WO2024016397A1 - 来自枯草芽孢杆菌的裂解性多糖单加氧酶及其应用 - Google Patents

来自枯草芽孢杆菌的裂解性多糖单加氧酶及其应用 Download PDF

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WO2024016397A1
WO2024016397A1 PCT/CN2022/111015 CN2022111015W WO2024016397A1 WO 2024016397 A1 WO2024016397 A1 WO 2024016397A1 CN 2022111015 W CN2022111015 W CN 2022111015W WO 2024016397 A1 WO2024016397 A1 WO 2024016397A1
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bslpmo10a
mlg
substrates
activity
polysaccharide monooxygenase
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王谦
孙小宝
王佳堃
刘建新
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浙江大学
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/14—Pretreatment of feeding-stuffs with enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase

Definitions

  • the invention belongs to the field of enzymology, and specifically relates to a lytic polysaccharide monooxygenase from Bacillus subtilis and its application.
  • Plant lignocellulose is the most abundant source of biomass in nature, with an annual output of approximately 10 11 -10 12 tons (Centore, Hochman, & Zilberman, 2014).
  • non-starch polysaccharides such as cellulose and hemicellulose and lignin are the main components, giving plants rigidity and resistance to insect and fungal attack.
  • These biopolymers need to be completely broken down into monomers and oligomers before they can be effectively used.
  • the highly complex and recalcitrant structure in lignocellulose acts as an obstacle to biodegradation.
  • Glycoside hydrolases including cellulases, xylanases, and glucanases, have been widely used in industry, especially in the development of food/feed additives.
  • GHs Glycoside hydrolases
  • lytic polysaccharide monooxygenases LPMOs, EC 1.14.99.53-56
  • LPMO lytic polysaccharide monooxygenases
  • LPMOs are classified into the auxiliary active family (AAs) (Levasseur, Drula, Lombard, Coutinho, & Henrissat, 2013).
  • AAs auxiliary active family
  • LPMOs have been found in bacteria, fungi, viruses, insects and archaea that act on cellulose, chitin, starch and other polysaccharide substrates (Lombard, Golaconda Ramulu, Drula, Coutinho, & Henrissat, 2014) . They are divided into 8 families according to their sequence and substrate specificity, AA9-11 and AA13-17.
  • LPMOs have a ⁇ -sheet topology, and some of them have ⁇ -sheets connected by loops of variable length (Hemsworth, Henrissat, Davies, & Walton, 2014; Harris et al., 2010; Leggio et al., 2015). Given their flat substrate-binding surfaces, these enzymes are suitable for binding and cleavage of crystalline polysaccharides, providing more attack sites for subsequent hydrolysis of GHs (Hemsworth, Johnston, Davies, & Walton, 2015; Johansen, 2016; Zhang et al .,2019).
  • xylan acts on the two main hemicellulose components of plant lignocellulose (Correa et al., 2019; Couturier et al., 2018; Wilsontner et al., 2019; Jung, Song, Kim, &Bae,2015; et al., 2022; Zerva, Pentari, Grisel, Berrin, & Topakas, 2020) and mannan (Li et al., 2021a; Limsakul et al., 2020) degrade less LPMO.
  • Bacillus subtilis is generally recognized as safe (GRAS) by the U.S. Food and Drug Administration (FDA).
  • GRAS GRAS
  • FDA U.S. Food and Drug Administration
  • Bacillus subtilis plays an important role in gastrointestinal health in humans and animals.
  • Bacillus subtilis improved the growth performance, immune response, heat stress tolerance, and disease resistance of poultry (Wang et al., 2022).
  • this microorganism has been proven to improve rumen development and the health of newborn Holstein calves (Wang et al., 2022). Therefore, it will be of great significance to mine new LPMOs from Bacillus subtilis.
  • the technical problem to be solved by this invention is: how to mine new LPMOs from Bacillus subtilis.
  • the technical solution of the present invention is: a lytic polysaccharide monooxygenase BsLPMO10A from Bacillus subtilis, consisting of 455 amino acid residues, and its amino acid sequence is shown in SEQ ID No. 1.
  • the gene encoding the above-mentioned cleavage polysaccharide monooxygenase BsLPMO10A has a variety of different coding sequences due to the degeneracy of codons.
  • nucleotide sequence of the gene is shown in SEQ ID No. 2.
  • a recombinant bacterium containing the above-mentioned expression vector A recombinant bacterium containing the above-mentioned expression vector.
  • the hemicellulose substrate is lichenin, ⁇ -glucan, locust bean gum, konjac glucomannan, beech wood xylan and betaine arabinose.
  • the cleavage polysaccharide monooxygenase BsLPMO10A of the present invention is used to promote the decomposition of polysaccharides in cooperation with glycoside hydrolase.
  • the present invention has the following beneficial effects:
  • BsLPMO10A Bacillus subtilis
  • the novel AA10LPMO derived from Bacillus subtilis (BsLPMO10A) of the present invention is subjected to heterologous expression and functional characteristics.
  • Recombinant BsLPMO10A has optimal activity at 50°C and pH 5.0.
  • Substrate specificity studies showed that the enzyme exhibits a broad spectrum of active substrates, including MLG-L, MLG-B, LBG, KGM, BX, especially for polysaccharides linked by ⁇ -1,4 glycosidic bonds, including ⁇ -(Glc1 ⁇ 4Glc), ⁇ -(Xyl1 ⁇ 4Xyl) and ⁇ -(Man1 ⁇ 4Man).
  • BsLPMO10A showed activity on arabinan for the first time in this study.
  • BsLPMO10A mainly releases natural oligosaccharides with a degree of polymerization (DP) of 3-5 and C1 oxidized oligosaccharides from DP3ox to DP6ox from mixed-linked glucan and beech xylan .
  • DP degree of polymerization
  • Its synergistic effect with a series of glycoside hydrolases showed that BsLPMO10A significantly accelerated the saccharification of glucan, xylan, cellulose and chitin.
  • the synergistic effect of BsLPMO10A and GHs was further verified through the degradation of six natural feeds.
  • FIG. 1 Phylogenetic tree of BsLPMO10A and other characterized AA10LPMOs in the database. Multiple sequence analysis of functionally characterized LPMOs was performed using MEGA11, followed by phylogenetic analysis using the build/test maximum likelihood method. BsLPMO10A (GenBank number: QDC27792.1).
  • FIG. 1 SDS-PAGE and zymogram analysis.
  • A Full-length BsLPMO10A and
  • B BsLPMO10A(CD); lane M, protein marker.
  • C Zymogram analysis: I, MLG-L; II, MLG-B; III, LBG; IV, KGM; V, BX; VI, PASC; VII, colloidal chitin.
  • FIG. 3 Biochemical characterization of BsLPMO10A.
  • A Optimal temperature and
  • Figure 4 Degradation of dextran by BsLPMO10A.
  • A HPAEC-PAD chromatogram of oligosaccharides released from MLG-L and MLG- by BsLPMO10A;
  • B-C MALDI-TOF-MS of oligosaccharides released by BsLPMO10A from MLG-L (B) and MLG-B (C) Analysis;
  • D HPAEC-PAD chromatogram of MLG-L and MLG-B degradation products by BsLPMO10A and glucanase (IDSGLUC5-38).
  • the inset shows the sum of native and C1 oxidation products from MLG-L and MLG-B by BsLPMO10A and glucanase (IDSGLUC5-38).
  • G1 glucose
  • FIG. 5 Degradation of BX by BsLPMO10A.
  • A HPAEC-PAD chromatogram of oligosaccharides released from BX by BsLPMO10A;
  • B HPAEC-PAD chromatogram of BX degradation products by BsLPMO10A and xylanase (IDSXYN11-1).
  • IDSXYN11-1 The inset shows the sum of natural products and C1 oxidation products generated from BX by BsLPMO10A and xylanase (IDSXYN11-1).
  • FIG. 1 HPAEC-PAD chromatogram of other polysaccharide degradation products by BsLPMO10A.
  • A KGM and LBG;
  • B SBA;
  • C Pectin and PGA;
  • D PASC;
  • E Chitin.
  • Figure 7 Synergistic effect of BsLPMO10A and GHs on polysaccharide degradation.
  • A Reducing sugars released from MLG-L by BsLPMO10A and glucanase (IDSGLUC5-38);
  • B Reducing sugars released from BX by BsLPMO10A and xylanase (TfXYN11-1);
  • C BsLPMO10A and mannose Reducing sugars released from LBG by glycanase (BsMAN26-3);
  • D Reducing sugars released from PASC by BsLPMO10A and cellulase (IDSGLUC5-11);
  • E Reducing sugars released by BsLPMO10A and chitinase (BtCHI18-1) from Reducing sugars released from chitin.
  • Statistical analysis between synergistic effects and GH alone effect groups was performed using t test. *, P ⁇ 0.05;
  • Figure 8 Synergistic effect of BsLPMO10A and GHs on the degradation of natural lignocellulosic raw materials.
  • A Peanut shell;
  • B Soybean straw;
  • C Leymus chinensis straw;
  • D Rice straw;
  • E Rice husk;
  • F Wheat straw.
  • Figure 9 Amino acid alignment and structure prediction of BsLPMO10A.
  • A Amino acid alignment of BsLPMO10A and other AA10LPMOs;
  • B-C WebLogo analysis of key residues involved in the "histidine scaffold”;
  • D Homology modeling of BsLPMO10A.
  • Bacillus subtilis W800N, Escherichia coli BL21(DE3) and pET-30a(+) are conventional commodities and are stored in this laboratory.
  • Icelandic moss lichen (MLG-L), barley beta-glucan (MLG-B), locust bean gum (LBG), konjac glucomannan (KGM), beech xylan (BX), beet arabinan (SBA), laminarin, colloidal chitin, citrus pectin with about 60% esterification degree, citrus polygalacturonic acid (PGA), and oligosaccharides were purchased from Megazyme (Wicklow, Ireland).
  • Phosphoric acid swollen cellulose is prepared from microcrystalline cellulose (Zhang, Cui, Lynd, & Kuang, 2006).
  • Microcrystalline cellulose (MCC), medium, kanamycin, and isopropylthio- ⁇ -D-galactopyranoside (IPTG) were purchased from Sangon (Shanghai, China).
  • Peanut shells, soybean straw, Leymus chinensis, rice straw, rice husk and wheat straw were collected from local farms (Zhejiang, China).
  • the structural homology model of BsLPMO10A was generated using the I-TASSER server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/download/).
  • the MEGA 11 neighbor-joining method was used to construct a phylogenetic tree, and the bootstrap method was used for phylogenetic testing.
  • the number of bootstrap replications was 1000 times.
  • the substitution type is amino acid
  • the model is Jones-Taylor-Thornton (JTT) model.
  • JTT Jones-Taylor-Thornton
  • the results are rendered using ESPript 3.0 (Robert & Gouet, 2014).
  • WebLogo3 was used to analyze the conserved amino acid sequences of each site (http://weblogo.threeplusone.com/).
  • Genomic DNA from Bacillus subtilis W800N was isolated using the Rapid Bacterial Genomic DNA Isolation Kit (Sangon, Shanghai, China) and used as a template for PCR amplification of the BsLPMO10A gene.
  • the full length (SEQ ID No. 2) and catalytic domain (SEQ ID No. 3) of BsLPMO10A were obtained using BsLPMO10A-SacI and BsLPMO10A-XhoI/BsLPMO10A(CD)-XhoI primers (Table 1).
  • the resulting fragment was inserted into pET-30a(+) between SacI and XhoI and then transformed into E. coli BL21(DE3) by heat shock.
  • the recombinant strains BL21/pET-30a(+)/BsLPMO10A and BL21/pET-30a(+)/BsLPMO10A(CD) were induced by IPTG, sonicated and used with HisTrap TM Start protein purification system and purification FF column (GE Healthcare BioSciences, USA) (Zhou et al., 2020) were used for purification. Purified enzyme was passed through 10-kDa Millipore Centrifugal filters (Merck Millipore, Burlington, MA, USA) further filtered to remove imidazole. Subsequently, the enzyme was analyzed by SDS-PAGE (15% electrophoresis gel and 4% stacking gel) (Laemmli, 1970).
  • Zymography was performed using separation gels containing 0.5% (w/v) of various polysaccharide substrates. After electrophoresis, the gel was renatured with methanol:water (1:3) solution for 15 min. After renaturation, the gel was immersed in 0.05M sodium acetate buffer (pH 5.5) and incubated at 37°C for 30 min. The gel was then stained with 1 mg/mL Congo red solution and washed with water and 1 M NaCl until a clear band was visible. and fixed with acetic acid.
  • BsLPMO10A was incubated in a 3-fold molar excess of CuSO on ice for 60 min for copper saturation pretreatment.
  • Use 10-kDa Millipore A centrifugal filter (Merck Millipore, Burlington, MA, USA) removed unbound copper, and the enzyme was resuspended in 0.05 M sodium acetate buffer (pH 5.5).
  • the Bradford method (Bradford, 1976) was used to determine enzyme concentration. Unless otherwise stated, subsequent experiments used copper saturase for activity determination.
  • DNS 3,5-Dinitrosalicylic acid assay: DNS analysis (Bailey, Biely, & Poutanen, 1992) was performed to determine BsLPMO10A activity. Briefly, 15 ⁇ L ( ⁇ 1.5 ⁇ M) of purified enzyme was mixed with 60 ⁇ L of 5 mg/mL various polysaccharide substrates (MLG-L, MLG-B, LBG, KGM, BX, SBA, laminarin, PASC, MCC, colloidal Butyrin, pectin and PGA) were dissolved in 0.05M sodium acetate buffer (pH 5.5) and reacted at 37°C and 200rpm for 24h. Then, add 75 ⁇ L DNS and boil the solution for 10 min. After cooling to room temperature, the absorbance was measured at 540 nm using spectrophotometry.
  • BsLPMO10A 15 ⁇ L ( ⁇ 1.5 ⁇ M) BsLPMO10A was incubated with 60 ⁇ L 5 mg/mL LBG solution at different temperatures (30-80°C) for 24 h, and the DNS method was used.
  • the optimal pH value of BsLPMO10A was determined by incubating the enzyme in various pH buffer solutions (citrate/phosphate buffer at pH 2.2-8.0 and 0.2M Tris-HCl buffer at pH 8.0-9.0) at 37°C for 24h. To be sure. The highest activity was determined as 100%.
  • the enzyme was mixed with different concentrations of ascorbic acid (0.5, 1, 1.5 and 3mM) and H 2 O 2 (0.5, 1, 2.5, 5, 10) in 5 mg/mL LBG solution. and 50mM) at 37°C for 24h, and activity was measured using the DNS method.
  • Ion chromatography with pulsed amperometric detector [HPAEC-PAD, ICS3000, separation column CarboPac PA200 (3 ⁇ 250mm) and CarboPac PA200 guard column (3 ⁇ 50mm)] (Dionex, Thermo) was then used as described in the literature ( Westereng et al., 2013) were measured.
  • BsLPMO10A catalysis was first used, followed by a GH to efficiently convert unreacted polysaccharide substrates into their monomers and dimers. Briefly, 150 ⁇ L ( ⁇ 15 ⁇ M) BsLPMO10A was incubated with 600 ⁇ L 5 mg/mL of various polysaccharide substrate solutions at 37°C for 24 h, and then the enzyme was inactivated by boiling for 5 min.
  • the solution was then centrifuged at 12,000 rpm for 15 min, and the supernatant was further hydrolyzed using 50 ⁇ L ( ⁇ 0.8 ⁇ M) of IDSGLUC5-38 or 50 ⁇ L ( ⁇ 0.5 ⁇ M) of IDSXYN11-1 at 37°C for 24 h, and then boiled for 5 min to inactivate the enzyme. .
  • the solution was centrifuged at 12,000 rpm for 15 min and used for HPAEC-PAD analysis, equipped with CarboPac PA1 (3 ⁇ 250mm) and CarboPac PA1 (3 ⁇ 50mm) guard columns (Dionex, Thermo).
  • Natural substrate assay Before activity assay, the natural lignocellulosic raw materials of peanut shells, soybean straw, Leymus chinensis, rice straw, rice husk and wheat straw were ground and treated with NaOH as previously described (Cao et al., 2021) for processing. To study the effect of BsLPMO10A or various GHs on the decomposition of natural lignocellulose, 500 ⁇ L ( ⁇ 20 ⁇ M) BsLPMO10A or ( ⁇ 0.5 ⁇ M) GHs were used to digest 0.1 g of various lignocellulose in 3 mL citrate/phosphate buffer (pH 6.0). Pretreated raw material samples and reacted at 25°C for 96h.
  • Insoluble material was rinsed 10 times with ddH 2 O and used for cellulose and hemicellulose determination according to the National Renewable Energy Laboratory (NREL) method (Sluiter et al., 2011).
  • NREL National Renewable Energy Laboratory
  • BsLPMO10A consists of 455 amino acid residues (SEQ ID No. 1), including an AA10 catalytic domain and fibronectin type III (FNIII) module. SignalP5.0 prediction indicates that the first 40 amino acids at the N-terminus are signal peptides.
  • Homology modeling shows that BsLPMO10A contains an immunoglobulin G (IgG)-like ⁇ -sandwich fold and a histidine scaffold sequence containing copper ions ( Figure 9). Two conserved histidines form a T-shaped "histidine scaffold", His41 and His130, located on the surface of the catalytic domain, representing a typical feature of LPMO.
  • AA10LPMO is believed to catalyze substrates mainly in C1-oxidation mode and can be further classified into C1- and C1/C4-oxidation subfamilies (Li et al .,2021b).
  • BsLPMO10A clustered with other C1-oxidized LPMOs from the AA10 family ( Figure 1).
  • BsLPMO10A co-occurs with 12 other LPMOs in the B. subtilis genome
  • BsLPMO10A shows relatively low similarity to these enzymes (33.70-62.63%) but higher similarity to the uncharacterized AA10 protein from Bacillus genus (99.53%)(WP_001065159.1).
  • approximately 30 enzymes in the AA10 family mainly of bacterial origin, have been characterized in the CAZy database. Compared with these functionally studied AA10LPMOs, BsLPMO10A was found to be more consistent with B.
  • BsLPMO10A is a mesophilic enzyme activated by a certain amount of ascorbic acid or H 2 O 2
  • the molecular weights of purified BsLPMO10A and its truncated catalytic domain [BsLPMO10A(CD)] are ⁇ 56 (A in Figure 2) and ⁇ 34 kDa (B in Figure 2), respectively. Both enzymes are active against MLG-L, MLG-B, LBG, KGM, BX and SBA (Fig. 2, C). It is worth noting that compared with only the catalytic domain, the full-length BsLPMO10A containing the catalytic domain and FNIII was more effective against MLG-L (P ⁇ 0.001), MLG-B (P ⁇ 0.001), LBG (P ⁇ 0.05) and KGM. (P ⁇ 0.01) (Table 2) has higher catalytic activity.
  • FNIII domains are widely found in bacterial, yeast, plant and animal proteins. It has been reported that Bacillus spp., Bacillus cereus (Mutahir et al., 2018) and Bacillus thuringiensis (Manjeet, Madhuprakash, Mormann, Moerschbacher, & Podile, 2019) contain one or more FNIII domains. However, their functions and detailed mechanisms are poorly understood and sometimes controversial. Knocking out the FNIII domain (also known as the X1 domain) did not change the catalytic efficiency of TfAA10B (Arfi, & Umbreit, 2014; Kruer-Zerhusen et al., 2017). In contrast, Zhou et al.
  • BsLPMO10A may preferentially act on certain polysaccharides composed of glucose or mannose units, followed by polysaccharides composed of xylose or arabinose (Table 2 and Figure 2, C).
  • the FNIII domain greatly contributes to substrate binding and/or catalytic efficiency.
  • wild-type BsLPMO10A showed significantly higher activity on "preferred" substrates including Iceland moss lichen, barley ⁇ -glucan, locust bean gum, and konjac glucomannan ( P ⁇ 0.05).
  • BsLPMO10A The optimal temperature and pH of BsLPMO10A are 50°C and 5.0, respectively ( Figure 3A and 3B), indicating that BsLPMO10 is a mesophilic enzyme active under neutral and weakly acidic conditions, which is consistent with most previously reported LPMO exhibits an optimal temperature range of 40–60°C (Li et al., 2021a; Li, Dilokpimol, Lucas, & de Vries, 2022; Zhang, Chen, Long, & Ding, 2021) consistent with this.
  • the Aspergillus oryzae AA17 enzyme LPMO-AoAA17 showed an optimal temperature of 100°C and was even active after autoclaving at 121°C and 15 psi (Bhatia & Yadav, 2021). It is well known that copper is critical to the oxidative activity of LPMO, and the use of EDTA chelation to remove Cu 2+ will lead to a decrease in LPMO activity and thermal stability (Gusakov, Bulakhov, Demin, & Sinitsyn, 2017; Semenova et al., 2019). Therefore, BsLPMO10A was saturated with copper before all assays performed in this study.
  • FIG. 3 C shows that ascorbic acid significantly promotes BsLPMO10A activity (P ⁇ 0.01) and acts as an electron donor in the oxidation reaction, which is consistent with previous reports (Stepnov et al., 2021).
  • some reducing agents including 1,2-dihydroxybenzene (Zhang et al., 2021) and tannic acid (Frommhagen et al., 2016) are insensitive to LPMO activity.
  • O 2 is considered to be a co-matrix participating in the LPMO oxidation reaction (Vaaje-Kolstad et al., 2010).
  • H 2 O 2 rather than O 2 drives the co-substrate of LPMO (Hegnar et al., 2019).
  • 1 or 2.5mM H 2 O 2 significantly increased BsLPMO10A activity by 70.7% (P ⁇ 0.001) or 49.5% (P ⁇ 0.01), respectively.
  • an inhibitory effect was observed when the H 2 O 2 concentration exceeded 5 mM (Fig. 3, D).
  • adding excess H 2 O 2 can negatively affect the enzyme and deplete electron donors (Calderaro et al., 2020), thereby inhibiting LPMO activity (Bissaro et al. ., 2017; Kuusk et al., 2018; Petrovic et al., 2018).
  • BsLPMO10A releases native and C1-oxidized oligosaccharides from the substrate, especially polysaccharides linked through ⁇ -1,4 glycosidic bonds
  • BsLPMO10A dextran-like substrates were used for HPAEC-PAD and MALDI-TOF-MS analyses.
  • the results showed that BsLPMO10A was able to release C1 oxidized and native (non-oxidized) oligosaccharides from MLG-L or MLG-B ( Figure 4, A).
  • the elution peaks with retention times from 5 to 17 min correspond to the degree of polymerization (DP ) ranged from 2 to 6, while the peaks observed at 17 to 25 min corresponded to C1 oxidation products (Correa et al., 2019; Stepnov et al., 2021; Zhang et al., 2021).
  • C4-oxidized LPMO mainly shows a typical curve at a retention time of 30-35 min, corresponding to a large peak of C1/C4 double oxidation products at about 40 min (Zarah Forsberg et al., 2014; Isaksen et al., 2014; Sun et al., 2020).
  • BsLPMO10A is an AA10LPMO oxidized on the C1 carbon ( Figure 4 A and 4 B).
  • the molecular weight of the oligosaccharide was verified using MALDI-TOF-MS.
  • B in Figure 4 and C in Figure 4 show an m/z signal of 505 ([M+H Da]+ ) or 527, 689, 851 and 1013 ([M+Na Da]+) products corresponding to cellotriose (DP3), cellotetraose (DP4), cellopentaose (DP5) and cellohexose (DP6).
  • Signals with m/z values of 543, 705, 867 and 1029 correspond to oxidized cellotriose (DP3ox), cellotetraose (DP4ox) with terminal C1-specific aldonic acid , cellopentaose (DP5ox) and cellohexose (DP6ox) (Phillips, Beeson, Cate, & Marletta, 2011; 3 to 6.
  • a ruminal microbial glucanase (IDSGLUC5-38), which efficiently converts the glucan substrate into glucose and cellobiose, was used (Cao et al ., 2021).
  • IDSGLUC5-38 ruminal microbial glucanase
  • MLG-L released 3127 ⁇ 535 ⁇ mol/L natural products and 3100 ⁇ 139 ⁇ mol/LC1 oxidation products.
  • BsLPMO10A was released from MLG-B More natural products (645 ⁇ 137 ⁇ mol/L) and C1 oxidation products (1260 ⁇ 58 ⁇ mol/L) (D in Figure 4).
  • MLG-B and MLG-L are classified as mixed bond glucans (MLG),
  • MLG mixed bond glucans
  • the backbone of both substrates consists of ⁇ -D-glucose residues linked by (1 ⁇ 4) and (1 ⁇ 3) bonds.
  • the proportion and distribution of these ⁇ -(1 ⁇ 4) and ⁇ -(1 ⁇ 3) bonds differ between MLGs.
  • BsLPMO10A is inactive against ⁇ -(1 ⁇ 3)-linked laminarin (Table 2), it was inferred that the enzyme attacks ⁇ -(1 ⁇ 4) linkages to release native and C1-oxidized cellooligosaccharides.
  • the average ratio of ⁇ -(1 ⁇ 4): ⁇ -(1 ⁇ 3) bonds of MLG-B and MLG-L is approximately 3:1 and 2:1 respectively (https://www.megazyme.com), which Consistent with MLG-B releasing more product than MLG-L (D in Figure 4).
  • BsLPMO10A was found to release native and C1-oxidized xylo-oligosaccharides from BX (Fig. 5, A).
  • BX is a branched xylan containing one xylose unit in the main chain and decorated with approximately 13% GlcA-OMe in the side chain (Table 2) (https://www.megazyme.com/Xylan- Beechwood-purified).
  • MALDI-TOF-MS analysis showed that the m/z signals released by BsLPMO10A were 547 ([M+H Da]+) or 569 and 701 ([M+Na Da]+) or 759, 891 and 1023 ([M+GlcA -OMe+Na Da]+) correspond to linear-/branched-xyltoterose (DP4), xypentose (DP5), and xylhexaose (DP6), respectively.
  • DP4 linear-/branched-xyltoterose
  • DP5 xypentose
  • DP6 xylhexaose
  • the m/z signal is 585([M+16+NaDa]+), 695([M+16+H Da]+) or 753([M+GlcA-OMe+16+H Da]+) or 511, 775 , 907 and 1039 (m/z values of [M+GlcA-OMe+16+Na Da]+) corresponding to C1 oxidation of linear/branched-xylobiose (DP2), xylotetraose (DP4), xylopentan sugar (DP5) and xylhexaose (DP6) (B in Figure 5).
  • DP2 linear/branched-xylobiose
  • DP4 xylotetraose
  • DP5 xylopentan sugar
  • DP6 xylhexaose
  • rumen microbial xylanase IDSXYN11-1 efficiently converts xylan into xylose and xylobiose (Wang, 2015a), both of which are used for the breakdown of BX.
  • IDSXYN11-1 rumen microbial xylanase
  • TtLPMO9A TtLPMO9B, TtLPMO9E, TtLPMO9G, TtLPMO9T, and TtLPMO9U
  • BeWX beech glucuronoxylan
  • AGX spruce arabic glucuronoxylan
  • acGX acetylated glucuronoxylan
  • TtLPMO9A TtLPMO9A
  • TtLPMO9E TtLPMO9G
  • TtLPMO9G TtLPMO9G
  • BsLPMO10A also showed catalytic effects on KGM and SBA, producing native and C1-oxidized oligosaccharides ( Figures 6A and 6B).
  • the activity of this enzyme on PASC, chitin, pectin, and PGA was difficult to detect (Fig. 6, C-E).
  • AA10LPMOs can catalyze chitin and/or PASC substrates, such as BaAA10A from Bacillus licheniformis (Forsberg et al., 2014), LmLPMO10 from Listeria monocytogenes (Paspaliari et al., 2015 ) and SgLPMO10F from Streptomyces griseus (Nakagawa et al., 2015). Therefore, these LPMOs were previously classified as chitin-binding protein 21 (CBP21) or carbohydrate-binding module 33 (CBM33).
  • CBP21 chitin-binding protein 21
  • CBM33 carbohydrate-binding module 33
  • KpLPMO10A from Kitasatospora papulosa was first reported to act on xylan, which consists of ⁇ -(1 ⁇ 4)-D-xylose residues (Correa et al., 2019). More surprisingly, PcAA10 derived from Paenibacillus curdlanolyticus showed activity against a range of chitin, cellulose (Avicel and ⁇ -cellulose) and hemicellulose (birch xylan, oat xylan and ivory nut manna glycan) substrate activity (Limsakul et al., 2020).
  • the BsLPMO10A developed in this study showed strong activity against hemicellulose decomposition, such as MLG, xylan, mannan, and arabinan (Table 2), exhibiting a wider range than PcAA10 Active substrate spectrum.
  • activity against mannans is commonly observed in AA9LPMO (Agger et al., 2014; Calderaro et al., 2020; Liu, Olson, Wu, Broberg, & Sandgren, 2017)
  • BsLPMO10A and PcAA10 show little The mannan-degrading ability was found in AA10LPMO.
  • BsLPMO10A showed activity against arabinan for the first time in this study (Table 2 and Figure 6, B).
  • the dispersed state of polysaccharides or the formation of aggregates may also contribute to the catalytic efficiency of LPMO.
  • a recent study reported that the oxidation effect of AoLPMO9A/B (Chen, Zhang, Long, & Ding, 2021) and MtLPMO9B (Sun et al., 2021) decreased sharply, while the crystallinity index increased.
  • the catalytic efficiency of BsLPMO10A also differs among substrates consisting of ⁇ -1,4-linked glucose.
  • BsLPMO10A is more active on soluble substrates including Icelandic moss lichen and barley ⁇ -glucan, followed by partially soluble PASC and insoluble MCC (Table 2). To sum up, the opposite is true for BsLPMO10A.
  • BsLPMO10A promotes the degradation of various polysaccharides and natural lignocellulosic biomass
  • BsLPMO10A was used to synergistically degrade a range of polysaccharides with GH. Although negligible reducing sugars were released after treatment with BsLPMO10A alone, this enzyme was observed to accelerate the breakdown of MLG-L, BX, PASC, and chitin (Fig. 7). After 72 hours of co-reaction, compared with the GH alone group, the reducing sugars in MLG-L, BX, PASC and chitin in the synergistic group were 3176 ⁇ 97, 7436 ⁇ 165, 649 ⁇ 44 and 2604 ⁇ 130 ⁇ mol/L respectively.
  • LPMOs Due to their widespread use in polysaccharide depolymerization, LPMOs are widely used to cooperate with GHs for substrate degradation. Over the past decade, LPMOs from AA9-11 and 13-16 have been shown to have synergistic effects with a variety of polysaccharides, including cellulose (Harris et al., 2010; Xin, Blossom, Lu, & Felby, 2022), geometric Butin (Nakagawa et al., 2015; Paspaliari et al., 2015), xylan (Correa et al., 2019; Jung et al., 2015) and pectin (Sabbadin et al., 2021). However, BsLPMO10A was found to significantly promote a variety of polysaccharides, especially hemicelluloses, in which the backbone contains ⁇ -1,4 glycosidic bonds, regardless of the type of sugar ring (Table 2 and Figure 6).
  • BsLPMO10A was used together with glucanase IDSGLUC5-38, mannanase BsMAN26-3 and xylanase TfXYN11-1 for the breakdown of feed.
  • the synergistic effect of BsLPMO10A, glucanase, xylanase, and mannanase significantly depolymerized cellulose and hemicellulose (P ⁇ 0.05).
  • the raw material of natural plant origin consists of cellulose, hemicellulose, pectin and lignin.
  • LPMO-AoAA17 an AA17 LPMO derived from Aspergillus oryzae was found to act on lignin by cleaving ⁇ -O-4 bonds (Bhatia & Yadav, 2021).

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Abstract

提供了一种来自枯草芽孢杆菌的裂解性多糖单加氧酶BsLPMO10A，该裂解性多糖单加氧酶的氨基酸序列如SEQ ID NO.1所示。还提供了编码该酶的基因，含有该基因的表达载体，含有该表达载体的重组菌，以及该酶在促进半纤维素底物降解、促进多糖底物β-1,4糖苷键断裂、和与糖苷水解酶协同促进多糖的分解上的应用。

Description

来自枯草芽孢杆菌的裂解性多糖单加氧酶及其应用 技术领域
本发明属于酶学领域，具体涉及一种来自枯草芽孢杆菌的裂解性多糖单加氧酶及其应用。
背景技术
植物木质纤维素是自然界中最丰富的生物质来源，年产量约为10 11-10 12吨(Centore,Hochman,&Zilberman,2014)。其中，非淀粉多糖，如纤维素和半纤维素以及木质素是主要成分，赋予植物刚性和抗昆虫和真菌侵袭的能力。在有效利用之前，这些生物聚合物需要完全分解成单体和低聚物。不幸的是，木质纤维素中高度复杂和顽固的结构成为生物降解的障碍。糖苷水解酶(GHs)，包括纤维素酶、木聚糖酶和葡聚糖酶，已广泛用于工业，特别是用于食品/饲料添加剂的开发。然而，仅由于天然多糖结构复杂，尤其是晶体结构中链之间形成的氢键，因此，仅仅使用GHs难以彻底分解天然多糖。
最近发现，裂解性多糖单加氧酶(LPMOs，EC 1.14.99.53-56)作为一种新型氧化酶，可催化顽固多糖的氧化裂解。LPMO是铜离子依赖性酶家族，可提高传统GHs的生物质解聚效率，并通过氧化和裂解多糖底物促进天然碳循环。根据碳水化合物活性酶数据库(CAZy,http://www.cazy.org/)，LPMO被归类为辅助活性家族(AAs)(Levasseur,Drula,Lombard,Coutinho,&Henrissat,2013)。迄今为止，在细菌、真菌、病毒、昆虫和古生菌中发现了大量作用于纤维素、几丁质、淀粉和其他多糖底物的LPMO(Lombard,Golaconda Ramulu,Drula,Coutinho,&Henrissat,2014)。根据其序列和底物特异性分为8个家族，AA9-11和AA13-17。大多数LPMO为β-折叠拓扑结构，其中某些LPMO为β-折叠通过可变长度的环状结构连接(Hemsworth,Henrissat,Davies,&Walton,2014；Harris et al.,2010；Leggio et al.,2015)。鉴于其平坦的底物结合表面，这些酶适用于结晶多糖的结合和裂解，为GHs的后续水解提供更多的进攻位点(Hemsworth,Johnston,Davies,&Walton,2015；Johansen,2016；Zhang et al.,2019)。
在过去十年中，人们在挖掘和表征纤维素和几丁质活性LPMO方面做出了巨大努力(Forsberg et al.,2014；Nakagawa et al.,2015；Paspaliari,Loose,Larsen,&Vaaje-Kolstad,2015)。相比之下，作用于植物木质纤维素两种主要半纤维素成分催化木聚糖(Correa et al.,2019；Couturier et al.,2018；Hüttner et al.,2019；Jung,Song,Kim,&Bae,2015；
Figure PCTCN2022111015-appb-000001
et al.,2022；Zerva,Pentari,Grisel,Berrin,&Topakas,2020)和甘露聚糖(Li et al.,2021a；Limsakul et al.,2020)降解的LPMO较少。
大多数芽孢杆菌属物种是非致病性的，并且一直是异源蛋白质表达的有吸引力的宿主，这主要是因为它们能够分泌功能性酶。在所有芽孢杆菌中，枯草芽孢杆菌被美国食品和药物管理局(FDA)普遍认为是安全的(GRAS)。作为益生菌，枯草芽孢杆菌在人类和动物的胃肠道健康中发挥着重要作用。例如，枯草芽孢杆菌改善了家禽的生长性能、免疫反应、热应激耐受性和抗病性(Wang et al.,2022)。同时，该微生物已被证实可以改善瘤胃发育和新生荷斯坦犊牛的健康状况(Wang et al.,2022)。因此，从枯草芽孢杆菌中挖掘新型LPMO将具有重要意义。
发明内容
本发明所要解决的技术问题为：如何从枯草芽孢杆菌中挖掘新型LPMO。
本发明的技术方案为：一种来自枯草芽孢杆菌的裂解性多糖单加氧酶BsLPMO10A，由455个氨基酸残基构成，其氨基酸序列如SEQ ID No.1所示。
编码上述裂解性多糖单加氧酶BsLPMO10A的基因，由于密码子的简并性，存在多种不同的编码序列。
进一步地，所述基因的核苷酸序列如SEQ ID No.2所示。
一种表达载体，含有上述所述的基因。
一种重组菌，含有上述所述的表达载体。
本发明的裂解性多糖单加氧酶BsLPMO10A在促进半纤维素底物降解上的应用。
进一步地，所述半纤维素底物为地衣多糖、β-葡聚糖、刺槐豆胶、魔芋葡甘露聚糖、山毛榉木木聚糖和甜菜碱阿拉伯糖。
本发明的裂解性多糖单加氧酶BsLPMO10A在促进多糖底物β-1,4糖苷键断裂上的应用。
本发明的裂解性多糖单加氧酶BsLPMO10A在与糖苷水解酶协同促进多糖的分解上的应用。
缩略语和关键术语定义
AA               辅助活性家族
BX               山毛榉木聚糖
CAZy             水化合物活性酶数据库
DNS              3,5-二硝基水杨酸
FDA              美国食品和药物管理局
FNⅢ             纤连蛋白Ⅲ型
GHs              糖苷水解酶
GRAS             普遍认为安全
HPAEC-PAD        脉冲安培检测器离子色谱
HRP              辣根过氧化物酶(HRP
IPTG             异丙基硫代-β-D-吡喃半乳糖苷
KGM              魔芋葡甘露聚糖
LBG              刺槐豆胶
MALDI-TOF-MS     基质辅助激光解析电离飞行时间质谱
MCC              微晶纤维素
MLG-B            大麦β-葡聚糖
MLG-L            冰岛苔藓地衣多糖
PASC             磷酸溶胀纤维素
PGA              柑橘聚半乳糖醛酸
SBA              甜菜阿拉伯聚糖
与现有技术相比，本发明具有以下有益效果：
本发明的源自枯草芽孢杆菌(BsLPMO10A)的新型AA10LPMO，对它进行异源表达和功能特征。重组BsLPMO10A在50℃和pH5.0时具有最佳活性。底物特异性研究表明，该酶表现出广泛的活性底物谱，包括MLG-L、MLG-B、LBG、KGM、BX，特别是对于通过β-1,4糖苷键连接的多糖，包括β-(Glc1→4Glc)、β-(Xyl1→4Xyl)和β-(Man1→4Man)。与此同时BsLPMO10A在本研究中首次显示出对阿拉伯聚糖的活性。HPAEC-PAD和MALDI-TOF-MS分析表明，BsLPMO10A主要从混合连接葡聚糖和山毛榉木聚糖中释放聚合度(DP)为3-5的天然寡糖和从DP3ox到DP6ox的C1氧化寡糖。它与一系列糖苷水解酶的协同作用表明BsLPMO10A显着加速了葡聚糖、木聚糖、纤维素和几丁质的糖化。此外，通过六种天然饲料的降解进一步验证了BsLPMO10A和GHs的协同作用。
附图说明
图1.BsLPMO10A和数据库中其他表征的AA10LPMO的系统发育树。使用MEGA11对功能表征的LPMO进行多序列分析，然后使用构建/测试最大似然法进行系统发育分析。BsLPMO10A(GenBank编号：QDC27792.1)。
图2.SDS-PAGE和酶谱分析。(A)全长BsLPMO10A和(B)BsLPMO10A(CD)；泳道M，蛋白Marker。(C)酶谱分析：Ⅰ、MLG-L；Ⅱ、MLG-B；Ⅲ、LBG；Ⅳ、KGM；Ⅴ、BX；Ⅵ、PASC；Ⅶ、胶体几丁质。
图3.BsLPMO10A的生化特征。(A)最适温度和(B)最适pH值。抗坏血酸(C)和H 2O 2(D)对BsLPMO10A活性的影响；CK，对照组。数据代表平均值±SD(n＝4)。使用t检验对CK和实验组之间进行统计分析。**，P<0.05；***，P<0.001。
图4.BsLPMO10A对葡聚糖的降解。(A)BsLPMO10A从MLG-L和MLG-释放的寡糖的HPAEC-PAD色谱图；(B-C)BsLPMO10A对MLG-L(B)和MLG-B(C)释放的寡糖进行MALDI-TOF-MS分析；(D)BsLPMO10A和葡聚糖酶(IDSGLUC5-38)对MLG-L和 MLG-B降解产物的HPAEC-PAD色谱图。插图显示了BsLPMO10A和葡聚糖酶(IDSGLUC5-38)从MLG-L和MLG-B得到的天然和C1氧化产物的总和。G1，葡萄糖；G2，纤维二糖；G3，纤维三糖；G4，纤维四糖；G5，纤维五糖；G6，纤维六糖；G1ox，C1-氧化葡萄糖；G2ox。C1-氧化纤维二糖。
图5.BsLPMO10A对BX的降解。(A)BsLPMO10A从BX释放的寡糖的HPAEC-PAD色谱图；(B)BsLPMO10A和木聚糖酶(IDSXYN11-1)对BX降解产物的HPAEC-PAD色谱图。插图显示了由BsLPMO10A和木聚糖酶(IDSXYN11-1)从BX产生的天然产物和C1氧化产物的总和。X1，木糖；X2，木二糖；X3、木三糖；X4，木四糖；X5，木戊糖；X6，木六糖；X1ox，C1-氧化木糖。
图6.BsLPMO10A对其他多糖降解产物的HPAEC-PAD色谱图。(A)KGM和LBG；(B)SBA；(C)果胶和PGA；(D)PASC；(E)几丁质。
图7 BsLPMO10A和GHs对多糖降解的协同作用。(A)BsLPMO10A和葡聚糖酶(IDSGLUC5-38)从MLG-L释放的还原糖；(B)BsLPMO10A和木聚糖酶(TfXYN11-1)从BX释放的还原糖；(C)BsLPMO10A和甘露聚糖酶(BsMAN26-3)从LBG释放的还原糖；(D)BsLPMO10A和纤维素酶(IDSGLUC5-11)从PASC释放的还原糖；(E)BsLPMO10A和几丁质酶(BtCHI18-1)从几丁质释放的还原糖。数据代表平均值±SD(n＝4)。使用t检验对协同作用和GH单独作用组之间的进行统计分析。*，P<0.05；**，P<0.05；***，P<0.001。
图8.BsLPMO10A和GHs对天然木质纤维素原料降解的协同作用。(A)花生壳；(B)大豆秸秆；(C)羊草秸秆；(D)水稻秸秆；(E)水稻壳；(F)小麦秸秆。数据代表平均值±SD(n＝4)。使用Tukey-Kramer检验进行多重比较。大小写字母分别表示纤维素或半纤维素含量差异(P<0.05)。
图9 BsLPMO10A的氨基酸比对和结构预测。(A)BsLPMO10A和其他AA10LPMO的氨基酸比对；(B-C)WebLogo分析“组氨酸支架”中涉及的关键残基；(D)BsLPMO10A的同源建模。
具体实施方式
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为从商业渠道购买得到的。
一、菌株、载体和化学品
枯草芽孢杆菌W800N、大肠杆菌BL21(DE3)和pET-30a(+)为常规商品，本实验室 保存。冰岛苔藓地衣多糖(MLG-L)、大麦β-葡聚糖(MLG-B)、刺槐豆胶(LBG)、魔芋葡甘露聚糖(KGM)、山毛榉木聚糖(BX)、甜菜阿拉伯聚糖(SBA)、海带多糖、胶体几丁质、约60％的酯化度柑橘果胶、柑橘聚半乳糖醛酸(PGA)，寡糖购自Megazyme(Wicklow,Ireland)。磷酸溶胀纤维素(PASC)由微晶纤维素制备(Zhang,Cui,Lynd,&Kuang,2006)。微晶纤维素(MCC)、培养基、卡那霉素和异丙基硫代-β-D-吡喃半乳糖苷(IPTG)购自Sangon(中国上海)。花生壳、大豆秸秆、羊草、水稻秸秆、水稻壳和小麦秸秆采集自当地农场(中国浙江)。
二、实验方法
(1)序列分析
将各枯草芽孢杆菌菌株的全部基因组序列以FASTA格式上传至在线软件dbCAN HMMdb v10.0(https://bcb.unl.edu/dbCAN2/blast.php)进行功能注释(Zhang et al.，2018)。然后将从注释结果中获得的候选基因提交到Pfam数据库(http://pfam.xfam.org/search#tabview＝tab0)，以预测潜在的域和功能。使用Clustal Omega多序列比对程序(http://www.ebi.ac.uk/Tools/msa/clustalo/)对LPMOs进行氨基酸比对。利用I-TASSER服务器(http://zhanglab.ccmb.med.umich.edu/I-TASSER/download/)生成了BsLPMO10A的结构同源模型。利用MEGA 11邻接法构建系统发育树，采用bootstrap法进行系统发育测试，bootstrap复制次数为1000次。取代类型为氨基酸，模型为Jones-Taylor-Thornton(JTT)模型。结果使用ESPript 3.0渲染(Robert&Gouet,2014)。利用WebLogo3对各位点的氨基酸保守序列进行分析(http://weblogo.threeplusone.com/)。
(2)基因克隆、表达、纯化和凝胶电泳
使用快速细菌基因组DNA分离试剂盒(生工，上海，中国)分离来自枯草芽孢杆菌W800N的基因组DNA，并作为BsLPMO10A基因PCR扩增的模板。BsLPMO10A的全长(SEQ ID No.2)和催化结构域(SEQ ID No.3)均使用BsLPMO10A-SacI和BsLPMO10A-XhoI/BsLPMO10A(CD)-XhoI引物获得(表1)。将所得片段插入到SacI和XhoI之间的pET-30a(+)中，然后通过热激转化到大肠杆菌BL21(DE3)中。重组菌株BL21/pET-30a(+)/BsLPMO10A和BL21/pET-30a(+)/BsLPMO10A(CD)进行IPTG诱导、超声处理和使用配备HisTrap TM的
Figure PCTCN2022111015-appb-000002
start蛋白纯化系统和纯化FF柱(GE Healthcare BioSciences，美国)(Zhou等人，2020)进行纯化。纯化的酶通过10-kDa Millipore
Figure PCTCN2022111015-appb-000003
离心过滤器(Merck Millipore,Burlington,MA,USA)进一步过滤以去除咪唑。随后，对酶进行SDS-PAGE(15％电泳胶和4％浓缩胶)(Laemmli，1970)分析。使用含有0.5％(w/v)的各种多 糖底物的分离凝胶进行酶谱分析。电泳完成后，将凝胶用甲醇:水(1:3)溶液复性15min，复性后凝胶浸入0.05M乙酸钠缓冲液(pH5.5)中并在37℃下孵育30min。然后将凝胶用1mg/mL刚果红溶液染色，用水和1M NaCl洗涤，直至透明条带可见。并用乙酸固定。
表1引物列表
Figure PCTCN2022111015-appb-000004
注：划线为酶切位点
(3)活性测定
在活性测定之前，BsLPMO10A在冰上于3倍摩尔浓度过量的CuSO 4中孵育60min，进行铜饱和预处理。使用10-kDa Millipore
Figure PCTCN2022111015-appb-000005
离心过滤器(Merck Millipore,Burlington,MA,USA)去除未结合的铜，然后将酶重新悬浮在0.05M乙酸钠缓冲液(pH5.5)中。Bradford方法(Bradford,1976)用于确定酶的浓度。除非另有说明，后续试验使用铜饱和酶用于活性测定。
3,5-二硝基水杨酸(DNS)测定：进行了DNS分析(Bailey,Biely,&Poutanen,1992)以确定BsLPMO10A活性。简而言之，将15μL(～1.5μM)纯化酶与60μL 5mg/mL各种多糖底物(MLG-L、MLG-B、LBG、KGM、BX、SBA、海带多糖、PASC、MCC、胶体几丁质、果胶和PGA)溶解在0.05M乙酸钠缓冲液(pH 5.5)中，在37℃和200rpm下反应24h。然后，加入75μL DNS并将溶液煮沸10min。冷却至室温后，使用分光光度法在540nm处测定吸光度。
(4)生化表征
为了估计BsLPMO10A的最适温度，将15μL(～1.5μM)BsLPMO10A与60μL 5mg/mL LBG溶液在不同温度(30-80℃)下孵育24h，并使用DNS方法。BsLPMO10A的最适pH值是通过将酶在各种pH缓冲溶液(pH 2.2-8.0的柠檬酸盐/磷酸盐缓冲液和pH 8.0-9.0的0.2M Tris-HCl缓冲液)中在37℃孵育24h来确定的。最高活性确定为100％。为了研究抗坏血酸和H 2O 2对BsLPMO10A的影响，酶在5mg/mL LBG溶液中与不同浓度的抗坏血酸(0.5、1、1.5和3mM)和H 2O 2(0.5、1、2.5、5、10和50mM)在37℃下孵育24h，并使用DNS方法测量活性。
(5)BsLPMO10A的催化产物
为了探索BsLPMO10A的产物和氧化活性，将1.5μM蛋白溶液与各种含有1.5mM抗坏血酸的多糖底物(MLG-L、MLG-B、LBG、KGM、BX、SBA、昆布多糖、PASC、胶体几丁质、果胶和PGA)在37℃下孵育24h。然后使用带有脉冲安培检测器离子色谱[HPAEC-PAD、ICS3000，分离柱为CarboPac PA200(3×250mm)以及CarboPac PA200保护柱(3×50mm)](Dionex，Thermo)，如文献中所述(Westereng et al.,2013)进行测定。
为了估计总氧化产物，先使用BsLPMO10A催化，再用一种GH将未反应完多糖底物有效地转化为它们的单体和二聚体。简而言之，将150μL(～15μM)BsLPMO10A与600μL 5mg/mL的各种多糖底物溶液在37℃下孵育24h，然后通过煮沸5min使酶失活。然后将溶液以12,000rpm离心15min，并使用50μL(～0.8μM)的IDSGLUC5-38或50μL(～0.5μM)的IDSXYN11-1在37℃下进一步水解上清液24h，然后煮沸5min使酶失活。将溶液以12,000rpm离心15min液用于HPAEC-PAD分析，配备CarboPac PA1(3×250mm)和CarboPac PA1(3×50mm)保护柱(Dionex,Thermo)。对于MALDI-TOF-MS分析，将1μL上述上清液与1μL溶解在含有10mM乙酸钠的50％乙腈溶液中的2,5-二羟基苯甲酸基质混合，并在样品板上干燥。然后在线性正离子模式下在300-1800的m/z范围内进行测量，使用60.0Hz的频率(UltrafleXtreme，Bruker，Germany)进行10次累积200次拍摄。按照(Kobayashi,Kamiya,&Enomoto,1996)的描述制备氧化的单糖和二糖，稍作修改。简而言之，将含有14mg碘的15mL甲醇溶液缓慢加入10mL 20mg/mL样品溶液(溶于ddH2O)中，并在40℃下孵育15min。然后，在室温(25℃)下加入5mL含有0.2g KOH的甲醇溶液，然后在40℃下加热1h，直到碘颜色褪去。通过加入20mL冰水将混合物冷却，并在N 2下进一步干燥。最后，在使用前将样品溶解在2mL ddH 2O中。
(6)BsLPMO10A和GHs对多糖和天然木质纤维素原料降解的协同作用
为了研究BsLPMO10A和GHs对多糖的协同作用，将7.5μL(～0.75μM)BsLPMO10A和7.5μL(～0.05μM)我们小组先前获得的各种GHs进行反应，包括瘤胃微生物葡聚糖酶(IDSGLUC5-38，原称Cel5A-h38)(Cao et al.,2021),Thermobifida fusca木聚糖酶(TfXYN11-1，原称TFX)(Wang et al.,2015a),瘤胃微生物木聚糖酶(IDSXYN11-1，原称XYN-LXY)(Wang et al.,2015b)，枯草芽孢杆菌甘露聚糖酶(BsMAN26-3，原称BSM)(Gao et al.,2019)，瘤胃微生物纤维素酶(IDSGLUC5-11，原称Cel5A-h11)(He,Jin,Cao,Mi,&Wang,2019)和几丁质酶(BtCHI18-1，原称BtCHI1)(Wu et al.,2022)，反应体系中含有1.5mM抗坏血酸，于37℃中进行孵育。在2、4、8、12、24、48和72h取等分试样， 并使用DNS方法评估还原糖。
天然底物测定：在活性测定之前，将花生壳、大豆秸秆、羊草、水稻秸秆、水稻壳和小麦秸秆的天然木质纤维素原料研磨并用NaOH如前所述方法(Cao et al.,2021)进行处理。为了研究BsLPMO10A或各种GHs对天然木质纤维素分解的影响，使用500μL(～20μM)BsLPMO10A或(～0.5μM)GHs在3mL柠檬酸盐/磷酸盐缓冲液(pH 6.0)中消解0.1g各种预处理的原料样品，并在25℃下反应96h。为了研究BsLPMO10A和GHs对天然木质纤维素分解的协同作用，使用500μL(～20μM)BsLPMO10A和(～0.5μM)GHs在3mL柠檬酸盐/磷酸盐缓冲液(pH 6.0)中消化0.1g各种预处理的原料样品，并在25℃下反应96h。反应结束时，将每种混合物以12,000rpm的转速离心15min，弃去上清液。不溶性材料用ddH 2O冲洗10次，并根据国家可再生能源实验室(NREL)方法(Sluiter et al.,2011)用于纤维素和半纤维素测定。将干燥的天然木质纤维素添加到10mL离心管中，并添加8mL 72％硫酸。在30℃孵育1h后，将最终硫酸浓度稀释至4％，并将混合物置于121℃高压灭菌锅中，孵育1h。样品中的纤维素和半纤维素被水解成单糖，并分别使用苔黑酚(Albaum,&Umbreit,1947)和蒽酮比色法(Hedge,&Hofreiter,1962)进行量化。
三、结果分析
(1)序列分析
序列分析表明，BsLPMO10A由455个氨基酸残基(SEQ ID No.1)组成，包括一个AA10催化结构域和纤连蛋白Ⅲ型(FNⅢ)模块。SignalP5.0预测表明，N端的前40个氨基酸是信号肽。同源性模型表明，BsLPMO10A含有一个免疫球蛋白G(IgG)样β-三明治折叠和一个含有铜离子的组氨酸支架序列(图9)。两个保守的组氨酸形成一个T形的“组氨酸支架”，His41和His130，位于催化结构域的表面，代表了LPMO的典型特征。“组氨酸支架”和几个附近的芳香族氨基酸残基被确定为底物结合或催化位点相关(Book et al.,2014；Labourel et al.,2020；Span&Marletta,2015；Wu et al.,2013；Zhou,Qi,Huang,&Zhu,2019)。与C1-、C4-或C1/C4-氧化模式的AA9酶不同，AA10LPMO被认为主要以C1-氧化模式催化底物，并可进一步分类为C1-和C1/C4-氧化亚家族(Li et al.,2021b)。在这项研究中，系统发育树分析表明，BsLPMO10A与来自AA10家族的其他C1氧化LPMO聚集在一起(图1)。尽管BsLPMO10A和枯草芽孢杆菌基因组中的其他12种LPMO共存，但BsLPMO10A与这些酶的相似性相对较低(33.70-62.63％)，但与来自芽孢杆菌属的未表征的AA10蛋白的相似性较高(99.53％)(WP_001065159.1)。迄今为止，在CAZy数据库中已经对AA10家族中的约30种酶进行了表征，这些酶主要来源于细菌。与这些功能研究的AA10LPMO相 比，BsLPMO10A与地衣芽孢杆菌DSM 13 BlLPMO10A(GenBank编号：AAU39477.1)(Courtade et al.,，2020)和解淀粉芽孢杆菌DSM 7 BaLPMO10A(GenBank编号：CBI42985.1)(Hemsworth et al.,2013)的相似度最高，相似度分别为73.4和63.5％，但这两者都是几丁质活性的LPMO。
(2)BsLPMO10A是一种嗜温酶，由一定量的抗坏血酸或H 2O 2激活
纯化的BsLPMO10A及其截短的催化结构域[BsLPMO10A(CD)]的分子量分别为～56(图2中A)和～34kDa(图2中B)。两种酶都对MLG-L、MLG-B、LBG、KGM、BX和SBA有活性(图2中C)。值得注意的是，与仅催化结构域相比，包含催化结构域和FNⅢ的全长BsLPMO10A对MLG-L(P<0.001)、MLG-B(P<0.001)、LBG(P<0.05)和KGM(P<0.01)(表2)催化活性更高。FNⅢ结构域广泛存在于细菌、酵母、植物和动物蛋白中。据报道，芽孢杆菌属、蜡状芽孢杆菌(Mutahir et al.,2018)和苏云金芽孢杆菌(Manjeet,Madhuprakash,Mormann,Moerschbacher,&Podile,2019)包含一个或多个FNⅢ结构域。然而，人们对其功能和详细机制知之甚少，有时还存在争议。敲除FNⅢ结构域(也称为X1结构域)并未改变TfAA10B的催化效率(Arfi,&Umbreit,2014；Kruer-Zerhusen et al.,2017)。相比之下，周等人(Zhou,Irwin,Escovar-Kousen,&Wilson,2004)表明没有FNⅢ结构域的截短Cel9A-90的催化活性降低到野生型的43％。令人惊讶的是，两种酶TfAA10B和Cel9A-90均来自褐色嗜热裂孢菌。以往的研究表明，FNⅢ模块有利于稳定酶的构象，提高可溶性和不溶性底物的活性(Han,Shang-Guan,&Yang,2019；Kataeva et al.,2002；Mutahir et al.,2018；Nguyen et al.,2021)。关于在不同底物上的不同性能，我们推测BsLPMO10A可能优先作用于某些由葡萄糖或甘露糖单元组成的多糖，其次是由木糖或阿拉伯糖组成的多糖(表2和图2中C)。在这种情况下，FNⅢ结构域极大地有助于底物结合和/或催化效率。因此，与截短酶相比，野生型BsLPMO10A对“优选”底物(包括冰岛苔藓地衣多糖、大麦β-葡聚糖、刺槐豆胶和魔芋葡甘露聚糖)显示出明显更高的活性(P<0.05)。对于“不太优选”的底物，例如山毛榉木聚糖和甜菜阿拉伯聚糖，去除FNⅢ结构域不会导致催化活性的严重降低，导致与野生型和截短的BsLPMO10A的活性相当。在最近的一项研究中，据报道FNⅢ结构域可能通过提高其对可溶性底物的亲和力(较低的Km值)来增加酶活性(Nguyen et al.,2021)。然而，FNⅢ结构域在酶性能中的详细机制仍有待解决。在本研究研究的多糖中，BsLPMO10A和BsLPMO10A(CD)对PASC、MCC、胶体几丁质、果胶或PGA均无活性。因此，采用全长BsLPMO10A进行进一步研究，因为它在各种多糖的分解中具有高活性。
表2.全长和截短BsLPMO10A的比活性
Figure PCTCN2022111015-appb-000006
数据代表平均值±SD(n＝4)。ND，未检测到。使用t检验进行BsLPMO10A和BsLPMO10A(CD)之间的统计分析。*，P<0.05；**，P<0.05；***，P<0.001
BsLPMO10A的最适温度和pH分别为50℃和5.0(图3中A和3中B)，表明BsLPMO10是一种在中性和弱酸性条件下具有活性的中温酶，这与大多数先前报道的LPMO表现出最佳温度范围为40–60℃(Li et al.,2021a；Li,Dilokpimol,Kabel,&de Vries,2022；Zhang,Chen,Long,&Ding,2021)一致。值得注意的是，观察到源自高温堆肥宏基因组的MgLPMO10显示出最高的熔化温度(Tm＝83℃)和高达80℃的催化活性(Tuveng et al.,2020)。更引人注目的是，米曲霉AA17酶LPMO-AoAA17显示出100℃的最佳温度，在121℃和15psi高压灭菌后甚至具有活性(Bhatia&Yadav,2021)。众所周知，铜对LPMO的氧化活性至关重要，使用EDTA螯合去除Cu 2+会导致LPMO活性和热稳定性降低(Gusakov,Bulakhov,Demin,&Sinitsyn,2017；Semenova et al.,2019)。因此，在本研究中进行的所有测定之前，对BsLPMO10A进行了铜饱和。随后，确定了抗坏血酸和H 2O 2对BsLPMO10A活性的影响。图3中C显示抗坏血酸显着促进BsLPMO10A活性(P<0.01)，在氧化反应中充当电子供体，这与先前报道的一致(Stepnov et al.,2021)。然而，包括1,2-二羟基苯(Zhang et al.,2021)和单宁酸(Frommhagen et al.,2016)在内的一些还原剂对LPMO活性不敏感。同时，O 2被认为是参与LPMO氧化反应的共基质(Vaaje-Kolstad et al., 2010)。令人惊讶的是，最近证明H 2O 2而不是O 2驱动LPMO的共底物(Hegnar et al.,2019)。在本研究中，1或2.5mM H 2O 2分别显着提高了BsLPMO10A活性70.7％(P<0.001)或49.5％(P<0.01)。然而，当H 2O 2浓度超过5mM时观察到抑制作用(图3中D)。尽管一定量的H 2O 2会促进LPMO催化，但添加过量的H 2O 2会对酶产生负面影响并耗尽电子供体(Calderaro et al.,2020)，从而抑制LPMO活性(Bissaro et al.,2017；Kuusk et al.,2018；Petrovic et al.,2018)。
(3)BsLPMO10A从底物中释放天然和C1氧化的寡糖，特别是通过β-1,4糖苷键连接的多糖
根据BsLPMO10A的底物范围，葡聚糖样底物用于HPAEC-PAD和MALDI-TOF-MS分析。结果表明，BsLPMO10A能够从MLG-L或MLG-B中释放C1氧化和天然(非氧化)寡糖(图4中A)。然而，由于存在β-1,3-和β-1,4-糖苷键((Eklof,Shojania,Okon,McIntosh,&Brumer,2013)，保留时间为5至17min的洗脱峰对应于聚合度(DP)范围为2至6的天然产物，而在17至25min观察到的峰对应于C1氧化产物(Correa et al.,2019；Stepnov et al.,2021；Zhang et al.,2021)。据报道，C4氧化的LPMO主要在30-35min的保留时间内表现出典型的曲线，在约40min时对应于C1/C4双氧化产物的大峰(Zarah Forsberg et al.,2014；Isaksen et al.,2014；Sun et al.,2020)。在这项研究中，没有检测到对应于双氧化产物的信号，因此，我们推测BsLPMO10A是一种在C1碳上氧化的AA10LPMO(图4中A和4中B)。为了更多地了解催化产物的组成和裂解位点，使用MALDI-TOF-MS验证了寡糖的分子量。图4中B和4中C显示m/z信号为505([M+H Da]+)或527、689、851和1013([M+Na Da]+)的产物对应于纤维三糖(DP3)、纤维四糖(DP4)、纤维五糖(DP5)和纤维六糖(DP6)。m/z值为543、705、867和1029([M+16+Na Da]+)的信号对应于具有末端C1特异性醛糖酸的氧化纤维三糖(DP3ox)、纤维四糖(DP4ox)、纤维五糖(DP5ox)和纤维六糖(DP6ox)(Phillips,Beeson,Cate,&Marletta,2011；X.Zhang et al.,2021)。结果表明，BsLPMO10A主要释放C1氧化和天然寡糖，DP值为3至6。为了化学计量确定BsLPMO10A的总氧化活性，使用一种瘤胃微生物葡聚糖酶(IDSGLUC5-38)，可有效地将葡聚糖底物转化为葡萄糖和纤维二糖(Cao et al.,2021)。反应24h后，MLG-L释放出3127±535μmol/L天然产物和3100±139μmol/LC1氧化产物。值得注意的是，与MLG-L相比，BsLPMO10A从MLG-B中释放出更多的天然产物(645±137μmol/L)和C1氧化产物(1260±58μmol/L)(图4中D)。MLG-B和MLG-L被归类为混合键葡聚糖(MLG)，这两种底物的主链由通过(1→4)和(1→3)键连接的β-D-葡萄糖残基组成。然而，这些β- (1→4)和β-(1→3)键的比例和分布在MLG之间有所不同。由于BsLPMO10A对β-(1→3)连接的海带多糖无活性(表2)，这推断该酶攻击β-(1→4)键以释放天然和C1-氧化的纤维寡糖。MLG-B和MLG-L的β-(1→4)：β-(1→3)键的平均比例分别约为3：1和2：1(https://www.megazyme.com)，这与MLG-B比MLG-L释放更多产品一致(图4中D)。
类似地，发现BsLPMO10A从BX中释放天然和C1氧化的低聚木糖(图5中A)。BX是一种支链木聚糖，在主链中包含一个木糖单元，并在侧链中装饰有约13％GlcA-OMe(表2)(https://www.megazyme.com/Xylan-Beechwood-purified)。MALDI-TOF-MS分析表明，BsLPMO10A释放的m/z信号为547([M+H Da]+)或569和701([M+Na Da]+)或759、891和1023([M+GlcA-OMe+Na Da]+)分别对应于直链-/支链-木四糖(DP4)、木戊糖(DP5)和木六糖(DP6)。m/z信号为585([M+16+NaDa]+)、695([M+16+H Da]+)或753([M+GlcA-OMe+16+H Da]+)或511、775、907和1039([M+GlcA-OMe+16+Na Da]+的m/z值)对应于C1氧化的线性/支链-木二糖(DP2)、木四糖(DP4)、木五糖(DP5)和木六糖(DP6)(图5中B)。此外，瘤胃微生物木聚糖酶(IDSXYN11-1)有效地将木聚糖转化为木糖和木二糖(Wang,2015a)都用于BX的分解。反应24h后，产生959±56μmol/L的天然产物和387±161μmol/L C1氧化产物(图5中C)。有趣的是，来自BX的C1氧化/天然产物的比率为0.40，低于从MLG-B(0.99)和MLG-L(1.95)获得的比率(图4中D)，这可能是由于它偏爱β-(Glc1→4Glc)其次是β-(Xyl1→4Xyl)(表2)。如上所述，多糖主链内的糖环单元和连接类型与LPMO催化密切相关。同时，被修饰的侧链被认为在氧化反应中起作用。在最近的一项研究中，
Figure PCTCN2022111015-appb-000007
及其同事(2022)研究了来自子囊菌类Thermothielavioides terrestrisLPH172的六种LPMO(TtLPMO9A、TtLPMO9B、TtLPMO9E、TtLPMO9G、TtLPMO9T和TtLPMO9U)在山毛榉葡萄糖醛酸木聚糖(BeWX)、云杉阿拉伯葡萄糖醛酸木聚糖(AGX)和桦木的乙酰化葡糖醛酸木聚糖(acGX)底物上的生化特性。他们发现六个TtLPMO9中的三个(TtLPMO9A、TtLPMO9E和TtLPMO9G)在BeWX上具有活性，它含有较少的与PASC结合的侧链。对于具有更复杂分支的木聚糖底物，仅测试的两种(TtLPMO9A和TtLPMO9E)或一种LPMO(TtLPMO9G)分别对AGX和acGX有活性。
此外，BsLPMO10A还表现出对KGM和SBA的催化作用，产生天然和C1氧化的寡糖(图6中A和6中B)。然而，该酶对PASC、几丁质、果胶和PGA的活性难以检测(图6中C-E)。据文献报道，AA10LPMOs能够催化几丁质和/或PASC底物，例如来自地衣芽孢 杆菌的BaAA10A(Forsberg et al.,2014),来自单核细胞增生李斯特菌的LmLPMO10(Paspaliari et al.,2015)和来自灰色链霉菌的SgLPMO10F(Nakagawa et al.,2015)。因此，这些LPMO过去被归类为几丁质结合蛋白21(CBP21)或碳水化合物结合模块33(CBM33)。然而，除了几丁质和纤维素氧化外，首次报道来自Kitasatospora papulosa的KpLPMO10A作用于木聚糖，木聚糖由β-(1→4)-D-木糖残基组成(Correa et al.,2019)。更令人惊讶的是，源自Paenibacillus curdlanolyticus的PcAA10显示出对一系列几丁质、纤维素(Avicel和α-纤维素)和半纤维素(桦木木聚糖、燕麦木聚糖和象牙坚果甘露聚糖)底物的活性(Limsakul et al.,2020)。与先前报道的LPMO不同，本研究中开发的BsLPMO10A显示出对半纤维素分解的强大活性，例如MLG、木聚糖、甘露聚糖和阿拉伯聚糖(表2)，表现出比PcAA10更宽的活性底物谱。尽管在AA9LPMO中普遍观察到针对甘露聚糖的活性(Agger et al.,2014；Calderaro et al.,2020；Liu,Olson,Wu,Broberg,&Sandgren,2017)，但BsLPMO10A和PcAA10显示出很少见的甘露聚糖降解能力在AA10LPMO中发现。更有趣的是，据我们所知，BsLPMO10A在本研究中首次显示出对阿拉伯聚糖的活性(表2和图6中B)。除了糖环、糖苷键和修饰的侧链外，多糖的分散状态或聚集体的形成也可能有助于LPMO的催化效率。最近的一项研究报告称，AoLPMO9A/B(Chen,Zhang,Long,&Ding,2021)和MtLPMO9B(Sun et al.,2021)的氧化效应急剧下降，同时结晶度指数增加。BsLPMO10A的催化效率在由β-1,4连接的葡萄糖组成的底物中也有所不同。我们推测BsLPMO10A对可溶性底物(包括冰岛苔藓地衣多糖和大麦β-葡聚糖)的活性更高，其次是部分可溶性PASC和不溶性MCC(表2)。综上所述，BsLPMO10A反之亦然。
(4)BsLPMO10A促进多种多糖和天然木质纤维素生物质的降解
鉴于其对多种底物的广泛催化活性，BsLPMO10A被用于与GH协同降解一系列多糖。尽管仅用BsLPMO10A处理后释放的还原糖可忽略不计，但观察到该酶加速了MLG-L、BX、PASC和几丁质的分解(图7)。共同反应72h后，和单独的GH组相比，协同组MLG-L、BX、PASC和几丁质中的还原糖分别为3176±97、7436±165、649±44和2604±130μmol/L，分别为单独组的1.47、1.56、1.44和1.25倍(P<0.001)。对于LBG的分解，与单独的内切甘露聚糖酶水解相比，BsLPMO10A和内切甘露聚糖酶共反应72h后，还原糖产量增加了11.68±7.85％(P＝0.12)(图7中C)。值得注意的是，尽管BsLPMO10A本身并没有从PASC或几丁质中释放出类似的寡糖(图6中D和6中E)，但它显着促进了相关GH对这两种底物的分解(图7中D和7中E)。由于它们在多糖解聚中的广泛用途，LPMOs被广泛用于与GHs协同进行底物降解。在过去十年中，来自AA9-11和13-16的LPMO已被证明与多种多 糖具有协同作用，包括纤维素(Harris et al.,2010；Xin,Blossom,Lu,&Felby,2022)、几丁质(Nakagawa et al.,2015；Paspaliari et al.,2015)、木聚糖(Correa et al.,2019；Jung et al.,2015)和果胶(Sabbadin et al.,2021)。然而，发现BsLPMO10A显着促进多种多糖，尤其是半纤维素，其中主链包含β-1,4糖苷键，而与糖环种类无关(表2和图6)。
随后，BsLPMO10A与葡聚糖酶IDSGLUC5-38、甘露聚糖酶BsMAN26-3和木聚糖酶TfXYN11-1一起用于饲料的分解。为了消除木质素对催化过程的阻碍作用，我们用2％NaOH对原料进行预处理以去除其中的木质素，并获得含有28-38％纤维素和18-32％半纤维素的碱处理样品(Paritosh et al.,2021)。这些结果表明LPMO对半纤维素表现出强烈的活性，但对纤维素几乎没有活性(图8)。仅用BsLPMO10A处理后，除大豆秸秆外，所有测试基质中的半纤维素组合物均显着下降(P<0.05)，如葡聚糖、木聚糖、甘露聚糖和阿拉伯聚糖降解的强大能力所示(表2)。例如，BsLPMO10A和甘露聚糖酶之间的协同作用显着降低(P<0.05)半纤维素组成，在花生壳中降低了10.61±6.18％(图8中A)，在水稻壳中降低了8.19±3.85％(P<0.05)。图8中E)。BsLPMO10A和葡聚糖酶的协同作用显着降低(P<0.05)半纤维素组成在拉毛果秸秆(图8中C)和水稻壳中的9.65±4.04％(图8中E)。BsLPMO10A和木聚糖酶之间的协同作用显着降低(P<0.05)半纤维素组成在大豆秸秆中降低了17.67±4.68％(图8中B)，在水稻壳中降低了10.00±4.14％(图8中E)和小麦秸秆中的16.16±2.79％(图8中F)。此外，BsLPMO10A、葡聚糖酶、木聚糖酶和甘露聚糖酶的协同作用显着解聚了纤维素和半纤维素(P<0.05)。天然植物来源的原料由纤维素、半纤维素、果胶和木质素组成。由于大豆秸秆、拉毛果秸秆、水稻壳和小麦秸秆中的木聚糖和葡聚糖相对丰富(Balan et al.,2008；Wu et al.,2018)，木聚糖酶和葡聚糖酶与甘露聚糖酶处理相比，处理对它们的分解更有效，半纤维素减少了9.65-17.67％(图8中B、8中C、8中E和8中F)。此前有报道称，LPMO会导致底物出现缺口并引发纤维结构的解体，导致链断裂和原始纳米纤维的释放(Li et al.,2021b；Villares et al.,2017；Zhang et al.,2017)。因此，LPMO处理的木质纤维素表现出强烈的原纤化。使用原子力显微镜进行的详细形态观察表明，C1-/C4-氧化的LPMO直接结合到纳米晶纤维素的表面以降解底物(Eibinger,Sattelkow,Ganner,Plank,&Nidetzky,2017；Moreau et al.,2019)，为BsLPMO10A分解天然底物提供了详细的证据。与多糖氧化LPMO不同的是，一种源自米曲霉的AA17LPMO(LPMO-AoAA17)被发现通过切割β-O-4键对木质素起作用(Bhatia&Yadav,2021)。

Claims (9)

  1. 一种来自枯草芽孢杆菌的裂解性多糖单加氧酶BsLPMO10A，由455个氨基酸残基构成，其氨基酸序列如SEQ ID No.1所示。
  2. 编码权利要求1所述的裂解性多糖单加氧酶BsLPMO10A的基因。
  3. 根据权利要求2所述的基因，其特征在于，所述基因的核苷酸序列如SEQ ID No.2所示。
  4. 一种表达载体，含有权利要求2或3所述的基因。
  5. 一种重组菌，含有权利要求4所述的表达载体。
  6. 权利要求1所述的裂解性多糖单加氧酶BsLPMO10A在促进半纤维素底物降解上的应用。
  7. 根据权利要求6所述的应用，其特征在于，所述半纤维素底物为地衣多糖、β-葡聚糖、刺槐豆胶、魔芋葡甘露聚糖、山毛榉木木聚糖和甜菜碱阿拉伯糖。
  8. 权利要求1所述的裂解性多糖单加氧酶BsLPMO10A在促进多糖底物β-1,4糖苷键断裂上的应用。
  9. 权利要求1所述的裂解性多糖单加氧酶BsLPMO10A在与糖苷水解酶协同促进多糖的分解上的应用。
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