CN109609529B - Preparation method of oligomerization feruloyl esterase based on foldon mediation - Google Patents
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Abstract
The invention discloses a preparation method of oligomerization feruloyl esterase based on foldon mediation, which designs, optimizes and synthesizes a gene fragment His-fae-linker-foldon; and (3) carrying out induction culture on the pPIC9K/His-fae-linker-foldon engineering bacteria, and separating and purifying to obtain the oligomerization feruloyl esterase. The invention applies the characteristic that the fusion of the target protein and the foldon structural domain can spontaneously form a tripolymer structure, and designs a linker to finish the fusion expression of the ferulic acid esterase in pichia pastoris so as to improve the catalytic performance of the ferulic acid esterase; adding de-starch wheat bran into a culture medium in the expression culture process of pPIC9K/His-fae-linker-foldon engineering bacteria to improve the expression quantity of ferulic acid esterase; the oligomerization ferulic acid esterase is stored in a special compound stabilizer, and the enzyme activity stability is greatly improved.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a preparation method of oligomerization FAE based on foldon mediation.
Background
In the plant cell wall, phenolic acid substances (such as ferulic acid, caffeic acid, p-coumaric acid, etc.) are connected with arabinosyl or galactosyl of polysaccharide (such as arabinoxylan, fructose) through ester bond, or are connected with lignin through ether bond to form a dense network structure. Feruloyl esterases (FAE. c 3.1.1.73), a subset of carboxylesterases, hydrolyze the ester bonds connecting ferulic acid to polysaccharides in plant cell walls, releasing ferulic acid. The FAE can cooperate with lignocellulose degrading enzymes, such as xylanase, cellulase and ligninase, to destroy the dense net structure of the lignocellulose and promote the degradation of the lignocellulose. However, the catalytic performance of FAE reported at present needs to be improved, and modification of enzyme is needed, for example, the ferulic acid esterase O42807 disclosed in the university report of Huaqiao (natural edition) (2016.37(2): p.224-229) of Chenyun Hua, etc. is expressed in the GS115 of Pichia pastoris, and the Aspergillus niger FAE gene codon optimization and high-efficiency expression in the Pichia pastoris, disclosed in the microbiological report (2017.44(5): p.1065-1073) of plum soldier, etc. are also needed. The invention patent with application number 201410724759.4 discloses a preparation method of recombinant FAE, which comprises the steps of utilizing ferulic acid esterase O42807, artificially synthesizing a gene FAE thereof after codon optimization, constructing a pAO815/FAE expression vector, constructing a pPIC9K/FAE expression vector and converting the pPIC9K/FAE expression vector into pichia pastoris GS115 for expression to obtain the recombinant FAE, wherein SDS-PAGE analysis shows that the fermentation supernatant is a single strip, the apparent molecular weight is 42kDa, the enzyme activity is 4.7U/mL, and the specific enzyme activity is 31.4U/mg. The recombinant FAE obtained by the invention has improved expression level compared with FAE O42807, but the expression level is still very low. Protein engineering techniques typified by site-directed mutagenesis and directed evolution have successfully screened for enzymes with optimized properties, but these methods still have difficulties in engineering enzymes, such as the establishment of mutant libraries and extensive screening efforts. The performance of the enzyme can be improved by immobilization or chemical modification, but the defects that the enzyme is inactivated in the immobilization process, the first immobilization cost is high, the reaction with a macromolecular substrate is difficult and the like exist.
The structure of a protein determines its function, and proteins can be engineered by protein engineering to obtain new functions of the protein. Oligomerization is a common way in which many proteins associate themselves into oligomers to gain a functional advantage. Oligomerization can provide various functional advantages for target enzymes, such as improved thermostability, pH tolerance, protein molecular structure stability, catalytic performance, and the like. foldon is a small 27-residue (GYIPEAPRDGQAYVRKDGEWVLLSTFL, shown in SEQ ID No. 2) β -propeller-like trimer consisting of a monomeric β -hairpin fragment, originally identified at the C-terminus of bacteriophage T4 fibrin, which can be artificially linked to a target enzyme to alter its properties by genetic fusion. Oligomers induced by COMP and foldon generally lead to an increase in thermostability. Can, etc. (Can,
a type III anti-freeze protein primer exhibits associated with amplified thermal stability biochemistry,2013.52(48): p.8745-8752.) the foldon is linked with the anti-freeze protein to form the oligomerized homo-anti-freeze protein and increase the concentration of the anti-freeze protein bound on the surface of the ice crystal, so that the activity of the antifreeze protein is obviously increased. Wang et al (Wang X, Ge H, Zhang D, et al. Oligomerization triggered by fold: a simple method to enhance the catalytic effectiveness of a lipase and an xylanase [ J]BMC Biotechnology,2017.17(1):57: p112-120) completed intracellular expression of foldon-ELPs-lichenase/xylanase in E.coli, k of the enzymecat/KmThe FAE with large molecular weight and relatively complex structure is expressed in colon bacillus by the applicant after being respectively improved by 4.2 times and 3.0 times, but the enzyme activity is low, an inclusion body is formed, and the application prospect is not good.
Disclosure of Invention
The following presents a simplified summary of embodiments of the invention in order to provide a basic understanding of some aspects of the invention. It should be understood that the following summary is not an exhaustive overview of the invention. It is not intended to determine the key or critical elements of the present invention, nor is it intended to limit the scope of the present invention. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is discussed later.
According to one aspect of the present invention, there is provided a method for preparing oligomerization feruloyl esterase based on foldon mediation, comprising: designing a linker sequence, optimizing a gene sequence His-fae-linker-foldon, adding SnaB I and Not I enzyme cutting sites at two ends respectively, and artificially synthesizing a gene fragment His-fae-linker-foldon; carrying out double digestion on pPIC9K plasmid and His-fae-linker-foldon by SnaB I and Not I, connecting the plasmid pPIC9K with gene fragment His-fae-linker-foldon by T4 ligase, transforming into E.coli DH5 alpha competent cells, screening pPIC9K/His-fae-linker-foldon plasmid positive clones and electrically transforming into P.pastoris GS115 (Pichia pastoris) for expression to obtain pPIC9K/His-fae-linker-foldon engineering bacteria; the pPIC9K/His-fae-linker-foldon engineering bacteria are induced to express in a culture medium, and oligomerized ferulic acid esterase is obtained by separation and purification.
Specifically, the construction of the pPIC9K/His-fae-linker-foldon engineering bacteria specifically comprises the following processes:
the method comprises the steps of designing a linker with an amino acid sequence shown as SEQ ID No.1, screening FAEO42807 with high enzyme activity in a UniProt database, optimizing a gene sequence His-fae-linker-foldon according to a nucleotide sequence, a His, a linker and a foldon gene sequence corresponding to the amino acid sequence of the FAEO42807 and codon preference use frequency of pichia pastoris, adding SnaB I and Not I enzyme cutting sites at two ends respectively, and artificially synthesizing a gene segment His-fae-linker-foldon. The pPIC9K plasmid and His-fae-linker-foldon are subjected to double digestion by SnaB I and Not I, after double digestion fragments are recovered by gel, the plasmid pPIC9K and the gene fragment His-fae-linker-foldon are connected by T4 ligase and transformed into E.coli DH5 alpha competent cells, positive clones are screened out for Sangon sequencing, and the recombinant plasmid with correct sequencing is named as pPIC9K/His-fae-linker-foldon plasmid.
Transformation of pPIC9K/His-fae-linker-foldon plasmid into Pichia pastoris specifically included: digesting pPIC9K/His-FAE-linker-foldon with SacI, transforming the linearized fragment into P.pastoris GS115 competence by an electric transformation method, coating the competent fragment on an MD plate to screen recombinants, taking a colony growing well on the MD plate, dibbling the colony to an YPD shake flask by using toothpicks, culturing for 16-18 h, extracting genome DNA, and performing PCR identification by using universal primers 5 'AOX and 3' AOX to obtain pPIC9K/His-FAE-foldon engineering bacteria for expressing FAE. The MD medium and YPD medium were prepared according to the manual of Pichia manipulation expression (Invitrogen, USA).
The pPIC9K/His-fae-foldon engineering bacteria are induced to express as follows: (1) inoculating the recombinant engineering bacteria into a seed culture medium. The culture conditions are as follows: culturing at 28-30 ℃ and 150-250 rpm for 16-18 h. The seed culture medium comprises the following components: BMGY medium. (2) Centrifuging the culture solution obtained in the step (1) at 2-4 ℃ and 3500-4500 rpm for 4-6 min, collecting cells, adding the cells into an induction culture medium, and culturing under the following conditions: culturing at 28-30 ℃ and 150-250 rpm for 96-100 hours. The induction medium comprises BMMY medium and 1-2% (mass percent) of de-starch wheat bran. The preparation method of the de-starched wheat bran comprises the following steps: drying fresh wheat bran at 105 deg.C for 4 hr, pulverizing, removing starch with amylase according to Oushaiyi method, oven drying sample, pulverizing, and sieving with 80 mesh sieve (Oushaiyi, Zhang-ning, in vitro experiment research on free radical scavenging of wheat bran enzymolysis product [ J ]. Nutrition, 2005,27(1): 25-29.). BMGY medium and BMMY medium were prepared according to the Pichia pastoris expression Manual (Invitrogen, USA).
The culture medium comprises an induction culture medium containing de-starch wheat bran. The principle of adding the de-starch wheat bran into the induction culture medium is as follows: the choice of culture substrate is critical to whether the microorganism is able to produce FAE. When glucose, xylose, lactose, maltose and xylitol are used as substrates, the microorganisms cannot produce FAE due to the repression of glucose catabolites. Mixed carbon sources containing large amounts of esterified ferulic acid, such as wheat bran, corn bran, brewer's grains, beet pulp and the like, are most suitable for use as substrates for the production of FAE ([1] Benoit I, Danchin E, Bleichrodt R, et al Biotechnical applications and potentials of functional groups based on the prediction, classification and biochemical sensitivity [ J ]. Biotechnical Lett,2008,30(3): slab 396). The yield of FAE is reduced by 81.8 percent compared with the untreated corncob which is used as a carbon source by using the corncob without the ferulic acid ester bond through the deep liquid culture of oxysporum; when free ferulic acid is added into the de-esterified corncob, the yield of FAE is only improved by 1.5 times, and the result shows that the ferulic acid ester bond in the wood fiber is a factor for inducing F.oxysporum to produce FAE (Topakas E, Christakopoulos P.Production and partial characterization of alkali cellulose esters by Fusarium oxysporum reduced synergistic bacterium batch culture [ J ]. World J Microbiol Biotechnol,2004,20(3):245 and 250). the FAE engineering bacteria reported in the literature are all selected from BMGY seed culture medium and MY induction culture medium, and the constructed FAE engineering bacteria have low activity. The invention combines theoretical guidance and experimental basis, selects the de-starch wheat bran to be added into the BMMY induction culture medium, and obviously improves the enzyme activity.
The process of separating and purifying the fusion protein expressed by the pichia pastoris comprises the following steps: centrifuging at the temperature of 2-4 ℃ and the rpm of 8000-12000 for 8-12 min to obtain supernatant, namely crude enzyme liquid, and purifying the crude enzyme liquid by using a Ni chromatographic column to obtain oligomerization FAE.
In addition, in the research process, the enzyme activity stability is closely related to a storage mode, and the ferulic acid esterase is not reported to be stored in a stabilizer in the prior art, so that the oligomerized FAE enzyme solution with good stability can be obtained by storing the obtained oligomerized ferulic acid esterase in a special composite stabilizer on the basis of a large amount of experimental and theoretical derivation. The composite stabilizer comprises the following components in percentage by mass: 4-6% of xylanase (not less than 20000U/mL), 2-4% of cellulase (not less than 10000U/mL) and 0.02-0.04% of MgSO4And 7-9% of PEG400, wherein the solvent is deionized water. Compared with the prior art which only stores in a low-temperature refrigerator, the enzyme activity of the method is greatly improved in stability.
The principle of the composite stabilizer for keeping enzyme activity is as follows: common enzyme solutions are stored at low temperature or added with common protective agents such as glycerol, PEG and the like, and do not aim at specific target enzymes. For plant cell walls with complex network structures, research results show that the plant cellulose cannot be effectively degraded by using FAE singly, and the degradation of the plant cellulose can be greatly improved only through the synergistic effect of the FAE and other cell wall degrading enzymes. FAEs secreted by A.niger are reported to release FA by themselves, but by the addition of xylanase, the FA release is increased by nearly 24-fold. Topakas et al reported that the feruloyl esterase StFaeC produced by B.thermophilus synergistically acts with xylanase to release 10 times as much FA from plant fiber as it would without xylanase. ([1]Faulds CB,Williamson G.Release of ferulic acid from wheat bran by a ferulic acid esterase(FAE-III)from Aspergillus niger[J].Appl Microbiol Biot,1995,43(6):1082-1087.[2]Topakas E,Vafiadi C,Stamatis H,et al.Sporotrichum thermophile type C feruloyl esterase(StFaeC):purification,characterization,and its use for phenolic acid(sugar)ester synthesis[J]Enzyme Microb Tech,2005,36(5-6): 729-. The invention supposes that the wood fiber degrading enzymes such as xylanase and the like are mate of FAE, and on the basis, the invention screens out the wood fiber degrading enzymes containing xylan through experimentsCarbohydrase, cellulase, MgSO4And PEG400 composite stabilizer, which can keep the activity of oligomerization FAE liquid enzyme stable. The storage stability of the enzyme is related to the existence form of the enzyme, most of the enzyme is relatively stable in a solid state, and liquid enzyme is easily polluted by microorganisms in the storage process, loses the enzyme activity and cannot be stored for a long time. In the actual industrial production process, the cost of freeze drying the enzyme is higher, and the addition of the composite stabilizer has low cost and easy operation. Therefore, the composite stabilizer is used for improving the storage stability of the liquid FAE, and has important significance for practical application of the liquid FAE.
According to the invention, a pPIC9K/His-FAE-linker-foldon plasmid is constructed based on foldon genes and a linker is designed by self and is transformed into Pichia pastoris GS115, the engineering bacteria is induced and expressed in a culture medium containing de-starch wheat bran, and oligomerization FAE with high catalytic efficiency is obtained by separation and purification. Storing oligomerized FAE in a medium containing xylanase, cellulase and MgSO4And PEG400 composite stabilizer, and can obtain oligomerization FAE enzyme solution with good stability. Compared with the prior art, the method has the following advantages:
1. a linker sequence is designed by self, extracellular expression of oligomerization FAE based on foldon in Pichia pastoris GS115 is realized, and the FAE is modified by applying the characteristic that a trimer can be formed spontaneously by fusing a target protein and a foldon structural domain, so that the catalytic performance of the FAE is improved.
2. According to the invention, the amylolytic wheat bran is creatively added into the pPIC9K/His-FAE-foldon engineering bacteria culture medium, so that the enzyme activity of FAE is improved, and compared with a culture medium without the amylolytic wheat bran, the enzyme activity of FAE is improved by 1.13 times.
3. The invention creatively stores the obtained oligomerization FAE in a composite stabilizer, and the formula comprises the following components: 5% of xylanase (more than or equal to 20000U/mL), 3% of cellulase (more than or equal to 10000U/mL) and MgSO4(0.03%) and PEG400 (8%). The oligomerization FAE obtained by purification is added into a composite stabilizer and stored at 4 ℃, the enzyme activity residual rate reaches 96.2 percent after 20 days, and compared with a control group which is only stored at low temperature and is not added with the stabilizer, the enzyme activity residual rate is improved by 1.12 times.
The oligomerization FAE based on foldon is expressed in Pichia pastoris GS115, and the engineering bacteria are inducedThe culture medium contains de-starch wheat bran in the expression process, so that the substrate affinity (K) of the oligomerized FAE with the enzyme activity of 8110U/mL, the specific enzyme activity of 76.2U/mg and the oligomerization FAE can be obtainedm) And catalytic efficiency (k)cat/Km) Compared with the monomer FAE of a control group, the monomer FAE is respectively improved by 5.42 times and 24.6 times; and the oligomerization FAE obtained by purification is stored in a composite stabilizer and placed at 4 ℃, and the enzyme activity residual rate reaches 96.2 percent after 20 days.
In conclusion, the method for improving the FAE catalytic efficiency is simple and efficient, does not need to know the 3D structure of the enzyme in detail in advance, and has good application prospect.
Drawings
The invention is further illustrated by the following figures and examples.
FIG. 1 shows the construction of pPIC9K/His-fae plasmid (A) and pPIC9K/His-fae-linker-foldon plasmid (B).
FIG. 2 is a diagram showing the results of double digestion (Not I, SnaB I) of the recombinant plasmid pPIC9K/His-fae with pPIC9K/His-fae-linker-foldon, in which: m: marker; 1: pPIC 9K/His-fae; 2: pPIC 9K/His-fae-linker-foldon.
FIG. 3(A) is a graph showing the results of the expression of the pPIC9K/His-fae plasmid, in which: m is protein marker; 1 is pPIC9K/His-FAE fermentation supernatant, mon-FAE in the figure is the product expressed by pPIC9K/His-FAE plasmid.
FIG. 3(B) is a graph showing the results of expression of the product from the pPIC9K/His-fae-linker-foldon plasmid, in which: m is protein marker; 1 is pPIC9K/His-FAE-linker-foldon fermentation supernatant and shown as oligo-FAE as expressed from pPIC9K/His-FAE-linker-foldon plasmid.
FIG. 4 is a graph showing the effect of de-amylosed wheat bran on the enzyme activity of olig-FAE in the induction medium.
FIG. 5 shows the optimum temperature of recombinant FAE.
FIG. 6 shows the temperature stability of recombinant FAE, (A) mon-FAE, (B) olig-FAE.
FIG. 7 is the optimum pH of recombinant FAE.
Fig. 8 is pH stability of recombinant FAE.
FIG. 9 is a TEM image of olig-FAE.
FIG. 10 is a graph of the effect of polyols on the thermal stability of olig-FAE.
FIG. 11 is a graph showing the effect of other enzymes on the thermostability of olig-FAE enzyme solutions.
FIG. 12 is MgSO4Influence on the thermostability of olig-FAE enzyme solutions.
Detailed Description
The detailed process of the preparation method of FAE based on foldon-mediated oligomerization according to the present invention will be described with reference to the accompanying drawings.
1.1 Main Material
1.1.1 strains and plasmid E.coli DH 5. alpha. purchased from Biotechnology engineering (Shanghai) Ltd; pastoris GS115, vector pPIC9K were purchased from Invitrogen, usa.
1.1.2 Main reagents and media: restriction enzymes (SnaB I, Not I, Sac I), T4 ligase (ThermoFisher, USA); plasmid extraction kit, glue recovery kit (bio-engineering (Shanghai) Co., Ltd.); a standard protein marker (10-170kD), an SDS-PAGE gel preparation kit and a Coomassie brilliant blue staining kit (Shanghai Biyuntian biotechnological research institute); ferulic acid methyl ester, PEG400, xylanase (more than or equal to 20000U/mL), cellulase (more than or equal to 10000U/mL) (Sigma company in America); other reagents are all domestic or imported analytical pure products. MD medium, YPD seed medium, BMGY seed medium, and BMMY induction medium were prepared according to the manual of Pichia manipulation expression (Invitrogen, USA). Transmission electron microscope (japan electronics corporation).
1.2 construction of expression vectors
FAEO42807 with higher enzyme activity is screened from a UniProt database, a gene sequence His-fae-linker-foldon is designed and optimized according to a nucleotide sequence corresponding to an amino acid sequence of the FAEO42807, a His, a linker and foldon gene sequence and codon preference use frequency of pichia pastoris, SnaB I enzyme cutting sites and Not I enzyme cutting sites are respectively designed at two ends, and a gene fragment His-fae-linker-foldon is artificially synthesized. The pPIC9K plasmid and His-fae-linker-foldon were double-digested with SnaB I and Not I, the double-digested fragments were recovered from the gel, plasmid pPIC9K and gene fragment His-fae-linker-foldon were ligated with T4 ligase and transformed into E.coli DH 5. alpha. competent cells, positive clones were selected and sequenced (by Shanghai Sangon Corp.), and the correctly sequenced recombinant plasmid was named pPIC9K/His-fae-linker-foldon plasmid. In the embodiment, the linker in the artificially synthesized gene segment His-fae-linker-foldon has 52 amino acid sequences (shown as SEQ ID No. 1):
GAGGAGGAGGAGGAGGSGGSGGSGGSGGSGGSGGSGGAGGAGGAGGAGGAGG。
1.3 expression and purification of recombinant FAE in Pichia pastoris
Digesting pPIC9K/His-fae-linker-foldon with SacI, transforming a linearized fragment into P.pastoris GS115 competence by using an electric transformation method, coating the competent fragment on an MD plate to screen recombinants, taking a colony growing well on the MD plate, dibbling the colony to an YPD shake flask by using a toothpick, culturing for 16-18 h, extracting genome DNA, and carrying out PCR identification and determination by using universal primers 5 'AOX and 3' AOX to obtain the pPIC9K/His-fae-linker-foldon engineering bacteria. In addition, pPIC9K/His-fae engineering bacteria were constructed in the same manner as a control group.
1.5 study of recombinant FAE Medium
(1) Inoculating the recombinant engineering bacteria into a seed culture medium. The culture conditions are as follows: culturing at 28 ℃ and 200rpm for 16-18 h. The seed culture medium comprises the following components: BMGY medium. (2) Centrifuging the culture solution after the step (1) at 4 ℃ and 4000rpm for 5min, collecting cells, adding the cells into an induction culture medium, and culturing under the following conditions: culturing at 28 ℃ and 200rpm for 96-100 hours. The induction culture medium comprises BMMY culture medium added with de-starch wheat bran with different concentrations. The influence of the starch-removed wheat bran on the activity of the FAE enzyme during the expression of the recombinant engineering bacteria is researched. BMGY seed medium and BMMY induction medium were prepared according to the Pichia pastoris expression Manual (Invitrogen, USA).
1.4 separation and purification of Pichia pastoris expressed fusion protein
And (3) after obtaining culture solution for induction expression, centrifuging at 4 ℃ and 10000rpm for 10min, obtaining supernatant fluid which is crude enzyme solution, purifying the crude enzyme solution by using a Ni chromatographic column to obtain oligomerization FAE, determining the protein mass concentration by using a Bradford method after purification, and analyzing the purified enzyme solution by SDS-PAGE. And (3) measuring the activity of the FAE enzyme by using a high performance liquid chromatography. The blank control was a boiling inactivated enzyme solution. The enzyme activity unit is defined as that 1 enzyme activity unit is the amount of enzyme required for generating 1 mu mol of ferulic acid under the conditions of 25 ℃ and pH value of 6.0 for 1 min.
1.5 investigation of enzymatic Properties of recombinant FAE
1.5.1 determination of optimum reaction temperature
Holding 250 μ L of enzyme solution for 5min, adding 250 μ L of methyl ferulate solution (prepared from Na with pH of 6.0)2HPO4And (4) -preparing a citric acid buffer solution), reacting for 10min at 40-65 ℃, and determining the enzyme activity of the recombinant FAE. The relative enzyme activity was calculated with the highest enzyme activity measured as 100%.
1.5.2 determination of temperature stability
And (3) placing the enzyme solution at 30-50 ℃, preserving the heat for 12h, sampling every 3h to determine the residual enzyme activity, and calculating the relative enzyme activity by taking the enzyme activity determined by preserving the heat for 0h as 100%.
1.5.3 determination of optimum reaction pH
Holding 250 μ L of enzyme solution at 25 deg.C for 5min, adding 250 μ L of methyl ferulate solution (0.2mol/L Na)2HPO4Adjusting the pH value of the citric acid buffer solution to be 3.0-8.0), reacting for 10min at 25 ℃, and measuring the enzyme activity of the recombinant FAE. The relative enzyme activity was calculated with the highest enzyme activity measured as 100%.
1.5.4 determination of pH stability
Placing the enzyme solution in 0.2mol/L, pH Na with the value of 3.0-8.02HPO4And (3) preserving heat for 2h at 25 ℃ in a citric acid buffer solution, taking 250 mu L of heat preservation enzyme solution, adding 250 mu L of ferulic acid methyl ester solution into the heat preservation enzyme solution, reacting for 10min at 25 ℃, measuring the residual enzyme activity of the recombinant FAE, and calculating the relative enzyme activity by taking the 0h enzyme activity of each pH value as 100%.
1.5.5 Effect of Metal ions on recombinant FAE enzyme Activity
Mixing the enzyme solution with various metal ions (Na) containing 10mmol/L+,K+,Mn2+,Fe2+,Cu2+,Mg2+,Ca2+,Zn2+) Na of (2)2HPO4-citric acid buffer (pH 6.0), incubation at 25 ℃ for 2h, measuring residual enzyme activity, calculating relative enzyme activity with enzyme activity measured without addition of metal ions as 100%.
1.5.6 determination of dynamic constant of recombinant FAE 8 tubes were filled with 200g/L ferulic acid methyl ester solution, 0.2mol/L Na with pH of 6.02HPO4-citric acid buffer solution, keeping the temperature at 25 ℃ for 3min, sequentially adding samples by using the same tube of enzyme solution, sequentially measuring the enzyme activity when the enzyme acts for 1-30min, then calculating the ratio of the enzyme activity to the reaction time, and keeping the ratio stable in a certain time, wherein the enzyme action is a first-order reaction in the time, and the time can be determined as K measurementmValue sum VmaxThe reaction time of (2). With different concentrations of substrate, Na at 0.2mol/L, pH6.02HPO4Reacting in a citric acid buffer system at 25 ℃ for a certain time, measuring enzyme activity, calculating corresponding reaction speed, and obtaining K by using a double reciprocal method of a Mie equationmValue and Vmax。
1.6 Transmission Electron microscopy analysis of recombinant FAE
A suitable concentration of 5. mu.L of protein sample was added dropwise to a 200 mesh copper grid coated with a carbon support film and incubated for 5min, and the excess protein sample was blot dried with filter paper. The grid was washed with two 20 μ L water and print dried followed by 25 μ L of 2% uranyl acetate droplets for 30 seconds, after which the print was dried and the sample was observed using a transmission electron microscope at 100kV and an electron micrograph was recorded using a CCD camera.
1.7 screening of stabilizers and optimization of the concentration
1.7.1 Effect of stabilizers on the thermostability of oligomerized FAE
Separately adding different concentrations of: (1) alcohols: PEG400, glycerol, propylene glycol, sorbitol; (2) xylanase, cellulase and pectinase. Placing in 25 deg.C water bath, sampling after 0h and 2h, cooling in ice bath, measuring enzyme activity, and calculating enzyme activity residual rate. Enzyme solution without stabilizer is used as control group.
1.7.2 determination of storage stability
Storing the enzyme solution added with the composite stabilizer and the enzyme solution of a control group in a refrigerator at normal temperature and 4 ℃, measuring the enzyme activity at regular intervals, and measuring for 20 days. Calculating the enzyme activity residual rate by taking the enzyme activity of 0d as 100 percent. Enzyme solution without stabilizer is used as control group.
2. Results and analysis
2.1 PCR identification and screening of recombinant FAE transformants
PCR was verified using 5 'AOX 1 and 3' AOX1 primers using pPIC9K/His-fae and pPIC9K/His-fae-linker-foldon genomes as templates, and PCR products were detected by 1% gel electrophoresis, the results are shown in FIG. 2. It was shown that the desired genes His-fae and His-fae-linker-foldon had successfully integrated into the genomes of these transformants.
2.2 expression and purification of recombinant FAE
The positive transformants were selected, cultured and induced to express according to 1.3, the expression level was measured, 1 transformant with the highest enzyme production activity in the experiment was selected, and the fermentation supernatant was purified by Ni column and analyzed by SDS-PAGE. As shown in fig. 3(a), M is protein marker; the 1 is pPIC9K/His-FAE fermentation supernatant, mon-FAE in the graph is the product expressed by pPIC9K/His-FAE plasmid, and as can be seen from the graph in FIG. 3(A), a clear band is obtained at 40kD, and a miscellaneous band is hardly generated, which primarily indicates that mon-FAE is successfully expressed. FIG. 3(A) shows that the apparent relative molecular mass of mon-FAE is significantly greater than its theoretical value of 29.97kD, mainly due to the glycosylation site at the N-terminus of the FAE protein sequence, whereas Pichia pastoris, when expressing a foreign protein, modifies it by glycosylation, thereby increasing the relative molecular mass. FIG. 3(B) shows that M is protein marker; 1 is pPIC9K/His-FAE-linker-foldon fermentation supernatant, while the shown olig-FAE is the product expressed by pPIC9K/His-FAE-linker-foldon plasmid, and as can be seen from the graph in FIG. 3(B), a band appears at 170kD in the supernatant fermentation broth. The reason why the apparent relative molecular mass of olig-FAE is significantly larger than its theoretical value of 99.12 is the same as that of mon-FAE.
The enzyme activities and specific activities of mon-FAE and olig-FAE were compared, as shown in Table 1. Table 1 shows that the enzyme activity of olig-FAE is improved by 42.4 times and the specific activity is improved by 3.73 times compared with that of mon-FAE.
TABLE 1 enzymatic and specific Activity of recombinant FAE
| Protein | Enzyme activity (U/L) | Specific activity (U/mg) |
| mon-FAE | 187 | 16.1 |
| olig-FAE | 8110 | 76.2 |
2.3 study of recombinant FAE Medium
The influence of the de-starch wheat bran on the activity and specific activity of the FAE enzyme during the expression of the recombinant engineering bacteria is researched, and the experimental result is shown in FIG. 4.
Experimental results show that the enzyme activity of olig-FAE is increased and then decreased along with the increase of the addition amount of the de-starch wheat bran, the highest expression amount can be 8110U/mL when the addition amount of the de-starch wheat bran is 1.5 percent (W/W), and the expression amount is 3800U/mL when the de-starch wheat bran is not added, compared with a culture medium without the de-starch wheat bran, the enzyme activity is improved by 1.13 times, and the fact that the addition of the de-starch wheat bran to a BMMY induction culture medium can improve the expression amount of the enzyme activity.
2.4 study of the enzymatic Properties of olig-FAE
2.4.1 determination of optimum temperature and temperature stability
The optimum temperature for the enzyme reaction at 40-65 ℃ was investigated, and the results are shown in FIG. 5. As can be seen from fig. 5: the optimum temperature of both mon-FAE and olig-FAE is 50 ℃, and the enzyme activities of both are higher before 50 ℃, and the relative enzyme activities are over 90 percent. After the temperature is over 50 ℃, the enzyme activities of the two enzymes are reduced rapidly, and probably the enzyme is inactivated along with the increase of the temperature.
After the recombinant FAE is subjected to heat preservation for 0, 3, 6, 9 and 12 hours at the temperature of 30-55 ℃, the residual enzyme activity of the recombinant FAE is measured, and the result is shown in FIG. 6. At the temperature of 30-55 ℃, the enzyme activity of both the mon-FAE and the olig-FAE is gradually reduced along with the extension of the heat preservation time, but the decline trend of the olig-FAE is gentler than that of the mon-FAE. After the temperature of 50 ℃ of the mon-FAE is kept for 3h, the enzyme activity is basically lost, and after the temperature of 50 ℃ of the olig-FAE is kept for 3h, the residual enzyme activity is 78.0%. Therefore, the temperature stability of olig-FAE is greatly improved compared with mon-FAE.
2.4.2 determination of optimum pH and pH stability
The pH optimum results for the recombinant enzyme are shown in FIG. 7. As can be seen from FIG. 7, the optimum pH of mon-FAE was 6.0; the optimum pH of olig-FAE was 5.0. The results of the recombinase pH stability are shown in FIG. 8. As can be seen from fig. 8, the stability of mon-FAE increases with increasing pH at pH 3.0 to 6.0, the pH stability of mon-FAE is highest at pH6.0, and the stability of mon-FAE gradually decreases at pH greater than 6.0; the stability of olig-FAE increases with increasing pH at pH 3.0-5.0, the highest pH stability of olig-FAE at pH 5.0, and the gradually decreasing stability of olig-FAE at pH greater than 5.0.
2.4.3 Effect of Metal ions on olig-FAE
The effect of metal ions on the activity of the recombinase is shown in Table 2, Mn2+、Zn2+、Ca2+、Mg2+、Fe3+、Cu2+Has an accelerating effect on it, K+Has certain inhibiting effect on olig-FAE.
TABLE 2 Effect of Metal ions on olig-FAE
2.4.4 determination of kinetic constants
Kinetic constants were determined at 25 ℃ and are shown in Table 3. Wherein the K of olig-FAEm(0.713. + -. 0.02) with mon-FAE (4.55)+/-0.07) is smaller, which shows that the substrate affinity of the olig-FAE is improved by 5.38 times compared with that of the mon-FAE; k of olig-FAEcat/Km(11.8 +/-1.12) is larger than mon-FAE (0.461 +/-0.63), which shows that the catalytic efficiency of olig-FAE is obviously improved compared with mon-FAE, and the improvement multiple is 24.6 times; meanwhile, k of olig-FAEcat、VmaxCompared with mon-FAE, the total content of the amino acid is improved.
TABLE 3 determination of kinetic constants of recombinant FAE
2.5 analysis of recombinant FAE by Transmission Electron microscopy
The structural properties of the olig-FAE protein at 25 ℃ were further investigated using transmission electron microscopy images, and to facilitate observation of the olig-FAE protein structure, the samples were diluted to 0.15mg/ml, and the electron microscopy results are shown in FIG. 9. In fig. 9 it is shown that particles with nearly spherical morphology aggregate together in the negative stain envelope, forming an oligomerized structure.
2.6 Effect of Complex stabilizers
2.6.1 Effect of different substances on the stability of olig-FAE
7 substances are selected as stabilizing agents of the enzyme solution. Placing in 50 deg.C water bath, sampling after 0h and 2h, cooling in ice bath, measuring enzyme activity, and calculating enzyme activity residual rate. Enzyme solution without stabilizer is used as control group. The results of the thermal stability test are shown in FIGS. 10 to 12.
The thermal stability of the low-concentration propylene glycol, sorbitol, PEG400 and glycerol to the olig-FAE is increased along with the increase of the concentration, the thermal stability is increased firstly and then reduced, when the concentration of the PEG400 is 8%, the stability of the enzyme activity is increased, and the relative enzyme activity is 108%. The xylanase and the cellulase can obviously keep the thermal stability influence of the olig-FAE, and when the xylanase is 5 percent and the addition amount of the cellulase is 3 percent, the enzyme activity is relatively kept stable. The additive with stabilizing effect on olig-FAE, the optimum addition amount and the enzyme activity residual rate thereof were obtained by a single factor test, as shown in Table 4.
TABLE 4 Effect of stabilizers on the thermal stability of liquid olig-FAE
The results in table 4 show that, under the same experimental conditions, the residual enzyme activity of the control group without any stabilizer is 65.3%, and the thermal stability of the olig-FAE is significantly improved by adding the stabilizer shown in table 4 alone.
2.6.2 determination of storage stability
Place olig-FAE in composite stabilizer (5% xylanase, 3% cellulase, 0.03% MgSO)48% PEG400), storing the mixture in a refrigerator at normal temperature and 4 ℃ for 20 days respectively, measuring the enzyme activity at intervals of 1-4 days, and researching the storage stability of the enzyme under different storage conditions, wherein experimental results show that the stability of the enzyme can be maintained in the storing process by adding the composite stabilizer. The enzyme activity of the olig-FAE enzyme solution added with the composite stabilizer is 96.2% of the initial enzyme activity after 20 days at 4 ℃, and the blank control only can preserve 45.5% of the initial enzyme activity, so that the enzyme activity is improved by 1.11 times.
3. Conclusion
The oligomerization FAE based on foldon is expressed in Pichia pastoris GS115, and the culture medium of the engineering bacteria contains de-starch wheat bran in the induction expression process, so that the obtained enzyme activity is 8110U/mL, the specific enzyme activity is 76.2U/mg, and the substrate affinity (K) of the oligomerization FAE ism) And catalytic efficiency (k)cat/Km) Compared with the monomer FAE of a control group, the FAE is respectively improved by 5.38 times and 24.6 times; and the oligomerization FAE obtained by purification is stored in a composite stabilizer at 4 ℃, and the enzyme activity residual rate reaches 96.2 percent after 20 days.
In conclusion, the method for improving the catalytic efficiency of the feruloyl esterase FAE is simple and efficient, does not need to know the 3D structure of the enzyme in detail in advance, and has good application prospect.
It should be emphasized that the term "comprises/comprising" when used herein, is taken to specify the presence of stated features, elements, steps or components, but does not preclude the presence or addition of one or more other features, elements, steps or components.
In addition, the method of the present invention is not limited to be performed in the time sequence described in the specification, and may be performed in other time sequences, in parallel, or independently. Therefore, the order of execution of the methods described in this specification does not limit the technical scope of the present invention.
While the present invention has been disclosed above by the description of specific embodiments thereof, it should be understood that all of the embodiments and examples described above are illustrative and not restrictive. Various modifications, improvements and equivalents of the invention may be devised by those skilled in the art within the spirit and scope of the appended claims. Such modifications, improvements and equivalents are also intended to be included within the scope of the present invention.
Sequence listing
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<120> preparation method of oligomerization feruloyl esterase based on foldon mediation
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<213> Artificial Sequence (Artificial Sequence)
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Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly
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Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser
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Gly Gly Ser Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Ala Gly
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Claims (6)
1. A preparation method of oligomerization ferulic acid esterase based on foldon mediation is characterized in that: the method comprises the following steps:
designing a linker sequence shown as SEQ ID No.1, designing a gene sequence His-fae-linker-foldon according to a nucleotide sequence corresponding to an amino acid sequence of ferulic acid esterase FAEO42807, adding SnaB I enzyme cutting sites and Not I enzyme cutting sites at two ends respectively, and artificially synthesizing a gene fragment His-fae-linker-foldon;
carrying out double digestion on pPIC9K plasmid and His-fae-linker-foldon by SnaB I and Not I, connecting the plasmid pPIC9K with gene fragment His-fae-linker-foldon by T4 ligase, constructing and screening pPIC9K/His-fae-linker-foldon plasmid positive clones, and carrying out electric transformation to P.pastoris GS115 for expression to obtain pPIC9K/His-fae-linker-foldon engineering bacteria;
the pPIC9K/His-fae-linker-foldon engineering bacteria are induced and expressed in a culture medium, and the induced expression adopts an induced culture medium with the following components: BMMY culture medium, 1-2% de-starch wheat bran; separating and purifying to obtain oligomerized feruloyl esterase; storing oligomerized ferulic acid esterase in a composite stabilizer, wherein the composite stabilizer comprises the following components in parts by mass: 4-6 parts of xylanase with the xylanase content being more than or equal to 20000U/mL, 2-4 parts of cellulase with the cellulase content being more than or equal to 10000U/mL, and 0.02-0.04 part of MgSO (MgSO) by weight47-9 parts of PEG 400.
2. The method of claim 1, wherein: the induced expression of the pPIC9K/His-fae-linker-foldon engineering bacteria in a culture medium specifically comprises the following steps: inoculating pPIC9K/His-fae-linker-foldon engineering bacteria into a seed culture medium, and culturing conditions are as follows: culturing at 28-30 ℃ and 150-250 rpm for 16-18 h; centrifuging the culture solution cultured by the seed culture medium at 2-4 ℃ and 3500-4500 rpm for 4-6 min, collecting cells, adding the cells into the induction culture medium, and culturing under the following conditions: culturing at 28-30 ℃ and 150-250 rpm for 96-100 hours.
3. The method of claim 2, wherein: the seed culture medium is BMGY culture medium.
4. The method of claim 1, wherein: the pPIC9K/His-fae-linker-foldon engineering bacteria comprise the following preparation processes: designing and optimizing a gene sequence His-fae-linker-foldon according to a nucleotide sequence, a His, a linker and a foldon gene sequence corresponding to an amino acid sequence of FAEO42807 and codon preference use frequency of pichia pastoris, respectively adding SnaB I enzyme cutting sites and Not I enzyme cutting sites at two ends, and artificially synthesizing a gene fragment His-fae-linker-foldon;
the pPIC9K plasmid and His-fae-linker-foldon were digested simultaneously with SnaB I and Not I, plasmid pPIC9K and gene fragment His-fae-linker-foldon were ligated with T4 ligase and transformed into E.coli DH 5. alpha. competent cells, positive clones selected and sequenced to give pPIC9K/His-fae-linker-foldon plasmid positive clones, which were then electroporated into P.pastoris GS115.
5. The method of claim 1, wherein: transformation of pPIC9K/His-fae-linker-foldon plasmid positive clones into P.pastoris GS115 specifically included: digesting pPIC9K/His-fae-linker-foldon with Sac I, transforming the linearized fragment to P.pastoris GS115 competence by an electric transformation method, coating the competent fragment on an MD plate to screen recombinants, taking a colony growing well on the MD plate, dibbling the colony to an YPD shake flask by using toothpicks, culturing for 16-18 h, extracting genome DNA, and performing PCR identification by using universal primers 5 'AOX and 3' AOX to obtain pPIC9K/His-fae-linker-foldon engineering bacteria expressing ferulic acid esterase.
6. The method of claim 1, wherein: the separation and purification step comprises the following steps: centrifuging at 0-5 ℃ and 8000-12000 rpm for 8-12 min to obtain supernatant, namely crude enzyme liquid, and purifying the crude enzyme liquid by using a Ni chromatographic column to obtain the oligomerization feruloyl esterase.
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