A substrate-binding domain-based processive endoglucanase fusion protein and its application

Abstract

A continuous endoglucanase fusion protein based on a substrate binding domain and its application, the amino acid sequence is shown in SEQ ID NO: 1. The comprehensive enzyme activity of the fusion protein VvCBM1‑F3 after Linker optimization was significantly improved by 3.7 times compared with the original enzyme, showing excellent catalytic efficiency. The optimum pH and optimum temperature of the fusion protein are maintained at 6.0 and 60°C, respectively, which are consistent with the original enzyme, providing a strong guarantee for its stability and adaptability in practical applications. Although the thermal stability is reduced, the affinity of the fusion protein for insoluble substrates has been significantly enhanced, which makes it more advantageous in processing complex substrates. The hydrolysis products are mainly cellobiose and cellotriose, which facilitates subsequent biotransformation and utilization. In summary, the fusion protein described in the present invention has broad application prospects and important practical value in the field of cellulose enzymatic hydrolysis.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to a continuous endoglucanase fusion protein based on a substrate binding domain and an application thereof. Background Art

[0002] Cellulose is the most abundant renewable resource on earth. The use of enzyme degradation technology to convert cellulosic biomass into biofuels and chemicals can not only effectively cope with the energy crisis, but also has important significance for promoting the sustainable development of society. However, the large number of enzymes required in the current cellulose degradation process has led to high enzyme application costs, which seriously restricts the cellulose biorefining process and is a bottleneck factor for the commercialization of cellulosic ethanol and high-value chemicals. Persistent endoglucanases have both endo- and exo-functionality, and have higher thermal stability and lower product inhibition than exo-glucanases. They have great application value in reducing the enzyme cost of cellulose bioconversion and promoting the large-scale application of cellulose enzymatic hydrolysis technology. Persistent endoglucanases are usually composed of multiple domains. In addition to the catalytic domain (CD), they usually contain one or more substrate binding modules (carbohydrate binding module, CBM). Usually, CBM can assist the recognition and binding of enzymes to substrate molecules, improve the affinity between enzymes and substrates, and thus improve the catalytic reaction efficiency of enzymes. In addition, when CBM is adsorbed on the fiber surface, it can promote the breaking of hydrogen bonds inside the microfibrils and reduce the crystallinity of crystalline cellulose, thereby increasing the accessibility of cellulose and making it easier to be hydrolyzed by the catalytic module. In the continuous endoglucanase, CBM not only affects the substrate specificity and catalytic efficiency of the enzyme, but also has a multi-faceted impact on the continuous catalytic action of the enzyme. However, the existing continuous endoglucanase still has problems such as weak exoenzyme activity and poor continuous hydrolysis ability. There are also few successful cases reported on the fusion of CBM with the catalytic domain to improve the catalytic efficiency of cellulase, which affects the application of the enzyme in low-cost and efficient degradation of cellulose. In addition, considering that the sequential direct fusion of CBM may lead to the weakening or loss of enzyme function, it is generally believed that introducing a linker between the gene sequences of the two domains is the most effective optimization method. Therefore, by fusing different substrate binding domains and optimizing the connecting peptide to obtain a persistent endoglucanase with high catalytic activity and high sustained hydrolysis ability, we can not only deepen our understanding of the structure and function of cellulase, but also provide support for further rational design and modification of it to obtain cellulase components that can meet the efficient conversion of biomass. Summary of the invention

[0003] Technical problem to be solved: The present invention provides a substrate-binding domain-based persistent endoglucanase fusion protein and its application. The persistent endoglucanase EG5C-1 from Bacillus subtilis is used as the research object. A series of EG5C-1 fusion proteins are successfully constructed by using gene fusion technology. Through the careful design of the linker, a fusion protein with improved hydrolysis activity on insoluble cellulose substrates is obtained. V CBM1-F3.

[0004] Technical solution: A processive endoglucanase fusion protein based on substrate binding domain V CBM1-F3, the amino acid sequence is shown in SEQ ID NO:1.

[0005] The gene expressing the above fusion protein has a nucleic acid sequence as shown in SEQ ID NO:2.

[0006] An expression vector containing the above gene.

[0007] The above expression vector is a plasmid, a phage or a virus.

[0008] A host cell containing the above vector.

[0009] The host cell is Escherichia coli, yeast, Bacillus, Lactobacillus, Aspergillus or Trichoderma.

[0010] An enzyme preparation comprising the above fusion protein.

[0011] A mutant of the gene sequence encoding the fusion protein, wherein the protein encoded by the mutant gene has at least 80% sequence homology with the amino acid sequence shown in SEQ ID NO: 1 and retains endoglucanase activity.

[0012] Application of the above fusion protein in degrading cellulose substrate.

[0013] The present invention uses conventional techniques and methods used in the field of genetic engineering and molecular biology, for example, the methods described in Molecular cloning: a laboratory manual, 3rd ED. (Sambrook, 2001) and Current protocols in molecular biology (Ausubel, 2003). These general references provide definitions and methods known to those skilled in the art. However, those skilled in the art can adopt other conventional methods, experimental schemes and reagents in the art on the basis of the technical solutions described in the present invention, without being limited to the specific embodiments of the present invention.

[0014] Beneficial effects: The continuous endoglucanase EG5C-1 fusion protein of the present invention exhibits significantly improved enzyme activity, specifically, the activity on the cellulose substrates Avicel and PASC increased by 2.2 times and 1.3 times, respectively. In addition, the fusion protein after Linker optimization V CBM1-F3, its comprehensive enzyme activity (FPAase) is significantly increased by 3.7 times compared with the original enzyme, showing excellent catalytic efficiency. It is worth noting that the optimal pH and optimal temperature of the fusion protein are maintained at 6.0 and 60 ° C, respectively, which is consistent with the original enzyme, providing a strong guarantee for its stability and adaptability in practical applications. Although the thermal stability is reduced, the affinity of the fusion protein for insoluble substrates has been significantly enhanced, which makes it more advantageous in processing complex substrates. The hydrolysis products are mainly cellobiose and cellotriose, which facilitates subsequent biotransformation and utilization. In summary, the fusion protein described in the present invention has broad application prospects and important practical value in the field of cellulose enzymatic hydrolysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the fusion protein structure composed of CBMs from different strains and the processive endoglucanase EG5C-1.

[0016] Figure 2 Analysis of expression components of recombinant fusion protein: supernatant (SN); precipitate (P).

[0017] Figure 3 Schematic diagram of the structures of EG5C-1-VcCBM1, EG5C-1-EX and their derivatives with different linkers added.

[0018] Figure 4 SDS-PAGE of purified EG5C-1-VcCBM1 (A) and EG5C-1-EX (B) fusion proteins with different linkers.

[0019] Figure 5 Effects of temperature and pH on fusion protein V Effects of iodine on the activity and stability of CBM1-F3, (A) optimum temperature, (B) temperature stability, (C) optimum pH, (D) pH stability.

[0020] Figure 6 Original enzyme EG5C-1 and its fusion protein V Ratio of soluble and insoluble reducing sugars produced by CBM1-F3.

[0021] Figure 7 Original enzyme EG5C-1 and its fusion protein VAnalysis of hydrolysis products of different cellulosic substrates by CBM1-F3. DETAILED DESCRIPTION

[0022] Example 1 Construction of processive endoglucanase EG5C-1 fusion protein

[0023] To improve the source Bacillus subtilis The activity of the processive endoglucanase EG5C-1 (amino acid sequence as shown in SEQ ID NO: 3, nucleic acid sequence as shown in SEQ ID NO: 4) of the GH5 family was enhanced by fusing CBMs from different sources to the C-terminus of EG5C-1, such as Figure 1 As shown, the primers used are as follows:

[0024] Table 1 Primer sequences

[0025]

[0026] Using the processive endoglucanase EG5C-1 gene as a template and referring to the Vazyme biological product and operation manual, the site-directed mutation sequence was amplified from the whole plasmid. Dpn After enzyme digestion, the PCR product was transformed into Escherichia coli competent cells by heat shock method. E. coli BL21(DE3), and spread on LB agar plates containing 100 μg / mL kanamycin sulfate, and cultured at 37°C for 14-16 h. The mutation results were verified by sequence determination (completed by Anhui General Biological Company) to obtain the corresponding cellulase fusion protein.

[0027] Example 2 Expression and purification of processive endoglucanase EG5C-1 and fusion protein

[0028] The fusion protein constructed in Example 1 and the original enzyme EG5C-1 were inoculated into 50 mL of LB liquid medium, and kanamycin sulfate was added to a final concentration of 100 μg / mL. The culture was carried out at 180 rpm and 37 °C overnight. The seed solution of the overnight culture was inoculated into 50 mL of fresh LB liquid medium at a 2% inoculum, and the culture was carried out at 180 rpm and 37 °C until the OD 600 When the pH value was between 0.6 and 1.0, the inducer IPTG (isopropyl-β-D-thiogalactoside) (final concentration 0.1 mmol / L) was added and the expression was induced at 25 °C for 24 h.

[0029] Take the fermentation liquid of induced expression, centrifuge at 12000 rpm for 20 min, discard the supernatant, and then resuspend the bacteria with 50 mM Na2HPO4-KH2PO4 (pH 6.0) buffer, ultrasonically disrupt, and perform SDS-PAGE electrophoresis detection. The concentration of concentrated gel is 4%, the concentration of separation gel is 12.5%, the sample and loading buffer are mixed in a ratio of 3:1, and the sample is loaded in a boiling water bath for 5 min for electrophoresis. The initial voltage of the electrophoresis instrument is set to 120 V. When the sample moves to the separation gel, the voltage is increased to 230 V until the sample moves to the bottom of the electrophoresis tank and the electrophoresis is terminated.

[0030] The results are as follows Figure 2 As shown in the figure, seven recombinant fusion proteins were successfully heterologously expressed in Escherichia coli. Compared with the supernatant of the bacterial suspension containing the empty pET28a vector, the fusion proteins EG5C-1-3c-3b, EG5C-1-3c, EG5C-1-3b, EG5C-1- Hc CBM6, EG5C-1- Sd CBM6, EG5C-1- V CBM1 and EG5C-1-EX had obvious bands around 96 kDa, 42 kDa, 40 kDa, 64 kDa, 51 kDa, 42 kDa and 61 kDa, respectively, which were consistent with the theoretically predicted molecular weights, indicating that the cellulase EG5C-1 and each fusion protein were successfully induced to express.

[0031] Example 3 Determination of enzyme activity of processive endoglucanase EG5C-1-CBMs fusion protein

[0032] Definition of enzyme activity unit: Under the reaction conditions of 60 ℃ and pH 6.0, the amount of enzyme required to produce 1 μmol of reducing sugar from the substrate per minute is one unit (U).

[0033] (1) Determination of endocellulase activity: Using sodium carboxymethyl cellulose (CMC-Na) as the substrate, a 1% (g / mL) CMC solution was first prepared using Na2HPO4-KH2PO4 buffer (50 mM, pH 6.0). The reaction system was a mixture of 1.5 mL of preheated 1% CMC substrate and 500 μL of appropriately diluted enzyme solution, which was reacted at 60 °C for 10 min. 3 mL of DNS reagent was added to terminate the reaction and the cell was placed in a boiling water bath for 5 min. After rapid cooling to room temperature, the absorbance was measured at 540 nm.

[0034] (2) Exocellulase activity assay: Using microcrystalline cellulose (Avicel) as the substrate, first use Na2HPO4-KH2PO4 buffer (50 mM, pH 6.0) to prepare a 10% (g / mL) Avicel solution. The reaction system is 1.5 mL of preheated 1% Avicel substrate mixed with 500 μL of appropriately diluted enzyme solution, placed at 60 °C for 30 min, and the test tube is quickly placed in ice water to cool and terminate the reaction. Centrifuge at 12000 rpm to remove insoluble substrates, then add 3 mL of DNS reagent to terminate the reaction process and place it in a boiling water bath for 5 min. After quickly cooling to room temperature, the absorbance is measured at 540 nm.

[0035] The results are shown in Table 2. The fusion proteins EG5C-1-3c-3b, EG5C-1-3c, EG5C-1-3b, EG5C-1- Hc CBM6 and EG5C-1- Sd The activity of CBM6 on the soluble substrate CMC was significantly lower than that of the original enzyme EG5C-1. However, for the insoluble substrates Avicel and PASC, the activities of each fusion protein were different. Compared with the original enzyme EG5C-1, EG5C-1-3c-3b showed a slight increase in activity on Avicel and PASC, while the activities of EG5C-1-3c and EG5C-1-3b on these two substrates were significantly reduced. Hc CBM6 and EG5C-1- Sd CBM6 showed reduced activity on Avicel and PASC substrates. V CBM1 had little effect on the enzyme activity of the CMC substrate, but increased the activity of the Avicel and PASC substrates by 2.2 and 1.3 times, respectively. When the expansin EX was fused with EG5C-1, although the activity of the CMC and Avicel substrates was improved, especially the activity of Avicel was increased by 1.7 times compared with the original enzyme, the activity of the PASC substrate was significantly reduced. Based on the above results, we selected EG5C-1- V CBM1 and EG5C-1-EX were further studied.

[0036] Table 2 Substrate specificity of the original enzyme EG5C-1 and its fusion protein

[0037]

[0038] Example 4 Linker to EG5C-1- V Effects of CBM1 and EG5C-1-EX fusion proteins

[0039] like Figure 3As shown, EG5C-1- V CBM1 and EG5C-1-EX genes were used as templates to construct connecting peptides of different lengths. V CBM1 and EG5C-1-EX genes were used as templates to amplify the sequence of the whole plasmid. The primers used were as follows:

[0040] Table 3 Primer sequences

[0041]

[0042] Example 5 Construction, expression and purification of fusion proteins with different linkers

[0043] According to the method in the above embodiment 2, different linkers V CBM1-F1, V CBM1-F2, V CBM1-F3, V CBM1-F4, V CBM1-R1, V CBM1-R2, V CBM1-R3, V The fusion proteins of CBM1-R4 and EX-F1, EX-F2, EX-F3, EX-F4, EX-R1, EX-R2, EX-R3, and EX-R4 were expressed and purified. The results of SDS-PAGE are shown in Figure 4 As shown, the fusion protein V The CBM1 series (F1 to F4 and R1 to R4) showed a clear band at approximately 42 kDa, whereas the EX series fusion proteins (F1 to F4 and R1 to R4) showed a distinct band at approximately 62 kDa.

[0044] Example 6 Determination of enzyme activity with different linker fusion proteins added

[0045] According to the enzyme activity assay method of Example 3 above, the enzyme activity of fusion proteins containing different linkers was assayed using three different substrates, CMC, Avicel and PASC. The results are shown in Tables 4 and 5. VFor CBM1 and its derived fusion proteins, all fusion proteins showed a certain degree of decrease in activity on the soluble substrate CMC compared to the original enzyme EG5C-1, while on the insoluble substrates Avicel and PASC, we observed that too long or too short connecting peptides (F4 or R4 and F1 or R1) would have an adverse effect on the activity of the fusion proteins. On the contrary, the addition of connecting peptides of appropriate length (F2 or R2 and F3 or R3) can enhance the activity of the enzyme on insoluble substrates. In particular, V The activity of CBM1-F3 fusion protein on Avicel was higher than that of EG5C-1- V CBM1 increased by about 1.7-fold, and activity on PASC increased by about 1.4-fold; V The CBM1-R3 fusion protein also showed similar activity enhancement, and its activity against Avicel and PASC was comparable to that of EG5C-1- V CBM1 increased by 1.5-fold and 1.2-fold, respectively. For EG5C-1-EX and its fusion variants, although their activities on Avicel were generally enhanced, their activities on the PASC substrate showed the same results as the parent protein EG5C-1-EX, showing a clear downward trend. In addition, we also evaluated the total enzyme activity of these fusion proteins on filter paper. The results showed that EG5C-1- V The hydrolysis activity of CBM1 and its fusion proteins with different linkers on filter paper was significantly improved, especially V The activity of CBM1-F3 increased by about 3.7 times compared with the original enzyme EG5C-1. In contrast, the activity of the fusion protein with the addition of expansin on the filter paper decreased significantly.

[0046] Table 4 EG5C-1- Vc Substrate specificity of CBM1 and its different linker fusion proteins

[0047]

[0048] Table 5 Substrate specificity of EG5C-1-EX and its different linker fusion proteins

[0049]

[0050] Example 7 Optimal Fusion Protein V Study on the Enzymatic Properties of CBM1-F3

[0051] 1. Optimum temperature and temperature stability

[0052] With CMC as substrate, the purified enzyme solution was reacted at 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, and 90 ℃, respectively, and the changes in enzyme activity at different temperatures were investigated to determine the optimal reaction temperature. The purified enzyme solution was placed in a water bath at 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, and 90 ℃, and the residual activity of the enzyme after incubation for 2 h under different temperature conditions was measured to determine the changes in the thermal stability of the enzyme. The highest enzyme activity was taken as 100%, and the relative enzyme activity was calculated in turn, and a curve of enzyme activity changing with temperature was drawn.

[0053] As shown in Figure 5, A and Figure 5 As shown in B, the fusion enzyme V The optimum temperature of CBM1-F3 and the original enzyme EG5C-1 is 60℃. The stability curve of VvCBM1-F3 in the entire temperature gradient is always lower than that of the original enzyme EG5C-1. After incubation at 60℃ for 2h, the residual enzyme activity of the fusion enzyme dropped to 75%, while that of the original enzyme remained above 80%.

[0054] 2. Optimum pH and pH stability

[0055] Different pH buffers were prepared: citric acid-sodium citrate (pH 3.0-6.0), Na2HPO4-KH2PO4 (pH 6.0-8.0) and glycine-sodium hydroxide (pH 8.0-9.0). Using CMC as the substrate, the activity changes of the enzyme under different pH values ​​(3.0-9.0) were measured to determine the optimal reaction pH. In order to investigate the effect of different pH on enzyme activity, the purified enzyme solution was placed in buffers of different pH values, and the residual activity of the enzyme after incubation for 2 hours under different pH conditions was measured to determine the changes in the pH stability of the enzyme. The highest enzyme activity was taken as 100%, and the relative enzyme activity was calculated in turn, and a curve of enzyme activity changing with pH was drawn.

[0056] The results are as follows Figure 5 C and Figure 5 As shown in D, the fusion enzyme V The optimum pH of CBM1-F3 and the original enzyme EG5C-1 is 6.0, and the pH stability of the fusion enzyme is consistent with that of the original enzyme in the gradient range of pH 3.0-pH 9.0, indicating that the fusion substrate binding domain does not have a negative impact on the pH stability of the enzyme.

[0057] 3. Determination of kinetic parameters

[0058] The kinetic parameters of processive endoglucanase EG5C-1 and its fusion protein were studied using sodium carboxymethylcellulose (CMC) and phosphoric acid swollen cellulose (PASC) as substrates.

[0059] (1) CMC mother solution (20 mg / mL) was prepared into different gradient concentrations of CMC (0-20 mg / mL) in Na2HPO4-KH2PO4 buffer (50 mM, pH 6.0) as substrate, and the initial hydrolysis activity of the enzyme was determined under the optimal reaction conditions (pH 6.0, 60 ℃).

[0060] (2) The PASC stock solution (200 mg / mL) was diluted to different gradient concentrations (0-200 mg / mL) in Na2HPO4-KH2PO4 buffer (50 mM, pH 6.0) as the substrate, and the initial hydrolysis activity of the enzyme was measured under the optimal reaction conditions (pH 6.0, 60 ℃).

[0061] According to the Michaelis-Menten equation, the data were fitted with a nonlinear regression equation using GraphPad Prism 10 software to obtain the Michaelis constant. K m and maximum reaction speed V max, calculated k cat and k cat / K m value.

[0062] The results are shown in Table 6. V CBM1-F3 acts on the soluble substrate CMC K m The value increased from 13.78 mg / mL of the original enzyme to 16.47 mg / mL, indicating that after fusion of the substrate binding domain CBM1, V CBM1-F3 has a reduced affinity for CMC. However, its catalytic efficiency k cat / K m The value was reduced to 7.6 mL / mg / s, which was 43% lower than that of the original enzyme. This result was consistent with the previous enzyme activity measurement results. After the substrate domain was fused, the catalytic activity of almost all fusion enzymes towards CMC was reduced. However, for the insoluble substrate PASC, the fusion enzyme showed completely different properties. V CBM1-F3 K m The value decreased from 28.44 mg / mL of the original enzyme to 7.17 mg / mL, indicating that its affinity for insoluble substrates was significantly enhanced. At the same time, its catalytic efficiency k cat / K m The value also increased significantly to 2.00 mL / mg / s, which was nearly 2.6 times higher than that of the original enzyme. This result confirmed that the fusion enzyme V CBM1-F3 has high activity in degrading crystalline cellulose.

[0063] Table 6 Original enzyme EG5C-1 and its fusion protein V Kinetic parameters of CBM1-F3

[0064]

[0065] Example 8 Fusion Protein V Determination of binding and persistence parameters of CBM1-F3

[0066] 1. Determination of persistence parameters

[0067] Add 500 μL of appropriately diluted enzyme solution to 1.5 mL of 1% (g / mL) PASC substrate, and react at 45 °C for 30 min, 60 min, 120 min, and 180 min. After the reaction is completed, the reaction solution is immediately placed in a boiling water bath for 5 min to terminate the reaction process. Subsequently, the reaction solution is centrifuged at 12000 rpm for 10 min, and the resulting supernatant is the soluble reducing sugar. The precipitate is washed 3 to 4 times with Na2HPO4-KH2PO4 buffer (50 mM, pH 6.0) to remove the residual supernatant. The washed precipitate is mixed with an equal volume of buffer and resuspended to obtain the insoluble reducing sugar.

[0068] Definition of sustainability: PASC is used as a substrate and is determined by measuring the ratio of soluble reducing sugars to insoluble reducing sugars produced during the degradation of the substrate by the enzyme. The content of reducing sugars in the supernatant and precipitate at different reaction time points is measured by the DNS method to calculate the ratio of soluble to insoluble reducing sugars.

[0069] The results are as follows Figure 6 As shown, the processive endoglucanase EG5C-1 still maintains significant processive catalytic activity after fusion with the CBM1 substrate domain. V The ratio of the soluble reducing end to the insoluble reducing end of CBM1-F3 increased from 2.64 to 5.47 as the reaction time was extended from 30 min to 180 min. However, the highest value of this ratio of the original enzyme after 180 min of reaction was only 3.41. This result shows that the fusion of CBM1 did not weaken the enzyme's continuous catalytic ability, but significantly improved its continuous catalytic ability. Further observations found that V The CBM1-F3 fusion enzyme showed better sustained catalytic performance in the later stage of the catalytic reaction, especially after more than 120 min. This phenomenon may be attributed to the fact that the substrate binding domain CBM1 enhances the adsorption of the enzyme to crystalline cellulose, thereby improving the catalytic efficiency.

[0070] 2. Determination of binding parameters

[0071] According to the Langmuir isotherm adsorption model, a series of enzyme solutions with different concentrations (0-30 μM) were first prepared and mixed with 10 mg of Avicel substrate. The mixture was placed on an ice bath shaker at 200 rpm for 2 h to maintain the activity of the enzyme and prevent the effect of temperature on the reaction. After the reaction, the mixture was centrifuged at 12000 rpm for 10 min and the supernatant was collected. The protein concentration of the supernatant in each group was then determined using the BCA kit and recorded as q 1. At the same time, the free total protein concentration of the corresponding control group was determined and recorded as q , the amount of bound protein is recorded as q ad= q - q 1. Using GraphPad Prism 10 software, the amount of free protein [ q ] as the horizontal axis, the amount of bound protein [ q ad] as the ordinate, and fitted the regression line equation. Finally, according to the Langmuir formula [ q ad= q max* q / ( K d+ q )], and the maximum amount of binding protein was calculated by fitting the data q max and dissociation constant K d.

[0072] The results are shown in Table 7. V The dissociation constant of CBM1-F3 ( K d ) is 34.48 μM, which has a lower dissociation constant than the original enzyme EG5C-1 of 51.32 μM, indicating that the fusion of the substrate binding domain significantly improves the affinity between the enzyme and the substrate. In addition, V The maximum binding capacity of CBM1-F3 for the substrate Avicel ( q max ) and the partition coefficient ( α ) also showed significant enhancement, among which the distribution coefficient directly reflects the ability of the enzyme to bind to the substrate, which was 2.5 times higher than that of the original enzyme.

[0073] Table 7 Original enzyme EG5C-1 and its fusion protein V Binding capacity parameters of CBM1-F3

[0074]

[0075] Example 9 Fusion Protein VAnalysis of hydrolysate of CBM1-F3

[0076] In order to determine the hydrolysis products of the fusion protein, CMC, Avicel, and PASC substrates were reacted with the enzyme at 45 °C for 3 h (for CMC) and 6 h (for Avicel and PASC), respectively. After the reaction, the enzyme was inactivated by boiling, followed by centrifugation at 12,000 rpm for 10 min, and the supernatant was collected. The supernatant was mixed with pre-cooled anhydrous ethanol, shaken, and left overnight to precipitate and remove impurities. The sample was dried, and then an appropriate amount of pure water was added to dissolve the hydrolyzate. Finally, the hydrolyzate was analyzed by thin layer chromatography (TLC). Similar steps were used to investigate the hydrolysis products of cellooligosaccharides (cellobiose to cellopentaose). First, the enzyme was reacted at 45 °C for 1 h. The hydrolyzate was then operated and analyzed according to the above-mentioned method for treating the hydrolyzate.

[0077] TLC thin layer chromatography detection:

[0078] (1) Preparation of the developing agent: n-butanol, acetic acid and water were mixed in a volume ratio of 3:2:1 to prepare the developing agent. Before the experiment, the developing agent was added to the developing tank and left for about 30 minutes to ensure that the solvent was evenly distributed in the tank to avoid edge effects;

[0079] (2) Sample spotting: Standards (10 mg / mL): glucose (G1), cellobiose (G2), cellotriose (G3), cellotetraose (G4), cellopentaose (G5), 1 μL; test samples, 3 μL;

[0080] (3) Perform two spreading operations at room temperature, each lasting about 1.5 h;

[0081] (4) Color development and analysis: Use 5% sulfuric acid ethanol solution as the color developer. Place the chromatography plate under color development conditions, that is, heat it at 120°C for 5 minutes to color the sugars.

[0082] The results are as follows Figure 7 As shown, the fusion enzyme V The enzymatic hydrolysis products of CMC, Avicel and PASC by CBM1-F3 were mainly cellobiose and cellotriose. V CBM1-F3 had no further degradation ability for cellobiose, and the final hydrolysis products of cellotriose, tetrasaccharide and pentasaccharide were also cellobiose and cellotriose, which was consistent with the degradation pattern of the original enzyme EG5C-1. These results indicate that the fusion substrate binding domain did not change the hydrolysis characteristics and product specificity of the processive endoglucanase EG5C-1.

[0083] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A substrate binding domain-based processive endoglucanase fusion protein, characterized in that: The amino acid sequence is shown in SEQ ID NO:

1.

2. A gene expressing the fusion protein according to claim 1, characterized in that: The nucleic acid sequence is shown in SEQ ID NO:

2.

3. An expression vector containing the gene according to claim 2.

4. The expression vector according to claim 3, characterized in that The expression vector is a plasmid or a virus.

5. A host cell containing the vector according to claim 3.

6. The host cell according to claim 5, characterized in that The host cell is Escherichia coli or Bacillus.

7. An enzyme preparation comprising the fusion protein of claim 1.

8. Use of the fusion protein according to claim 1 in degrading cellulose substrates.

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