CN112142832A - Streptomyces cellooligosaccharide transport protein gene - Google Patents

Abstract

The invention discloses a Streptomyces cello-oligosaccharide transporter gene and belongs to the field of biotechnology. In the present invention, the Streptomyces cello-oligosaccharide transporter gene is connected to the pET-28a vector, and then transferred into the competent target strain and induced to express and cultured, so as to successfully construct an engineering strain that efficiently expresses the Streptomyces cello-oligosaccharide transporter. Thermodynamic experiments have proved that it can efficiently bind cellooligosaccharides, especially cellobiose, which provides a tool for further utilization of cellulose in the industry.

Description

A Streptomyces cellooligosaccharide transporter gene

technical field

The invention relates to the field of biotechnology, in particular to a Streptomyces cello-oligosaccharide transporter gene.

Background technique

With the increasing depletion of fossil energy and people's increasing attention to environmental pollution, energy crisis and other issues, the search for clean renewable energy has become a current research hotspot. Lignocellulose is a complex composed of a variety of organic polymer compounds with high crystallinity and degree of polymerization, mainly formed by cellulose, hemicellulose and lignin interconnected by covalent and non-covalent bonds. With abundant reserves, low cost, clean and environmental protection, it is favored by scientists from all over the world.

Cellulases, including exo-1,4-beta-D-glucanases (CBH), endo-1,4-beta-D-glucanases (endo-beta-glucanases, CBH) glucanases, EG), β-glucosidase (endo-β-glucosidases, GE), can hydrolyze cellulose and hemicellulose into monosaccharides, of which exo-1,4-β-D-glucanase is derived from Both ends of cellulose hydrolyze crystalline cellulose to release cellobiose; endo-1,4-β-D-glucanase acts on the β-1,4 glycosidic bond inside cellulose to randomly hydrolyze amorphous cellulose , release various cello-oligosaccharides; β-glucosidase, also known as cellobiase, mainly hydrolyzes cellobiose and cello-oligosaccharides to release glucose. Cellobiose has inhibitory effect on exo-1,4-β-D-glucanase and endo-1,4-β-D-glucanase, while glucose has inhibitory effect on β-glucosidase, When the glucose concentration in the hydrolyzate increases, the hydrolysis efficiency of cellulose decreases. Therefore, the lower enzymatic hydrolysis efficiency of lignocellulose and the higher use cost of cellulase are the bottlenecks restricting the development and utilization of lignocellulose, a renewable energy source.

The direct utilization of cellooligosaccharides in lignocellulose pretreatment solution is one of the ways to effectively reduce the cost of enzymatic hydrolysis and fermentation of cellulose and realize efficient fermentation and conversion of lignocellulose. However, cellooligosaccharide is a macromolecular substance and cannot pass through the cell membrane by simple diffusion such as free diffusion, and must be assisted by transporters. In the current industrial fermentation microbial strains, there is no cellooligosaccharide transporter, and it does not have the ability to directly utilize cellooligosaccharide for fermentation.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a Streptomyces cello-oligosaccharide transporter gene and protein, to solve the problems existing in the above-mentioned prior art, by transferring the Streptomyces cello-oligosaccharide transporter into the target strain to obtain efficient utilization of cellulose, In particular, the engineered bacteria of cellobiose have laid the foundation for the industrial utilization of cellulose.

For achieving the above object, the present invention provides the following scheme:

The present invention provides a Streptomyces cello-oligosaccharide transporter gene, and the nucleotide sequence of the oligosaccharide transporter gene is shown in SEQ ID NO.1.

The present invention also provides an oligosaccharide transporter expressed by the Streptomyces cello-oligosaccharide transporter gene, and the amino acid sequence of the oligosaccharide transporter is shown in SEQ ID NO. 2.

The present invention also provides a method for constructing a cellulose fermentation engineering bacteria, which comprises constructing an expression vector for the Streptomyces cello-oligosaccharide transporter gene, transforming a competent target strain with the vector, and then inducing the transformed target strain. Streptomyces cellooligosaccharide protein expression in strains.

The present invention also provides an engineering bacterium constructed by the construction method.

The invention also provides an application of the engineering bacteria in cellulose fermentation.

Further, the cellulose is cellooligosaccharide.

Further, the cello-oligosaccharide includes one or more of cellobiose, cellotriose, cellotetraose and cellopentose.

Further, the cello-oligosaccharide is cellobiose.

The present invention discloses the following technical effects:

In the present invention, by transferring the gene of Streptomyces cello-oligosaccharide transporter into competent target strains and inducing expression culture, an engineering strain with high-efficiency expression of Streptomyces cello-oligosaccharide transporter is successfully constructed, and it is proved by thermodynamic experiments that it can efficiently combine Cello-oligosaccharides, especially cellobiose, provide a tool for further industrial utilization of cellulose.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings required in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some of the present invention. In the embodiments, for those of ordinary skill in the art, other drawings can also be obtained according to these drawings without any creative effort.

Fig. 1 is the standard curve of bovine serum albumin;

Figure 2 is the SDS-polyacrylamide gel electrophoresis analysis of the expression product, wherein lane 1 is Standardprotein Marker (Low); lane 2 is purified Ssc-SCAB;

Figure 3 shows the microcalorimetric differential scanning results of the interaction between the substrate binding protein Ssc-SCAB and various cellooligosaccharides, where A is the DSC analysis of Ssc-SCAB and its interaction with glucose; B is the DSC analysis of Ssc-SCAB and its interaction with fiber DSC analysis of disaccharide; C is the DSC analysis of Ssc-SCAB and its combination with cellotriose; D is the DSC analysis of Ssc-SCAB and its combination with cellotetraose; E is the DSC analysis of Ssc-SCAB and its combination with cellopentose ;

Figure 4 is the isothermal titration curve of cellooligosaccharide and substrate binding protein Ssc-SCAB at 35°C, where A is cellobiose titration Ssc-SCAB; B is cellotriose titration Ssc-SCAB; C is cellotetraose titration Ssc -SCAB; D is cellopentose titration Ssc-SCAB.

Detailed ways

Various exemplary embodiments of the present invention will now be described in detail, which detailed description should not be construed as a limitation of the invention, but rather as a more detailed description of certain aspects, features, and embodiments of the invention.

It should be understood that the terms described in the present invention are only used to describe particular embodiments, and are not used to limit the present invention. Additionally, for numerical ranges in the present disclosure, it should be understood that each intervening value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value in that stated range is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, 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 relates. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials in connection with which the documents are referred. In the event of conflict with any incorporated document, the content of this specification controls.

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 invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the present invention. The description and examples of the present application are only exemplary.

As used herein, "comprising," "including," "having," "containing," and the like, are open-ended terms, meaning including but not limited to.

The materials, reagents, etc. used in the present invention can be obtained from commercial sources unless otherwise specified.

Example 1 Transformation of Escherichia coli with Ssc-scaB gene

1. Codon optimization and recombinant plasmid construction

According to literature reports (The CebE/MsiK Transporter is a Doorway to the Cello-oligosaccharide-mediated Induction of Streptomyces scabies Pathogenicity, Jourdan S et al, 2016 and Several Archaeal Homologs of Putative Oligopeptide-BindingProteins Encoded by Thermotoga maritima Bind Sugars, Nanavati D M et al, 2006 and Cellobiose Uptake in the Hyperthermophilic Archaeon Pyrococcus furiosus IsMediated by an Inducible, Konings S M et al, 2001 and Cellodextrin Utilization by Bifidobacterium breveUCC 2003, Pokusaeva K et al, 2011 and HyperthermophilicThermotoga Species Differ with Respect to Specific Carbohydrate Transporters and Glycoside Hydrolases, Frock AD et al, 201 the Subtle MutationsDriving Host Sensing by the Plant Pathogen Streptomyces scabies, SamuelJourdan AI M F et al., 2017), BlastX alignment analysis was performed on the gene sequences from Streptomyces scabies, and the encoded amino acid sequences were subjected to protein domain prediction and Signal peptide analysis, which was named Ssc-scaB. According to the analysis of the gene sequence, it was constructed into the pET-28a expression vector by Kelei Biotechnology Co., Ltd. to obtain a DNA molecule solution.

2. Miniprep plasmids from E. coli

(1) Take 1.5 mL of overnight cultured bacterial solution into an EP tube with a volume of 2 mL, centrifuge at 12,000 rpm for 1 min, and discard the supernatant.

(2) Add 250 μL of pre-chilled P1 solution (add RNase A before use) to the EP tube, and use a vortex shaker to resuspend the cells.

(3) Add 250 μL of pre-cooled P2 solution to the EP tube, and gently turn up and down 6-8 times to fully lyse the cells.

(4) Add 350 μL of pre-cooled P3 solution to the EP tube, immediately turn it up and down gently 6-8 times, mix well, and centrifuge at 12000 rpm for 10 min.

(5) Transfer the supernatant to the adsorption column CP3, centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column CP3 back into the collection tube.

(6) Add 500 μL of deproteinized solution PD to the adsorption column CP3, centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column CP3 back into the collection tube.

(7) Add 600 μL of de-rinsing solution PW to the adsorption column CP3, centrifuge at 12000 rpm for 1 min, discard the waste liquid in the collection tube, and put the adsorption column CP3 back into the collection tube.

(8) Repeat step 7.

(9) Put the adsorption column CP3 back into the collection tube, centrifuge at 12,000 rpm for 2 minutes, transfer the adsorption column CP3 to a clean EP tube, add 50 μL of sterile water to the middle of the adsorption membrane, stand at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 2 minutes. The plasmid solution was collected into EP tubes and stored at -20°C for later use.

3. Preparation of E. coli competent cells by calcium chloride-magnesium chloride method (E.coli Rosetta(DE3))

(1) Take out the E. coli strains preserved in glycerol from -40°C, dip the bacterial solution with an inoculating loop in the ultra-clean workbench, streak it on an antibiotic-free LA plate, and incubate at 37°C overnight.

(2) Pick a single colony with good growth from the LA plate, inoculate it into a straight-shaped flask containing 5 mL of LB medium, and cultivate overnight at 37° C. and 200 rpm.

(3) Transfer to a conical flask containing 100 mL of LB medium at 1% inoculum volume (v/v), cultivate at 37° C., 200 rpm for 2-3 hours, and the OD600 of the bacterial solution is 0.4-0.6.

(4) Use an ice bath to cool the cultured bacterial liquid, and the ice bath time is about 30 minutes, with occasional mixing in the middle.

(5) Dispense the fully cooled bacterial solution into pre-cooled, sterile polypropylene tubes with a volume of 50 mL in the ultra-clean bench, centrifuge at 5000 rpm for 10 min at 4°C, and discard the supernatant.

(6) Add 35 mL of pre-cooled calcium chloride-magnesium chloride solution to the polypropylene tube, tap gently to resuspend the cells, centrifuge at 5000 rpm for 5 min at 4°C, and discard the supernatant.

(7) Repeat step 6 twice.

(8) Add 4 mL of pre-cooled glycerol-calcium chloride solution to the polypropylene tube, tap gently to resuspend the cells, and dispense the competent cells into sterile, pre-cooled EP tubes on ice, 100 μL per tube , refrigerate at -80°C.

4. DNA molecule transforms E. coli competent cells

(1) Take out the E. coli competent cells from -80°C, quickly insert them into ice, and let the E. coli competent cells melt naturally in the ice.

(2) Pipette an appropriate amount of the DNA molecule solution prepared in step 1 with a micropipette and add it to an EP tube containing 100 μL of E. coli competent cells. After mixing, let it stand in an ice bath for 30 min.

(3) Quickly put the EP tube into a 42°C water bath, and heat shock for 90s under the condition of standing.

(4) Immediately after the end of the heat shock, take out the EP tube and let it stand in an ice bath for 3 min.

(5) Add 200 μL of SOC medium to the EP tube, and culture at 37° C. and 200 rpm for 1 h.

(6) Take an appropriate volume of the culture and spread it on the LA plate containing the corresponding antibiotics, and place it in a 37°C constant temperature incubator to invert for 12-16 hours.

Example 2 Inducible expression of recombinant substrate binding protein

(1) Pick well-grown single colonies from the transformation plates and put them into straight-shaped flasks containing 5 mL of LB medium (containing 50 μg/mL kanamycin, 25 μg/mL chloramphenicol), at 37°C, 200 rpm Incubate for 10h.

(2) Transfer to conical flasks containing 65mL LB medium (containing 50μg/mL kanamycin, 25μg/mL chloramphenicol) according to the inoculum amount of 1% (v/v), at 37°C, Incubate overnight at 200 rpm.

(3) Transfer to conical flasks containing 1L LB medium (containing 50 μg/mL kanamycin, 25 μg/mL chloramphenicol) at 1% (v/v) inoculum respectively, at 37°C, Incubate at 200rpm for 2-3h, and add IPTG with a final concentration of 1mmol/L when the OD600 of the bacterial solution reaches 0.4-0.6.

(4) The recombinant strain E.coli Rosetta(DE3)/pET-Ssc-scaB was cultured at 16℃ and 180rpm for 20h to induce the 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 6 L bacterial liquid, centrifuge at 8000 rpm for 10 min to collect the bacterial cells.

(2) Resuspend the cells with 300mL Lysis buffer (pH 8.0, imidazole 10mmol/L, sodium chloride 300mmol/L, sodium dihydrogen phosphate 50mmol/L), add 3mL 25mg/mL lysozyme, mix well and put on ice Let stand for 20 minutes.

(3) Using an ultrasonic cell disruptor in the ice-water mixture to disrupt the cells, the total disruption time is 20 minutes, the working time is 5s, the intermittent time is 5s, and the disruption power is 30%.

(4) After the cell disruption, the recombinant strain E.coliRosetta(DE3)/pET-Ssc-scaB was centrifuged at 4°C and 12000rpm for 30min, and the supernatant after centrifugation was the crude protein solution.

(5) Pipette 2 mL of Ni-NTA fillers into the chromatography column respectively, and fully equilibrate the chromatographic column with Lysis buffer (pH 8.0, imidazole 10 mmol/L, sodium chloride 300 mmol/L, sodium dihydrogen phosphate 50 mmol/L). Ni-NTA filler.

(6) Mix the crude protein solution with the Ni-NTA filler equilibrated with Lysis buffer, and place it at 10°C and 150 rpm for 1 h to fully bind the recombinant substrate-binding protein containing histidine tag to the Ni-NTA filler.

(7) Transfer the mixture to the chromatography column and empty the liquid, add 10 mL of pre-cooled Washbuffer (pH 8.0, sodium chloride 300 mmol/L, sodium dihydrogen phosphate 50 mmol/L, purify Bbr to the chromatography column) -The concentration of imidazole was 20 mmol/L for CLDE and Ssc-SCAB, and the concentration of imidazole for purification of Pfu-CBTA and Tma-CBTA was 40 mmol/L), gently resuspend Ni-NTA, and empty the liquid in the column, repeat 4 times .

(8) After thorough washing, add 10 mL of pre-cooled Elution buffer (pH 8.0, imidazole 250 mmol/L, sodium chloride 300 mmol/L, sodium dihydrogen phosphate 50 mmol/L) to the column to elute the substrate-binding protein, and collect The eluate was repeated 3 times, and the eluate containing the substrate-binding protein was stored at 4°C.

(9) After the elution of the substrate-binding protein is completed, the Ni-NTA packing is washed several times with Elution buffer, and stored with 20% ethanol solution after washing. When purifying the same substrate-binding protein, the Ni-NTA packing can be reused 3-5 times, and the Ni-NTA packing can be regenerated after the binding rate decreases.

2. Desalting of Substrate Binding Proteins

(1) Transfer the eluate containing the substrate-binding protein to a 30kDa ultrafiltration tube. When the eluate is large, it can be transferred in multiple times. Centrifuge at 5000rpm for 30min at 4°C, and discard the waste liquid in the collection tube. .

(2) When the eluate in the ultrafiltration tube is about 500 μL, add buffer A solution (pH 7.0, 50 mM Tris-HCl, 100 mM NaCl) to the ultrafiltration tube, centrifuge at 5000 rpm for 30 min at 4°C, and discard the collection tube in the waste liquid.

(3) Repeat step 2 twice.

(4) Transfer the solution containing the substrate-binding protein in the ultrafiltration tube and the ultrafiltrate in the collection tube to a clean EP tube and a straight bottle, respectively, and store at 4°C.

3. Drawing and Quantitative Analysis of Protein Standard Curve

Pipette 100μL of bovine serum albumin BSA standard (2mg/mL), add 1900μL of ddH20 and mix well, dilute the BSA standard in a 1.5mL EP tube with diluent (ddH2O, 0.9% NaCl or PBS) according to Table 2-4, Three parallels for each concentration.

Table 1 Determination of bovine serum protein standard curve

Pipette 20 μL of the diluted BSA standard solution into 1.5 mL EP tubes, and make three parallels for each concentration. Add 1 mL of rewarmed Broadford Dye Reagent to each tube, mix well, and react at room temperature for 5 min. Take 200 μL of each and add 96 Orifice plate, use a microplate reader to measure the absorbance value at 600nm, calculate the average value of each concentration and subtract the average value of Blank, take the protein content as the abscissa and the reading of OD600 as the ordinate, draw the standard curve of the BSA standard solution (figure 1).

Quantification of purified substrate-binding protein: Take 20 μL of the target protein solution of different dilutions and add 1 mL of rewarmed Broadford Dye Reagent, mix well, react at room temperature for 5 min, add 200 μL of each to the ELISA plate, read the absorbance value of OD600, Substitute into the standard curve to calculate the mass concentration of the target protein, and the mass concentration is 6.288107 mg/ml.

Example 4 SDS-PAGE analysis of substrate binding proteins

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is used to analyze the expression of substrate-binding protein and the molecular weight and purification degree of purified substrate-binding protein. The experimental steps are as follows:

(1) Wash the glue maker, glue comb, glass plate and Spacer, and assemble them. Check the tightness with water. If the tightness is good, discard the water in the glass plywood layer and dry it with filter paper.

(2) Prepare the separating gel and stacking gel solutions according to Table 2-5. After mixing, pour the separating gel solution into the interlayer of the glass plate. After pouring to a suitable height, add 1 mL of absolute ethanol and let it stand at room temperature for 30 minutes.

(3) After the separation gel has solidified, discard the absolute ethanol in the glass interlayer, blot it dry with filter paper, quickly pour the concentrated gel solution, fill the glass plate interlayer and insert the glue comb, and let it stand at room temperature for 30 minutes.

(4) After the concentrated gel is solidified, put the gel into the electrophoresis tank, rinse the gel hole with electrode buffer, and perform pre-electrophoresis with a constant current of 10 mA for 15 min.

Table 2 SDS-polyacrylamide gel formulation

(5) Mix 30 μL of substrate-binding protein solution with 10 μL of 4×protein loading buffer, take a boiling water bath for 30 min, and centrifuge at 12000 rpm for 10 min.

(6) Take 4-10 μL of the supernatant respectively and add it to the polyacrylamide gel well. After the sample is loaded, electrophoresis is carried out at a constant voltage of 120V until the bromophenol blue band reaches the bottom of the polyacrylamide gel.

(7) After electrophoresis, pry the glass plate open, take out the polyacrylamide gel carefully, put it into Coomassie brilliant blue R-250 staining solution for staining, and stain at 25°C and 60rpm for 30min.

(8) After dyeing, discard the Coomassie brilliant blue R-250 dyeing solution, rinse with water, add an appropriate amount of decolorizing solution to decolorize, decolorize at 25°C, 60 rpm, until the polyacrylamide gel has no obvious background color, and several times are required in the middle. Change the destaining solution.

(9) After the polyacrylamide gel is fully destained, use the imager Gel DocTM XR+ to obtain an ideal SDS-polyacrylamide gel electrophoresis image.

As shown in Figure 2, the induced recombinant strains were purified by ultrasonic blasting respectively, and obvious protein characteristic bands appeared at the corresponding molecular weights, and Ssc-SCAB (Lane 2) appeared characteristic protein bands at 46.6kDa The band indicates that we have successfully expressed these four substrate-binding proteins, and the purified Ssc-SCAB protein solution is of high purity.

Example 5 Determination of the affinity between substrate binding proteins and cellooligosaccharides by isothermal titration calorimetry (ITC)

1. Various Cellooligosaccharide Titration Substrate Binding Proteins

(1) Dilute the substrate-binding protein solution to 0.1-0.2 mmol/L with the ultrafiltrate in the last collection tube and prepare various cellooligosaccharide solutions with a concentration of about 10 times the concentration of the substrate-binding protein.

(2) Vacuum degassing at 4°C for 10-20min, and set each parameter of isothermal titration.

Titration conditions for the substrate binding protein Ssc-SCAB:

The titrated concentration of substrate-binding protein was 0.1 mmol/L, the titrated concentration of various cellooligosaccharides was 4 mmol/L, the volume of the injection needle was 40 μL, the suction filtration time of the injection needle was 5 s, and the duration of each titration was 6 s. The volume of the first drop is 0.1 μL, the volume of the second to 20 drops is 1.5 μL, a total of 20 drops, the interval between two drops is 120 s, the sample cell temperature is 35 °C, the reference energy value is 10 μcal/sec, and the initial extension time is 1 min. The needle stirring speed was 500 rpm, and the feedback mode was low.

2. Thermodynamic parameters of substrate-binding proteins

Using Origin ITC 200 software for data analysis and curve fitting, in an isothermal titration experiment, ITC can determine the affinity constant Kb, the stoichiometric number n, the binding enthalpy change ΔH and entropy change ΔS, according to the experimentally measured thermodynamics The Gibbs free energy ΔG can be calculated from the parameters and the Gibbs-Helmholtz equation.

3. Results

Isothermal titration calorimetry (ITC) was used to determine the binding properties of substrate-binding proteins to cellooligosaccharides in buffer A. When isothermal titration was performed at 30°C or 50°C, equal volumes of cellooligosaccharides were dropped into cells filled with substrate binding. in the sample cell of the protein (the first drop is smaller in volume). If the substrate-binding protein binds to cellooligosaccharide, heat is released and recorded to form an energy peak. The first few drops generate more energy, and the peak value is higher. The binding protein tends to be saturated, less and less energy is generated, and the peak is lower and lower. When the substrate binding protein in the sample cell is completely saturated, the recorded peak is the heat generated by the dilution of the cellooligosaccharide, stoichiometric The number n also does not change; if the substrate binding protein is not bound to the cellooligosaccharide, there is only a small peak due to the exothermic dilution of the dilution. Under the same titration conditions, the heat of dilution generated when various cello-oligosaccharides were titrated with buffer A was measured as a blank, and the background was subtracted point-to-point during data analysis.

Isothermal titration calorimetry can directly determine the binding enthalpy change (ΔH), entropy change (ΔS), binding constant (K _b ) and stoichiometric number (n) when the substrate-binding protein is bound to cellooligosaccharide. , and at the same time, the Gibbs free energy change (ΔG) and _transition temperature (T) of the binding reaction can be calculated according to the thermodynamic parameters measured experimentally. The substrate-binding protein Pfu-CBTA was titrated with cellobiose, cellotriose, cellotetraose, and cellopentose, respectively. The titration results were analyzed by curve fitting using a binding site model. According to the obtained dissociation constant K _d , It is suggested that all substrate-binding proteins have different degrees of affinity for their respective substrates, and some substrate-binding proteins still have weak heat capacity changes after isothermal titration saturation, which may be due to the interaction between cellooligosaccharides and substrate-binding proteins. Caused by nonspecific interactions and/or dilution of cellooligosaccharides.

DSC thermodynamic analysis was performed using the substrate-binding protein Ssc-SCAB with a final concentration of 0.022 mmol/L, and the results are shown in Figure 3 . After the substrate binding protein Ssc-SCAB is combined with cellobiose, cellotriose and cellotetraose, the Tm value is higher than that of Ssc-SCAB without cello-oligosaccharide, indicating that cellobiose, cellotriose and cellotetraose are closely related to cellobiose, cellotriose and cellotetraose. Following Ssc-SCAB binding, an internal conformational change of Ssc-SCAB is induced. Using the non-dimorphic mode in the analysis software, the thermal denaturation process of the substrate-binding protein Ssc-SCAB was fitted and analyzed. The results are shown in Figure 3. Tehoff enthalpy ΔH _V and calorimetric enthalpy ΔH (see Table 3 for details).

Table 3 DSC thermodynamic parameters of Ssc-SCAB

It can be seen from Table 3 that the ratio of △H>△Hv and △H/△Hv of the substrate binding protein Ssc-SCAB is equal to 3.15, indicating that Ssc-SCAB is a monomer protein and contains more than two domains. , Ssc-SCAB has only one heat capacity scanning peak during the denaturation process, there are two possible reasons: one is that the multiple domains inside Ssc-SCAB have the same or similar melting temperature Tm and enthalpy change ΔH during the denaturation process , and the denaturation processes of multiple domains are independent of each other and do not interfere with each other, and the heat capacity scanning peaks overlap; the second is that the molecular weights of each domain are different, and the heat capacity peaks formed when the domains with small molecular weights are subjected to microcalorimetric differential scanning It is not obvious, and it does not appear within the set coordinate range. Substrate binding protein Ssc-SCAB and its complexes with various cellooligosaccharides have the relationship of △H _V ﹤△H, indicating that the denaturation process of substrate binding protein Ssc-SCAB is a non-dimorphic transition mode. After Ssc-SCAB was combined with cellobiose, cellotriose and cellotetraose, both ΔH _V and ΔH increased, the increase of _{ΔHV was more obvious, the ratio of ΔH V /} ΔH increased, and the unfolded The temperature range is narrower than that of Ssc-SCAB without cellooligosaccharide in the natural state, indicating that after Ssc-SCAB forms a complex with cellooligosaccharide, the synergistic effect of the non-dimorphic denaturation process of Ssc-SCAB is significantly enhanced, and the directional dimorphic process is significantly enhanced. trend of changing state patterns.

Combining Table 3 and Figure 3, it can be seen that cellobiose (Fig. 3-B), cellotriose (Fig. 3-C), and cellotetraose (Fig. 3-D) bind to the native substrate-binding protein Ssc-SCAB, so , the stability of the complex formed by cellobiose, cellotriose, cellotetraose and Ssc-SCAB was improved, and the respective Tm values were higher than that of the substrate-binding protein Ssp-SCAB in the natural state without cello-oligosaccharide. After glucose was added to Ssc-SCAB, its Tm value was 48.96 °C, which was almost unchanged compared with the Tm value of 48.66 °C of the substrate-binding protein Ssc-SCAB in its native state without cellooligosaccharide binding, indicating that the substrate-binding protein Ssc-SCAB SCAB did not bind glucose (Fig. 3-A). The Tm value of the substrate-binding protein Ssc-SCAB and cellopentose is 51.64℃, and the total enthalpy becomes 2.62×10 ⁵ cal/mol and the Tm of the substrate-binding protein Ssc-SCAB in the native state of unbound cello-oligosaccharide The total enthalpy change was 2.32×10 ⁵ cal/mol compared with the temperature of 48.66°C, which showed little change, indicating that the substrate-binding protein Ssc-SCAB did not bind to cellopentose (Fig. 3-E). The slight change in enthalpy change may be due to the large molecular weight of cellopentose, which has a non-specific interaction with Ssc-SCAB, which can be further verified by isothermal titration calorimetry in the later stage.

The substrate-binding protein Ssc-SCAB was titrated with a cello-oligosaccharide solution with a final concentration of 4 mmol/L, and the binding thermodynamic properties of Ssc-SCAB were determined. The results are shown in Figure 3 . Ssc-SCAB can be combined with cellobiose, cellotriose and cellotetraose, and the results of isothermal titration calorimetry are consistent with those of microcalorimetry. The titration results were fitted and analyzed using a binding site model, and the binding thermodynamic parameters are shown in Table 4.

Table 4 Thermodynamic parameters of the interaction between the substrate binding protein Ssc-SCAB and cellooligosaccharide

It can be seen from Table 4 that Ssc-SCAB binds to cellobiose, cellotriose and cellotetraose, and has the strongest affinity for cellobiose. The binding constant K _b is (1.32±0.09)×10 ⁶ M ^-1 . It has the weakest affinity for cellotriose, and the binding constant K _b is (1.22±0.04)×10 ⁶ M ^-1 .

Table 5 Thermodynamic parameters of substrate binding protein Ssc-SCAB

From Table 4 and Table 5, it can be seen that Ssc-SCAB has the strongest affinity for cellobiose (Fig. 4-A), and its dissociation constant K _d measured at 35 °C is 0.76 μM, and cellotriose (Fig. 4-B) Next, with a dissociation constant of 0.82 μM, the affinity for cellotetraose (Fig. 4-C) was the weakest with a dissociation constant of 1.06 μM. It can be seen from Table 5 that the binding entropy changes (ΔS) of Ssc-SCAB and various cellooligosaccharides (except cellopentose (Fig. 4-D)) are all less than 0, indicating that Ssc-SCAB and various cellooligosaccharides ( The binding reaction of Ssc-SCAB and various cello-oligosaccharides (except cellopentose) is a spontaneous forward irreversible process under the experimental conditions; the change in binding enthalpy (ΔH) is less than 0, indicating that Ssc-SCAB and various cello-oligosaccharides (except cellopentose) have a positive effect. The binding reaction is an exothermic reaction under the experimental conditions; △S, △H, and △G are all less than 0. It can be judged that under the experimental conditions, the substrate-binding protein Ssc-SCAB interacts with various cellooligosaccharides (fiberoligosaccharides). The binding reaction of pentasaccharide) is an enthalpy-driven forward spontaneous irreversible reaction, and the temperature T plays an active role. The stronger the affinity between cellooligosaccharide and the substrate-binding protein, the more stable the cellooligosaccharide-substrate-binding protein complex is formed, and vice versa, the easier the complex is to dissociate. When the Gibbs function ΔG of the complex is less than 0, the complex is stable, and the larger the negative value, the more stable it is.

The above-mentioned embodiments are only to describe the preferred modes of the present invention, but not to limit the scope of the present invention. Without departing from the design spirit of the present invention, those of ordinary skill in the art can make various modifications to the technical solutions of the present invention. Variations and improvements should fall within the protection scope determined by the claims of the present invention.

sequence listing

<110> Guangxi Academy of Sciences

<120> A Streptomyces cellooligosaccharide transporter gene

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1365

<212> DNA

<213> 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

<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 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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