette at a bandwidth of 2 nm and a scan rate of 100 nm/min. Each spectrum is the average of three scans and are subtracted from their respective blanks [27]. The spectra were then deconvoluted to determine the relative percentages of α-helices, β-sheets, and random structures in the protein using Dichro Web [26].

2.7 Effect of reEriceXIP on the Hydrolysis of Beechwood Xylan by Xylanases TfxA_CD214 (0.51 U, 200 µL) and reBaxA454 (0.51 U, 200 µL) were incubated with beechwood xylan (0.5%, 500 µL) at 50 °C for 4 h. The inhibition sample was prepared as follows: TfxA_CD214 (0.51 U, 200 µL) and reBaxA454 (0.51 U, 200 µL) were incubated with reEriceXIP (0.25 mg, 250

Fig. 2  Inhibitory activity of reEriceXIP on family GH11 xylanases

µL) at 30 °C for 30 min; the two mixtures were added into beechwood xylan solution (1.0%, 250 µL) and incubated at 50 °C for 4 h, respectively. To terminate hydrolysis, samples were boiled for 5 min and then immediately cooled on ice. These samples were centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatants were analyzed by highperformance liquid chromatography (Waters 2695) with the evaporative light scattering detection (Waters 2424) [28] The injection volume of samples was 20 µL. X1–X6 were used as standard.

3 Results and Discussion 3.1 Cloning and Recombinant Expression of the ricexip Gene The ricexip gene was amplified from the rice genome and then sequenced. The gene has an 873 bp open reading frame, encoding for 290 amino acids with a predicted molecular mass of 32.0 kDa (Fig. 1). The 28 amino acids in the N-terminus were determined as the signal peptide through the analysis of SignalP 4.1 Server. Three potential domains that bind to family GH11 xylanase were identified [7]. The ricexip gene showed 99% identity with the gene in GenBank (AP014967.1). The ricexip was ligated into the pCold TF vector and the positive clones were confirmed through PCR. The pColdricexip plasmids were transformed into E. coli BL21 (DE3), and positive clones were obtained. The ColdXIP-5 transformant was induced with IPTG at 15 °C for 24 h. The recombinant inhibitor reEriceXIP was secreted into the culture medium and retained in the cell cytoplasm of ColdXIP-5. E. coli was utilized as a host system because of its ease of use, fast growth rates, relatively low cost of production, and adaptability to large-scale expression [29]. Nowadays, many

Fig. 3  SDS-PAGE (a) and Western blot (b) assay of reEriceXIP. Lane 1: E. coli BL21 (DE3) with pCold TF vector induced by IPTG at 15 °C for 24 h; Lane 2: purified reEriceXIP by high-affinity ­Ni2+-charged resin; Lane 3: ultrasonicated supernatant of ColdXIP-5 induced by IPTG at 15 °C for 24 h; lane 4: ColdXIP-5 induced by IPTG at 15 °C for 24 h; Lane 5: fermentation supernatant of ColdXIP-5 induced by IPTG at 15 °C for 24 h. M: protein marker

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strategies, including high-throughput expression screening, was studied for its expression effectively [30]. The pCold TF vector presents many advantages over the pET series vector, such as providing a cold-shock technology and a folding chaperone Trigger Factor for high-level expression of soluble recombinant proteins. The cold-shock technology requires decreasing culture temperature from 37 to 15 °C during the induction period, can suppress the expression of other cellular proteins and temporarily halt overall cell growth [31]. Trigger Factor is the ribosome-associated chaperone in bacteria and features a molecular weight of 48 kDa. When combined with a target protein, the Trigger Factor can facilitate correct folding and efficient soluble production [32–34].

3.2 Inhibitory Activity Assay As shown in Fig. 2, the inhibitory rates of reEriceXIP on TfxA_CD214, TfxA_CD309, TfxA_CD310, TfxA_ CD526, TfxA_CD162, and TfxA_CD311 were 28.21, 27.10, 17.11 26.19, 11.86, and 10.25% (TfxA_CD mutant series), respectively; the inhibitory rates on reBaxA454 and reBaxA199 (BaxA mutant series) were 50.42 and 5.08%, respectively; and the inhibitory rates on AnxA, TLx, and TRx (fungal xylanases) were 23.68, 1.07, and 28.14%, respectively. reEriceXIP showed high inhibitory activity on xylanases of TfxA_CD214, TfxA_CD309, TfxA_CD526, reBaxA454, AnxA, and TRx. Results verified the previous observation that reEriceXIP is active against family GH 11 xylanases [2]. TfxA_CD214 and reBaxA454 were selected for further study because of their high sensitivity to reEriceXIP.

3.3 Purification, SDS‑PAGE, and Western Blot Analysis of reEriceXIP After adding IPTG at 15 °C for 24 h, the protein concentration of the fermentation supernatant of ColdXIP-5 was 0.096  mg/mL. reEriceXIP was purified by N ­ i2+ affinity chromatography. SDS-PAGE (Fig. 3a) and Western blot (Fig. 3b) analysis revealed that the molecular weight of the reEriceXIP was approximately 87.8 kDa. The ricexip gene encoded a protein with a molecular mass of 32.0 kDa. The reEriceXIP produced under the control of the cspA lac promoter was combined with the Trigger Factor chaperone, which increased the molecular weight of reEriceXIP by 54 kDa.

3.4 Effects of Dose, Time, and Temperature on Inhibitory Activity of reEriceXIP The inhibiting rate of reEriceXIP on TfxA_CD214 and reBaxA454 increased with increasing reEriceXIP dose

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Fig. 4  Effects of dose (a), time (b), and temperature (c) on inhibitory activity of reEriceXIP. a The doses of reEriceXIP are 0, 0.057, 0.114, 0.171, 0.228 and 0.285  mg. The unit of TfxA_CD214 and reBaxA454 is 0.3 U. b The unit of TfxA_CD214 and reBaxA454 is 0.3  U. The doses of reEriceXIP that interacted with TfxA_CD214 and reBaxA454 are 0.228 and 0.171 mg, respectively. c For optimum inhibitory temperature assay, the highest activity was taken as 100%

Inhibiting the Catalytic Activity of Family GH11 Xylanases by Recombinant Rice… Fig. 5  Fluorescence spectrum analysis and Stern–Volmer plots of fluorescence quenching of reEriceXIP on TfxA_CD214 (a) and reBaxA454 (b). a Final concentrations of reEriceXIP from a to k are 0, 0.02, 0.04, 0.07, 0.09, 0.12, 0.15, 0.18, 0.20, 0.23, and 0.25 µM, respectively. b Final concentrations of reEriceXIP from a to k are 0, 0.02, 0.05, 0.08, 0.11, 0.14, 0.17, 0.20, 0.24, 0.27, and 0.30 µM, respectively

before the optimal mass (Fig. 4a). reEriceXIP at 0.228 mg exerted the highest inhibitory effect on TfxA_CD214 (0.3 U), with a dose ratio of 2.2:1. reEriceXIP at 0.171  mg demonstrated the best inhibitory effect on reBaxA454 (0.3 U), with a dose ratio of 1:1. These results suggest that reBaxA454 is more sensitive to reEriceXIP than TfxA_ CD214, which is consistent with inhibitory assay results. The highest inhibitory rate of reEriceXIP was reached after 30  min of interaction with the TfxA_CD214 or reBaxA454. reEriceXIP drastically inhibited the catalytic activity of TfxA_CD214 after 20–30 min of interaction. By contrast, it showed strong inhibitory activity on reBaxA454 after 30–40 min of interaction (Fig. 4b). The reEriceXIP inhibitory rate on xylanases increased with increasing temperature, reached the maximum for

TfxA_CD214 and reBaxA454 at 40 °C, and then decreased with increasing temperature (Fig. 4c).

3.5 Fluorescence Spectroscopic Analysis of Interaction Between Xylanase and reEriceXIP The fluorescence emission spectra of the samples upon excitation at 280 nm were recorded from 300 to 450 nm. Having an excitation wavelength of 280 nm, the spectrum change reflected the environmental change of tryptophan residues present in xylanase. As shown in Fig. 5, the maximum fluorescence spectrum of TfxA_CD214 and reBaxA454 was at 330 nm and changed with increasing reEriceXIP concentration, and decreasing fluorescence intensity [35]. As shown in Fig. 5a, b, according to the formula (1), ­Kq was 7.91 × 1013 L/mol/s for TfxA_CD214 and 1.36 × 1014 L/

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Fig. 6  Double Log plot of fluorescence quenching of TfxA_CD214 (a) and reBaxA454 (b) by reEriceXIP

mol/s for reBaxA454. These values are > 2.0 × 1010 L/mol/s, suggesting static quenching. As shown in Fig. 6a, b, according to formula (2), ­KA was 0.627 L/µmol for TfxA_CD214 and 0.750 L/µmol for reBaxA454, with binding sites n (290 K) of 0.739 and 0.798, respectively. The binding site between xylanases and reEriceXIP was approximately one, and the combination of both was unstable.

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Fig. 7  Circular dichroism (CD) spectral analysis of TfxA_CD214 (a) and reBaxA454 (b) with reEriceXIP

TfxA_CD214 incubated with reEriceXIP showed a predominantly random of the protein (Fig. 7a, Supplemental file Table 2). The CD spectrum of reBaxA454 showed minimum wavelengths of 208 and 222 nm, indicating a predominant α-helical structure of the protein. The CD spectrum of reBaxA454 that interacted with reEriceXIP showed a minimum wavelength of 218 nm, indicating the formation of a β-sheet-rich structure (Fig. 7b, Supplemental file Table 2).

3.6 CD Spectroscopic Analysis

3.7 Effect of reEriceXIP on the Hydrolysis of Beechwood Xylan by Xylanases

The CD spectrum of TfxA_CD214 and its incubation with reEriceXIP showed that β-sheet structure of the TfxA_ CD214 protein declined predominantly. The α-helical of reBaxA454 predominantly turn into a beta-sheet. The CD spectrum of TfxA_CD214 showed a minimum wavelength of nearly 217 nm and a strong maximum wavelength nearly 196 nm, indicating a predominant β-sheet of the enzyme structure as previously reported [26]. The CD spectrum of

Hydrolysis products released from beechwood xylan by xylanases and xylanases with reEriceXIP were detected by HPLC-ELSD (Fig. 8). TfxA_CD214 and TfxA_CD214 with reEriceXIP released X2–X5 from beechwood xylan with X3 as the major product (Fig. 8a, Supplemental file Table 3). Meanwhile, the hydrolysates released from beechwood xylan by reBaxA454 and reBaxA454 with reEriceXIP were

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Inhibiting the Catalytic Activity of Family GH11 Xylanases by Recombinant Rice… Fig. 8  HPLC-ELSD analysis of hydrolysis products from beechwood xylan. a Beechwood xylan was hydrolyzed by TfxA_ CD214 and TfxA_CD214 with reEriceXIP. b Beechwood xylan was hydrolyzed by reBaxA454 and reBaxA454 with reEriceXIP. X xylose, X2 xylobiose, X3 xylotriose, X4 xylotetraose, X5 xylopentaose, X6 xylohexaose

X2–X6, and the major product was different (Fig. 8b, Supplemental file Table 3). reEriceXIP inhibited the hydrolysis of beechwood xylan by TfxA_CD214 and reBaxA454 and decreased the amounts of hydrolysates. However, types of hydrolysis products were not changed by reEriceXIP. The hydrolysates of beechwood xylan released by immobilized recombinant TfxA expressed in E. coli BL21 using the pET 30 (a) + vector were X2–X5, with X3 as the major product [36]. The hydrolysis product from birchwood xylan by recombinant TfxA expressed in Pichia pastoris GS115 were X1–X6 with X2 as main product [37]. The BaxA mutant (reBaxA50) released X1–X5 from beechwood,

birchwood, and oat spelt xylan, with X3 as the major product, respectively [38]. XOs are value-added oligosaccharides produced from several agricultural by-products [39]. With the exception of common beneficial effects of prebiotics, XOS possess antioxidant properties, immune stimulations, and positive influences on the development of colon cancer [40–42]. XOs are used as prebiotics and feed additives in many countries [43, 44]. XIP is a naturally occurring protein that affects the hydrolysis of xylan and reduces the application effects of xylanase. Therefore, the xylanase that is tolerant to XIP holds big advantage at hydrolysing natural substrates, such

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as corn cobs, wheat bran, rice husks, sugarcane bagasse, and wheat straw.

4 Conclusion The ricexip gene was cloned and successfully expressed in E. coli BL21 (DE3) fused with the Trigger Factor chaperone. reEriceXIP showed different inhibitory activities on family GH11 xylanases and change the second structure of TfxA_ CD214 and reBaxA454. Moreover, the binding site between xylanases (TfxA_CD214 and reBaxA454) and reEriceXIP was approximately one. reEriceXIP inhibited the hydrolysis of beechwood xylan by TfxA_CD214 and reBaxA454 and decreased the global concentration of hydrolytes, but it did not change the types of hydrolysates. Acknowledgements  This work was supported by the National Natural Science Foundation of China (No. 31672462) and the Zhejiang Provincial Natural Science Foundation of China (No. LY15C200013).

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