CN112142832A - Streptomyces cellooligosaccharide transport protein gene - Google Patents
Streptomyces cellooligosaccharide transport protein gene Download PDFInfo
- Publication number
- CN112142832A CN112142832A CN202011123905.XA CN202011123905A CN112142832A CN 112142832 A CN112142832 A CN 112142832A CN 202011123905 A CN202011123905 A CN 202011123905A CN 112142832 A CN112142832 A CN 112142832A
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- CN
- China
- Prior art keywords
- cellooligosaccharide
- scab
- ssc
- transport protein
- ala
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
The invention discloses a streptomycete cellooligosaccharide transport protein gene, and belongs to the technical field of biology. The invention successfully constructs the engineering strain for efficiently expressing the streptomyces cellooligosaccharide transport protein by connecting the streptomyces cellooligosaccharide transport protein gene to a pET-28a carrier and then transferring the streptomyces cellooligosaccharide transport protein gene to a competent target strain for induced expression culture, and the thermodynamics proves that the streptomyces cellooligosaccharide transport protein gene can be efficiently combined with cellooligosaccharides, particularly cellobiose, thereby providing a tool for further utilizing cellulose in the industry.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a streptomycete cellooligosaccharide transport protein gene.
Background
With the gradual depletion of fossil energy and the improvement of attention of people to problems such as environmental pollution and energy crisis, the search for clean renewable energy becomes a current research hotspot. Lignocellulose is a complex composed of various organic high molecular compounds and having high crystallinity and polymerization degree, mainly formed by mutually connecting cellulose, hemicellulose and lignin through covalent bonds and non-covalent bonds, and is favored by scientists in various countries due to abundant reserves, low cost, cleanness and environmental protection.
Cellulases comprising exo-1, 4- β -D-glucanases (CBH), endo-1, 4- β -D-glucanases (EG), β -Glucosidases (GE) which can hydrolyze cellulose, hemicellulose to monosaccharides, wherein exo-1, 4- β -D-glucanases hydrolyze crystalline cellulose from both ends of cellulose, releasing cellobiose; the endo-1, 4-beta-D-glucanase acts on beta-1, 4 glycosidic bonds in the cellulose, and randomly hydrolyzes amorphous cellulose to release various cellooligosaccharides; beta-glucosidase, also known as cellobiase, primarily hydrolyzes cellobiose, cellooligosaccharides, releasing glucose. Cellobiose has an inhibitory effect on exo-1, 4-beta-D-glucanase and endo-1, 4-beta-D-glucanase, while glucose has an inhibitory effect on beta-glucosidase, and the hydrolysis efficiency of cellulose decreases as the concentration of glucose in the hydrolysate increases. Therefore, the lignocellulose hydrolysis efficiency is low, and the use cost of the cellulase is a bottleneck for restricting the development and utilization of lignocellulose which is a renewable energy source.
The direct utilization of cellooligosaccharide in lignocellulose pretreatment liquid is one of the ways of effectively reducing the cost of cellulose enzymolysis and fermentation and realizing the efficient fermentation and conversion of lignocellulose. However, cellooligosaccharide is a macromolecular substance and cannot pass through cell membranes in a simple diffusion manner such as free diffusion, and the assistance of a transport protein is necessary. Currently, industrial fermentation microbial strains do not have cellooligosaccharide transporters and do not have the ability to directly utilize cellooligosaccharides for fermentation.
Disclosure of Invention
The invention aims to provide a streptomycete cellooligosaccharide transport protein gene and protein, which are used for solving the problems in the prior art, and engineering bacteria capable of efficiently utilizing cellulose, especially cellobiose, are obtained by transferring the streptomycete cellooligosaccharide transport protein into a target strain, so that a foundation is laid for realizing industrial utilization of the cellulose.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a streptomycete cellooligosaccharide transport protein gene, wherein the nucleotide sequence of the oligosaccharide transport protein gene is shown as SEQ ID No. 1.
The invention also provides an oligosaccharide transport protein expressed by the streptomyces cellooligosaccharide transport protein gene, and the amino acid sequence of the oligosaccharide transport protein is shown as SEQ ID No. 2.
The invention also provides a construction method of the cellulose fermentation engineering bacteria, which comprises the steps of constructing an expression vector of the streptomycete cellooligosaccharide transport protein gene, transforming a competent target strain by using the vector, and inducing expression of the streptomycete cellooligosaccharide protein in the transformed target strain.
The invention also provides the engineering bacteria constructed by the construction method.
The invention also provides application of the engineering bacteria in cellulose fermentation.
Further, the cellulose is cellooligosaccharide.
Further, the cellooligosaccharide comprises one or more of cellobiose, cellotriose, cellotetraose and cellopentaose.
Further, the cellooligosaccharide is cellobiose.
The invention discloses the following technical effects:
the invention successfully constructs the engineering strain for efficiently expressing the streptomyces cellooligosaccharide transport protein by transferring the streptomyces cellooligosaccharide transport protein gene into a competent target strain and carrying out induced expression culture, and proves that the streptomyces cellooligosaccharide transport protein gene can be efficiently combined with cellooligosaccharide, particularly cellobiose through thermodynamic experiments, thereby providing a tool for further utilizing cellulose in the industry.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 is a bovine serum albumin standard curve;
FIG. 2 shows SDS-polyacrylamide gel electrophoresis analysis of the expression product, wherein lane 1 is Standard protein Marker (Low); lane 2 is purified Ssc-SCAB;
FIG. 3 is a microcalorimetric differential scanning quantitation result of the interaction of the substrate binding protein Ssc-SCAB with various cellooligosaccharides, where A is Ssc-SCAB and its DSC analysis with glucose; b is Ssc-SCAB and DSC analysis of the Ssc-SCAB and cellobiose; c is Ssc-SCAB and DSC analysis of the Ssc-SCAB and cellotriose; d is DSC analysis of Ssc-SCAB and fiber tetrasaccharide thereof; e is Ssc-SCAB and DSC analysis of the Ssc-SCAB and fiber pentasaccharide;
FIG. 4 is a plot of an isothermal titration of cellooligosaccharide and substrate binding protein Ssc-SCAB at 35 deg.C, where A is the cellobiose titration Ssc-SCAB; b is cellotriose titration Ssc-SCAB; c is fiber tetrasaccharide titration Ssc-SCAB; d is fiber pentasaccharide titration Ssc-SCAB.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
The materials, reagents and the like used in the present invention can be obtained commercially unless otherwise specified.
Example 1 transformation of Escherichia coli with Ssc-scaB Gene
1. Codon optimization and recombinant plasmid construction
The sequences of Proteins encoding Proteins from The cellulose-oligosaccharide-polypeptide-modified expression of Streptomyces plasmids, journal S et al, 2016 and sequence engineering Homologs of purified oligo-polypeptide-bound Proteins Encoded by heterologous markers Bind to polynucleotides, Nanavatii D M et al, 2006 and clone update in The heterologous polypeptide microorganism bacterium vector, plasmid vector DNA, Kognis S M et al, 2001 and clone update in The heterologous polypeptide vector DNA, yeast plasmid, strain S M et al, 2001 and clone update nucleotide sequences, protein sequences from Streptomyces polypeptides, strain C2003, K2011 and strain DNA, protein sequences from Streptomyces strains, strain DNA, DNA sequences from Streptomyces strains, DNA sequences, this was named Ssc-scaB. According to the analysis of gene sequence, the DNA molecule solution is obtained by constructing the pET-28a expression vector by Colei Biotechnology Limited liability company.
2. Coli miniprep plasmid
(1) 1.5mL of overnight-cultured bacterial suspension was added to a 2 mL-volume EP tube, centrifuged at 12000rpm for 1min, and the supernatant was discarded.
(2) To the EP tube was added 250. mu.L of a pre-cooled P1 solution (RNase A was added before use), and the cells were resuspended using a vortex shaker.
(3) 250 μ L of pre-cooled P2 solution was added to the EP tube and gently turned up and down 6-8 times to lyse the cells sufficiently.
(4) Add 350. mu.L of pre-cooled P3 solution to the EP tube, gently tip up and down 6-8 times immediately, mix well, centrifuge at 12000rpm for 10 min.
(5) Transferring the supernatant to adsorption column CP3, centrifuging at 12000rpm for 1min, discarding the waste liquid in the collection tube, and returning adsorption column CP3 to the collection tube.
(6) Adding 500 μ L deproteinized solution PD into adsorption column CP3, centrifuging at 12000rpm for 1min, discarding waste liquid in the collection tube, and placing adsorption column CP3 back into the collection tube.
(7) Adding 600 μ L of the removing-rinsing liquid PW 3 into adsorption column CP3, centrifuging at 12000rpm for 1min, discarding the waste liquid in the collection tube, and returning adsorption column CP3 into the collection tube.
(8) And 7, repeating the step.
(9) The adsorption column CP3 was returned to the collection tube, centrifuged at 12000rpm for 2min, the adsorption column CP3 was transferred to a clean EP tube, 50. mu.L of sterile water was added to the middle of the adsorption membrane, allowed to stand at room temperature for 2min, centrifuged at 12000rpm for 2min, the plasmid solution was collected in the EP tube, and stored at-20 ℃ for further use.
3. Preparation of competent cells of Escherichia coli by calcium chloride-magnesium chloride method (E.coli Rosetta (DE3))
(1) The glycerol preserved e.coli strain was taken out from-40 ℃, dipped in the bacterial solution with an inoculating loop in a clean bench, streaked on an antibiotic-free LA plate, and cultured overnight at 37 ℃.
(2) Well-grown single colonies were picked from LA plates, inoculated into a straight flask containing 5mL of LB medium, and cultured overnight at 37 ℃ and 200 rpm.
(3) Inoculating into 100mL LB medium-containing Erlenmeyer flask according to 1% (v/v), culturing at 37 deg.C and 200rpm for 2-3h, and adjusting bacterial liquid OD600 to 0.4-0.6.
(4) And cooling the cultured bacterial liquid by using an ice bath for about 30min, and occasionally mixing uniformly.
(5) The well-cooled bacterial solution was dispensed into pre-cooled, sterile polypropylene tubes with a volume of 50mL in a clean bench, centrifuged at 5000rpm for 10min at 4 ℃ and the supernatant discarded.
(6) 35mL of a precooled calcium chloride-magnesium chloride solution was added to the polypropylene tube, the resuspended cells were gently tapped, centrifuged at 5000rpm for 5min at 4 ℃ and the supernatant was discarded.
(7) Repeat step 6 twice.
(8) Add 4mL of pre-cooled glycerol-calcium chloride solution to the polypropylene tube, gently tap the resuspended cells, dispense competent cells on ice into sterile, pre-cooled EP tubes, 100. mu.L per tube, and refrigerate at-80 ℃.
Transformation of competent cells of E.coli with DNA molecules
(1) Taking out the escherichia coli competent cells from-80 ℃, quickly inserting the escherichia coli competent cells into ice, and naturally melting the escherichia coli competent cells in the ice.
(2) And (3) sucking a proper amount of the DNA molecular solution prepared in the step (1) by using a micropipette, adding the DNA molecular solution into an EP tube containing 100 mu L of escherichia coli competent cells, uniformly mixing, and standing for 30min in an ice bath.
(3) The EP tube was quickly placed in a water bath at 42 ℃ and heat-shocked for 90s while standing.
(4) Immediately taking out the EP tube after the heat shock is finished, and standing for 3min in ice bath.
(5) Add 200. mu.L of SOC medium into the EP tube, incubate at 37 ℃ and 200rpm for 1 h.
(6) Taking a proper volume of culture, spreading the culture on LA plates containing corresponding antibiotics, and placing the LA plates in a constant-temperature incubator at 37 ℃ for inverted culture for 12-16 h.
Example 2 inducible expression of recombinant substrate binding proteins
(1) From the transformation plate, well-grown single colonies were picked up and cultured at 37 ℃ and 200rpm for 10 hours in a straight flask containing 5mL of LB medium (containing 50. mu.g/mL kanamycin and 25. mu.g/mL chloramphenicol), respectively.
(2) Each of the cells was inoculated in an amount of 1% (v/v) into a conical flask containing 65mL of LB medium (containing 50. mu.g/mL kanamycin and 25. mu.g/mL chloramphenicol), and cultured overnight at 37 ℃ and 200 rpm.
(3) The cells were inoculated in 1% (v/v) amounts into Erlenmeyer flasks containing 1L of LB medium (containing 50. mu.g/mL kanamycin and 25. mu.g/mL chloramphenicol), and cultured at 37 ℃ and 200rpm for 2-3 hours, and IPTG was added to the final concentration of 1mmol/L when the OD600 of the cells was from 0.4 to 0.6.
(4) The recombinant strain E.coli Rosetta (DE3)/pET-Ssc-scaB was cultured at 180rpm for 20 hours at 16 ℃ for inducible expression of the target protein.
Example 3 purification of recombinant substrate binding proteins
1. Purification of recombinant substrate binding proteins
(1) After inducing the expression of 6L of bacterial liquid, centrifuging at 8000rpm for 10min, and collecting the thallus.
(2) The cells were resuspended in 300mL lysine buffer (pH 8.0, imidazole 10mmol/L, sodium chloride 300mmol/L, sodium dihydrogen phosphate 50mmol/L), 3mL lysozyme 25mg/mL was added, mixed well and allowed to stand on ice for 20 min.
(3) And (3) breaking cells in an ice-water mixture by using an ultrasonic cell breaker, wherein the total cell breaking time is 20min, the working time is 5s, the intermittence time is 5s, and the cell breaking power is 30%.
(4) After the cell breaking is finished, after the cell breaking of the recombinant strain E.coli Rosetta (DE3)/pET-Ssc-scaB is finished, the cell breaking liquid is centrifuged for 30min at 12000rpm at 4 ℃, and the centrifuged supernatant is crude protein liquid.
(5) 2mL of the Ni-NTA filler was pipetted into the column, and the Ni-NTA filler in the column was fully equilibrated with lysine buffer (pH 8.0, imidazole 10mmol/L, sodium chloride 300mmol/L, sodium dihydrogen phosphate 50 mmol/L).
(6) Mixing the crude protein solution with Ni-NTA filler balanced by lysine buffer, standing at 10 deg.C and 150rpm for 1 hr to allow the histidine-tag-containing recombinant substrate binding protein to be fully bound with the Ni-NTA filler.
(7) The mixtures were transferred to a column and the liquid was drained, 10mL of precooled Wash buffer (pH 8.0, 300mmol/L sodium chloride, 50mmol/L sodium dihydrogen phosphate, 20mmol/L imidazole concentration for Bbr-CLDE and Ssc-SCAB purification, 40mmol/L imidazole concentration for Pfu-CBTA and Tma-CBTA purification) was added to the column, the Ni-NTA was resuspended gently, the liquid in the column was drained and repeated 4 times.
(8) After washing sufficiently, 10mL of precooled Elution buffer (pH 8.0, imidazole 250mmol/L, sodium chloride 300mmol/L, sodium dihydrogen phosphate 50mmol/L) was added to the column to elute the substrate-bound protein, and the eluate was collected and repeated 3 times, and the eluate containing the substrate-bound protein was stored at 4 ℃.
(9) After the Elution of the substrate binding protein is finished, the Ni-NTA filler is washed for multiple times by using Elution buffer, and is preserved by using a 20% ethanol solution after the washing is finished. When the same substrate binding protein is purified, the Ni-NTA filler can be reused for 3-5 times, and the Ni-NTA filler can be regenerated after the binding rate is reduced.
2. Desalting of substrate binding proteins
(1) Transferring the eluent containing the substrate binding protein to a 30kDa ultrafiltration tube, transferring the eluent for multiple times when the eluent is more, centrifuging for 30min at 5000rpm under the condition of 4 ℃, and discarding waste liquid in a collecting tube.
(2) When the volume of the eluate in the ultrafiltration tube was about 500. mu.L, buffer A solution (pH 7.0, 50mM Tris-HCl, 100mM NaCl) was added to the ultrafiltration tube, and the mixture was centrifuged at 5000rpm for 30 minutes at 4 ℃ to discard the waste liquid in the collection tube.
(3) Repeat step 2 twice.
(4) The solution containing the substrate binding protein in the ultrafiltration tube and the ultrafiltrate in the collection tube were transferred to a clean EP tube and straight bottle, respectively, and stored at 4 ℃.
3. Drawing and quantitative analysis of protein standard curve
100 μ L of bovine serum albumin BSA standard (2mg/mL) was pipetted, 1900 μ L of ddH20 was added and mixed well, and the BSA standard was diluted with a diluent (ddH2O, 0.9% NaCl or PBS) in 1.5mL EP tubes according to tables 2-4, in triplicate for each concentration.
TABLE 1 bovine serum albumin Standard Curve assay
Respectively absorbing 20 mu L of diluted BSA standard solution, adding into 1.5mL of EP tubes, performing three parallels for each concentration, respectively adding 1mL of rewarming Broadford Dye Reagent into each tube, uniformly mixing, reacting at room temperature for 5min, adding 200 mu L of the diluted BSA standard solution into a 96-well plate, measuring the absorbance value at 600nm by using a microplate reader, calculating the average value of each concentration, subtracting the average value of Blank, and drawing a standard curve of the BSA standard solution by taking the protein content as the abscissa and the OD600 reading as the ordinate (figure 1).
Quantification of purified substrate binding protein: respectively taking 20 mu L of target protein solution with different dilution times, adding 1mL of rewarming Broadford Dye Reagent, uniformly mixing, reacting for 5min at room temperature, respectively taking 200 mu L of the rewarming Dye Reagent, adding to an ELISA plate, reading the absorbance value of OD600, substituting into a standard curve to calculate the mass concentration of the target protein, wherein the mass concentration is 6.288107 mg/mL.
EXAMPLE 4 SDS-PAGE analysis of substrate binding proteins
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the expression of the substrate binding protein and the molecular weight and degree of purification of the purified substrate binding protein, the experimental procedure was as follows:
(1) and (3) cleaning the glue maker, the glue comb, the glass plate and the Spacer, assembling, detecting the tightness by using water, discarding the water in the glass clamping plate layer if the tightness is good, and sucking the water by using filter paper.
(2) And preparing a separation gel and a concentrated gel solution according to the table 2-5, uniformly mixing, pouring the separation gel solution into the interlayer of the glass plate, pouring to a proper height, adding 1mL of absolute ethyl alcohol, and standing at room temperature for 30 min.
(3) And after the separation gel is solidified, removing the absolute ethyl alcohol in the glass interlayer, sucking the absolute ethyl alcohol by using filter paper, quickly filling a concentrated gel solution, inserting a gel comb after filling the glass plate interlayer, and standing the gel comb for 30min at room temperature.
(4) After the concentrated gel is solidified, putting the gel into an electrophoresis tank, washing gel holes by using electrode buffer solution, and performing pre-electrophoresis for 15min by using a constant current of 10 mA.
TABLE 2 SDS-Polyacrylamide gel formulations
(5) mu.L of the substrate binding protein solution was mixed with 10. mu.L of 4 × protein loading buffer, and centrifuged in a boiling water bath for 30min at 12000rpm for 10 min.
(6) Adding 4-10 μ L of the supernatant into the polyacrylamide gel hole, and performing electrophoresis at constant voltage of 120V after the sample loading is finished until the bromophenol blue band reaches the bottom of the polyacrylamide gel.
(7) After electrophoresis, the glass plate is pried open, polyacrylamide gel is carefully taken out, and the polyacrylamide gel is placed into Coomassie brilliant blue R-250 staining solution for staining, and is stained at 25 ℃ and 60rpm for 30 min.
(8) After dyeing is finished, discarding Coomassie brilliant blue R-250 dyeing liquid, washing with clear water, adding a proper amount of decolorizing liquid for decolorizing, decolorizing at 25 ℃ and 60rpm until no obvious background color exists in polyacrylamide gel, and replacing the decolorizing liquid for many times in the middle.
(9) After the polyacrylamide Gel is sufficiently decolorized, an ideal SDS-polyacrylamide Gel electrophoresis image is obtained by using an imager Gel DocTM XR +.
As shown in FIG. 2, ultrasonic cell-breaking purification is performed on the induced recombinant strain, a distinct protein characteristic band appears at the corresponding molecular weight, and a characteristic protein band appears at 46.6kDa of Ssc-SCAB (Lane 2), which indicates that the four substrate binding proteins are successfully expressed and the purity of the purified Ssc-SCAB protein solution is high.
Example 5 Isothermal Titration Calorimetry (ITC) determination of the affinity between substrate binding proteins and cellooligosaccharides
1. Various cellooligosaccharide titration substrate binding proteins
(1) And (3) diluting the substrate binding protein solution to 0.1-0.2 mmol/L by using ultrafiltrate in the last collecting pipe respectively and preparing various cellooligosaccharide solutions with the concentration about 10 times that of the substrate binding protein.
(2) Vacuum degassing at 4 ℃ for 10-20 min, and setting various parameters of isothermal titration.
Titration conditions for the substrate binding protein Ssc-SCAB:
the titration concentration of the substrate binding protein is 0.1mmol/L, the titration concentration of various cellooligosaccharides is 4mmol/L, the volume of a sample injection needle is 40 mu L, the suction filtration time of the sample injection needle is 5s, the duration of each titration is 6s, the volume of the 1 st drop is 0.1 mu L, the volume of the 2 nd drop to the 20 th drop is 1.5 mu L, 20 drops in total, the interval time of every two drops is 120s, the temperature of a sample cell is 35 ℃, the reference energy value is 10 mu cal/sec, the initial extension time is 1min, the stirring speed of the sample injection needle is 500rpm, and the feedback mode is low.
2. Thermodynamic parameters of substrate binding proteins
Data analysis and curve fitting are carried out by using Origin ITC 200 software, in a single isothermal titration experiment, the ITC can measure an affinity constant Kb, a stoichiometric number n, a combined enthalpy change delta H and entropy change delta S, and Gibbs free energy delta G can be calculated according to thermodynamic parameters measured by the experiment and a Gibbs-Helmholtz equation.
3. Results
The binding properties of the substrate binding protein to cellooligosaccharides in buffer A were determined using Isothermal Titration Calorimetry (ITC) by dropping cellooligosaccharides into separate pools filled with substrate binding protein (the first drop, which is smaller in volume) at equal volumes at either 30 ℃ or 50 ℃. If the substrate binding protein is combined with the cellooligosaccharide, heat is released and an energy peak is recorded, the first drops generate more energy and have higher peak values, the substrate binding protein tends to be saturated with the continuous dropping of the cellooligosaccharide into the sample pool, the generated energy is less and less, the peak values are lower and lower, when the substrate binding protein in the sample pool is completely saturated, the recorded peak values are the heat generated by the dilution of the cellooligosaccharide, and the stoichiometric number n cannot be changed any more; if the substrate binding protein does not bind to cellooligosaccharide, there is only a minor peak due to the dilution exotherm. Under the same titration conditions, the heat of dilution generated when various cellooligosaccharides are titrated to buffer A is measured and used as a blank, and the background is subtracted from the point at the time of data analysis.
The isothermal titration calorimetry can directly measure the change amount of binding enthalpy (delta H), the change amount of entropy (delta S) and the binding constant (K) generated when the substrate binding protein is combined with the cellooligosaccharideb) And a stoichiometric number (n), and simultaneously calculating Gibbs free energy change (delta G) and transition temperature (T) of the binding reaction according to thermodynamic parameters measured by experimentsRotating shaft). Respectively titrating substrate binding protein Pfu-CBTA by using cellobiose, cellotriose, cellotetraose and cellopentaose, performing curve fitting operation analysis on titration results by using a binding site mode, and obtaining a dissociation constant KdIt is suggested that all substrate binding proteins have different degrees of affinity for their respective substrates, and some substrate binding proteins have a slight amount of heat capacity change after saturation of isothermal titration, probably due to nonspecific interactions of cellooligosaccharides with substrate binding proteins and/or dilution of cellooligosaccharides.
DSC thermodynamic analysis was performed using the substrate binding protein Ssc-SCAB at a final concentration of 0.022mmol/L, and the results are shown in FIG. 3. After the substrate binding protein Ssc-SCAB is combined with cellobiose, cellotriose and cellotetraose, the Tm value is lower than that of the unbound celloThe sugar has a higher Ssc-SCAB, indicating that the combination of cellobiose, cellotriose and cellotetraose with Ssc-SCAB induces a change in the internal conformation of Ssc-SCAB. Fitting analysis of the substrate binding protein Ssc-SCAB thermal denaturation process using a non-binary pattern in the analysis software, the results of which are shown in FIG. 3, gave the van-T Hoff enthalpy Δ H of the Ssc-SCAB and its complexes with various cellooligosaccharidesVAnd caloric enthalpy Δ H (see table 3 for details).
TABLE 3 DSC thermodynamic parameters of Ssc-SCAB
From Table 3 it can be seen that the substrate binding proteins Ssc-SCAB have a. DELTA.H & gt. DELTA.Hv and the ratio of. DELTA.H/. DELTA.Hv equals 3.15, indicating that Ssc-SCAB is a monomeric protein and contains more than two domains inside, and from FIG. 3 it can be seen that Ssc-SCAB has only one heat capacity scan peak during denaturation for two possible reasons: the method comprises the following steps that firstly, a plurality of structural domains in the Ssc-SCAB have the same or similar dissolution temperature Tm and enthalpy change delta H in the denaturation process, the denaturation processes of the structural domains are independent and not interfered with each other, and heat capacity scanning peaks are overlapped; secondly, the difference of the molecular weight of each structural domain is large, the heat capacity peak formed by the structural domain with small molecular weight when the differential scanning of microcalorimetry is carried out is not obvious, and the structural domain does not appear in the set coordinate range. The substrate binding protein Ssc-SCAB and the compound formed by the substrate binding protein Ssc-SCAB and various cellooligosaccharides have delta HVThe relationship < Δ H, indicating that the denaturation process of the substrate binding protein Ssc-SCAB is in a non-dyadic transition mode. After Ssc-SCAB binding cellobiose, cellotriose, cellotetraose,. DELTA.HVAnd Δ H are both increasedVThe increase range of (A) is more obvious, Delta HVThe increased ratio of/. DELTA.H, and the narrower temperature range for unfolding than the native, unconjugated cellooligosaccharide Ssc-SCAB, indicates that the Ssc-SCAB is not denatured bimodally after it forms a complex with cellooligosaccharideThe process has obviously strengthened synergistic effect and has the trend of two-state mode transformation.
As is clear from Table 3 and FIG. 3, cellobiose (FIG. 3-B), cellotriose (FIG. 3-C), and cellotetraose (FIG. 3-D) bind to the natural substrate binding protein Ssc-SCAB, and therefore, the stability of the complex formed by cellobiose, cellotriose, and cellotetraose with Ssc-SCAB is improved, and the respective Tm values are higher than those of the natural substrate binding protein Ssp-SCAB to which cellooligosaccharide has not been bound. After addition of glucose, Ssc-SCAB showed a Tm of 48.96 ℃ with little change compared to the Tm of 48.66 ℃ for Ssc-SCAB, a native state substrate binding protein that is not bound to cellooligosaccharides, indicating that Ssc-SCAB does not bind glucose (FIG. 3-A). The Tm value of the substrate binding protein Ssc-SCAB and the cellopentaose is 51.64 ℃, and the total enthalpy of heat becomes 2.62X 105cal/mol Tm value 48.66 ℃ with natural substrate binding protein Ssc-SCAB not bound to cellooligosaccharide, total enthalpy change 2.32X 105The change is small compared with cal/mol, which indicates that the substrate binding protein Ssc-SCAB is not combined with the fiber pentasaccharide (FIG. 3-E), the slight change of the Tm value and the total calorimetric enthalpy change of the substrate binding protein Ssc-SCAB is probably due to the fact that the molecular weight of the fiber pentasaccharide is large and nonspecific effect is generated with the Ssc-SCAB, and the later period can be further verified by isothermal titration calorimetry.
The binding thermodynamics of Ssc-SCAB were determined by titrating the substrate binding proteins Ssc-SCAB separately with cellooligosaccharide solutions at a final concentration of 4mmol/L, as shown in FIG. 3. The Ssc-SCAB can be combined with cellobiose, cellotriose and cellotetraose, and the result of isothermal titration calorimetry is consistent with that of microcalorimetric differential scanning calorimetry. The titration results were fit analyzed using a binding site model, and the thermodynamic parameters of each binding are shown in table 4.
TABLE 4 thermodynamic parameters for the interaction of the substrate binding protein Ssc-SCAB with cellooligosaccharides
As is clear from Table 4, Ssc-SCAB binds to cellobiose, cellotriose and cellotetraose, and has affinity for cellobioseStrongest force, binding constant KbIs (1.32 +/-0.09) × 106M-1The lowest affinity for cellotriose, binding constant KbIs (1.22 +/-0.04) multiplied by 106M-1。
TABLE 5 thermodynamic parameters of the substrate binding proteins Ssc-SCAB
As can be seen from tables 4 and 5, the affinity of Ssc-SCAB for cellobiose (FIG. 4-A) is strongest, and its dissociation constant K is measured at 35 ℃d0.76. mu.M, cellotriose (FIG. 4-B) and a dissociation constant of 0.82. mu.M, with the weakest affinity for cellotetraose (FIG. 4-C) and a dissociation constant of 1.06. mu.M. As can be seen from Table 5, the binding entropy change (Δ S) of Ssc-SCAB and various cellooligosaccharides (except cellopentasaccharide (FIG. 4-D)) was less than 0, indicating that the binding reaction of Ssc-SCAB and various cellooligosaccharides (except cellopentasaccharide) is a spontaneous forward irreversible process under the present experimental conditions; the change of binding enthalpy (Δ H) is less than 0, indicating that the binding reaction between Ssc-SCAB and various cellooligosaccharides (except cellopentasaccharide) is exothermic under the experimental conditions; since Δ S, Δ H, and Δ G are all less than 0, it can be judged that the binding reaction of the substrate binding protein Ssc-SCAB with various cellooligosaccharides (excluding cellopentasaccharide) is an irreversible reaction spontaneously proceeding in a positive direction driven by enthalpy under the present experimental conditions, and the temperature T plays an active role. The stronger the affinity between the cellooligosaccharide and the substrate-binding protein, the more stable the cellooligosaccharide-substrate-binding protein complex formed, and conversely, the more easily the complex dissociates. When the gibbs function Δ G of the complex is less than 0, the complex is stable, and the more negative the complex is.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Sequence listing
<110> Guangxi academy of sciences
<120> a streptomycete cellooligosaccharide transport protein gene
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1365
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgcgagcac gtaccctccc gcactcccgg ctccagtcgg gcgggggcag ccgaatcgcc 60
cgcaggacgc gcaagacggt ggtcatcgcg gccgtcgccg cgctgggcgc agggctgctg 120
gccggctgtg ccgacgacgg caaggacgag gaaggcggct cgtcggacgg cggcggcggt 180
ggcaagacca agatcacgct gggcctcttc ggcaccgcgg gcttcgagga gtccggtctg 240
tacaaggagt acgagaaact ccacccggac gtcgacatcc agcagaccgt cgtggagcgg 300
aacgagaact actaccccgc gctcctcaac cacctgacca ccggcagcgg cctccaggac 360
atccagatgg tcgaggtcgg caacatcgcc gagatcgtcg gaacccagtc cgacaagctg 420
ctcgacctgt cgaagtacgg caaggagagc gactacctgc cctggaagtg gagccagggc 480
tcgacctccg gcggccagac cgtcgcgctg ggcaccgacg tcggtccgat ggccatctgc 540
taccgcaagg acctcttcga ggccgccggt ctgccctccg accgcgagga ggtcggcaag 600
ctgtggaccg gcagctggga caagttcgtc gacgccggca accagtacaa gaagaaggcg 660
cccaagggca ccaccttcct ggactccccc ggcggtctgc tgcaggcgat cctgagcagt 720
gagaaggacc gcttctacga cgcctcgggc aaggtcatct acaagacgaa cccggcagtg 780
aagtcggcgt tcgacctcac ggccaaggcc gccaaggccg ggctggtcgg gaaccagacg 840
cagttccagc cggcgtggga caccacgatc gccaacagca agttcgccgc gatgtcctgc 900
ccgccgtgga tgctcggcta catcaagggc aagtcgaagc ccgaggcggc cggcaagtgg 960
gacatcgccc aggcgccgaa gtccggcaac tggggcggct ccttcctctc ggtgcccaag 1020
aacggcaaga acgccgagga ggccgcgaag ctggccgcct ggttgaccgc gccggagcag 1080
caggcgaagc tcttcgccgt acagggcagc ttccccagca ccccggccgc ctacgactcg 1140
gccgcggtga aggacgcgaa gaacgacatg accggtgacg cgccgatcgg cacgatcttc 1200
gccgaggccg ccaagaacat cccggtccag acgatcggcc cgaaggacca gatcatccag 1260
cagggcctga ccgacaacgg cgtgatcctg gtgacccagg gcaagtcggc ctcggatgcc 1320
tggaagaacg ccgtcaagac catcgacaac gcactggaca agtga 1365
<210> 2
<211> 454
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Arg Ala Arg Thr Leu Pro His Ser Arg Leu Gln Ser Gly Gly Gly
1 5 10 15
Ser Arg Ile Ala Arg Arg Thr Arg Lys Thr Val Val Ile Ala Ala Val
20 25 30
Ala Ala Leu Gly Ala Gly Leu Leu Ala Gly Cys Ala Asp Asp Gly Lys
35 40 45
Asp Glu Glu Gly Gly Ser Ser Asp Gly Gly Gly Gly Gly Lys Thr Lys
50 55 60
Ile Thr Leu Gly Leu Phe Gly Thr Ala Gly Phe Glu Glu Ser Gly Leu
65 70 75 80
Tyr Lys Glu Tyr Glu Lys Leu His Pro Asp Val Asp Ile Gln Gln Thr
85 90 95
Val Val Glu Arg Asn Glu Asn Tyr Tyr Pro Ala Leu Leu Asn His Leu
100 105 110
Thr Thr Gly Ser Gly Leu Gln Asp Ile Gln Met Val Glu Val Gly Asn
115 120 125
Ile Ala Glu Ile Val Gly Thr Gln Ser Asp Lys Leu Leu Asp Leu Ser
130 135 140
Lys Tyr Gly Lys Glu Ser Asp Tyr Leu Pro Trp Lys Trp Ser Gln Gly
145 150 155 160
Ser Thr Ser Gly Gly Gln Thr Val Ala Leu Gly Thr Asp Val Gly Pro
165 170 175
Met Ala Ile Cys Tyr Arg Lys Asp Leu Phe Glu Ala Ala Gly Leu Pro
180 185 190
Ser Asp Arg Glu Glu Val Gly Lys Leu Trp Thr Gly Ser Trp Asp Lys
195 200 205
Phe Val Asp Ala Gly Asn Gln Tyr Lys Lys Lys Ala Pro Lys Gly Thr
210 215 220
Thr Phe Leu Asp Ser Pro Gly Gly Leu Leu Gln Ala Ile Leu Ser Ser
225 230 235 240
Glu Lys Asp Arg Phe Tyr Asp Ala Ser Gly Lys Val Ile Tyr Lys Thr
245 250 255
Asn Pro Ala Val Lys Ser Ala Phe Asp Leu Thr Ala Lys Ala Ala Lys
260 265 270
Ala Gly Leu Val Gly Asn Gln Thr Gln Phe Gln Pro Ala Trp Asp Thr
275 280 285
Thr Ile Ala Asn Ser Lys Phe Ala Ala Met Ser Cys Pro Pro Trp Met
290 295 300
Leu Gly Tyr Ile Lys Gly Lys Ser Lys Pro Glu Ala Ala Gly Lys Trp
305 310 315 320
Asp Ile Ala Gln Ala Pro Lys Ser Gly Asn Trp Gly Gly Ser Phe Leu
325 330 335
Ser Val Pro Lys Asn Gly Lys Asn Ala Glu Glu Ala Ala Lys Leu Ala
340 345 350
Ala Trp Leu Thr Ala Pro Glu Gln Gln Ala Lys Leu Phe Ala Val Gln
355 360 365
Gly Ser Phe Pro Ser Thr Pro Ala Ala Tyr Asp Ser Ala Ala Val Lys
370 375 380
Asp Ala Lys Asn Asp Met Thr Gly Asp Ala Pro Ile Gly Thr Ile Phe
385 390 395 400
Ala Glu Ala Ala Lys Asn Ile Pro Val Gln Thr Ile Gly Pro Lys Asp
405 410 415
Gln Ile Ile Gln Gln Gly Leu Thr Asp Asn Gly Val Ile Leu Val Thr
420 425 430
Gln Gly Lys Ser Ala Ser Asp Ala Trp Lys Asn Ala Val Lys Thr Ile
435 440 445
Asp Asn Ala Leu Asp Lys
450
Claims (8)
1. A streptomycete cellooligosaccharide transport protein gene is characterized in that the nucleotide sequence of the oligosaccharide transport protein gene is shown as SEQ ID No. 1.
2. The oligosaccharide transporter protein encoded by the streptomyces cellooligosaccharide transporter gene of claim 1, wherein the amino acid sequence of the oligosaccharide transporter protein is set forth in SEQ ID No. 2.
3. A construction method of cellulose fermentation engineering bacteria, which is characterized in that the construction method comprises the steps of constructing an expression vector of the streptomycete cellooligosaccharide transport protein gene in claim 1, transforming a competent target strain by using the vector, and inducing expression of the streptomycete cellooligosaccharide protein in the transformed target strain.
4. An engineered bacterium constructed by the construction method of claim 3.
5. The use of the engineered bacterium of claim 4 in cellulose fermentation.
6. The use of claim 5, wherein the cellulose is cellooligosaccharide.
7. The use of claim 6, wherein the cellooligosaccharide comprises one or more of cellobiose, cellotriose, cellotetraose, and cellopentaose.
8. The use of claim 6, wherein the cellooligosaccharide is cellobiose.
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