CN106520733B

CN106520733B - Beta-xylosidase enzyme aggregate and preparation method thereof

Info

Publication number: CN106520733B
Application number: CN201610910450.3A
Authority: CN
Inventors: 李爽; 徐天旺; 杨晓锋
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2016-10-19
Filing date: 2016-10-19
Publication date: 2020-09-22
Anticipated expiration: 2036-10-19
Also published as: CN106520733A

Abstract

The invention discloses a preparation method of a beta-xylosidase endoenzyme aggregate, which comprises the following steps: (1) splicing the connecting peptide and the amphiphilic short peptide to construct an expression vector; the amphiphilic short peptide is ELK 16; (2) connecting the beta-xylosidase coding gene with the expression vector, and converting the beta-xylosidase coding gene into receptor bacteria to obtain engineering bacteria capable of expressing the beta-xylosidase-short peptide fusion protein; (3) and (3) performing induced expression on the engineering bacteria, performing wall breaking treatment on cells, centrifuging and/or filtering to obtain a precipitate, namely the beta-xylosidase endoenzyme aggregate. Compared with the ThXylC obtained by conventional expression, the ThXylC-ELK16 active enzyme aggregate obtained by the invention has improved specific enzyme activity and catalytic efficiency, and the heat tolerance of the ThXylC-ELK16 active enzyme aggregate is also obviously improved. The method has great potential for popularization and application in production.

Description

Beta-xylosidase enzyme aggregate and preparation method thereof

Technical Field

The invention belongs to the fields of bioengineering and biocatalysis. The invention particularly relates to a method for greatly improving specific enzyme activity, catalytic efficiency and heat tolerance of beta-xylosidase (ThXylC) by adding amphiphilic short peptide at the tail end of the beta-xylosidase.

Background

In industrial and agricultural production, enzyme with better heat tolerance is often required as a catalyst for reaction. This is because an enzyme having good heat resistance has the following advantages as compared with an enzyme at ordinary temperature: (1) the reaction rate is accelerated; according to the van't Hoff law, the reaction rate response is improved by 2-4 times when the temperature is increased by 10 ℃ in a proper temperature range. (2) The catalysis process is carried out at high temperature, can improve the solubility and the availability of substrates such as starch, cellulose, lipid and the like, and reduce the viscosity of compounds so as to be beneficial to substance diffusion and mixing; (3) when the reaction temperature is higher than 50 ℃, the growth chance of mixed bacteria is greatly reduced, thereby reducing the pollution of microorganisms to products and being beneficial to simplifying the separation steps of the products. Based on the advantages, the enzyme with good heat tolerance has wide application prospect in the fields of food, chemical industry, pharmacy, energy development, environmental protection and the like.

Generally, there are 3 methods for obtaining enzymes with good heat tolerance, (1) screening from natural extreme microorganisms (Sharma PK, et al, Gene 2012,491:264-₆KD and 18A (Lin Z, Zhou B, Wu W, equivalent. Faraday disorders, 2013,166:243-256.) to optimize the catalytic properties of the enzyme. For example, a Self-assembly short peptide (SAP) is expressed at the N-terminal fusion of monooxygenase (lipoxygenase), and the thermal stability of the enzyme at 50 ℃ is prolonged from 10min to 46min (Lu XY, et al, appl. Microbiol. Biotechnol.2013,97, 9419-9427). In the previous study of the subject group, the half-life of the nitrilase at 50 ℃ was also successfully prolonged from 13h to 56h by introducing the 18A short peptide at the C-terminal of the nitrilase (Yang X, et al, RSC Advances 2014,4: 60675-84). However, these methods of expressing a short peptide by fusion to the end of a target protein only partially improve the stability of the enzyme, and do not affect the optimum reaction temperature of the enzyme, that is, the optimum enzymatic reaction temperature does not change.

Disclosure of Invention

The object of the present invention is to provide a method for increasing the heat tolerance of beta-xylosidase, where the heat tolerance comprises the optimum enzymatic reaction temperature of the enzyme and the heat stability of the enzyme. More specifically, the invention realizes the improvement of the heat tolerance of the beta-xylosidase by fusing and expressing the amphiphilic short peptide ELK16 at the C end or the N end of the beta-xylosidase.

It is another object of the present invention to provide a method for obtaining β -xylosidase activity aggregates having a better temperature tolerance.

The purpose of the invention is realized by the following technical scheme:

(1) splicing the connecting peptide and the amphiphilic short peptide to construct an expression vector; the amphiphilic short peptide is ELK 16;

(2) connecting the beta-xylosidase coding gene with the expression vector, and converting the beta-xylosidase coding gene into receptor bacteria to obtain engineering bacteria capable of expressing the beta-xylosidase-short peptide fusion protein;

(3) and (3) performing induced expression on the engineering bacteria, performing wall breaking treatment on cells, centrifuging and/or filtering to obtain a precipitate, namely the beta-xylosidase endoenzyme aggregate ThXylC-ELK 16.

The expression vector is a vector that can be overexpressed in a microorganism.

The connecting peptide sequence comprises connecting peptide used for expressing common connecting fusion protein, including but not limited to PT-linker (PTPPTTTPPTTPTPTP), (GGGGS)₃、(Gly)₆、(Gly)₈、(EAAAK)n(n＝1-3)。

The splicing sequence of the connecting peptide and the amphiphilic short peptide can be connecting peptide-amphiphilic peptide or amphiphilic peptide-connecting peptide, the corresponding amphiphilic peptide is fused with beta-xylosidase for expression, and the amphiphilic peptide can be positioned at the C end or the N end of the beta-xylosidase.

The nucleotide sequence of the connecting peptide spliced with the amphiphilic short peptide is shown as SEQ ID No. 1.

The beta-xylosidase gene is not strictly required to be an open reading frame of the beta-xylosidase coding gene, as long as the sequence is inserted into an expression vector to be transcribed and translated into a beta-xylosidase coding amino acid sequence normally. Preferably the open reading frame of the gene encoding the β -xylosidase. The beta-xylosidase gene may be selected from, but is not limited to, Thermoanaerobacterium aoteraense SCUT 27.

The recipient bacterium described in step (3) may be any one as long as the expression vector described in step (1) can be expressed in vivo, including but not limited to e.coli DH5 α, e.coli JM109, e.coli JM110, e.coli TOP10, e.coli BL21, e.coli BL21(DE3), e.coli BL21(DE3)/pLysS, e.coli BL21Rosetta (DE3), preferably e.coli BL21(DE 3).

And (4) washing the precipitate by using a buffer solution for purification.

The transformation method described in step (2) includes, but is not limited to, heat shock method, CaCl₂Methods, electrotransformation methods, and the like.

The cell wall breaking treatment method in step (3) includes, but is not limited to, ultrasonication, high pressure disruption, lysozyme disruption, and the like, and the ultrasonication method is preferred.

The enzymatic kinetic parameters of ThXylC and ThXylC-ELK16 were determined and characterized enzymatically using p-nitrophenyl-beta-D-xyloside (pNPX) as substrate. The reaction system is as follows: the pH value of the buffer solution is 4.0-8.0, the initial concentration of the buffer solution is 1-10 mmol/L of p-nitrophenyl-beta-D-xyloside (pNPX), the optimal concentration is 4mmol/L, the reaction temperature is 45-85 ℃, and the optimal temperatures of ThxylC and ThxylC-ELK16 are 65 ℃ and 70 ℃ respectively.

The buffer solution is not particularly limited as long as the pH value of the buffer solution can be effectively adjusted, and the buffer solution includes, but is not limited to, acetic acid-sodium acetate buffer solution, potassium phosphate buffer solution and phosphate buffer solution. Potassium phosphate buffer at a pH of 6.5 is preferred.

Compared with the prior art, the invention has the following beneficial effects:

(1) the beta-xylosidase is induced to aggregate in escherichia coli by the amphiphilic short peptide, and the active beta-xylosidase aggregate with the purity of about 95 percent can be obtained by conventional cell disruption centrifugation. Because the active aggregate has strong mechanical operation stability and can bear ultrasound, high pressure or other strong physical cell crushing processes, a protein sample with higher purity can be obtained through simple cell crushing and centrifugal operation, and the affinity purification of the active aggregate is simpler than that of the original soluble expressed beta-xylosidase (marked as ThXylC).

(2) Compared with ThXylC, the ThXylC-ELK16 obtained by the invention has poor water solubility and is beneficial to long-term storage; the catalytic property is obviously improved, the industrial application of the beta-xylosidase is promoted, and the method has wide application prospect.

(3) Compared with the ThxylC obtained by conventional expression, the ThxylC-ELK16 active enzyme aggregate obtained by the invention has improved specific enzyme activity and catalytic efficiency, and obviously improved heat tolerance (including optimal catalytic reaction temperature and temperature stability).

Drawings

FIG. 1 is a schematic diagram of the pET-ELK plasmid.

FIG. 2SDS-PAGE analysis of beta-xylosidase expression purification (A) ThXylC protein samples, M, protein molecular weight standards; 1, E.coli BL21(DE3)/pET-xylC1 thalli ultrasonication and cracking of the crude enzyme liquid (supernatant); 2, the crude enzyme solution after heat treatment; 3, passing liquid of nickel column affinity chromatography; 4, collecting the obtained sample by affinity chromatography; and 5, desalting the sample. (B) A sample of ThXylC-ELK16 protein, M, a protein molecular weight standard; 1, E.coli BL21(DE3)/pET-xylC2-ELK thalli supernatant after ultrasonic disruption and cracking; 2, the precipitation part (containing the target protein) after the ultrasonic disruption; 3, ThXylC-ELK16 obtained after 2 buffer washes.

FIG. 3 is a pH optimum curve for ThXylC-ELK16 and ThXylC according to the present invention.

FIG. 4 is a graph showing the optimum enzymatic reaction temperature curves for ThXylC-ELK16 and ThXylC according to the present invention.

FIG. 5 is a graph of temperature stability for ThXylC-ELK16 and ThXylC of the present invention.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The beta-xylosidase gene is derived from Thermoanaerobacterium thermophilum thermoanaerobacter aethioense SCUT27, and the ID of the gene in GenBank database is KX 372717. In the following examples, the experimental procedures without specifying the conditions were carried out according to the conditions described in the manual of molecular cloning.

Example 1

Construction of expression vectors containing encoded short peptides

Based on the amino acid sequence of ELK16 (LELELKLKLELELKLK, see SEQ ID:2), the corresponding DNA sequence containing a stop codon was synthesized at the gene synthesis company. Wherein a PT-linker (PTPPTTTPPTTPTPTP) sequence is introduced at the upstream of an ELK16 sequence, HindIII and XhoI enzyme cutting sites are introduced at two ends of a synthetic sequence, and the complete gene sequence is shown in SEQ ID: 1. Plasmid pET-30a (+) is used as a basic plasmid, HindIII and XhoI are used for double enzyme digestion, the DNA sequence of ELK16 which is correspondingly cut by enzyme is connected, a connection product is transformed into E.coli DH5 alpha competent cells, and a positive transformant is identified through colony PCR, enzyme digestion and sequencing analysis, so that an expression vector pET-ELK is obtained, wherein the expression vector pET-ELK is shown in figure 1.

Example 2

Construction of beta-xylosidase expressing strains

1. Amplification of beta-xylosidase gene

Beta-xylosidase gene (GenBank: KX372717) was amplified using genomic DNA of Thermoanaerobacterium aoteraense SCUT27 as a template, and the following PCR primers were used:

XylC-F：5′-TCGGCTCATATGGAATACCATGTGGCTAAAA-3' (see SEQ ID:3), the underlined bases are NdeI cleavage sites.

XylC-R：5′-TAGCAACTCGAGAGAAGAGCCCCAAACTTTTATGTAATTATTTCCT-3' (see SEQ ID:4), the underlined bases are XhoI cleavage sites.

XylC-E-R：5'-CTGTTCAAGCTTCCAAACTTTTATGTAATTATTTCCT-3' (see SEQ ID:5), the underlined bases are HindIII sites.

The primer pairs XylC-F/XylC-R and XylC-F/XylC-E-R are respectively used for amplification to obtain beta-xylosidase genes xylC1 and xylC2 with the length of about 2 kb.

2. Construction of recombinant expression vectors

Carrying out double enzyme digestion on purified PCR products xylC1 and pET30a (+) by Nde I and XhoI respectively, purifying and connecting enzyme digestion products, and transferring the enzyme digestion products into a recipient bacterium E.coli BL21(DE3) to obtain a conventional expression bacterium E.coli BL21(DE3)/pET30-xylC1 of beta-xylosidase.

Carrying out double enzyme digestion on purified PCR products xylC2 and pET-ELK by Nde I and HindIII respectively, purifying and connecting enzyme digestion products, and transferring the enzyme digestion products into a receptor bacterium E.coli BL21(DE3) to obtain a beta-xylosidase aggregate expression bacterium E.coli BL21(DE3)/pET30-xylC 2-ELK.

Example 3

Obtaining of soluble β -xylosidase (ThXylC) and active β -xylosidase aggregates (ThXylC-ELK16) expressed conventionally.

Recombinant engineering bacteria E.coli BL21(DE3)/pET-xylC1 and E.coli BL21(DE3)/pET30-xylC2-ELK cultured overnight are respectively inoculated in a fresh LB liquid culture medium containing 50 mu g/ml kanamycin in a ratio of 1:100, and cultured at 37 ℃ and 250rpm until the cell density OD is reached₆₀₀About 0.5 to 0.7, and β -xylosidase was induced by adding IPTG at a final concentration of 1mmol/L, followed by further culturing at 30 ℃ and 180rpm for 24 hours.

The cells were collected by centrifugation at 8000g and 4 ℃ in 10ml of PB buffer (50mmol/L phosphate buffer, 50mmol/L sodium chloride, pH7.5) per 1g of wet cells, and disrupted by sonication. High speed centrifugation at 15000g for 20 minutes.

E.coli BL21(DE3)/pET-xylC1, centrifuging and discarding the precipitate, firstly, preserving the heat of the supernatant part at 65 ℃ for 30min to inactivate the heat-labile protein sample, and purifying and desalting the supernatant after centrifuging the sample by adopting a conventional nickel column affinity chromatography to obtain the purified conventional beta-xylosidase (which is recorded as ThxylC, and shown in figure 2A). The recovery of the purified protein samples for each step is shown in Table 1.

TABLE 1ThXylC protein sample purification

Coli BL21(DE3)/pET30-xylC2-ELK cell lysate was centrifuged, the supernatant was discarded, and the precipitated sample was further purified as follows (FIG. 2B): as shown in table 2.

(1) The pellet was washed by thoroughly resuspending it in a PB buffer (pH7.5) of the same volume as the cell resuspension (10 ml PB buffer per 1g of wet cells), and then placed on ice for 10 min;

(2)15000g, centrifuging for 25min at 4 ℃ and removing the supernatant;

(3) repeating the

steps

1 and 2 once;

(4) the pellet was resuspended in an appropriate amount of 100mmol/L potassium phosphate buffer (pH 6.5) and stored at 4 ℃.

TABLE 2 purification of ThXylC-ELK16 protein

The result obtained here is the catalytically active ss-xylosidase aggregate ThXylC-ELK 16.

Example 4

Enzymatic kinetic study of beta-xylosidase

Enzyme kinetics study the enzyme kinetics parameters of ThXyl C and ThXyl C-ELK16 obtained in example 3 were determined using p-nitrophenyl- β -D-xyloside (pNPX) as a substrate by adding 20. mu.l of pNPX to 170. mu.l of 100mmol/L potassium phosphate buffer pH6.5 to a final concentration of 1mmol/L to 10mmol/L, adding 10. mu.L of enzyme solution, reacting at 65 ℃ for 5min, and rapidly adding 600. mu.L of 1M Na₂CO₃The reaction was terminated. Absorbance values were determined at 405nm by interrogating p-nitrophenol (pNP) with A₄₀₅The amount of pNP catalytically produced was obtained from the standard curve between.

The beta-xylosidase activity unit is defined as the amount of enzyme required for (U)1min to catalytically release 1. mu. mol pNP.

The enzyme kinetic parameters of ThXylC and ThXylC-ELK16 calculated according to the Michaelis equation are shown in Table 3.

TABLE 3 kinetic parameters of different expression forms of beta-xylosidase

Example 5

Characterization of enzymatic Properties of beta-xylosidase

1. Study of optimum reaction pH

pH 4.0-5.5: prepared from 100mmol/L acetic acid-sodium acetate buffer solution.

pH 5.5-8.0: prepared from 100mmol/L potassium phosphate buffer.

Purified ThXylC and ThXylC-ELK16 enzyme activities were measured in different pH buffers, respectively. The results are shown in FIG. 3, where the respective enzyme activities at pH6.5 were 100%. Both ThXylC and ThXylC-ELK16 had optimum reaction pH of 6.5, and ThXylC-ELK16 had higher catalytic activity than ThXylC under extreme pH conditions.

2. Study of optimum reaction temperature

The enzyme activities of purified ThXylC and ThXylC-ELK16 were determined at different temperatures. The results are shown in FIG. 4, in which the optimum reaction temperature for ThXylC is 65 ℃ and the optimum reaction temperature for ThXylC-ELK16 is increased to 70 ℃ based on the highest catalytic enzyme activity of 100%. And in a high-temperature region (70-80 ℃), the ThXylC-ELK16 has obviously higher catalytic enzyme activity compared with ThXylC.

3. Investigation of temperature stability

The purified ThXylC and ThXylC-ELK16 were incubated at 65 ℃ and 70 ℃ for different periods of time, respectively, to determine the residual enzyme activity, and the results are shown in FIG. 5, where the initial enzyme activity was 100%. The enzyme activity retention rate of ThXylC is about 27.5% after the ThXylC is insulated for 48 hours at the temperature of 65 ℃, and the enzyme activity is almost completely lost after the ThXylC is insulated for about 6 hours at the temperature of 70 ℃. The active aggregation particles ThXylC-ELK16 of the beta-xylosidase are insulated for 48 hours at 65 ℃, the enzyme activity retention rate still reaches 55%, and the residual enzyme activity still remains 27.8% after the particles are insulated for 48 hours at 70 ℃.

As can be seen from the above experimental results, β -xylosidase activity aggregate particles (ThxylC-ELK16) obtained by adding amphiphilic short peptide ELK16 to the C-terminal of β -xylosidase (ThxylC) have catalytic efficiency (specific enzyme activity, K16)_cat/K_m) The heat tolerance (the most suitable catalytic reaction temperature and the heat stability) is obviously improved, and an application basis is provided for further industrial popularization and application.

Claims

1. A preparation method of beta-xylosidase enzyme aggregate is characterized by comprising the following steps:

(1) splicing the connecting peptide and the amphiphilic short peptide to construct an expression vector; the amphiphilic short peptide is ELK 16; the nucleotide sequence of the connecting peptide spliced with the amphiphilic short peptide is shown as SEQ ID NO. 1;

(2) connecting the beta-xylosidase coding gene with the expression vector, and converting the beta-xylosidase coding gene into receptor bacteria to obtain engineering bacteria capable of expressing the beta-xylosidase-short peptide fusion protein; the beta-xylosidase gene is selected from Thermoanaerobacterium anoteraense SCUT27, and the accession number is GenBank: KX 372717;

(3) and (3) performing induced expression on the engineering bacteria, performing wall breaking treatment on cells, centrifuging and/or filtering to obtain a precipitate, namely the beta-xylosidase endoenzyme aggregate.

2. The method of claim 1, wherein the recipient bacteria include, but are not limited to, e.coli dh5 α, e.coli jm109, e.coli jm110, e.coli top10, e.coli bl21, e.coli BL21(DE3), e.coli BL21(DE3)/pLysS, e.coli BL21Rosetta, e.coli BL21Rosetta (DE 3).

3. The method according to claim 1 or 2, wherein the precipitate of step (3) is purified by washing with a buffer.

4. A β -xylosidase endoenzyme aggregate prepared according to the method of any one of claims 1 to 3.