WO2022156402A1 - 一种1,3/1,4-木聚糖酶mlx1034及其编码基因与应用 - Google Patents

一种1,3/1,4-木聚糖酶mlx1034及其编码基因与应用 Download PDF

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WO2022156402A1
WO2022156402A1 PCT/CN2021/136034 CN2021136034W WO2022156402A1 WO 2022156402 A1 WO2022156402 A1 WO 2022156402A1 CN 2021136034 W CN2021136034 W CN 2021136034W WO 2022156402 A1 WO2022156402 A1 WO 2022156402A1
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xylanase
mlx1034
recombinant
recombinant expression
xylo
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张玉忠
赵芳
陈秀兰
孙海宁
宋晓妍
李平一
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山东大学
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    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
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Definitions

  • the invention relates to a 1,3/1,4-xylanase MLX1034 and its encoding gene and application, belonging to the technical field of biotechnology.
  • Xylan is the main component of plant hemicellulose, and its content is second only to cellulose.
  • Xylan in the cell wall of higher plants is a complex polypentacarbon sugar whose backbone is composed of xylose groups linked by ⁇ -1,4-glycosidic bonds, and some substituents are linked to the backbone by O2 or O3 atoms. Connected, common substituent groups include arabinosyl, galactosyl, glucuronic acid and the like. Therefore, the complete hydrolysis of such xylan requires the participation of multiple enzymes, of which endo-1,4-xylanase plays a major role.
  • Xylo-oligosaccharides refer to functional xylo-oligosaccharides composed of 2 to 7 xylose molecules.
  • beneficial bacteria such as bifidobacteria
  • xylo-oligosaccharides can selectively proliferate beneficial bacteria (such as bifidobacteria) in the intestinal tract and can improve the intestinal tract. It has the functions of lowering blood pressure, lowering blood sugar and cholesterol, and anti-cancer, so it is widely used in the fields of food, medicine, cosmetics and feed additives.
  • xylo-oligosaccharides are mainly prepared from raw materials such as corn cob, wheat bran and bagasse by biological enzymatic catalysis or chemical catalysis.
  • the xylan in the marine environment is mainly derived from the cell walls of red and green algae, and it is significantly different in structure from the xylan in higher plants.
  • the xylan derived from marine algae are all straight-chain homo-xylan, which are only composed of xylose groups connected by ⁇ -1,3-glycosidic bonds or ⁇ -1,4-glycosidic bonds, and their types include 1,3 - Xylan, 1,4-xylan and 1,3/1,4-xylan with mixed glycosidic linkages.
  • red algae the polysaccharides in the cell walls of algae of the order Palmariales and Nemaliales are mainly 1,3/1,4-xylan.
  • Palmaria palmata is a famous cold-water economic seaweed, which is rich in dietary fiber, protein and vitamins. Some compounds in its algae have anti-inflammatory and antioxidant activities. It is popular as a seafood food.
  • the structure of water-soluble 1,3/1,4-xylan extracted from Palmaria palmata is: 1,4-xylan backbone is disorderly distributed with discontinuous ⁇ -1,3 -Xylosidic bonds, the ratio of ⁇ -1,3-xylosidic bonds to ⁇ -1,4-xylosidic bonds is about 1:4.
  • 1,3/1,4-xylan derived from the algae Palmaria palmata has been commercialized.
  • the present invention provides a 1,3/1,4-xylanase MLX1034 and its encoding gene and application.
  • the 1,3/1,4-xylanase MLX1034 is an endonuclease derived from Polaribacter sp. Q13, which was deposited in China Typical on December 28, 2020 Culture Collection Center, preservation address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei province, preservation number: CCTCC NO: M 2020985.
  • the final product is xylo-oligosaccharide with a degree of polymerization greater than 1 and is mainly xylohexaose.
  • the encoding gene of the above-mentioned 1,3/1,4-xylanase MLX1034, the nucleotide sequence is shown in SEQ ID NO.2.
  • the encoding gene of the 1,3/1,4-xylanase MLX1034 is derived from Polaribacter sp. Q13, which was deposited in China Type Culture on December 28, 2020 Object Preservation Center, preservation address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei province, preservation number: CCTCC NO: M 2020985.
  • a recombinant expression vector comprising the encoding gene of 1,3/1,4-xylanase MLX1034.
  • the recombinant expression vector is constructed with plasmid pET-22b.
  • the recombinant expression vector is transformed into host cells to generate recombinant cells.
  • the host cell is Escherichia coli.
  • the Escherichia coli is Escherichia coli BL21(DE3).
  • a recombinant cell comprising the gene encoding 1,3/1,4-xylanase MLX1034.
  • a recombinant expression strain the strain MLX1034 is obtained by culturing recombinant cells.
  • the conditions for culturing the recombinant cells are:
  • LB liquid medium was used, the culture temperature was 37°C, and the shaker was cultured until the OD 600nm was 0.8-1.0.
  • IPTG is added for induction, and the recombinant expression strain is obtained by centrifugation.
  • the 1,3/1,4-xylanase MLX1034 is expressed by a recombinant expression strain.
  • a method for degrading 1,3/1,4-xylan comprising:
  • the nucleotide sequence shown in SEQ ID NO.2 is cloned into a plasmid as a recombinant vector, and the recombinant vector is transformed into a host cell to construct a recombinant cell; cultivate in a fermentation medium Recombinant cells to obtain recombinant expression strains; recombinant expression strains express 1,3/1,4-xylanase MLX1034, 1,3/1,4-xylanase MLX1034 to 1,3/1,4-xylanase Glycans are degraded.
  • a preparation method of xylo-oligosaccharide comprising:
  • the nucleotide sequence shown in SEQ ID NO.2 is cloned into a plasmid as a recombinant vector, and the recombinant vector is transformed into a host cell to construct a recombinant cell; cultivate in a fermentation medium Recombinant cells to obtain recombinant expression strains; recombinant expression strains express 1,3/1,4-xylanase MLX1034, 1,3/1,4-xylanase MLX1034 to 1,3/1,4-xylanase Glycans are degraded to yield xylo-oligosaccharides.
  • the degree of polymerization of the xylo-oligosaccharide is greater than 1.
  • the main component of the xylo-oligosaccharide is xylohexaose.
  • the plasmid is plasmid pET-22b.
  • the host cell is Escherichia coli, more preferably Escherichia coli BL21(DE3).
  • the conditions for culturing the recombinant cells in the fermentation medium are:
  • Adopt LB liquid medium culture temperature 37 °C, shaker culture, cultivate to OD 600nm ;
  • IPTG is added for induction, and the recombinant expression strain is obtained by centrifugation.
  • Polaribacter sp. Q13 in the present invention is isolated from the surface of red algae samples on Nelson Island, Antarctica, combined with the genome of Polaribacter sp. Q13 and its growth in 1,3/1,4-xylan medium
  • the extracellular proteome data in identified MLX1034 as an extracellular 1,3/1,4-xylanase secreted by Polaribacter sp. Q13.
  • the coding gene of MLX1034 is 1017bp in total, encoding 338 amino acid residues.
  • MLX1034 has a predicted molecular weight of 39.1kDa, contains an N-terminal signal peptide (Met1-Ser27), and has only one glycoside hydrolase family 26 (GH26) catalytic domain, which is an unreported protein in the GH26 family.
  • the recombinant expression strain was cultured and isolated and purified to obtain 1,3/1,4-xylanase MLX1034.
  • the 1,3/1,4-xylanase MLX1034 only has a strong effect on 1,3/1,4-xylan. Strong degradation activity, very low activity on 1,3-xylan, no activity on 1,4-xylan, indicating that 1,3/1,4-xylanase MLX1034 is a 1,3/1 , 4-xylan-specific degrading enzymes.
  • the 1,3/1,4-xylanase MLX1034 provided by the present invention is derived from Polaribacter sp. Q13, which is easy for heterologous expression and purification, and can efficiently and specifically degrade 1,3/1, 4-Xylan, which produces xylo-oligosaccharides with a degree of polymerization greater than 1,
  • 1,3/1,4-xylanase MLX1034 provided by the present invention degrades 1,3/1,4-xylan, the main component of the enzymatic hydrolysis product at each time point is xylohexaose And no xylose is produced, so it has obvious advantages in the optimization of the production process of xylo-oligosaccharides.
  • Figure 1 is the SDS-PAGE electrophoresis image of 1,3/1,4-xylanase MLX1034 after heterologous expression and purification;
  • M protein molecular weight standard (marker); 1034: purified 1,3/1,4-xylanase MLX1034;
  • Figure 2 is the effect curve of temperature on the activity of 1,3/1,4-xylanase MLX1034;
  • Fig. 3 is the influence curve of temperature on the stability of 1,3/1,4-xylanase MLX1034;
  • Figure 4 is the effect curve of pH on the activity of 1,3/1,4-xylanase MLX1034;
  • Fig. 5 is the influence curve of pH on the stability of 1,3/1,4-xylanase MLX1034;
  • Figure 6 is a high performance liquid chromatography (HPLC) chart of degradation products of 1,3/1,4-xylanase MLX1034 to 1,3/1,4-xylan;
  • Control control group, that is, the reaction system without enzyme.
  • a Polaribacter sp. Q13 deposited in the China Center for Type Culture Collection on December 28, 2020, deposit address: Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei province, preservation number: CCTCC NO: M 2020985 .
  • 1,3/1,4-xylan derived from palmate red algae, 1,3-/1,4- ⁇ 1/4, available from Elicityl.
  • 1,3-Xylan extracted from sea grapes.
  • 1,4-linked wheat arabinoxylan and beech xylan available from Megazyme.
  • TYS liquid medium 0.5wt% peptone, 0.1wt% yeast powder, 3wt% artificial seawater, pH 7.8.
  • Freshwater LB liquid medium 1wt% peptone, 0.5wt% yeast powder, 1wt% NaCl, three distilled water preparation.
  • Freshwater LB solid medium 1wt% peptone, 0.5wt% yeast powder, 1wt% NaCl, 1.5wt% agar powder, three distilled water preparation.
  • Embodiment 1 the acquisition of polar bacillus (Polaribacter sp.) Q13
  • the single colony of the above-mentioned seed preservation was streaked into 1,3/1,4-xylan solid medium, cultivated at a constant temperature of 20°C, and the strains that could degrade and utilize 1,3/1,4-xylan were isolated.
  • the guaranteed seed is recorded as Q13.
  • strain Q13 could grow and culture on 1,3/1,4-xylan solid medium for 3 to 4 days, and form yellow, moist, smooth surface and regular edge round colonies. Being a Gram-negative bacterium, the strain Q13 was identified as Polaribacter sp..
  • the above-mentioned Polaribacter sp. Q13 was deposited in the China Center for Type Culture Collection on December 28, 2020.
  • the preservation address is Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei province, and the preservation number is CCTCC NO: M 2020985.
  • Polaribacter sp. Q13 was inoculated into TYS liquid medium, cultured at 20°C for 24 hours, 1 mL of bacterial liquid was collected, and the bacterial cells were collected to extract genomic DNA according to the instructions of Biotech genome extraction kit (routine extraction steps). Whole genome sequencing was done by BGI.
  • SignalP-5.0 http://www.cbs.dtu.dk/services/SignalP/ was used to predict the signal peptide of MLX1034 to be Met1-Ser27, delete After the coding sequence of the signal peptide, the following two primers were designed:
  • PCR amplification was performed using F and R as primers and the genomic DNA of strain Q13 as template.
  • the amplification procedure was as follows: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 20sec, annealing at 55°C for 20sec, extension at 72°C for 45sec, 30 cycle; extension at 72°C for 10 min.
  • PCR amplification system (50 ⁇ L) is as follows:
  • the pET-22b vector (purchased from Novagen) was double digested with restriction enzymes NdeI and XhoI, and the digested product was subjected to 1 wt% agarose gel electrophoresis, and then the DNA recovery kit from Omega was used according to its Description of the recovery of the linearized pET-22b vector.
  • the amplified DNA fragment was ligated to the linearized pET-22b vector using In-Fusion (purchased from TaKaRa Company).
  • the ligation system (2.5 ⁇ L) was as follows:
  • the expression vector was transformed into Escherichia coli BL21(DE3) competent cells according to the heat shock transformation method in the "Molecular Cloning Experiment Guide”. ), let stand on ice for 30min; heat shock at 42°C for 90sec; quickly transfer to ice and let stand for 10min; add 200 ⁇ L fresh water LB liquid medium, water bath at 37°C for 1h; spread the cells to a final concentration of 100 ⁇ g/mL ampicillin Penicillin was cultured overnight at 37°C on freshwater LB solid plates.
  • the transformants were sent to BGI for sequencing verification.
  • the sequencing results showed that the 1,3/1,4-xylanase MLX1034 coding gene fragment was successfully inserted between the restriction sites NdeI and XhoI of pET-22b.
  • the insertion direction was Correct, and without base mutation, deletion and addition, the recombinant plasmid vector was named pET-22b-MLX1034.
  • Example 3 Heterologous expression and isolation and purification of 1,3/1,4-xylanase MLX1034
  • the constructed recombinant plasmid vector pET-22b-MLX1034 was transformed into Escherichia coli BL21(DE3) competent cells according to the heat shock transformation method in "Molecular Cloning Experiment Guide", and spread on the cells containing 100 ⁇ g/mL ampicillin The freshwater LB solid plates were cultured at 37°C overnight;
  • the seed liquid was transferred to fresh water LB liquid medium containing 100 ⁇ g/mL ampicillin at an inoculum amount of 1% (v/v), and cultivated at 37°C and 180 rpm until the OD 600nm was about 0.8-1.0 , IPTG (0.1 mM) was added, and the cells were induced at 20° C. and 100 rpm for 16 h to obtain bacterial liquid.
  • the purified 1,3/1,4-xylanase MLX1034 was subjected to SDS-PAGE electrophoresis to detect the purity, and the electrophoresis results are shown in FIG. 1 .
  • Example 4 Analysis of the enzymatic properties of 1,3/1,4-xylanase MLX1034
  • 1,3/1,4-Xylanase MLX1034 enzyme activity assay method is as follows:
  • Standard reaction system 90 ⁇ L xylan substrate (10 mg/mL, dissolved in 20 mM pH 7.0 phosphate buffer), 10 ⁇ L 1,3/1,4-xylanase MLX1034 enzyme solution.
  • Enzyme activity determination method The enzyme activity was determined by dinitrosalicylic acid (DNS) method. The specific operation is as follows: after the reaction system is reacted at 40 ° C for 10 min, add 100 ⁇ L of color developer DNS; then boil in water for 5 min, add 500 ⁇ L of three-distilled water, and use a microplate reader to measure OD 550nm ; The system of 1,4-xylanase MLX1034 enzyme solution was used as blank control.
  • DNS dinitrosalicylic acid
  • One unit of enzyme activity (U) is defined as the amount of enzyme required to produce 1 ⁇ mol of xylose per minute under this reaction system. A standard curve of product concentration was drawn with different concentrations of xylose.
  • 1,3/1,4-xylanase MLX1034 only has a strong degradation activity to 1,3/1,4-xylan, with a specific activity of 224.0 U/mg; - Very low activity of xylan, only 0.04U/mg; no activity on 1,4-xylan (including wheat arabinoxylan and beech xylan).
  • 1,3/1,4-xylanase MLX1034 maintains the enzyme activity above 80% at 30 to 45 °C, and the relative activity is the highest at 40 °C; 1,3/1,4 -Xylanase MLX1034 is relatively stable at 40°C, and keeps the residual activity of the enzyme above 80% within 60 minutes; at 50°C, the residual activity of the enzyme will decrease to less than 40% with the extension of the incubation time; at 60°C Extremely unstable, it will rapidly inactivate within 5 minutes.
  • 1,3/1,4-xylan was dissolved in 20mM sodium citrate buffer (pH 3.0-6.0), 20mM PBS buffer (pH 6.0-8.0), 20mM sodium citrate buffer (pH 6.0-8.0), respectively. Tris-HCl buffer (pH 8.0-9.0), 20 mM glycine-sodium hydroxide buffer (pH 9.0-11.0) and the reaction system was prepared. The reaction system was reacted at 40 °C for 10 min, and the enzymatic activity of 1,3/1,4-xylanase MLX1034 at different pH was determined. The highest enzymatic activity was defined as 100% to calculate the relative activity. The results are shown in Figure 4. .
  • the effect of pH on enzyme stability The reaction buffer and 1,3/1,4-xylanase MLX1034 enzyme solution were mixed in a volume ratio of 49:1 (volume ratio), the reaction buffer included 20mM citric acid Sodium buffer (pH 3.0 ⁇ 6.0), 20mM PBS buffer (pH 6.0 ⁇ 8.0), 20mM Tris-HCl buffer (pH 8.0 ⁇ 9.0), 20mM glycine-sodium hydroxide buffer (pH 9.0 ⁇ 11.0 ). After incubation at 4°C for 1 h, the residual activity of 1,3/1,4-xylanase MLX1034 was determined. The highest enzyme activity was defined as 100% to calculate the residual activity at different pH. The results are shown in Figure 5. .
  • Example 5 Degradation pattern analysis of 1,3/1,4-xylanase MLX1034 on 1,3/1,4-xylan
  • 1,3/1,4-xylan (5mg/mL, dissolved in 20mM pH7.0 phosphate buffer), 1,3/1,4-xylanase MLX1034 enzyme solution according to 99:1 (volume ratio), react at 30°C, take out the reaction solution after 6min, 10min, 1h, and 24h of reaction respectively, stop the reaction by boiling the reaction solution for 5min, and then filter the reaction solution with a 0.22 ⁇ m filter membrane to obtain the degradation product.
  • the degradation products were analyzed by high performance liquid chromatography (HPLC), the high performance liquid chromatography (HPLC) analysis column was Superdex30Increase 10/300GL (purchased from GE Healthcare), the detector was an evaporative light detector (ELSD), and the mobile phase was three. Distilled water, and roughly calibrated the retention time with 1,4-xylo-oligosaccharide (1,4X1-X6) as the standard. The results are shown in Figure 6.
  • the 1,3/1,4-xylanase MLX1034 provided by the present invention is an endo-xylanase
  • the degradation product is a xylo-oligosaccharide with a degree of polymerization greater than 1
  • the main component is xylohexaose

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Abstract

提供了一种1,3/1,4-木聚糖酶MLX1034及其编码基因与应用。该木聚糖酶MLX1034分离自极地杆菌(Polaribacter sp.)Q13，其氨基酸序列如SEQ ID NO.1所示，其编码基因的核苷酸序列如SEQ ID NO.2所示。该木聚糖酶MLX1034可以特异性地降解1,3/1,4-木聚糖，产生聚合度大于1的低聚木糖，并且理化性质稳定，可以在室温下进行反应，适合广泛应用于红藻低聚木糖的工业生产中，能够有效降低生产过程中的能源成本。

Description

一种1,3/1,4-木聚糖酶MLX1034及其编码基因与应用 技术领域
本发明涉及一种1,3/1,4-木聚糖酶MLX1034及其编码基因与应用，属于生物技术技术领域。
背景技术
木聚糖是植物半纤维素的主要成分，其含量仅次于纤维素。高等植物细胞壁中的木聚糖是复杂的多聚五碳糖，其骨架是由木糖基团通过β-1,4-糖苷键连接而成，一些取代基通过O 2或O 3原子与骨架相连，常见的取代基团包括阿拉伯糖基、半乳糖基、葡萄糖醛酸基等。因此，完全水解此类木聚糖需要多种酶的共同参与，其中内切型1,4-木聚糖酶起主要作用。
低聚木糖是指由2～7个木糖分子构成的功能性木寡糖，大量研究表明低聚木糖对肠道内有益菌(如双歧杆菌)具有选择性增殖作用，能够改善肠道菌群、增强机体免疫力，具有降血压、降低血糖和胆固醇以及抗癌等功能，因而在食品、医药、化妆品以及饲料添加剂等领域被广泛应用。目前，低聚木糖主要通过生物酶法催化或化学催化从玉米芯、麦麸、蔗渣等原料中进行制备。
海洋环境中的木聚糖主要来源于红藻和绿藻的细胞壁，与高等植物中的木聚糖在结构上存在明显差异。海洋藻类来源的木聚糖均为直链同型木聚糖，只由木糖基团通过β-1,3-糖苷键或β-1,4-糖苷键连接而成，其类型包括1,3-木聚糖、1,4-木聚糖以及混合糖苷键的1,3/1,4-木聚糖。在红藻中，掌形藻目(Palmariales)和海索面目(Nemaliales)藻类细胞壁中的多糖主要为1,3/1,4-木聚糖。其中，掌形藻目中的掌状红皮藻Palmaria palmata是一种著名的冷水性经济海藻，富含膳食纤维、蛋白质及维生素等，其藻体内某些化合物具有抗炎、抗氧化活性功能，作为海洋食品备受人们青睐。从掌状红皮藻Palmaria palmata中提取的水溶性1,3/1,4-木聚糖的结构为：1,4-木聚糖骨架中无序地分布着不连续的β-1,3-木糖苷键，β-1,3-木糖苷键与β-1,4-木糖苷键的比例约为1:4。目前，掌状红皮藻Palmaria palmata来源的1,3/1,4-木聚糖已经商品化。
由于藻类巨大的初级生产力，海洋中孕育着巨大的具有独特结构的1,3/1,4-木聚糖资源，利用这些资源可以制备区别于现有产品的新型低聚木糖，具有较好的应用前景。但是，目前仍缺乏高效的1,3/1,4-木聚糖酶来制备低聚木糖。
发明内容
针对现有技术的不足，本发明提供一种1,3/1,4-木聚糖酶MLX1034及其编码基因与应用。
本发明技术方案如下：
一种1,3/1,4-木聚糖酶MLX1034，氨基酸序列如SEQ ID NO.1所示。
根据本发明优选的，所述1,3/1,4-木聚糖酶MLX1034为内切酶，来自极地杆菌(Polaribacter sp.)Q13，极地杆菌Q13于2020年12月28日保藏于中国典型培养物保藏中心，保藏地址：湖北省武汉市武昌区八一路珞珈山，保藏编号：CCTCC NO：M 2020985。
本发明的1,3/1,4-木聚糖酶MLX1034降解1,3/1,4-木聚糖时，终产物为聚合度大于1的低聚木糖且主要为木六糖。
上述1,3/1,4-木聚糖酶MLX1034的编码基因，核苷酸序列如SEQ ID NO.2所示。
根据本发明优选的，所述1,3/1,4-木聚糖酶MLX1034的编码基因来源于极地杆菌(Polaribacter sp.)Q13，极地杆菌Q13于2020年12月28日保藏于中国典型培养物保藏中心，保藏地址：湖北省武汉市武昌区八一路珞珈山，保藏编号：CCTCC NO：M 2020985。
一种重组表达载体，该重组表达载体包含有1,3/1,4-木聚糖酶MLX1034的编码基因。
根据本发明，优选的，所述的重组表达载体用质粒pET-22b构建。
根据本发明，优选的，所述重组表达载体转化到宿主细胞中产生重组细胞。
根据本发明，优选的，所述的宿主细胞为大肠杆菌。
根据本发明，优选的，所述的大肠杆菌为大肠杆菌BL21(DE3)。
一种重组细胞，该重组细胞包含有1,3/1,4-木聚糖酶MLX1034的编码基因。
一种重组表达菌株，该菌株MLX1034是对重组细胞进行培养得到。
根据本发明，优选的，对重组细胞进行培养的条件为：
采用LB液体培养基，培养温度37℃，摇床培养，培养至OD 600nm为0.8～1.0。
根据本发明，优选的，培养至OD 600nm为0.8～1.0后，加入IPTG诱导，离心分离得到重组表达菌株。
根据本发明，优选的，所述的1,3/1,4-木聚糖酶MLX1034，由重组表达菌株表达得到。
根据本发明，上述1,3/1,4-木聚糖酶MLX1034在1,3/1,4-木聚糖降解中的应用。
一种1,3/1,4-木聚糖的降解方法，包括：
使用如SEQ ID NO.2所示的核苷酸序列，将所述核苷酸序列克隆到质粒中作为重组载体，该重组载体转化到宿主细胞中，构建得到重组细胞；在发酵培养基中培养重组细胞，得到重 组表达菌株；重组表达菌株表达得到1,3/1,4-木聚糖酶MLX1034，1,3/1,4-木聚糖酶MLX1034对1,3/1,4-木聚糖进行降解。
一种低聚木糖的制备方法，包括：
使用如SEQ ID NO.2所示的核苷酸序列，将所述核苷酸序列克隆到质粒中作为重组载体，该重组载体转化到宿主细胞中，构建得到重组细胞；在发酵培养基中培养重组细胞，得到重组表达菌株；重组表达菌株表达得到1,3/1,4-木聚糖酶MLX1034，1,3/1,4-木聚糖酶MLX1034对1,3/1,4-木聚糖进行降解，得到低聚木糖。
根据本发明，优选的，所述的低聚木糖聚合度大于1。
根据本发明，优选的，所述的低聚木糖的主要成分为木六糖。
根据本发明，优选的，所述的质粒为质粒pET-22b。
根据本发明，优选的，所述的宿主细胞为大肠杆菌，进一步优选为大肠杆菌BL21(DE3)。
根据本发明，优选的，在发酵培养基中培养重组细胞的条件为：
采用LB液体培养基，培养温度37℃，摇床培养，培养至OD 600nm；
优选的，培养至OD 600nm为0.8～1.0后，加入IPTG诱导，离心分离得到重组表达菌株。
本发明的技术特点：
本发明中的极地杆菌(Polaribacter sp.)Q13分离自从南极纳尔逊岛的红藻样品表面，结合极地杆菌(Polaribacter sp.)Q13的基因组及其在1,3/1,4-木聚糖培养基中的胞外蛋白组数据，确定了MLX1034为极地杆菌(Polaribacter sp.)Q13所分泌的胞外1,3/1,4-木聚糖酶。MLX1034的编码基因共1017bp，编码338个氨基酸残基。MLX1034的序列分析表明，该蛋白预测分子量为39.1kDa，含N端信号肽(Met1-Ser27)，仅具有一个糖苷水解酶第26家族(GH26)催化结构域，是GH26家族中一个尚未报道的具有新的序列的1,3/1,4-木聚糖酶。根据其基因序列设计引物并利用PCR技术从菌株Q13的基因组DNA中克隆了MLX1034的基因并重组至大肠杆菌，获得了MLX1034的重组表达菌株。培养重组表达菌株并分离纯化得到1,3/1,4-木聚糖酶MLX1034。1,3/1,4-木聚糖酶MLX1034仅对1,3/1,4-木聚糖具有很强的降解活性，对1,3-木聚糖的活性极低，对1,4-木聚糖没有活性，说明1,3/1,4-木聚糖酶MLX1034是一个1,3/1,4-木聚糖特异性降解酶。1,3/1,4-木聚糖酶MLX1034的最适酶活温度为40℃，在pH＝7.0的磷酸缓冲液中活性最高，表明1,3/1,4-木聚糖酶MLX1034是一个中温、中性酶。对1,3/1,4-木聚糖酶MLX1034酶解1,3/1,4-木聚糖后的产物进行高效液相色谱分析发现，该酶是一个严格的内切型木聚糖酶，其酶解产物在不同的反应时间点均主要为木六糖，无木糖产生，是从1,3/1,4-木聚糖中制备低聚木糖的有效工具酶。
本发明的有益效果：
1、本发明提供的1,3/1,4-木聚糖酶MLX1034，来自极地杆菌(Polaribacter sp.)Q13，易于异源表达与纯化，可以高效、特异性地降解1,3/1,4-木聚糖，产生聚合度大于1的低聚木糖，
并且理化性质稳定，可以在室温下进行反应，适合广泛应用于红藻低聚木糖的工业生产中，
能够有效降低生产过程中的能源成本。
2、本发明提供的1,3/1,4-木聚糖酶MLX1034在降解1,3/1,4-木聚糖时，在各个时间点的酶解产物的主要成分均为木六糖且无木糖产生，因此在低聚木糖的生产工艺优化过程中具有明显的优势。
附图说明：
图1为异源表达纯化后的1,3/1,4-木聚糖酶MLX1034的SDS-PAGE电泳图；
图中：M：蛋白质分子量标准品(marker)；1034：纯化后的1,3/1,4-木聚糖酶MLX1034；
图2为温度对1,3/1,4-木聚糖酶MLX1034的活性影响曲线；
图3为温度对1,3/1,4-木聚糖酶MLX1034的稳定性影响曲线；
图4为pH对1,3/1,4-木聚糖酶MLX1034的活性影响曲线；
图5为pH对1,3/1,4-木聚糖酶MLX1034的稳定性影响曲线；
图6为1,3/1,4-木聚糖酶MLX1034对1,3/1,4-木聚糖的降解产物的高效液相色谱(HPLC)图；
图中：Control：对照组，即无酶反应体系。
具体实施方式
下面结合附图和实施例对本发明作进一步说明，但本发明所保护范围不限于此。
生物材料来源：
一株极地杆菌(Polaribacter sp.)Q13，2020年12月28日保藏于中国典型培养物保藏中心，保藏地址：湖北省武汉市武昌区八一路珞珈山，保藏编号：CCTCC NO：M 2020985。
1,3/1,4-木聚糖：来源于掌状红藻，1,3-/1,4-≈1/4，Elicityl公司有售。
1,3-木聚糖：抽提自海葡萄。
1,4-连接的小麦阿拉伯木聚糖和山毛榉木聚糖：Megazyme公司有售。
TYS液体培养基：0.5wt％蛋白胨，0.1wt％酵母粉，3wt％人工海水配制，pH 7.8。
淡水LB液体培养基：1wt％蛋白胨，0.5wt％酵母粉，1wt％NaCl，三蒸水配制。
淡水LB固体培养基：1wt％蛋白胨，0.5wt％酵母粉，1wt％NaCl，1.5wt％琼脂粉，三蒸水配制。
实施例1、极地杆菌(Polaribacter sp.)Q13的获得
1、菌株初筛
将3g南极纳尔逊岛采集的红藻样品置于无菌平皿中，用20mL无菌海水冲洗表面附着细菌，并梯度稀释10～10 5倍；将稀释后的样品通过常规的稀释法或划线法均匀接种涂布于TYS固体培养基，于20℃恒温培养，直到TYS固体培养基上生长出形态各异的单菌落，将各个单菌落重复划线接种于TYS固体培养基，直至纯化得到单菌落，使用60％(v/v)甘油(3:7；甘油:菌液)将各个单菌落保存在-80℃。
2、菌株复筛
将上述保种的单菌落划线接种至1,3/1,4-木聚糖固体培养基，于20℃恒温培养，分离能够降解利用1,3/1,4-木聚糖的菌株，保种记为Q13。
通过初筛和复筛，筛选到菌株Q13可以在1,3/1,4-木聚糖固体培养基上生长培养3～4天后，形成黄色、湿润、表面光滑、边缘规则的圆形菌落，为革兰氏阴性细菌，所述菌株Q13经鉴定为极地杆菌(Polaribacter sp.)。
上述极地杆菌(Polaribacter sp.)Q13，2020年12月28日保藏于中国典型培养物保藏中心，保藏地址：湖北省武汉市武昌区八一路珞珈山，保藏编号：CCTCC NO：M 2020985。
实施例2、1,3/1,4-木聚糖酶MLX1034编码基因的克隆及重组质粒载体的构建
1、极地杆菌(Polaribacter sp.)Q13基因组DNA的提取和全基因组测序
将极地杆菌(Polaribacter sp.)Q13接种于TYS液体培养基，于20℃培养24h，取1mL菌液，收集菌体后参照百泰克公司基因组提取试剂盒说明书提取基因组DNA(常规提取步骤)。全基因组测序由华大基因完成。
2、引物设计与合成
根据全基因组测序得到的MLX1034基因序列及其编码的氨基酸序列信息，使用SignalP-5.0(http://www.cbs.dtu.dk/services/SignalP/)预测MLX1034的信号肽为Met1-Ser27，删除该信号肽的编码序列后，设计如下两条引物：
F:5’-AAGAAGGAGATATACATATGCAAGAGGTAAAACCTCGTTTT-3’
R:5’-TGGTGGTGGTGGTGCTCGAGTTTATTATTAAGGTGAATAAA-3’
引物由华大基因合成。
3、PCR扩增基因序列及扩增产物的回收
(1)以F和R为引物，以菌株Q13基因组DNA为模板，进行PCR扩增，扩增程序为：95℃预变性2min；95℃变性20sec，55℃退火20sec，72℃延伸45sec，30个循环；72℃延伸10min。
PCR扩增体系(50μL)如下：
Figure PCTCN2021136034-appb-000001
(2)对PCR扩增产物进行1wt％琼脂糖凝胶电泳，然后用Omega公司的DNA回收试剂盒按照其说明回收扩增出的DNA片段。
4、重组质粒载体的构建
(1)酶切克隆载体
使用限制性内切酶NdeI和XhoI对pET-22b载体(购自Novagen公司)进行双酶切，并对酶切产物进行1wt％琼脂糖凝胶电泳，然后用Omega公司的DNA回收试剂盒按照其说明回收线性化的pET-22b载体。
(2)DNA片段与线性化的载体连接
使用In-Fusion(购自TaKaRa公司)将扩增出的DNA片段连接到线性化的pET-22b载体上。
连接体系(2.5μL)如下：
DNA片段                  1μL
线性化pET-22b            1μL
5x In-Fusion             0.5μL
(3)将表达载体转化至大肠杆菌DH5α中
将表达载体按照《分子克隆实验指南》上的热激转化方法转化至大肠杆菌BL21(DE3)感受态细胞，具体操作为：将2.5μL连接体系加入至50μL大肠杆菌DH5α感受态细胞(购自TransGen)中，冰中静置30min；42℃热激90sec；快速转至冰中静置10min；加入200μL 淡水LB液体培养基，37℃水浴1h；将菌体涂布至含有终浓度100μg/mL氨苄青霉素的淡水LB固体平板上，37℃过夜培养。
将转化子送至华大基因进行测序验证，测序结果表明1,3/1,4-木聚糖酶MLX1034编码基因片段成功插入到pET-22b的酶切位点NdeI和XhoI之间，插入方向正确，且未发生碱基突变、缺失以及增加的情况，将重组质粒载体命名为pET-22b-MLX1034。
实施例3：1,3/1,4-木聚糖酶MLX1034的异源表达及分离纯化
1、1,3/1,4-木聚糖酶MLX1034在大肠杆菌BL21(DE3)中的异源表达
(1)将构建好的重组质粒载体pET-22b-MLX1034按照《分子克隆实验指南》上的热激转化方法转化至大肠杆菌BL21(DE3)感受态细胞，并涂布于含100μg/mL氨苄青霉素的淡水LB固体平板上，37℃过夜培养；
(2)在平板上挑取单菌落，接种至含100μg/mL氨苄青霉素的淡水LB液体培养基中，37℃过夜培养，得到种子液；
(3)将种子液按1％(v/v)的接种量转接至含100μg/mL氨苄青霉素的淡水LB液体培养基中，在37℃、180rpm条件下培养至OD 600nm约为0.8～1.0时，加入IPTG(0.1mM)，20℃、100rpm诱导16h，得到菌液。
(4)将菌液在4℃、7000rpm离心5min，收集菌体。
2、1,3/1,4-木聚糖酶MLX1034的分离纯化
(1)粗酶液的制备：将菌体重悬于Lysis buffer(50mM Tris-HCl,100mM NaCl,pH 8.0)中，使用压力破碎仪破碎细胞，于4℃、12000rpm离心60min，所得上清即为粗酶液；
(2)镍亲和层析
镍亲和柱用Lysis buffer预处理后，将粗酶液上样至镍亲和柱，然后用Wash buffer(50mM Tris-HCl,100mM NaCl,10mM Imidazole,pH 8.0)冲洗10个柱体积，最后用Elution buffer(50mM Tris-HCl,100mM NaCl,250mM Imidazole，pH 8.0)洗脱目的蛋白；
(3)凝胶过滤层析
用GF buffer(10mM Tris-HCl,100mM NaCl,pH 8.0)将Superdex 200Prep grade凝胶过滤层析柱平衡后，将镍亲和层析纯化后的样品浓缩后上样至层析柱，使用GF buffer洗脱；收集目的蛋白峰的峰尖样品，加10％甘油后-80℃保存备用；
(4)蛋白纯度检测
将纯化后的1,3/1,4-木聚糖酶MLX1034进行SDS-PAGE电泳检测纯度，电泳结果如图1所示。
由图1可知，纯化后1,3/1,4-木聚糖酶MLX1034在电泳凝胶上的位置与预测的分子量吻合，因此在大肠杆菌BL21(DE3)中成功异源表达了1,3/1,4-木聚糖酶MLX1034。
实施例4：1,3/1,4-木聚糖酶MLX1034的酶学性质分析
1,3/1,4-木聚糖酶MLX1034酶活性测定方法如下：
标准反应体系：90μL木聚糖底物(10mg/mL，溶于20mM pH 7.0的磷酸缓冲液)、10μL1,3/1,4-木聚糖酶MLX1034酶液。
酶活测定方法：采用二硝基水杨酸(DNS)法测定酶活性。具体操作为：将反应体系在40℃下反应10min后，加入100μL显色剂DNS；然后沸水浴5min，加入500μL三蒸水，使用酶标仪测定OD 550nm；以添加加热失活1,3/1,4-木聚糖酶MLX1034酶液的体系作为空白对照。
一个酶活力单位(U)定义为：此反应体系下，每分钟产生1μmol木糖所需的酶量。用不同浓度的木糖绘制产物浓度标准曲线。
依据以上酶活反应体系及测定方法，分析1,3/1,4-木聚糖酶MLX1034的酶学性质。
(1)底物特异性分析
以1,3/1,4-木聚糖、1,3-木聚糖以及1,4-连接的小麦阿拉伯木聚糖和山毛榉木聚糖为底物，测定1,3/1,4-木聚糖酶MLX1034对不同类型木聚糖的活性，分析1,3/1,4-木聚糖酶MLX1034的底物特异性，结果如表1所示。
由表1可知，1,3/1,4-木聚糖酶MLX1034仅对1,3/1,4-木聚糖具有很强的降解活性，比活力为224.0U/mg；对1,3-木聚糖的活性极低，仅仅为0.04U/mg；对1,4-木聚糖(包括小麦阿拉伯木聚糖及山毛榉木聚糖)没有活性。
表1 MLX1034的底物特异性
Figure PCTCN2021136034-appb-000002
(2)温度对酶活性及稳定性的影响
温度对酶活性的影响：在0～60℃范围内，反应体系分别在0℃、10℃、20℃、30℃、40℃、50℃、60℃条件下反应10min，测定1,3/1,4-木聚糖酶MLX1034在不同温度下的酶活性，将最高酶活性定义为100％来计算相对活性，结果如图2所示。
温度对酶稳定性的影响：将1,3/1,4-木聚糖酶MLX1034酶液分别在40℃、50℃、60℃下温育，在0～60min范围内，每隔10min测定1,3/1,4-木聚糖酶MLX1034的残余活性，将保温0min的酶活性定义为100％来计算保温不同时间的残余活性，结果如图3所示。
由图2和图3可知，1,3/1,4-木聚糖酶MLX1034在30～45℃内保持酶活性在80％以上，在40℃时相对活性最高；1,3/1,4-木聚糖酶MLX1034在40℃下相对稳定，60min内一直保持酶残余活性在80％以上；在50℃下，酶残余活性会随着保温时间延长，降低至40％以下；在60℃下极不稳定，5min内就会快速失活。
(3)pH对酶活性及稳定性的影响
pH对酶活性的影响：将1,3/1,4-木聚糖分别溶于20mM的柠檬酸钠缓冲液(pH 3.0～6.0)、20mM的PBS缓冲液(pH 6.0～8.0)、20mM的Tris-HCl缓冲液(pH 8.0～9.0)、20mM的甘氨酸-氢氧化钠缓冲液(pH 9.0～11.0)并配制反应体系。反应体系在40℃下反应10min，测定1,3/1,4-木聚糖酶MLX1034在不同pH下的酶活性，将最高酶活性定义为100％来计算相对活性，结果如图4所示。
pH对酶稳定性的影响：将反应缓冲液和1,3/1,4-木聚糖酶MLX1034酶液按照体积比49：1(体积比)的比例混合，反应缓冲液包括20mM的柠檬酸钠缓冲液(pH 3.0～6.0)、20mM的PBS缓冲液(pH 6.0～8.0)、20mM的Tris-HCl缓冲液(pH 8.0～9.0)、20mM的甘氨酸-氢氧化钠缓冲液(pH 9.0～11.0)。在4℃下温育1h，然后测定1,3/1,4-木聚糖酶MLX1034的残余活性，将最高酶活性定义为100％来计算不同pH下的残余活性，结果如图5所示。
由图4和图5可知，1,3/1,4-木聚糖酶MLX1034在pH为7时相对活性最高；1,3/1,4-木聚糖酶MLX1034在在酸性环境(pH 3.0～4.0)下不稳定，但在pH5.0～11.0条件下能够稳定存在。
实施例5：1,3/1,4-木聚糖酶MLX1034对1,3/1,4-木聚糖的降解模式分析
将1,3/1,4-木聚糖(5mg/mL，溶于20mM pH7.0的磷酸缓冲液)、1,3/1,4-木聚糖酶MLX1034酶液按照99：1(体积比)的比例混合，在30℃下反应，分别在反应6min，10min，1h，24h后取出反应液，将反应液沸水浴5min终止反应，再使用0.22μm滤膜过滤反应液，得到降解产物。
将降解产物进行高效液相色谱(HPLC)分析，高效液相色谱(HPLC)分析柱为Superdex30Increase 10/300GL(购自GE Healthcare公司)，检测器为蒸发光检测器(ELSD)，流动相为三蒸水，以1,4-木寡糖(1,4X1-X6)为标准品粗略地标定保留时间，结果如图6所示。
由图6可知，本发明提供的1,3/1,4-木聚糖酶MLX1034为内切型木聚糖酶，降解产物为聚合度大于1的低聚木糖，主要成分为木六糖；并且在反应0～24h内的各反应时间点，只产生低聚木糖，无木糖产生。

Claims (22)

  1. 一种1,3/1,4-木聚糖酶MLX1034，其特征在于，氨基酸序列如SEQ ID NO.1所示。
  2. 如权利要求1所述的1,3/1,4-木聚糖酶MLX1034，其特征在于，所述1,3/1,4-木聚糖酶MLX1034为内切酶，来自极地杆菌(Polaribacter sp.)Q13，极地杆菌Q13于2020年12月28日保藏于中国典型培养物保藏中心，保藏地址：湖北省武汉市武昌区八一路珞珈山，保藏编号：CCTCC NO：M 2020985。
  3. 权利要求1所述的1,3/1,4-木聚糖酶MLX1034的编码基因，其特征在于，核苷酸序列如SEQ ID NO.2所示。
  4. 如权利要求3所述的编码基因，其特征在于，所述1,3/1,4-木聚糖酶MLX1034的编码基因来源于极地杆菌(Polaribacter sp.)Q13，极地杆菌Q13于2020年12月28日保藏于中国典型培养物保藏中心，保藏地址：湖北省武汉市武昌区八一路珞珈山，保藏编号：CCTCC NO：M 2020985。
  5. 一种重组表达载体，其特征在于，该重组表达载体包含如SEQ ID NO.2所示的1,3/1,4-木聚糖酶MLX1034的编码基因。
  6. 根据权利要求5所述的重组表达载体，其特征在于，所述的重组表达载体用质粒pET-22b构建。
  7. 根据权利要求6所述的重组表达载体，其特征在于，所述重组表达载体转化到宿主细胞中产生重组细胞。
  8. 根据权利要求7所述的重组表达载体，其特征在于，所述的宿主细胞为大肠杆菌。
  9. 根据权利要求8所述的重组表达载体，其特征在于，所述的大肠杆菌为大肠杆菌BL21(DE3)。
  10. 一种重组细胞，其特征在于，该重组细胞包含如SEQ ID NO.2所示的1,3/1,4-木聚糖酶MLX1034的编码基因。
  11. 一种重组表达菌株，该菌株MLX1034是对权利要求10所述的重组细胞进行培养得到。
  12. 根据权利要求11所述的重组表达菌株，其特征在于，对重组细胞进行培养的条件为：
    采用LB液体培养基，培养温度37℃，摇床培养，培养至OD 600nm为0.8～1.0。
  13. 根据权利要求12所述的重组表达菌株，其特征在于，培养至OD 600nm为0.8～1.0后，加入IPTG诱导，离心分离得到重组表达菌株。
  14. 一种1,3/1,4-木聚糖酶MLX1034，由权利要求11所述的重组表达菌株表达得到。
  15. 权利要求14所述的1,3/1,4-木聚糖酶MLX1034在1,3/1,4-木聚糖降解中的应用。
  16. 一种1,3/1,4-木聚糖的降解方法，包括：
    使用如SEQ ID NO.2所示的核苷酸序列，将所述核苷酸序列克隆到质粒中作为重组载体，该重组载体转化到宿主细胞中，构建得到重组细胞；在发酵培养基中培养重组细胞，得到重组表达菌株；重组表达菌株表达得到1,3/1,4-木聚糖酶MLX1034，1,3/1,4-木聚糖酶MLX1034对1,3/1,4-木聚糖进行降解。
  17. 一种低聚木糖的制备方法，包括：
    使用如SEQ ID NO.2所示的核苷酸序列，将所述核苷酸序列克隆到质粒中作为重组载体，该重组载体转化到宿主细胞中，构建得到重组细胞；在发酵培养基中培养重组细胞，得到重组表达菌株；重组表达菌株表达得到1,3/1,4-木聚糖酶MLX1034，1,3/1,4-木聚糖酶MLX1034对1,3/1,4-木聚糖进行降解，得到低聚木糖。
  18. 根据权利要求17所述的低聚木糖的制备方法，其特征在于，所述的低聚木糖聚合度大于1。
  19. 根据权利要求17所述的低聚木糖的制备方法，其特征在于，所述的低聚木糖的主要成分为木六糖。
  20. 根据权利要求17所述的低聚木糖的制备方法，其特征在于，所述的质粒为质粒pET-22b。
  21. 根据权利要求17所述的低聚木糖的制备方法，其特征在于，所述的宿主细胞为大肠杆菌，进一步优选为大肠杆菌BL21(DE3)。
  22. 根据权利要求17所述的低聚木糖的制备方法，其特征在于，在发酵培养基中培养重组细胞的条件为：
    采用LB液体培养基，培养温度37℃，摇床培养，培养至OD 600nm为0.8～1.0；
    优选的，培养至OD 600nm为0.8～1.0后，加入IPTG诱导，离心分离得到重组表达菌株。
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