阅读说明：本技术 一种化学修饰的木聚糖酶及其制备方法和应用 (Chemically modified xylanase, preparation method and application thereof ) 是由 马江锋 姜岷 王寅竹 吴昊 董维亮 于 2021-08-27 设计创作，主要内容包括：本发明公开了一种化学修饰的木聚糖酶及其制备方法和应用,属于酶工程领域。利用NaIO-(4)的氧化特点,将右旋糖酐上的羟基氧化为醛基后对木聚糖酶进行化学修饰。修饰后的酶拥有右旋糖酐结构,可以与木质纤维素中纤维素结合,达到一种固定化作用,从而使酶活力提高了237%,同时拥有更高的最大反应速率Vmax,该方法克服了木聚糖酶在木质纤维素中反应速率低的缺点,提高了木聚糖酶的催化活性,具有广阔的工业应用前景。(The invention discloses a chemically modified xylanase, a preparation method and application thereof, belonging to the field of enzyme engineering. Using NaIO 4 The oxidation characteristic is that the xylanase is chemically modified after hydroxyl on the dextran is oxidized into aldehyde group. The modified enzyme has a dextran structure and can be combined with cellulose in lignocellulose to achieve an immobilization effect, so that the enzyme activity is improved by 237%, and the modified enzyme has a higher maximum reaction rate Vmax, the method overcomes the defect of low reaction rate of xylanase in lignocellulose, improves the catalytic activity of xylanase,has wide industrial application prospect.)

1. A chemically modified xylanase enzyme characterized by: the amino acid sequence of the xylanase is shown as SEQ ID NO. 1, the coding gene is shown as SEQ ID NO. 2, and the chemical modifier is dextran.

2. The chemically modified xylanase according to claim 1, characterized in that: the chemical modifier is dextran containing aldehyde group.

3. The chemically modified xylanase according to claim 1, characterized in that: the chemical modifier is coupled with xylanase through amino of amino acid.

4. The chemically modified xylanase according to claim 2, characterized in that: the dextran containing aldehyde group adopts NaIO₄Oxidizing the ortho-hydroxyl of dextran.

5. The chemically modified xylanase according to claim 2, characterized in that: the dextran containing aldehyde groups adopts 150kDa dextran.

6. The method of producing a chemically modified xylanase according to claim 1, characterized in that it comprises the following steps:

(1) preparation of xylanase

Connecting the xylanase coding gene with an expression vector pEt28a (+), and transforming the connection product into an escherichia coli BL21(DE3) cell to obtain a recombinant strain; culturing the recombinant strain for fermentation expression, crushing and centrifuging fermentation liquor, and then carrying out ammonium sulfate precipitation and ion exchange column purification on supernatant fluid to obtain xylanase liquid;

(2) preparation of dextran containing aldehyde group

2.4g NaIO in the dark₄Dissolving in 10mL pure water to obtain NaIO₄Pouring the solution into 10mL of dextran solution with the concentration of 2g/mL, stirring for 8 hours at room temperature in a dark place,

after the reaction is finished, 5mL of ethylene glycol is added for reaction for 2h, and then the reaction solution is dialyzed overnight in a dialysis bag of 8000-14000;

(3) preparation of xylanase chemical modifier

Taking 1mL of modifier and 2mL of xylanase enzyme solution, diluting to 10mL with PBS solution with pH7.5, adding 0.1g of xylan, reacting at 35 ℃ and pH 7.0 for 6h, adding 0.1g of sodium cyanoborohydride, continuing to react for 1h, and dialyzing at 4 ℃ for 4h to obtain the xylanase chemical modifier.

Technical Field

The invention belongs to the field of enzyme engineering, and particularly relates to chemically modified xylanase as well as a preparation method and application thereof.

Background

Xylan is the main component of plant hemicellulose, and is the polysaccharide which is most abundant in the natural world except cellulose. The xylan backbone is a polymer of β -D-vicinal xylopyranose residues linked by β -1, 4-glycosidic linkages. Xylans in plants are located between lignin and cellulose, with varying degrees of interaction between xylan and other components of the wood fiber. Xylan is mainly linked to lignin by covalent bonds and acts with cellulose by non-covalent bonds. One of the difficulties in the degradation of lignocellulose is that the complex structure of the xylanase causes difficult degradation, so that the xylanase is chemically modified to be connected with dextran, so that the xylanase can be combined with cellulose to achieve an immobilization effect, thereby improving the affinity and promoting the degradation of the lignocellulose. Although chemical modification of enzyme can change certain properties of enzyme and improve the use efficiency of enzyme, the structure of enzyme molecule is often destroyed in the modification process, and the activity of enzyme is reduced, so that the chemical modification conditions of enzyme, including modification time, temperature and pH, need to be optimized to reduce the loss of enzyme activity.

Disclosure of Invention

The invention provides a chemically modified xylanase, a preparation method and application thereof, the invention reasonably designs and optimizes the modification method, overcomes the defects of the prior art, and the xylanase chemically modified by the method has higher affinity to cellulose and improves the degradation effect of lignocellulose.

The first purpose of the invention is to provide a chemically modified xylanase, wherein the amino acid sequence of the xylanase is shown as SEQ ID NO. 1, the coding gene is shown as SEQ ID NO. 2, and the chemical modifier is dextran.

Furthermore, the chemical modifier is dextran containing aldehyde group, and is coupled with xylanase through amino group of amino acid.

Further, the dextran containing aldehyde groups is obtained by oxidizing the adjacent hydroxyl groups of the dextran with NaIO 4.

Further, the dextran containing aldehyde groups adopts 150kDa dextran. The method comprises the following steps: connecting the xylanase coding gene fragment with an expression vector pET28a (+), and transforming the connection product into escherichia coli BL21 to obtain a recombinant strain; culturing the recombinant strain, and inducing expression; and crushing and centrifuging fermentation liquor, and then salting out and purifying to obtain enzyme solution of the xylanase.

The second purpose of the invention is to provide a preparation method of chemically modified xylanase, which comprises the following steps:

(1) preparation of xylanase

Connecting the xylanase coding gene with an expression vector pEt28a (+), and transforming the connection product into an escherichia coli BL21(DE3) cell to obtain a recombinant strain; culturing the recombinant strain for fermentation expression, crushing and centrifuging the fermentation liquor, and then carrying out ammonium sulfate precipitation and ion exchange column purification on the supernatant to obtain xylanase liquid.

(2) Preparation of dextran containing aldehyde group

2.4g NaIO in the dark₄Dissolving in 10mL pure water to obtain NaIO₄The solution was poured into 10mL of 2g/mL dextran solution and stirred at room temperature in the dark for 8 h. After the reaction was completed and 5mL of ethylene glycol was added for 2h, the reaction solution was dialyzed overnight in 8000-14000 dialysis bags.

(3) Preparation of xylanase chemical modifier

The third purpose of the invention is to provide the application of the chemically modified xylanase for quickly degrading lignocellulose.

The beneficial technical effects of the invention are as follows: compared with unmodified xylanase, the modified xylanase and cellulose have certain binding force, and an immobilization effect is achieved, so that the xylanase has higher reaction rate in the decomposition process of lignocellulose, and the Km of the Michaelis equation is reduced, which shows that the affinity with xylan is also increased.

Drawings

FIG. 1 detection of enzyme Activity after modification reactions at different times

FIG. 2 detection of enzyme Activity after modification reactions at different pH

FIG. 3 detection of enzyme Activity after modification reactions at different temperatures

FIG. 4 UV absorption spectra of modified and unmodified enzymes.

FIG. 5 Michaelis equation for modified and unmodified enzymes

FIG. 6 electron micrograph of modified enzyme in combination with unmodified enzyme and lignocellulose

Detailed Description

The technical solutions in the embodiments of the present invention will be fully described below with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without any inventive step, are within the scope of the present invention.

Experimental reagents and materials

1. Bacterial strain and carrier: coli BL21(DE3) and expression vector pET28a were supplied by the university of industries of tokyo, south beige, and this material was also commercially available.

2. Enzymes and other biochemical reagents: DNA polymerase and dNTP were purchased from Nanjing NuoWeizan Biotech GmbH, and others were made of domestic reagents (all available from general Biochemical reagent Co., Ltd.)

3. Culture medium: LB culture medium: 10g/L of Peptone, 5g/L of Yeast extract and 10g/L of NaCl, and dissolving by distilled water. On the basis of the solid medium, 2% (w/v) agar was added.

Fermentation medium:

description of the drawings: the molecular biological experiments, which are not specifically described in the following examples, were carried out by referring to the specific methods listed in the manual of molecular cloning, laboratory Manual (third edition), or according to the kit and product instructions.

Expression and enzyme Activity detection of xylanases as described in example 1

A mutant fragment containing EcoRI and SalI was designed using Aspergillus niger XylB as a template, and ligated to pET28a (+) by one-step cloning to obtain the objective plasmid. The plasmid was transferred to Escherichia coli BL21(DE3), the transformant was spread on LB plates and cultured at 37 ℃ for 12 hours, and the single colony grown was the expression strain. And selecting a single colony, inoculating the single colony into 5mL of LB culture medium, culturing for 12h at 37 ℃ and 200rpm, further inoculating the single colony into 200mL of LB culture medium, performing amplification culture, and adding 0.1mM IPTG (isopropyl thiogalactoside) to induce and culture for 12h when the OD600 of the bacterial liquid is 0.6-0.8.

The XylB-F upstream primer is: 5'-ATGGGTCGCGGATCCGAATTCATGTTTAAATTTA AAAAAAATTTC-3'

The XylB-R upstream primer is: 5'-TGCGGCCGCAAGCTTGTCGACCCATACAGTCAC GTTAGAGCTA-3'

And (3) enzyme activity detection: reacting 0.2mL enzyme solution with 1mL 1% xylan solution with pH of 7.0 at 55 deg.C for 15min, adding 0.3mL LDNS and 0.3mL water into 0.1mL reaction solution, reacting in boiling water bath for 10min, diluting to 4mL, determining OD₅₄₀The reducing sugar content was calculated from a standard curve.

Definition of enzyme activity: the amount of enzyme required to break down xylan to yield 1. mu. mol of reducing sugars per minute is defined as one activity unit under certain conditions.

Fermentative production and purification of xylanases as described in example 2

Inoculating 50 mu L of the seed-preserving bacteria liquid into 5mL of LB culture medium, culturing at 37 ℃ and 200rpm for 12h, further inoculating into 200mL of fermentation culture medium, performing amplification culture, and adding 0.1mM IPTG to induce and culture for 12h when the OD600 of the bacteria liquid is 0.6-0.8. After fermentation, carrying out ultrasonic crushing on the fermentation liquor, centrifuging after crushing, taking supernatant, adding ammonium sulfate solution with the same volume and saturation of 100%, stirring for 2h at 4 ℃, standing for 6h, centrifuging to obtain precipitate, and adding PBS (phosphate buffer solution) with pH of 7.0 to dissolve the precipitate for storage. Example 3 methods and Condition optimization for chemical modification of xylanases

Oxidation of dextran: 2g dextran with molecular weight of 150kDa and 2.4g NaIO₄Dissolving in 10mL water respectively, and adding NaIO in dark₄Pouring into dextran solution, stirring and reacting for 6h at 30 ℃ in the dark, and adding 5mL of ethylene glycol to continue reacting for 2 h. Dialyzing overnight after the reaction is finished to obtain the dextran solution containing aldehyde groups, namely the modifier.

Determination of optimum modification time. Taking 1mL of modifier, using PBS (phosphate buffer solution) with pH value of 7.5 to fix the volume to 8mL, adding 0.2g of xylan, finally adding 2mL of enzyme solution, stirring at 30 ℃ for reacting for different times (4h, 8h, 12h, 16h, 20h and 24h), adding 0.1g of sodium cyanoborohydride, continuing to react for 1h, and dialyzing at 4 ℃ for 4h after the reaction is finished to obtain the xylanase modifier.

Optimal modified pH determination. Taking 1mL of modifier, using PBS with different pH values (5.5, 6.0, 6.5, 7.0, 7.5 and 8.0) to fix the volume to 8mL, adding 0.2g of xylan, finally adding 2mL of enzyme solution, stirring and reacting at 30 ℃ for 8h, adding 0.1g of sodium cyanoborohydride, continuing to react for 1h, and dialyzing at 4 ℃ for 4h after the reaction is finished to obtain the xylanase modifier.

And (4) determining the optimal modification temperature. Taking 1mL of modifier, using PBS (phosphate buffer solution) with pH value of 7.5 to fix the volume to 8mL, adding 0.2g of xylan, finally adding 2mL of enzyme solution, stirring and reacting for 8h at different temperatures (4 ℃, 20 ℃, 25 ℃, 30 ℃ and 35 ℃), adding 0.1g of sodium cyanoborohydride, continuing to react for 1h, and dialyzing for 4h at 4 ℃ after the reaction is finished to obtain the xylanase modifier.

Comparing the relative enzyme activities under different modification conditions, the modification time is preferably controlled to be 8h, the modification effect is not obvious when the modification time is too short, and the enzyme activity is greatly lost when the modification time is too long. The pH should be controlled to be neutral and alkaline, under which the enzyme molecules are in an unprotonated state and are easy to react, while the temperature should be kept at room temperature for the reaction to proceed.

The best modification method of xylanase comprises the following steps: taking 1mL of modifier, using PBS (phosphate buffer solution) with pH value of 7.5 to fix the volume to 8mL, adding 0.2g of xylan, finally adding 2mL of enzyme solution, stirring and reacting at 30 ℃ for 8h, adding 0.1g of sodium cyanoborohydride, continuing to react for 1h, and dialyzing at 4 ℃ after the reaction is finished for 4h to obtain the xylanase modifier.

Example 4 Meloidogyne equation for degradation of lignocellulose by chemical modification of xylanase

Respectively preparing lignocellulose solutions with different concentrations and pretreated by dilute sulfuric acid, reacting for 5min at 55 ℃, detecting the generation amount of reducing sugar, and calculating the Mie equation. The specific experimental results are as follows:

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