CN117737099A - Heat-resistant acid-resistant xylosidase gene, protein expressed by same and application thereof - Google Patents

Abstract

The invention discloses a heat-resistant acid-resistant xylosidase gene, an expression protein and application thereof, wherein the nucleotide sequence of the heat-resistant acid-resistant xylosidase gene is shown as SEQ ID NO.1, and the amino acid sequence of the heat-resistant acid-resistant xylosidase is shown as SEQ ID NO. 2. The modified gene codes the xylosidase which has the characteristics of extremely strong heat resistance and high activity under the condition of acidic pH, and has the highest enzyme activity under the condition of 70 ℃ and pH of 4.5, and the specific enzyme activity reaches 77.6 mu mol/mg min; has higher enzyme activity at 65-75deg.C and pH of 4.0-5.0. The xylosidase is suitable for degradation of xylan and xylooligosaccharide at 70 ℃ under acidic pH conditions, and has potential industrial application value.

Description

Heat-resistant acid-resistant xylosidase gene, protein expressed by same and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a heat-resistant acid-resistant xylosidase gene, and an expressed protein and application thereof.

Background

Xylan is the main component of plant cell wall hemicellulose, one of the most abundant renewable resources in nature except cellulose. Xylose is present in hemicellulose pentoses and is an important renewable resource to be developed. Xylosidases are a key class of enzymes that release xylose from xylan or xylo-oligosaccharides, and have a synergistic effect with xylanases, the final hydrolysate being xylose. The xylosidase can be used as a tool for preparing xylose produced by degrading xylan, can improve the utilization rate of lignocellulose raw materials, can be used as an efficient industrial enzyme preparation, and has potential application value.

The enzymolysis efficiency of the existing xylosidase under the high-temperature and acidic environment is mostly unsatisfactory. The optimum temperature of the xylosidase from Bacteroides ovatus is 25 ℃ and the optimum pH is 6.0; the optimum temperature of the xylosidase from Caulobacter crescentus is 45 ℃, the optimum pH is 6.0, and the two enzymes cannot adapt to high temperature; the optimum temperature of the xylosidase derived from Geobacillus sp.reached 70 ℃, but the optimum pH was 6.5, and acid resistance was not possible. The prior industrial production generally needs to be under high temperature and acidic conditions, and the prior xylosidase resources are severely limited in actual use, so that the application potential of the xylosidase resources in industries such as bioenergy, food additives, feed fermentation and the like can not be fully exerted.

Disclosure of Invention

The invention aims to: aiming at the problems existing in the prior art, the invention provides the heat-resistant acid-resistant xylosidase gene, the expressed protein of which is obviously improved in heat resistance and acid resistance, and hemicellulose enzymolysis can be carried out under some extreme conditions, so that the heat-resistant acid-resistant xylosidase gene is applied to hemicellulose degradation and has potential industrial application value.

The invention also provides a protein expressed by the heat-resistant acid-resistant xylosidase gene and application thereof.

The technical scheme is as follows: in order to achieve the above purpose, the invention provides a heat-resistant acid-resistant xylosidase gene, the nucleotide sequence of which is shown in SEQ ID NO. 1.

The invention relates to an expression protein of a heat-resistant acid-resistant xylosidase gene, which is xylosidase and has an amino acid sequence shown in SEQ ID NO. 2.

The primer pair used for amplifying the heat-resistant acid-resistant xylosidase gene is shown in SEQ ID NO. 3-4:

SEQ ID NO.3：TGCTCTAGAATGGCTCCTGAAATCAGTGT

SEQ ID NO.4：GGCCTCGAGTCAGTGATGGTGATGGTGATGTGATCT

TTCTACTTCTATCA。

wherein the expression protein comprises derived protein thereof, and the derived protein is derived protein with xylosidase activity formed by substitution, deletion and addition of one or more amino acid residues of amino acid shown in a sequence SEQ ID NO. 2.

The invention relates to a recombinant vector containing the heat-resistant acid-resistant xylosidase gene.

The invention relates to recombinant bacteria containing the heat-resistant acid-resistant xylosidase gene or the expression protein or the recombinant vector.

Wherein the recombinant escherichia coli DE3 is an initial strain.

The heat-resistant acid-resistant xylosidase gene or the expression protein is applied to enzymolysis of xylan, xylooligosaccharide and hemicellulose related substrates.

Wherein the hemicellulose related substrate is p-nitrophenol-beta-D-xylopyranoside. And p-nitrophenol-beta-D-xylopyranoside is active, i.e. is active on xylan and xylo-oligosaccharide by default. Wherein the enzymolysis reaction temperature is 65-75 ℃ and the pH value is 4.0-5.0.

Preferably, the enzymolysis reaction temperature is 70 ℃ and the pH is 4.5.

According to the invention, through database comparison, a conserved sequence in a Thermoanaerobacterium thermosaccharolyticum DSM 571 xylosidase sequence is taken as a framework, a random coiled part of a non-conserved sequence in an amino acid sequence is selected as a potential modification site, a proper site is selected according to a simulated three-dimensional structure, a heat-resistant amino acid is introduced to form disulfide bonds, the interaction of charges in the enzyme is optimized, and the active center of the enzyme is rigidized, so that the enzyme with better properties is obtained. The heat resistance and acid resistance of the enzyme are improved, the optimal temperature is improved, the optimal pH is reduced, and the enzyme activity is improved. The recombinase obtained by the research has important application value in the production of ethanol, butanol and beer brewing by pentasaccharide fermentation.

The beneficial effects are that: compared with the prior art, the invention has the following advantages:

the modified gene expressed protein is characterized in that the xylosidase has extremely strong heat resistance and high activity under the acidic pH condition, and has the highest enzyme activity under the conditions of 70 ℃ and pH 4.5, and the specific enzyme activity reaches 77.6 mu mol/mg min; the enzyme has higher enzyme activity at the temperature of 65-75 ℃ and the pH value of 4.0-5.0, and can be effectively used in fermentation under some special conditions.

The xylosidase has extremely high heat resistance, and the activity of the enzyme is kept more than 90% after incubation for 2 hours in a reaction system under the condition of 70 ℃; the acid resistance of the enzyme is good, and the enzyme activity is maintained to be more than 80% when the enzyme is incubated for 1h within the pH range of 4.0-5.5. Compared with the prior modification, the specific enzyme activity of the xylosidase is improved by about 11 percent, and the heat resistance is obviously improved from 60 ℃ to 70 ℃. The xylosidase obtained by the invention has greater superiority than the prior xylosidase, is suitable for degrading xylan or xylooligosaccharide at 70 ℃ under the conditions of high temperature and acidic pH, and has potential industrial application value.

Drawings

FIG. 1 is an SDS-PAGE protein electrophoresis of the xylosidase TtXyl A;

FIG. 2 is a graph of optimum temperature results for TtXYLA;

FIG. 3 is a graph of the optimum pH results for TtXyl A;

FIG. 4 is a graph of the thermal stability results of TtXYLA;

fig. 5 is a graph showing the pH stability of TtXylA.

Detailed Description

The invention is further described below with reference to examples and figures.

Materials, reagents and the like used in the following examples were obtained commercially unless otherwise specified.

Example 1

Preparation of xylosidase Gene TtXylA

The amino acid sequence (SEQ ID NO. 5) of Thermoanaerobacterium thermosaccharolyticum DSM 571 xylosidase (NCBI number: ADL 68512.1) is taken as a template, the base sequence of the amino acid sequence is SEQ ID NO.6, and a non-conserved site of the xylosidase is obtained through database comparison to serve as a potential transformation site; simultaneously, disulfide bonds are introduced through the amino acid preference of thermophilic xylosidase and the strategy of optimizing intramolecular interaction, so as to optimize the interaction of charges in the enzyme and rigidize the active center of the enzyme; and finally, setting various parameters of the modified enzyme, and modifying. The TtXyl A gene is prepared by modifying the amino acid sequence according to the codon preference of escherichia coli through codon optimization and artificial synthesis, and the gene sequence is shown as SEQ ID NO. 1.

Example 2

Subcloning of the xylosidase Gene TtXYLA

The xylosidase gene in example 1 was amplified by PCR using the following primer pair:

Tt-1：5’–CTAGCCATGGCATGATCAGCAAAAGCTTCTACGCAC-3’

Tt-2：5’–CCGCTCGAGCTTCATCCACAGAATAGAGGTAAC-3’

in the synthesis of the primer, tt-1 is introduced into the NCO I cleavage site, and Tt-2 is introduced into the Xho I cleavage site.

PCR reaction system: 1. Mu.L of P.Thermosaccharolyticum DSM 571 genomic DNA (total DNA extracted from DSMZ Strain collection, germany), 1. Mu.L of Tt-1, 1. Mu.L of Tt-2,9.5. Mu.L of ddH ₂ O，12.5μL prime STAR HSDNA Polymerase。

PCR reaction conditions: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30sec, annealing at 55℃for 30sec, extension at 72℃for 2min,35cycles; extending at 72 ℃ for 10min; preserving heat at 12 ℃.

The PCR products were checked for yield and specificity by 1% agarose gel electrophoresis and purified by PCR product recovery kit (BIOMIGA, shanghai), sequenced to correspond to SEQ ID NO. 1.

Example 3

Construction and verification of recombinant cloning and expression vector pET-20b-ttXyl A

The purified PCR product (prepared in example 2), pET-20b (Novagen), was digested with NCO I and Xho I, respectively, and the digested PCR and large fragment of the vector were recovered by agarose electrophoresis. The target fragment after the rubber cutting recovery was ligated with the vector by adding 1. Mu.L of 10 XLigase Buffer and 1. Mu.L of Ligase overnight at 16 ℃. Coli DH 5. Alpha. Was transformed with the ligation reaction product, and then plated on a Petri dish containing 100. Mu.g/mL Amp (ampicillin), and incubated at 37℃for 10-15 hours.

A plurality of single colonies were picked from the transformation plate and plasmids were extracted using the plasmid miniprep kit of BIOMIGA. The obtained plasmid was verified by double cleavage and the obtained recombinant plasmid was sequenced. Sequencing results show that the cloned target fragment (with the nucleotide length of 2043 bp) is inserted into the pET-28a vector, so that the expression vector pET-20b-ttXyl A is obtained, the DNA sequence of the TtXyl A is shown as SEQ ID NO.1, the amino acid sequence of the expressed protein (heat-resistant acid-resistant xylosidase) is shown as SEQ ID NO.2, and the protein is named as TtXyl A.

Example 4

The expression vector pET-20b-ttXylA (prepared in example 3) was transformed into a host bacterium E.coli BL21 (DE 3) (Novagen) by heat shock to obtain a recombinant bacterium containing a recombinant plasmid. Single colony recombinant bacteria were inoculated in 5mL Luria-Bertani (LB) medium containing 100. Mu.g/mL kanapecillin, and shake cultured at 37℃for 10h at 200 rpm. The 1mL of the bacterial liquid is inoculated into a 1000mL shaking flask containing 200mL of culture medium, shaking culture is carried out at 37 ℃ and 200rpm, when the absorbance reaches 0.4-0.6, 200 mu L of 1M IPTG is added, and the induction expression is carried out at 30 ℃ and 200rpm for 12h. The culture broth was centrifuged at 8000rpm for 10min at 4℃with a high-speed refrigerated centrifuge to collect the cells. The cells were recovered by washing with 50mL of ultrapure water and centrifuging at 8000rpm for 10min at 4℃followed by resuspension (0.5MNaCl,20mM Tris-HCl,5mM Imidazole,pH 7.9) with 20mL Binding Buffer, disruption of the cells with ultrasonic waves in an ice-water bath and centrifugation at 10000rpm for 20min at 4℃to give a crude extract containing xylosidase TtXyl A.

The crude extract was purified using a Ni-NTA affinity column (see His-Band kit, novagen). The purity of the purified enzyme was identified and the molecular weight was determined by SDS-PAGE, and the results are shown in FIG. 1, wherein 1 represents purified TtXyl A protein eluted with 1M imidazole (as pure enzyme solution for subsequent experiments). The molecular weight is about 75kDa, which is close to the theoretical value.

Example 5

Enzymatic Property analysis of recombinant xylosidase TtXyl A

The enzyme activity is defined as: under the optimal conditions, the enzyme amount required for the enzymolysis of the p-nitrophenol-beta-D-xylopyranoside to generate 1 mu mol of p-nitrophenol within 1min is 1U.

(1) Optimum temperature

The pure enzyme solution obtained in example 4 was diluted with 50mM imidazole-potassium hydrogen phthalate buffer solution having pH of 5.0, and the enzyme activity was measured using the enzyme solution diluted 10 times. The enzyme activity measurement reaction system is 200 mu L, and the enzyme activity measurement reaction system is prepared from 2mM p-nitrophenol-beta-D-xylometazole100. Mu.L of a furanoside (Sigma) solution, 90. Mu.L of 50mM potassium imidazole-phthalate buffer pH 5.0 and 10. Mu.L of a diluting enzyme solution; the pH of the reaction system was 5.0. After incubating the reaction system at 50-100deg.C (interval gradient set to 5 ℃ C.) for 10min, 600. Mu.L of 1MNA was added ₂ CO ₃ The reaction was terminated and the absorbance at 405nm was measured. The enzyme reaction was performed without adding a diluted enzyme solution, and the mixed solution under the same treatment conditions was used as a blank. The experiment is repeated for 3 times, an average value is drawn, and the research result shows that the xylosidase has the highest enzyme activity at 70 ℃; the absorbance of the enzyme activity reaction system at this temperature was set to 100% relative activity, and the absorbance of the enzyme activity reaction system at the other temperature and the absorbance of the highest enzyme activity system were set as relative activities, and the results are shown in FIG. 2. Wherein the enzyme activity is higher in the reaction system with the temperature of 65-75 ℃.

(2) Optimum pH

The pure enzyme solution obtained in example 4 was diluted with 50mM imidazole-potassium hydrogen phthalate buffer solution having pH of 5.0, and the enzyme activity was measured using the enzyme solution diluted 10 times. The enzyme activity assay system was 200. Mu.L, and consisted of 100. Mu.L of 2mM p-nitrophenol-beta-D-xylopyranoside (pH 3.5-6.5, interval gradient set to 0.5), 90. Mu.L of 50mM imidazole-potassium hydrogen phthalate buffer and 10. Mu.L of diluted enzyme solution, after incubating the reaction system at 70℃for 10min, 600. Mu.L of 1M Na was added ₂ CO ₃ The reaction was terminated, the absorbance at 405nm was measured, and the experiment was repeated three times to obtain an average value. The enzyme reaction was performed without adding a diluted enzyme solution, and the mixed solution under the same treatment conditions was used as a blank. The research result shows that the xylosidase has the highest enzyme activity at the pH value of 4.5; the relative activity of the enzyme activity reaction system at the pH value is 100%, and the ratio of the absorbance value of the enzyme activity reaction system at other pH values to the absorbance value of the highest enzyme activity system is shown in FIG. 3. The enzyme activity is higher under the condition of pH 4.0-5.0.

(3) Thermal stability of recombinant enzymes

The pure enzyme solution obtained in example 4 was diluted with 50mM imidazole-potassium hydrogen phthalate buffer having a pH of 4.5, and the enzyme activity was measured using the enzyme solution diluted 10 times. The temperature range is set to 65-85 ℃, and a temperature gradient is set at intervals of 5 ℃. The enzyme activity reaction system is set to 200 mu L, 10 mu L of purified enzyme solution diluted by 10 times and 90 mu L of 50mmol/L imidazole-potassium hydrogen phthalate buffer solution with pH of 4.5 are respectively added into a centrifuge tube, the mixture is placed into constant temperature metal baths with different temperature gradients and respectively kept for 30-120min (a time gradient is set every 30 min), and according to the different set keeping time, 100 mu L of 2mM p-nitrophenol-beta-D-xylopyranoside solution is respectively added after being taken out, and the mixture is reacted in a water bath with 70 ℃ for 10min, and the absorbance at 405nm is measured. Three parallel groups are arranged in each group, the enzyme reaction is carried out by adding no diluted enzyme liquid, the mixed liquid under the same treatment condition is used as a blank control, and the enzyme activity of the diluted enzyme liquid which is not subjected to heat preservation and reacts for 10min under the conditions of 70 ℃ and pH 4.5 is used as the relative activity of 100 percent. The research result shows that the enzyme activity is not obviously reduced after the treatment for 2 hours at 70 ℃, and the result is shown in figure 4, so that the xylosidase has better heat stability at 65-75 ℃.

(4) Recombinant enzyme pH stability

The pure enzyme solution obtained in example 4 was diluted with 50mM imidazole-potassium hydrogen phthalate buffer having a pH of 4.5, and the enzyme activity was measured using the enzyme solution diluted 10 times. The pH range was set to 4.0-8.5, with a pH gradient set every 0.5. The enzyme activity reaction system is set to 200 mu L,90 mu L of imidazole-potassium hydrogen phthalate buffer solution with the concentration of 50mmol/L and purified enzyme solution with the concentration of 10 mu L diluted by 10 times are respectively added into a centrifuge tube, after the mixture is kept warm for 1h in a constant temperature metal bath at 40 ℃, 100 mu L of 2mM of p-nitrophenol-beta-D-xylopyranoside solution is added, and the mixture is placed into a water bath at 80 ℃ for reaction for 10min, and OD is carried out ₄₀₅ Absorbance values were measured at nm. Three groups of the enzyme-catalyzed reaction were arranged in parallel, and the mixed solution under the same treatment conditions was used as a blank control. The enzyme activity was 100% by reaction at 70℃for 10min using a diluted enzyme solution without pH adjustment. The research result shows that the enzyme activity in the buffer system with the pH value of 4.0-5.5 is not obviously reduced after the water bath with the temperature of 70 ℃ is carried out for 1h, and the result is shown as figure 4, and the xylosidase has very good acid resistance under the condition with the pH value of 4.0-5.5 at the temperature of 70 ℃.

The xylosidase (template sequence was prepared as in examples 2-4) before modification was also determined by the method described above, and the results are shown in Table 1.

Table 1 results of comparison of TtXyl A with xylosidase before modification

As shown in Table 2, compared with the xylosidase before transformation, the specific enzyme activity of the xylosidase is improved by about 11%, the heat resistance is obviously improved from 60 ℃ to 70 ℃, and the acid resistance is improved to pH 4.5; meanwhile, the temperature stability is improved to a certain extent, and the method has better industrial application value.

Example 6

The specificity of the recombinant xylanase TtXyl A for CMC-Na, filter paper, p-nitrophenol-alpha-L-arabinofuranoside and p-nitrophenol-beta-D-xylopyranoside was studied, and the results are shown in Table 2.

The recombinant xylanase TtXyl A is respectively acted on CMC-Na, filter paper, p-nitrophenol-alpha-L-arabinofuranoside and p-nitrophenol-beta-D-xylopyranoside, and is measured according to the system of the example 5 at the optimal pH temperature, and the enzyme specificity is higher and only the p-nitrophenol-beta-D-xylopyranoside has activity after the enzyme activity measurement experiment in the example 5.

TABLE 2 enzymatic Activity of recombinant xylosidase TtXyl A against CMC-Na, filter paper, p-nitrophenol-alpha-L-arabinofuranoside and p-nitrophenol-beta-D-xylopyranoside

The xylosidase coded by the modified gene in the research has the characteristics of extremely strong heat resistance and high activity under the condition of acidic pH, the enzyme activity is highest under the condition of 70 ℃ and the pH of 4.5, and the specific enzyme activity reaches 77.6 mu mol/mg min; has higher enzyme activity at 65-75deg.C and pH of 4.0-5.0. The obtained recombinant xylosidase is suitable for degradation of xylan and xylooligosaccharide at 70 ℃ under acidic pH conditions, and has important application value in production of ethanol, butanol, beer brewing and the like by fermenting pentose.

Claims (10)

1. The heat-resistant acid-resistant xylosidase gene is characterized in that the nucleotide sequence of the heat-resistant acid-resistant xylosidase gene is shown as SEQ ID NO. 1.

2. An expression protein of a heat-resistant acid-resistant xylosidase gene as set forth in claim 1, wherein the expression protein is xylosidase and has an amino acid sequence as set forth in SEQ ID No. 2.

3. A primer for amplifying the thermostable acid-tolerant xylosidase gene according to claim 1, wherein the primer pair used is shown in SEQ ID No. 3-4:

SEQ ID NO.3：TGCTCTAGAATGGCTCCTGAAATCAGTGT

SEQ ID NO.4：GGCCTCGAGTCAGTGATGGTGATGGTGATGTGATCT

TTCTACTTCTATCA。

4. the expressed protein according to claim 2, wherein the expressed protein comprises a derivative protein thereof, and the derivative protein is a derivative protein with xylosidase activity, wherein the amino acid shown in the sequence SEQ ID NO.2 is substituted, deleted and added by one or more amino acid residues.

5. A recombinant vector comprising the thermostable acid-resistant xylosidase gene according to claim 1.

6. A recombinant bacterium comprising the thermostable xylosidase gene according to claim 1, the expression protein according to claim 2, or the recombinant vector according to claim 5.

7. The recombinant strain according to claim 6, wherein the recombinant strain E.coli DE3 is a starting strain.

8. Use of the thermostable acid-resistant xylosidase gene according to claim 1 or the expressed protein according to claim 2 for enzymatic hydrolysis of xylan, xylooligosaccharide, hemicellulose-related substrates.

9. Use according to claim 8, characterized in that the hemicellulose-related substrate is preferably p-nitrophenol- β -D-xylopyranoside.

10. The use according to claim 8 or 9, wherein the enzymatic hydrolysis reaction temperature is 65-75 ℃, pH 4.0-5.0.