CN109456954B

CN109456954B - Beta-glucosidase mutant with improved thermal stability and application thereof

Info

Publication number: CN109456954B
Application number: CN201811393398.4A
Authority: CN
Inventors: 刘玉焕; 曹立创; 黄欣; 李水凤; 覃宗敏; 孔伟
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2018-11-21
Filing date: 2018-11-21
Publication date: 2021-08-20
Anticipated expiration: 2038-11-21
Also published as: CN109456954A

Abstract

The invention discloses a beta-glucosidase mutant with improved thermal stability and application thereof. The invention provides a beta-glucosidase mutant with improved thermostability, which is obtained by mutation on the basis of a wild beta-glucosidase amino acid sequence, wherein the mutation site is T167I/A298G or T167I/V181F/A187E/A298G, and the amino acid sequences of the obtained mutants 1R2 and 3R1 are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2. The half-life of the beta-glucosidase mutants 1R2 and 3R1 at 50 ℃ is 15 times and 4800 times of that of the wild enzyme Ks5A7 respectively, the hydrolysis activity to cellobiose is 1.8 times and 1.6 times of that of the wild enzyme respectively, and high tolerance to glucose is still maintained. Therefore, the beta-glucosidase mutant can greatly improve the thermal stability of the beta-glucosidase and provides an application basis for the fields of food, biological energy, textile, paper making and medicine.

Description

Beta-glucosidase mutant with improved thermal stability and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a beta-glucosidase mutant with improved thermal stability and application thereof.

Background

Cellulose is the most widely distributed and abundant renewable resource on earth, and cellulose produced by photosynthesis worldwide every year is as much as 10¹¹-10¹²Ton. The degradation product glucose of cellulose is a raw material of bioethanol which can replace energy. Therefore, efficient degradation of cellulose is crucial to the development of alternative energy sources. Cellulases are a general term for a family of multicomponent enzyme systems that act synergistically to break down cellulose to yield cellobiose and cellooligosaccharides, which are ultimately hydrolyzed to glucose. Wherein the beta-glucosidase can hydrolyze cellobiose and cellooligosaccharide to generate glucose.

Beta-glucosidase (EC 3.2.1.21), known as beta-D-glucopyranoside hydrolase, is an enzyme that hydrolyzes the non-reducing terminal glycosidic bond of saccharides and glycosidic compounds, releasing the corresponding aglycone and glucose. Such enzymes are widely distributed in archaea, bacteria and eukaryotes and perform various biological functions, such as biomass conversion in microorganisms and animals, glycolipid and glycoside cleavage, lignification of plants, and oligosaccharide metabolism of cell walls, etc. The beta-glucosidase is widely applied to various fields of food, biological energy, textile, paper making, medicine and the like, and is used for degrading cellulose; hydrolyzing flavor precursors in fruit juice and fruit wine, releasing flavor active substances, and improving the flavor and quality of the beverage; hydrolyzing isoflavone aglycone to increase isoflavone content in food and feed; plays a great role in synthesizing oligosaccharide, nonionic surfactant alkyl glycoside and the like.

To further drive the industrial application of β -glucosidase, higher requirements are put on its existing performance, such as stable activity over a long period of time, tolerance to higher concentrations of glucose, high activity in extreme environments (extreme temperature or pH, etc.) or acceptance of different substrates (including non-natural substrates). Wherein, the thermal stability of the enzyme is very important for industrial application, the reaction speed of the enzyme is faster under the high temperature condition, the reaction period can be shortened, the space-time yield is improved, the cost is saved, and the pollution by other microorganisms in the reaction process can be avoided. The industrial reaction temperature for cellulose degradation is 50 ℃, while the thermal stability of the wild beta-glucosidase is poor, and the half-life period at 50 ℃ is only 1min, which is not beneficial to the application of the beta-glucosidase in industry. Therefore, research and exploration for improving the stability of the beta-glucosidase are important.

Disclosure of Invention

The invention aims to solve the technical problem of poor thermal stability of the existing beta-glucosidase, adopts a mutation mode, provides a beta-glucosidase mutant which is remarkably improved in thermal stability, remarkably improved in hydrolysis activity to cellobiose and still kept high tolerance to glucose.

It is a first object of the present invention to provide beta-glucosidase mutants with improved thermostability.

It is a second object of the present invention to provide a gene encoding the above mutant.

The third purpose of the invention is to provide a recombinant vector, a recombinant cell or a recombinant engineering bacterium carrying the mutant gene.

The fourth purpose of the invention is to provide a method for improving the thermal stability of beta-glucosidase.

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

the invention provides a beta-glucosidase mutant with improved heat stability, and the amino acid sequence of the mutant is shown in SEQ ID NO. 1.

Further, a beta-glucosidase mutant with improved thermal stability is provided, and the amino acid sequence of the mutant is shown as SEQ ID NO. 2.

The research of the invention finds that the thermal stability of the beta-glucosidase can be obviously improved by carrying out T167I/A298G site mutation on the amino acid sequence of the wild beta-glucosidase, and further, the thermal stability of the beta-glucosidase is improved more remarkably after two mutation sites V181F/A187E are added. The invention provides two beta-glucosidase mutants with improved thermal stability, the amino acid sequences of the beta-glucosidase mutants are respectively shown as SEQ ID NO.1 and 2, the beta-glucosidase mutants are obtained by mutation on the basis of the amino acid sequence of the wild beta-glucosidase, and the SEQ ID NO.1 comprises two mutation sites: T167I/A298G, SEQ ID NO.2 contains 4 mutation sites: T167I/V181F/A187E/A298G.

The invention also provides a beta-glucosidase mutant gene with improved thermal stability, which codes a mutant shown in SEQ ID NO. 1; specifically shown as SEQ ID NO. 3.

The invention also provides another beta-glucosidase mutant gene with improved thermal stability, which codes a mutant shown in SEQ ID NO. 2; specifically shown as SEQ ID NO. 4.

The wild-type beta-glucosidase Ks5A7 encoding gene is as shown in NCBI accession number: HV 348683.

The invention also provides a recombinant vector, a recombinant cell or a recombinant engineering bacterium carrying the mutant gene.

The invention also provides a method for improving the thermal stability of the beta-glucosidase, which is to perform the following mutation on the amino acid of the beta-glucosidase: T167I/A298G, namely the beta-glucosidase mutant 1R2 with remarkably improved thermostability is obtained.

Further, amino acids of β -glucosidase were mutated as follows: T167I/V181F/A187E/A298G, namely the beta-glucosidase mutant 3R1 with more remarkably improved thermal stability is obtained.

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

the invention provides beta-glucosidase mutants with improved thermal stability, the obtained beta-glucosidase mutants 1R2 and 3R1 respectively have 15 times and 4800 times of half-life period at 50 ℃ of wild enzyme Ks5A7, the hydrolysis activity to cellobiose is respectively 1.8 times and 1.6 times of that to wild enzyme, and high tolerance to glucose is still maintained. Therefore, the beta-glucosidase mutant with improved thermal stability is more suitable for industrial application and has wide application prospect in the fields of food, biological energy, textile, paper making and medicine.

Drawings

FIG. 1 is a line graph showing the thermostability of a mutant β -glucosidase and a wild-type enzyme at 50 ℃.

FIG. 2 is a line graph showing the effect of substrate concentration on the activity of a mutant β -glucosidase and the activity of wild-type enzyme when cellobiose is used as the substrate.

FIG. 3 is a line graph of the effect of glucose concentration on the activity of beta-glucosidase mutant and wild-type enzymes.

Detailed Description

The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 construction of a library of random mutations of beta-glucosidase

1. Using pET 28a (+) -tac plasmid (pET 28a (+) -tac-Ks5A7) carrying wild type beta-glucosidase gene as template, adopting low mutation rate (3mutations/kb) to carry out error-prone PCR (epPCR) on the gene, referring to

II Random Mutagenesis kit instructions for use.

Two rounds of mutation were performed in sequence:

the template for the first round of mutation was pET 28a (+) -tac-Ks5A7,

the second round mutation template was the plasmid pET 28a (+) -tac-1R2 obtained after the first round of mutation.

2. Specifically, the primers for the epPCR are shown in Table 1, and the reaction system for the epPCR is shown in Table 2:

TABLE 1 primers for constructing random mutation library of beta-glucosidase (primers for epPCR)

TABLE 2 EpPCR reaction System for constructing random mutation library of beta-glucosidase

The conditions for the epPCR reaction were: the first stage is as follows: pre-denaturation at 95 ℃ for 2 min; and a second stage: denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 1.5min for 30 cycles; and a third stage: extension at 72 ℃ for 10 min.

After the completion of the epPCR reaction, 5. mu.L of the epPCR reaction product was subjected to gel electrophoresis analysis and used

And Gel Extraction Kit is used for carrying out Gel recovery and purification on the epPCR product. The epPCR product after purification and the vector pET 28a (+) -tac were subjected to double digestion with Nde I and Xho I respectively under the conditions: the enzyme was cleaved at 37 ℃ for 1h, and the cleavage system is shown in Table 3:

TABLE 3 double digestion System for epPCR products and vectors

Purifying the enzyme digestion product obtained in the above step, wherein the epPCR fragment and the pET 28a (+) -tac vector are mixed in a molar ratio of 4: 1, mixing, and connecting for 1h in a constant-temperature water bath at 22 ℃, wherein the connecting system is shown in a table 4:

TABLE 4 ligation of the cleavage products and the vector

The ligation product obtained above was used by OMEGA

The MicroElute DNA Clean-Up Kit is used for recovery. mu.L of the ligation product was mixed with 100. mu.L of E.coli DH 5. alpha. electroporation competent cells, and placed in a sterile electric shock cup (0.2cm) and subjected to electric shock at a voltage of 2500V (for about 5ms), followed by addition of 900. mu.L of SOC medium, and the transformant was cultured with shaking at 37 ℃ and 180rpm for 1 hour. The culture was spread evenly on LB medium plates (supplemented with 50. mu.g/mL kanamycin and 20. mu.M IPTG), and incubated overnight in a 37 ℃ incubator to obtain a random mutation library. To evaluate the quality of the mutant library, 25 clones were randomly picked, incubated overnight at 37 ℃ and 200rpm, plasmids were extracted and sequenced, and the mutation rate was calculated.

Example 2 screening of a library of random mutations in beta-glucosidase

1. The library of mutations obtained in example 1 was spread on LB medium plates (supplemented with 50. mu.g/mL kanamycin and 20. mu.M IPTG), incubated overnight in a 37 ℃ incubator, and then incubated at room temperature for 1d to ensure sufficient expression of. beta. -glucosidase. Colonies on the plates were treated at high temperature to kill cells and inactivate beta-glucosidase without increased thermostability (first round mutation: 60 ℃ treatment for 40 min; second round mutation: 75 ℃ treatment for 40 min). The screening indicator was a 0.1M potassium phosphate solution of pH 6.0 containing 1g/L esculin, 2.5g/L ferric ammonium citrate and 5g/L agar, 10mL of the screening indicator was carefully poured onto the heat-treated colonies, and after incubation at room temperature for 10min, positive colonies were identified based on the formation of a black halo around the colonies. The positive colonies embedded in agar were removed with sterile toothpicks and used by the company OMEGA

Plasmid was extracted from the Plasmid Mini Kit, transformed into E.coli DH5 alpha for Plasmid amplification, purification and DNA sequencing.

2. From the wild enzyme Ks5A7, 2 mutants were obtained by the two rounds of selection: 1R2(T167I/A298G) and 3R1 (T167I/V181F/A187E/A298G).

Example 3 expression and purification of beta-glucosidase mutants

1. Expression of beta-glucosidase mutants

The plasmid for sequencing-confirmed mutation in example 2 was transformed into E.coli BL21(DE3), and a single colony was cultured overnight in LB liquid medium at 37 ℃ and 200 rpm. According to the following steps: 100, then transferred to LB liquid medium, and cultured with shaking at 37 ℃ and 200rpm until the cell density OD_600nmAt 0.85, IPTG was added to a final concentration of 0.8 mM. Culturing at 25 deg.C and 200rpm for 12 hr, centrifuging at 10000 Xg for 5min, and collecting thallus.

2. Purification of beta-glucosidase mutants

The beta-glucosidase mutant contains a C-terminal 6 XHis tag and is produced by Novagen

The Purification of the Purification Kit comprises the following specific steps:

(1) taking 100mL of induction culture, centrifuging, removing supernatant, adding 15mL of precooled 1 × Binding Buffer, mixing uniformly, crushing thalli by using ultrasonic waves, centrifuging for 8min at 10000 × g, and collecting supernatant;

(2) putting 4 mLHis-Bind resin in a filter column to form a purification column;

(3) washing the purification column with 10mL of deionized water, 15mL of 1 × Charge Buffer and 10mL of 1 × Binding Buffer in sequence;

(4) slowly adding the crushed thallus supernatant into a purification column along the tube wall, and naturally passing through the purification column under the action of gravity;

(5) washing the purification column by using 20mL of 1 multiplied by Binding Buffer and 12mL of 1 multiplied by Wash Buffer in sequence;

(6) eluting with 6mL of 1 × Elute Buffer, and collecting eluates in batches;

(7) detecting the protein purity in the eluate by 12% SDS-PAGE, and selecting and combining the batches with the purification of more than 95%;

(8) the target protein was replaced with 100mM potassium phosphate buffer pH 6.0 using an ultrafiltration tube having a Millipore molecular weight cut-off of 10kDa, and stored at 4 ℃ until use.

Example 4 thermostability, Cellobiose hydrolytic Activity and glucose tolerance of beta-glucosidase mutants

1. Determination of thermal stability of beta-glucosidase mutant at 50 deg.C

10U of wild enzyme Ks5A7 and its mutant were incubated at 50 deg.C, samples were taken at regular intervals, residual enzyme activity was determined using 5mM pNPG as substrate, and the reaction was carried out at pH 6.0(0.1M potassium phosphate) and 50 deg.C.

The results of measurement of thermostability at 50 ℃ of the β -glucosidase mutants are shown in table 5 and fig. 1, and it can be seen that the half-lives (t) of the β -glucosidase mutants 1R2 and 3R1 are shown (t)_1/2) The protein is respectively increased to 15min and 4800min from 1min before mutation, which are respectively 15 times and 4800 times of the wild enzyme Ks5A 7.

TABLE 5 enzymological Properties of beta-glucosidase mutants

2. Measurement of Cellobiose hydrolysis Activity of beta-glucosidase mutant

The cellobiose hydrolysis activity of the wild enzyme Ks5A7 and the beta-glucosidase mutants 1R2 and 3R1 is measured by taking 1% (w/v) cellobiose as a substrate under the conditions of pH 6.0 and 50 ℃, and the enzyme activity is measured by the following method:

the concentration of the purified β -glucosidase was measured using a protein quantification kit (Bradford method) with Bovine Serum Albumin (BSA) as a control.

The enzyme activity determination principle is as follows: the p-nitrophenol-beta-glucoside (pNPG) is colorless when dissolved in water, the glycosidic bond in the p-nitrophenol-beta-glucoside (pNPG) is hydrolyzed to generate p-nitrophenol (pNP), the maximum absorption peak is at 400-420 nm, and the absorbance is in direct proportion to the concentration of the pNP. Hydrolysis of the glycosidic bond of cellobiose produces two molecules of glucose, which in turn under the action of Glucose Oxidase (GOD) produces gluconic acid and hydrogen peroxide (H)₂O₂)，H₂O₂Reacting with 4-amino-pyriline (4-AAP) and phenol under the catalysis of Peroxidase (POD) to generate red quinoneimine. Quinoimines have a maximum absorption peak near 500nm, and the absorbance is proportional to the glucose concentration.

The enzyme activity determination method comprises the following steps: when pNPG was used as a substrate, the reaction system was 10. mu.L of the enzyme solution and 490. mu.L of a 5mM substrate solution (0.1M potassium phosphate solution, pH 6.0), mixed well, incubated at 50 ℃ for 10min, and 500. mu.L of 10% (w/v) Na was added₂CO₃The reaction was terminated. And taking out 300 mu L of reaction liquid, adding the reaction liquid into an enzyme label plate, measuring the light absorption value at 405nm, and calculating the enzyme activity unit according to a corresponding standard curve. Three replicates of each group were run with inactivated enzyme solution as blank control. When cellobiose is used as a substrate, the reaction system is 50. mu.L of the enzyme solution and 450. mu.L of 1% (w/v) cellobiose solution, and the reaction is terminated by uniformly mixing, incubating at 50 ℃ for 10min, and then incubating at 90 ℃ for 5 min. After cooling to room temperature, 10. mu.L of the reaction mixture was taken out and added to 750. mu.L of the reaction mixture of the glucose oxidation kit, followed by incubation at 37 ℃ for 15 min. After the reaction, 300. mu.L of the enzyme was taken out and added to an ELISA plate, and the absorbance was measured at 492nm, and the enzyme activity was calculated from the glucose standard curve. Three replicates of each group were run with inactivated enzyme solution as blank control.

The enzyme activity unit (1U) is defined as: the amount of enzyme required to catalyze the production of 1. mu. mol pNP or glucose in 1min under standard reaction conditions.

Drawing a p-nitrophenol (pNP) standard curve: a1.0 mM pNP stock solution was prepared using potassium phosphate buffer (0.1M, pH 6.0). As shown in Table 2, different volumes of pNP mother liquor and potassium phosphate buffer were added, mixed well, and 300. mu.L of 10% (w/v) Na was added₂CO₃. Measuring light absorption value (OD) at 405nm on an enzyme label plate with 300 mu L₄₀₅) Each reaction was performed 3 times in parallel and plotted, the formula of which is: 19.185x-1.6296, R²＝0.9995。

TABLE 6pNP Standard Curve Generation

Drawing a glucose standard curve: a glucose solution stock solution having a concentration of 2.5g/L was prepared from potassium phosphate buffer (0.1M, pH 6.0), and was diluted to glucose solutions having different concentrations as shown in Table 3. Adding 10 μ L diluted glucose solution into 750 μ L glucose kit test solution, respectively, mixing, incubating at 37 deg.C for 15min, and measuring its light absorption value (OD) at 492nm with 300 μ L_492nm) And drawing a curve, wherein the formula is as follows: 127.35x-0.9915, R²＝0.9975。

TABLE 7 plotting of glucose Standard Curve

As shown in Table 5, it can be seen that the specific enzyme activity of the wild enzyme Ks5A7 is 243.18U/mg, the specific enzyme activity of the mutant 1R2 is 437.32U/mg, and the specific enzyme activity of 3R1 is 399.68U/mg. Thus, the specific enzyme activities of the beta-glucosidase mutants 1R2 and 3R1 were 1.8 and 1.6 times higher than that of the wild enzyme Ks5A7, respectively, when 1% (w/v) cellobiose was used as the substrate.

3. Effect of Cellobiose concentration on beta-glucosidase mutants

The activity of the wild enzyme Ks5A7 and the activity of the beta-glucosidase mutants 1R2 and 3R1 are determined by taking cellobiose with different concentrations (0.1-5%, w/v) as a substrate, and the specific enzyme activity is calculated.

As shown in FIG. 2, it can be seen that, in accordance with the wild enzyme Ks5A7, the activities of the β -glucosidase mutants 1R2 and 3R1 increased with the increase in cellobiose concentration, and as high as 5% (w/v) cellobiose had no inhibitory effect on the activity of the β -glucosidase mutant.

4. Determination of glucose tolerance of beta-glucosidase mutants

Since the activity of Ks5A7 is activated by glucose, half maximal Inhibitory Concentration (IC) was chosen₅₀) Surrogate inhibition constant (K)_i) The tolerance capacity is measured and defined as 50 percent inhibitionGlucose concentration required for enzyme activity. The effect of different concentrations of glucose on the activity of the β -glucosidase Ks5A7 and its mutants was determined using 5mM pNPG as substrate and the reaction was performed at pH 6.0(0.1M potassium phosphate) and 50 ℃. The enzyme activity without glucose was set as 100% as a control.

As can be seen from the results in Table 5 and FIG. 3, the glucose tolerance of the β -glucosidase mutants 1R2 and 3R1 is not much different from that of the wild-type enzyme Ks5A 7.

The above detailed description is of the preferred embodiment for the convenience of understanding the present invention, but the present invention is not limited to the above embodiment, that is, it is not intended that the present invention necessarily depends on the above embodiment for implementation. It will be appreciated by those skilled in the art that any modifications to the invention, equivalent alterations to the materials selected for use in the invention, and the addition of additional components, selection of specific means, etc., are intended to be within the scope and disclosure of the invention.

Sequence listing

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atgaaattta atgaaaattt tgtttggggt gttgccacga gtagctatca gatcgaaggt 60

gcctataatg aagatggtcg cggtaaagat atttggggcg attattgtag cattccaggc 120

atcatctttg ataatcataa tggcgataaa gcgtgcgaac attattatcg ctataaagaa 180

gatgttgcca tcatgaaaga aatgggcatc aaagcgtatc gctttagtat tgcgtgggcc 240

cgcatctttc cggaaggcat cggcaaaatc aatcagaaag gtgtggattt ttatcataat 300

ttaatcgatg aactgatcaa aaatgatatt gttccgtatg tgaccctgtt tcattgggat 360

ttaccactgg aactggccga aaaaggcggc tggctgaatg atgattcagt ggaatggttt 420

aaagattatg cggaattttt tggtaaagaa tatggccata aaatcaaata tatcatgact 480

tttaatgaac cacagtgcat catcggtctg ggtttacagc agggcatcca tgcaccgggc 540

tttaaactgt ctcctaaaga agtgctgaaa tctacacata atttactgaa agctcacggc 600

gccgccgtta aagtgctgcg caaagttgct ccgaataccc agttaggcat tgctccgacg 660

tgcggcgttg ccttaccgat ctcagaaaat aaaaaagaca ttgaaattgc acgcaaacgc 720

tattttgata ttctggatct gaatgatgcg tatgtgtgga gcgtgagcct gtttttggac 780

ccaatcgtgt taggcgatta tccaaccaaa tattatgaac tgtataaaga acatttacct 840

aaaattacac aggaggacct gaaactgatc tcacagccgt tagattttct gggccagaat 900

atctataatg gctatcgtgt gagcgaagat gaaaatggca attatgtgta tcctaaacgc 960

aaagcaggtt atgatcatac ggatatgggt tggccaatta caccgtcagc cctgtattgg 1020

ggtcctcgct ttatctgcga acgctataat ctgccgtttt atattacgga aaatggctta 1080

gcctgtcatg atgttgtgag cttagataat aaagttcatg atcctaatcg catcgatttt 1140

ctgaataaat atctgctgga ttatagtcgc gcctcttgcg aaggttatga tattcgcggc 1200

tattttcagt ggtcactgat ggataatttt gaatggcgcg aaggctatag caaacgcttt 1260

ggtatggtgt atgtggattt tgaaacacag aaacgtacaa tcaaagatag cggttattgg 1320

tataaaaaag tgatcgaaga aaatggtgaa aatctg 1356

<210> 5

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

catatgaaat ttaatgaaaa ttttgtttgg gg 32

<210> 6

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ctcgagcaga ttttcaccat tttcttcgat cac 33

Claims

1. The beta-glucosidase mutant with improved heat stability is characterized in that the amino acid sequence of the mutant is shown as SEQ ID NO. 1.

2. The beta-glucosidase mutant with improved heat stability is characterized in that the amino acid sequence of the mutant is shown as SEQ ID NO. 2.

3. A mutant β -glucosidase gene with improved thermostability, which encodes the mutant of claim 1.

4. The mutant gene of claim 3, wherein the nucleotide sequence is represented by SEQ ID No. 3.

5. A mutant β -glucosidase gene with improved thermostability, which encodes the mutant according to claim 2.

6. The mutant gene of claim 5, wherein the nucleotide sequence is represented by SEQ ID No. 4.

7. A recombinant vector, a recombinant cell or a recombinant engineered bacterium carrying the mutant gene of any one of claims 3 to 6.