XYLANASE MUTANT AND ITS PREPARATION METHOD AND APPLICATION

Abstract

The present invention discloses a kind of xylanase mutant and its preparation method and application, which relates to the technical field of genomic engineering and genetic engineering. Such mutant includes one or more mutants obtained by taking xylanase HwXyl10A as female parent to conduct saturation mutagenesis to the site of Gly363. Specifically, relates to obtaining 19 mutants through site-directed mutagenesis, and then conducting yeast expression to them, after that, obtaining two mutants with significantly improved specific activity and thermal stability through screening of thermal stability and catalytic activity; the present invention can significantly improve the thermal stability and catalytic efficiency of xylanase through modifying the site of Gly363, and is of important guiding significance for improving the thermal stability and the catalytic efficiency of the 10th family of xylanases and other glycoside hydrolases as well as lays the foundation for its application in industrial production.

Claims

1. A kind of xylanase mutant with high specific activity and heat resistance, characterized in that consisting of one or more mutants obtained by taking xylanase HwXyl10A as female parent to complete Gly363 site-saturation mutagenesis.

2. The said xylanase mutant with high specific activity and heat resistance according to claim 1, characterized in that obtaining two xylanase mutants with high specific activity and heat resistance, i.e., HwXyl10A_G363R and HwXyl10A_G363K after taking xylanase HwXyl10A as female parent to conduct saturation mutagenesis to the site of Gly363.

3. The said xylanase mutant with high specific activity and heat resistance according to claim 2, characterized in that the amino acid sequence of the said HwXyl10A_G363R is as shown in SEQ ID NO: 4; the said amino acid sequence of the HwXy110A_G363K is as shown in SEQ ID NO:6.

4. A nucleotide sequence of the said xylanase mutant with high specific activity and heat resistance according claim 1.

5. The said nucleotide sequence according to claim 4, characterized in that having the nucleotide sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 5.

6. A kind of recombinant vector, characterized in that the said recombinant vector consists of the said nucleotide sequence according to claim 4.

7. A kind of recombinant strain, characterized in that consisting of the said recombinant vector according to claim 6.

8. A kind of recombinant strain, characterized in that consisting of the said recombinant vector according to claim 5 wherein said nucleotide sequence is integrated into the genome of said recombinant strain.

9. A preparation method for the said xylanase mutant with high specific activity and heat resistance according to claim 1, characterized in that consisting of following steps: Step 1: Obtain the nucleotide sequence of the xylanase mutant with high specific activity and heat resistance, and after amplification, transform the amplified products into DMT competent cells, and thus obtain the recombinant expression vector of xylan mutant; Step 2: Transform the obtained recombinant expression vector of xylan mutant into Pichia pastoris, and then induce expression, and thus obtain the recombinant strain of Pichia pastoris; Step 3: Ferment and cultivate the recombinant strain of Pichia pastoris to induce the expression of recombinant xylanase; Step 4: Recover and purify the xylanase mutant with high specific activity and heat resistance.

10. A preparation method for the said xylanase mutant with high specific activity and heat resistance according to claim 8, characterized in that the said Pichia pastoris is Pichia pastoris GS115.

11. An application of the said xylanase mutant with high specific activity and heat resistance according claim 1 in industries of feed or sugar production through degrading xylan by utilizing biomass.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0023] For the purpose of illustrating the embodiments of the present invention or the technical scheme in the prior art more clearly, the text below will briefly introduce the drawings required in the embodiments. It is obvious that the drawings described below are only certain embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without paying creative labor.

[0024] FIG. 1 shows the SDS-PAGE analysis of the xylanase mutant with high specific activity and heat resistance, wherein M represents the low molecular weight protein Marker; A, C and E represent the purified wild-type enzyme HwXyl10A, and the mutants, i.e., HwXyl10A_G363R and HwXyl10A_G363K; and B, D and F are HwXyl10A , HwXyl10A_G363R and HwXyl10A_G363K after de-N glycosylation respectively;

[0025] FIG. 2 shows the measurement results of optimal pH values of the wild-type xylanase and two xylanase mutants, i.e., HwXyl10A_G363R and HwXyl10A_G363K;

[0026] FIG. 3 shows the measurement results of pH stability of the wild-type xylanase and two mutants, i.e., HwXyl10A_G363R and HwXyl10A_G363K;

[0027] FIG. 4 shows the measurement results of optimal temperatures of the wild-type xylanase and two mutants, i.e., HwXyl10A_G363R and HwXyl10A_G363K;

[0028] FIG. 5 shows the measurement results of half-time (t½) of the wild-type xylanase and two mutants, i.e., HwXyl10A_G363R and HwXyl10A_G363K at the temperature of 80° C.;

[0029] FIG. 6 shows the measurement results of T50 values of the wild-type xylanase and two mutants, i.e., HwXyl10AG363R and HwXyl10A_G363K.

DESCRIPTION OF THE INVENTION

[0030] Various exemplary embodiments of the present invention will be described in detail herein. It should be noted that such detailed description cannot be deemed as a limitation to the present invention, but should be understood as a more detailed description related to certain aspects, characteristics, and embodiments of the present invention.

[0031] It should be understood that the terms described in the present invention are only used to describe specific embodiments rather than imposing restriction on the present invention. In addition, for the numerical range referred to in the present invention, it should be understood that it discloses each intermediate value between the upper limit and the lower limit of the range specifically. Furthermore, each smaller range between the intermediate value within any stated values or stated range and the intermediate value within any other stated values or stated range is also included in the present invention. The upper limits and the lower limits of these smaller ranges can be independently included or excluded from the range.

[0032] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art of the present invention. Although the present invention only describes the preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All bibliographies referred to in this specification are incorporated by reference, which are used to disclose and describe the methods and/or materials related to the mentioned bibliographies. In the event of conflict with any incorporated bibliographies, the contents of this specification shall prevail.

[0033] It is obvious to those of ordinary skill in the art that various improvements and variations can be made to the specific embodiments of the specification described herein without departing from the scope or spirit of the present invention. Furthermore, other embodiments obtained from the specification of the present invention are also obvious to those of ordinary skill in the art. And the specification and examples of this application are only exemplary.

[0034] For those words of “consist of”, “include”, “have”, and “contain”, etc. used herein are all open terms, which means including but not limited to.

[0035] Unless otherwise specified, the reagents or materials used in the following embodiments can be obtained through commercial channels.

[0036] The test materials used in the following embodiments:

[0037] 1. Strains and vectors: the expression host, i.e., Pichia pastoris GS115 is purchased from Invitrogen.

[0038] 2. Enzymes and other biochemical reagents: the high-fidelity polymerase is purchased from Fermentas, and the xylan of beech is purchased from Sigma. Others are domestic analytical reagents (all of which can be purchased from general biochemical reagent companies).

[0039] 3. Mediums:

[0040] 1) The YPD medium: contains 2% of glucose, 2% of peptone and 1% of yeast extract;

[0041] 2) LB medium: contains 1% of peptone, 0.5% of yeast extract, 1% of NaC1 and 1% of agar powder (solid);

[0042] 3) MD medium: contains 1.5% of agarose, 2% of glucose, 0.00004% of Biotin and 1.34% of YNB;

[0043] 5) BMGY medium: contains 2% of peptone, 1% of yeast extract, 1% of glycerine (V/V), 0.00004% of Biotin and 1.34% of YNB;

[0044] 6) BMMY medium: contains 2% of peptone, 1% of yeast extract, 1.34% of YNB, 0.5% methanol (V/V), and 0.00004% of Biotin.

EMBODIMENT 1

The Obtaining of the Coding Gene of the Xylanase Mutant with High Specific Activity and Heat Resistance

[0045] Taking the recombinant expression vector, i.e., pic9r-HwXyl10a, of the gene of xylanase HwXyl10a derived from Hortaea werneckii (the nucleotide sequence is as shown in SEQ ID NO: 1 and the amino acid sequence is as shown in SEQ ID NO: 2) as template, and then adopt the method of site-directed mutagenesis to conduct saturation mutagenesis to the site of Gly363, wherein, the primer design is as shown in Table 1. In addition, the bibliography related to the mutating method and the cloning method is (Improvement in catalytic activity and thermostability of a GH10 xylanase and its synergistic degradation of biomass with cellulase; You, et al., 2019).

TABLE-US-00001 TABLE 1 Primers for saturation mutagenesis to the site of Gly363 in xylanase HwXyl10A Size Primers Sequences (5′.fwdarw.3′) .sup.a (bp) Gly363- nnntgggatccaagagtaagtatcaac 27 PF Gly363- tatggatcccannnacctttccaggt 27 PR

EMBODIMENT 2

The Preparation of the Xylanase Mutant with High Specific Activity and Heat Resistance

[0046] Transforming the linear recombinant expression vector obtained by PCR of the embodiment 1 directly into the competence of DMT, and then verify the bacterial colony of PCR, and thus obtain the nucleotide sequences of 19 mutants of this site except Gly, after that, transform them into Pichia pastoris GS115 after the linearization of recombinant plasmids, and thus obtain the recombinant yeast strain GS115/HwXyl10A -G363X (wherein, X represents 19 amino acids except Gly).

[0047] Inoculating the strain of GS115 which contains recombinant plasmids into a 10 mL test tube with 2 mL of BMGY medium, and then cultivate it in a shaker at the temperature of 30° C. and at the rotational speed of 220 rpm for 48 hours, after that, centrifuge 3000 g of culture solution for 5 minutes, and then discard the supernatant and conduct sedimentation, after that, take 2 mL of BMMY medium containing 0.5% methanol to resuspend and complete induction and cultivation at the temperature of 30° C. and at the rotational speed of 220 rpm for 48 h. After that, take the supernatant to conduct enzyme activity detection, and thus screen out the mutants with improved thermal stability and the catalytic activity based upon comparing with the wild-type enzyme, i.e., HwXyl10A G363R (the amino acid sequence of which is as shown in SEQ ID NO: 4, and the nucleotide sequence is as shown in SEQ ID NO: 3) and HwXyl10A_G363K (the amino acid sequence of which is as shown in SEQ ID NO: 6 and the nucleotide sequence is as shown in SEQ ID NO: 5).

[0048] Amplifying the fermentation system of the wild-type GS115/HwXyl10A and the two mutants, i.e., GS115/HwXyl10A_G363R and GS115/HwXyl10A_G363K, wherein, inoculate them into the YPD medium to obtain culture solution of seed firstly, i.e., inoculate into a 1 L erlenmeyer flask with 300 mL BMGY medium according to the inoculum size of 1%, and then cultivate it in a shaker at the temperature of 30° C. and at the rotational speed of 220 rpm for 48 hours, after that, centrifuge 3000 g of culture solution for 5 minutes, and then discard the supernatant and conduct sedimentation, after that, take 100 mL of BMMY medium containing 0.5% methanol to resuspend and complete induction and cultivation at the temperature of 30° C. and at the rotational speed of 220 rpm. During the process, add 0.5 mL of methanol every 12 hours to keep the methanol concentration of the bacterial solution at 0.5%, meanwhile, take the supernatant to conduct enzyme activity detection. Finally, concentrate the supernatant to 20 mL, and purify the protein by anion exchange method to conduct measurement and comparison of enzymatic properties. After purifying the expressed xylanase, its protein content can account for more than 90% of the total protein (as shown in FIG. 1).

EMBODIMENT 3.

The Comparative Analysis of Enzymatic Properties of the Recombinant Xylanase Mutant with High Specific Activity and Heat Resistance and the Wild-Type Xylanase Mutant

[0049] 1. Measurement through DNS Method

[0050] The specific method is as follows: according to the optimum pH and at the optimum temperature of each, the reaction system of 1 mL of solution consists of taking 100 μL diluted enzyme solution and 900 μL of substrate, and then, reacting for 10 min, as well as adding 1.5 mL DNS to stop the reaction, after that, boiling for 5 min with boiling water. Then, measuring the OD value of 540 nm after cooling. Wherein, one unit of enzyme activity (U) is defined as the amount of enzyme required to decompose xylan, and thus produce 1 μmoL of reducing sugar per minute under the given conditions.

[0051] 2. The measurement of properties of the recombinant xylanase mutant with high specific activity and heat resistance and the wild-type xylanase mutant

[0052] (1) The Measurement Method for the Optimal pH Value and the pH Stability of the Recombinant Xylanase Mutant with High Specific Activity and Heat Resistance and the Wild-Type Xylanase Mutant

[0053] Completing enzymatic reaction on the purified xylanase mutant obtained in the embodiment 2 and the wild-type xylanase at different pH values (2.0-7.0), and thus measure their optimal pH values. In addition, the measurement of Xylanase activity of substrate xylan is completed at the temperature of 75° C. in 0.1 mol/L of buffer solution of citric acid-disodium hydrogen phosphate with different pH values (2.0, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7.0).

[0054] The results are as shown in FIG. 2, the optimal reaction pH values of the wild-type xylanase and the xylanase mutants are approximate and are between 4.0-4.5; wherein, the remaining enzyme activity of the wild-type xylanase and the xylanase mutants are measured after being processed at the pH values of 1.0-12.0 and at the treatment of 37° C. for 1 hour, and the results are as shown in FIG. 3, wherein, the pH stability of the wild-type xylanase is not significantly different from that of the two xylanase mutants, which stays between pH2.0-pH9.0 stably.

[0055] (2) The Measurement Method for the Optimum Temperatures of the Wild-Type Xylanase and the Xylanase Mutants

[0056] The measurement of optimal temperatures of the recombinant xylanase mutant with high specific activity and heat resistance and the wild-type xylanase is as follows: complete enzymatic reaction in the buffer solution system of 0.1mol/L citric acid-disodium hydrogen phosphate buffer solution (pH 4.5) and at different temperatures (35-90° C.).

[0057] The results are as shown in FIG. 4, which indicate that the optimal temperatures of the recombinant xylanase mutant with high specific activity and heat resistance and the wild-type xylanase (75° C.) are between 75° C. and 80° C., and the relative enzyme activity of two mutants is significantly increased based upon comparing with that of the wild-type enzyme at the high temperature of 80-90° C.

[0058] (3) The Measurement Method for the Thermal Stability of the Wild-Type Xylanase and the Mutants

[0059] The half-time (t½) at the temperature of 80° C.: The remaining enzyme activity of the mutants and the wild-type xylanase are measured after being treated at the temperature of 80° C. for different times, which is up to 30 minutes.

[0060] The measurement results of the half-time at the temperature of 80° C. are as shown in FIG. 5, which indicate that the t½ of the xylanase mutants HwXyl10A_G363R and HwXyl10A_G363K are 15 minutes and 20 minutes, respectively, which have been extended 0.88 times and 1.5 times than that of the wild-type xylanase (8 minutes), respectively, and the mutant HwXyl10A_G363K shows the best thermal stability.

[0061] T.sub.50: The xylanase mutant and the wild-type xylanase are treated at the temperature between 60° C. and 85° C. for half an hour, and the corresponding temperature when the enzyme activity remains half of the original represents the T.sub.50 value of the enzyme.

[0062] The measurement results of T.sub.50 are as shown in FIG. 6, which indicate that the T.sub.50 values of the xylanase mutants HwXyl10A_G363R and HwXyl10A_G363K are 78° C. and 80° C., respectively, which have been increased by 3° C. and 5° C. than that of the wild-type xylanase HwXyl10A (75° C.), respectively. Such results are consistent with the trend of measurement results of half-time. And the sequence of thermal stability is:

HwXyl10A_G363K>HwXyl10A_G363R>HwXyl10A .

[0063] (4) The Measurement Method for the Kinetic Parameters of the Recombinant Xylanase Mutant with High Specific Activity and Heat Resistance and the Wild-Type Xylanase

[0064] The measurement method refers to the bibliography of (A thermophilic and acid stable family-10 xylanase from the acidophilic fungus Bispora sp MEY-1. Extremophiles. 2009; 13:849-57. Luo, et al., 2009), which is used to measure the time of first-order reaction. In addition, the reaction time for measuring Km and Vmax is determined as 5 minutes. Furthermore, xylans with different concentrations (i.e., 1.25, 1.0, 0.8, 0.4, 0.2, 0.15 and 0.1 mg/mL) will be used as the substrate, and the enzyme activity will be measured under the optimal conditions (i.e., at the optimal temperature and pH value), and then calculate corresponding reaction rate, and thus figure out the K.sub.m value and the V.sub.max by means of the GraFit7 software.

[0065] The catalytic efficiency (k.sub.cat/K.sub.m) of the recombinant xylanase mutants with high specific activity and heat resistance under respective optimal conditions and taking xylan as the substrate is 3050 mL/s⋅mg and 3180 mL/s⋅mg, respectively, which has been increased by 3% and 7% than that of the wild-type xylanase (2970 mL/s⋅mg), respectively. And the specific activities of the recombinant xylanase mutants with high specific activity and heat resistance are 4030 U/mg and 4990 U/mg, respectively, which have been increased by 24% and 53% than that of the wild-type xylanase (U/mg) (see Table 2).

TABLE-US-00002 TABLE 2 The comparison table of the specific activity and the kinetic parameters between the xylanase mutants with high catalytic efficiency and the wild-type xylanase mutant specific K.sub.m k.sub.cat k.sub.cat/K.sub.m activity (mg/mL) (s.sup.−1) (mL/s .Math. mg) (U/mg) HwXyl10A 0.88 ± 0.13 2610 ± 74  2970 ± 383 3260 ± 62  HwXyl10A_ 0.98 ± 0.11 2990 ± 104 3050 ± 119 4030 ± 177 G363R HwXyl10A_ 1.13 ± 0.07 3590 ± 176 3180 ± 159 4990 ± 201 G363K

[0066] The above-mentioned embodiments only describe the preferred approaches of the present invention rather than imposing restriction on the scope of the present invention. Various variations and improvements of the technical scheme of the present invention made by those of ordinary skill in the art without departing from the design spirit of the present invention should fall within the protection scope determined by the claims of the present invention.