In this study, to improve the thermostability of recombinant xylanase XynC, molecular dynamics simulation analysis was performed based on the 3D structure of the enzyme to identify its flexible regions. FoldX software was employed to conduct virtual saturation mutagenesis on the sites within the enzyme's flexible regions, and the mutational stability of the enzyme was calculated. The structural stability of the mutations was evaluated using free energy change, leading to the identification of six mutation sites. These mutation sites were then expressed in Komagataella pastoris to verify the changes in thermostability of different mutant proteins. Finally, through screening via shake flask culture, two mutant proteins with enhanced thermostability—XynC-D57R and XynC-N88W—were obtained. After heat treatment at 80 °C for 1 hour, their residual enzyme activities were 49.4 % and 42.3 %, respectively. In contrast, the residual enzyme activity of the wild-type XynC was only 35.0 % after heat treatment at 80 °C for 1 hour. The thermostability of the mutants was significantly improved compared with that of the wild-type XynC.

Using DH5α-xynC / DH5α-xynC-CBM (Stored in our laboratory) as the template, mutagenic primers were designed based on the amino acids at the mutation sites, and the codon preference of Komagataella pastoris was checked. The specific primer sequences are shown in Table 1.1, and the designed primer sequences were synthesized by Tsingke Biotechnology Co., Ltd (Xi’an, China).

Site-directed mutagenesis was performed using DH5α-xynC as the template with primers listed in Table 1.1. The PCR reaction mixture and cycling conditions are detailed in Table 1.2 and Table 1.3, respectively.

A 10 μL aliquot of the PCR product was digested with Dpn I restriction enzyme (Takara, Beijing, China) to eliminate the methylated parental DNA template. The reaction was incubated at 37 °C for a minimum of 3 hours. The detailed digestion mixture (150 μL) is provided in Table 1.4.

Take 100 μL of DH5α competent cells and transfer them to an ice bath for 15~20 seconds. Add the PCR product to be transformed, with a volume equal to one-tenth of that of the competent cell suspension, mix well, and place in the ice bath for 30 minutes. Incubate the centrifuge tube in a 42 °C water bath for 60 seconds, then immediately transfer it to the ice bath for cooling for 2~3 minutes. Add 900 μL of fresh LB medium pre-warmed to 37 °C, and culture with shaking in a shaker for 45 minutes at 37 °C and 200 rpm. After centrifugation at 4000 rpm for 5 minutes, discard 900 μl of the supernatant and spread the remainder onto LB solid plates containing 25 μg/mL Zeocin®. Place the plate in a 37 °C constant-temperature incubator for overnight culture.

Pick well-isolated single colonies with uniform morphology on the plate, and inoculate it into 5 mL of LB medium at 37 °C and 200 rpm for overnight cultivation. The culture is submitted to Tsingke Biotechnology Co., Ltd (Xi’an, China) for sequencing.

The activity assay method of xylanase during thermostability screening is performed in accordance with the Chinese National Standards (GB/T 23874-2009). The strains with higher activity obtained from the screening process will be re-inoculated into 50 mL YPD medium for overnight cultivation. The supernatant from the shake flask culture will be aliquoted into centrifuge tubes and incubated in an 80°C water bath for 1 hour. The non-heat-treated xylanase was used as the control group, and its enzyme activity was defined as 100 %. The residual activity of all treatment groups is expressed as a percentage of the control group.

(1) Standard curve:

Take 2 mL of xylose standard solutions with concentrations of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 and 0.7 mg/mL. Add 2 mL of buffer and 5 mL of DNS, then vortex for 5 seconds, heat in boiling water bath for 5 minutes, cool to room temperature, and make up to 25 mL with up water. Use up water as the blank for zeroing. Detect the absorbance at 540 nm. Plot the standard curve with the concentration of xylose as the x-axis and the absorbance at 540 nm as the y-axis.

(2) Sample Enzyme Activity Assay

Mix the 20 μL enzyme sample with 20 μL of a 5 mg/mL Xylan Solution. Incubate at 37 °C for 30 minutes. Add 50 μL of DNS reagent, mix well, and heat in a boiling water bath for 5 minutes. Add 160 μL of up water, mix well, and centrifuge at 10,000×g for 1 minute at room temperature. Finally, take 200 μL of the reaction mixture and measure the absorbance at 540 nm. One unit of enzyme activity (U) is defined as the amount of enzyme required to release 1 μmol of reducing sugar per minute from a 5 mg/ml Xylan Solution under standard conditions (37 °C, pH 5.5).

Pick single colony into 100 mL LB medium containing 50 mg/L Zeocin®. The culture was then carried out at 37 °C at a speed of 200 rpm for 12~16 hours. The plasmid was extracted using E.Z.N.A.® Plasmid DNA Mini Kit I (Omega Bio-Tek, USA) in accordance with the instruction manual of the E.Z.N.A.® Plasmid DNA Mini Kit I (Omega Bio-Tek, USA).

The plasmid and Avr II restriction enzyme (Takara, Beijing, China) are mixed and added to a 1.5 mL centrifuge tube for the linearization reaction. The linearization reaction mixture is as shown in the table below:

After being mixed evenly, it was placed in a 37 °C constant temperature incubator for incubation for 3~5 hours. The products were verified by DNA agarose gel electrophoresis. All products were subsequently recovered and purified by phenol-chloroform extraction (phenol:chloroform:isoamyl alcohol = 25:24:1, pH 8.0, Thermo Fisher Scientific, USA).

Take 80 μl of freshly prepared GS115 Komagataella pastoris competent cells, place them on ice, add 10 μg of linearized plasmid, gently mix with a pipette gun, and transfer to a 2 cm electroporation cup. After incubation on ice for 10 minutes, use the 1652100 MicroPulser electroporation instrument (BIO-RAD, USA) to set the Komagataella pastoris mode, place the electroporation cup in the electroporation instrument, perform one pulse, immediately add 900 μl of 1 mol/L ice-cold sorbitol. Mix well by pipetting, then incubate at 30 °C, 200 rpm for 1 hour. Take 100 μL of the incubation product and spread it on a YPD agar plate containing 50 mg/L Zeocin®, place it at 30 °C for cultivation until a monoclonal colony grows.

Then, 100 μL of the product was spread onto YPD agar plates containing 50 mg/L Zeocin® to screen for positive transformants. The plates were incubated at 30 °C for 48~72 hours until single colonies appeared.

The tip of a sterilized toothpick was used to pick individual colonies, which were then inoculated onto YPD agar plates containing 1 mg/mL Zeocin®. The plates were incubated at 30 °C for 24~48 hours until single colonies formed.

Select well-isolated single colonies with uniform morphology from the high-concentration antibiotic culture plate, and inoculate them into 500 μL of YPD medium (in a 96-well U-bottom deep-well plate). After overnight cultivation at 30 °C and 200 rpm, their xylanase activities were determined according to the Chinese national standard (GB/T 23874-2009). Select the top 12 recombinant strains with the highest activity for further confirmation via shake flask culture.

This study constructed a heterologous expression system for Pro-Xylane Synthase via molecular cloning technology. It successfully achieved the functional verification of phosphite dehydrogenase (PTDH, encoded by the ptxD gene) and sorbitol dehydrogenase (RDH, encoded by the rbtD gene) in E.coli, and realized the functional reconstitution of PTDH and RDH in the recombinant Komagataella pastoris system. Through transformation and screening, a recombinant plasmid capable of characterizing catalytic activity by the production of coenzyme NADH was successfully constructed.

Based on previous research results, gene mining was conducted. Screening was performed from the aspects of sequence homology, protein solubility, and active site mutation status. Finally, Pro-xylane Synthase genes with high homology, good solubility, and no active site mutations—ptxD and rbtD—were obtained (the protein encoded by ptxD is abbreviated as PTDH, and the protein encoded by rbtD is abbreviated as RDH).

Retrieve the PTDH protein sequence (accession number: AAC71709.1) and RDH protein sequence (accession number: VIO69643.1) from the NCBI database, and entrust a company (Tsingke Biotechnology Co., Ltd (Xi’an, China)) to conduct gene synthesis. The target genes were cloned into the pET-28a plasmid vector via linearization, with the C-terminal HIS tag retained. The recombinant vectors pET-28a-ptxD and pET-28a-rbtD were constructed and transformed into E. coli DH5α to obtain pET-28a-ptxD-DH5α and pET-28a-rbtD-DH5α glycerol stocks.

Take 100 μL each of pET-28a-ptxD-DH5α and pET-28a-rbtD-DH5α bacterial solutions, and inoculate them into 50 mL LB medium containing 50 mg/L kanamycin sulfate. Culture at 37 °C with 200 rpm for 12~16 h, then streak on LB solid medium containing 50 mg/L kanamycin sulfate to isolate single colonies.

Pick single colonies with good morphology and uniform size, and inoculate them into 100 mL LB medium containing 50 mg/L kanamycin sulfate (to obtain sufficient recombinant plasmids). Culture at 37 °C with 200 rpm for 12~16 h. Extract plasmids using the E.Z.N.A.® Plasmid DNA Mini Kit I (Omega Bio-Tek, USA), and store the plasmids at -20 °C by freezing.

In this experiment, E. coli BL21(DE3) competent cells prepared by the CaCl₂ treatment method were used as the host strain. Recombinant plasmids were transformed into the competent cells via heat shock at 42 °C. The specific transformation process is as follows:

Take BL21(DE3) competent cells from the -80 °C refrigerator and all subsequent operations are performed on ice. Add 1 μL of recombinant plasmid, gently flick the centrifuge tube to mix, and incubate on ice for 30 min. Perform heat shock in a 42 °C water bath for 60 s, then quickly transfer to ice for cooling for 2 min (do not shake the centrifuge tube during this period). In an ultra-clean bench, add 900 μL of fresh LB medium without antibiotics, mix well, and place in a shaker for culture at 37 °C with 200 rpm for 30 min. Aspirate 200 μL of the bacterial solution, spread it on LB agar plates with kanamycin sulfate (50mg/L), and incubate in a 37 °C incubator for 12~16 h.

Pick single colonies of transformed E. coli from the LB agar plates, inoculate them into 50 mL LB medium, add kanamycin sulfate with a final concentration of 50 mg/L, and culture overnight (approximately 16 h) at 37 °C with 200 rpm to prepare the primary seed culture. Inoculate the primary seed culture into 500 mL LB medium at a 1% inoculation ratio, add kanamycin sulfate with a final concentration of 50 mg/L, and perform shake flask fermentation at 37 °C with 200 rpm.

Recombinant E. coli cells were cultivated in shake flasks until the optical density at 600 nm (OD₆₀₀) reached 0.6~0.8. Take 100 μL of the total bacterial solution before IPTG (isopropyl-β-D-thiogalactoside) induction (for SDS-PAGE sample preparation). Then add IPTG with a final concentration of 0.5 mM to the remaining bacterial solution, adjust the culture conditions to 25 °C with 200 rpm, and continue culturing for approximately 20 h. After cultivation, retain 100 μL of the total bacterial solution after IPTG induction (for SDS-PAGE sample preparation). Centrifuge the remaining bacterial solution at 4 °C with 4000 rpm for 30 min, discard the supernatant, collect the bacterial cells, weigh them, and store at -20 °C by freezing.

Prepare 50 mM Phosphate Buffer (PB), 50 mM Phosphate-Buffered Saline (PBS), and cell lysis buffer according to Tables 2.1, 2.2, and 2.3.

The harvested cell pellet was resuspended in cell lysis buffer at a ratio of 10 mL per gram of wet cells. TieChui E. coli Lysis Buffer was added to 10 % of the total volume, and the mixture was incubated on ice for 30 min. The suspension was then disrupted by ultrasonication for 20 min at 30% amplitude (5 s on / 8 s off). The lysate was centrifuged at 4000 rpm for 30 min at 4 °C using a refrigerated centrifuge. The supernatant was filtered through a 0.45 μm membrane filter, and a 100μL aliquot of the clarified lysate was collected for SDS–PAGE analysis. The remaining pellet was resuspended in an equal volume of up water, and another 100 μL aliquot of the resuspended pellet was retained for SDS–PAGE analysis.

The recombinant fusion protein expression vector carries a 6×HisTag, so Ni-NTA chromatography column packing was used for ion affinity column chromatography to purify the protein. The specific procedure is as follows:

1. Equilibrate the chromatography column packing: Open the lower plug of the chromatography column. After all 20 % ethanol (used for column storage) has flowed out, wash the column bed with approximately 3 column volumes of up water, then wash the column bed with approximately 3 column volumes of PBS column buffer.
2. Sample loading: Pour the lysate supernatant filtrate into the chromatography column, start sample loading, and collect the flow-through. Repeat sample loading once, and retain 100 μL of the lysate supernatant flow-through (Q1) for SDS-PAGE sample preparation.
3. Elute impurity proteins: After sample loading, elute impurity proteins with PBS column buffer. Use Coomassie Brilliant Blue G-250 (Thermo Fisher Scientific, USA) to detect whether the column bed is clean. When eluting with the last column volume of PBS column buffer, retain 100 μL of the impurity protein eluate (Q2) for SDS-PAGE sample preparation.
4. Prepare 500 mM imidazole eluent according to Table 2.4.

5. Elute the target protein: After all the last column volume of PBS column buffer (used for impurity elution) has flowed out, add 10 mL of eluent, let it stand for 10 min, open the plug, collect the first 4 mL of eluent, and retain 100 μL of the supernatant before desalting (for SDS-PAGE sample preparation).
6. Repeat step ⑤ 1~2 times.
7. Clean the column bed: After elution, clean the column bed with 5 mL of eluent, then wash the residual imidazole in the column bed with 6 column volumes of up water, and seal the column with 20 % ethanol.

Based on the molecular sieve principle, gel exclusion chromatography was used to remove high-concentration imidazole and sodium chloride from the target protein. The entire process was performed in an ice bath. The specific procedure is as follows:

1. Equilibrate the desalting column: Wash the residual 20 % ethanol in the desalting column with 3 column volumes (1 desalting column volume = 5 mL) of up water, then wash the desalting column with 10 column volumes of PB equilibrium buffer.
2. Sample loading: Pass 1 mL of the target protein through the column to allow the target protein to enter the desalting column. Due to the molecular sieve principle, small-molecular-weight salt ions flow out of the desalting column first, while the target protein is retained in the column.
3. Collect the target protein: Pass 1.5 mL of PB buffer through the column to elute and collect the target protein, and retain 100 μL of the sample after desalting (for SDS-PAGE sample preparation).
4. Repeat steps ①~③ to desalt all target proteins.
5. Clean the desalting column: Wash the desalting column with 3 column volumes of up water, and seal the column with 20 % ethanol.
6. Protein preservation: Add 50 % glycerol to the collected protein at a volume ratio of "protein: 50 % glycerol = 9:1", aliquot the protein, and store at -20 °C by freezing.

During the expression and purification of the target protein, the following samples were retained separately: total bacterial solution before induction, total bacterial solution after IPTG induction, lysate supernatant, lysate precipitate, lysate supernatant flow-through (Q1) after column passage, impurity protein eluate (Q2), sample before desalting, and sample after desalting. Protein expression was detected by SDS-PAGE electrophoresis. The specific procedure is as follows:

1. Preparation of polyacrylamide gel: Use the PI007 Automated Gradient Gel Casting Instrument (Tsingke Biotechnology Co., Ltd (Xi’an, China)) to prepare the gel.
2. Sample preparation: Add 20 μL of 5×protein loading buffer to each of the 8 retained samples (total bacterial solution before induction, total bacterial solution after induction, lysate supernatant, lysate precipitate, Q1, Q2, sample before desalting, sample after desalting), mix well, boil for 10 min (to remove metastable aggregates), then centrifuge at 10,000 ×g for 10 min.
3. Sample loading: Place the gel plate into the electrophoresis tank, add 1×SDS electrophoresis buffer until the comb is submerged, then pull out the comb vertically. Add 5 μL of protein Marker and 10 μL of protein sample into the gel wells in sequence.
4. Electrophoresis: After sample loading, connect the power supply and adjust the voltage to 90 V. When the Marker bands start to separate (indicating that the protein has entered the resolving gel), adjust the voltage to 130 V. Stop electrophoresis when the bromophenol blue band at the protein front migrates to the bottom of the gel.
5. Staining and observation: Stain with the TSP8112 Safe Protein Fast Staining Solution (No Destaining) (Tsingke Biotechnology Co., Ltd (Xi’an, China)). Rinse the electrophoresed protein gel twice with an appropriate amount of up water, shaking on a shaker for 1 min each time. Pour off the up water, add an appropriate amount of staining solution to cover the gel, and shake on a shaker for 10~15 min. Discard the staining solution, rinse the residual staining solution with up water, and observe the results on a white plate.

1. Standard curve: Take one microplate and add reagents according to the following specifications: Add 0, 1, 2, 3, 4, 5 μL of 1 μg/μL BSA to 6 wells (no protein standard solution in Well 0), add 195 μL of Coomassie Brilliant Blue G-250 solution (Thermo Fisher Scientific, USA) to each well, then add up water to make the total volume 200 μL. Mix well and let stand for 2 min, then detect the absorbance at 595 nm. Draw the standard curve with protein concentration as the x-axis and absorbance at 595 nm as the y-axis. (R²>0.9)
2. Determination of protein concentration in the sample to be tested: Take 5 μL of the desalted protein solution from affinity chromatography, add 195 μL of Coomassie Brilliant Blue G-250 solution (Thermo Fisher Scientific, USA), and mix well. After 2 min, use Well 0 of the standard curve as the blank control to detect the absorbance at 595 nm. Calculate the protein mass of the corresponding sample according to the standard curve, and divide by the total sample volume (5 μL) to obtain the sample concentration (unit: mg/mL).

One enzyme activity unit (U) is defined as the amount of Pro-xylane Synthase required to reduce NAD⁺ to produce 1 μmol of NADH per minute under optimal reaction conditions (optimal conditions for PTDH: 30 °C, pH=7.3; optimal conditions for RDH: 30 °C, pH=9.5).

Prepare the enzyme activity determination mixtures for PTDH and RDH according to Tables 2.5 and 2.6. The BSA added in the mixture serves to protect protein activity.

Use a microplate reader to determine the change in ultraviolet absorbance at 340 nm at 30 °C, with a detection time of 10 min. Use the sample without enzyme as the control, and perform three parallel experiments on the desalted protein.

Calculate the production of NADH according to the molar extinction coefficient of the reaction product NADH, then calculate the enzyme activity (U) and specific activity (U/mg) based on the NADH production.

The extinction coefficient (ε) of NADH at 340 nm = 6220 L·mol⁻¹·cm⁻¹.

The formula for calculating enzyme activity is:

Design the sequence for inserting the target genes ptxD and rbtD into the Multiple Cloning Site (MCS) of the pPIC9K plasmid, and insert a kexin sequence between the two target gene sequences. After verifying that there is no recognition site for QuickCut™ Sal Ⅰ (Takara, Beijing, China) (used in subsequent enzyme digestion) in the target gene sequences, entrust a company to synthesize the plasmid.

Take 100 μL of pPIC9K-RDH-PTDH-TOP10 bacterial solution, inoculate it into 50 mL low-salt LB medium containing 50 mg/L kanamycin sulfate, and culture at 37 °C with 200 rpm for 12~16 h. Then streak on low-salt LB solid medium containing 50 mg/L kanamycin sulfate to isolate single colonies.

Pick single colonies with good morphology and uniform size, inoculate them into 100 mL low-salt LB medium containing 50 mg/L kanamycin sulfate (to obtain sufficient recombinant plasmids), and culture at 37 °C with 200 rpm for 12~16 h. Extract plasmids using the E.Z.N.A.® Plasmid DNA Mini Kit I (Omega Bio-Tek, USA), determine the plasmid concentration, and store at -20 °C by freezing.

Construct the enzyme linearization mixture according to Table 2.7, and perform enzyme linearization at 30 °C for 6 h.

Extract linearized plasmids using the phenol-chloroform extraction method (phenol:chloroform:isoamyl alcohol = 25:24:1, pH 8.0, Thermo Fisher Scientific, USA) and verify the plasmids before and after enzyme linearization by Agarose Gel Electrophoresis. The steps of Agarose Gel Electrophoresis are as follows.

(1) Gel preparation

1. Take 0.3 g of agarose, add 30 mL of 1×TAE buffer solution, melt in microwave oven to make 1.0 % agarose gel solution.
2. Select a comb with suitable pore size and stand it vertically at one end of the plexiglass inner tank.
3. When the agarose gel solution is cooled to 50 ~ 60°C, add 3 μL of nucleic acid dye and mix thoroughly, pour the gel into the glue bed without stopping, 3 ~ 4 mm high, avoid bubbles, and solidify at room temperature.
4. After the solution is completely solidified, gently remove the comb, place the plexglass inner tank in the electrophoresis tank, and add about 700 mL of 1×TAE as electrophoresis buffer, 1~2 mm above the gel surface.

(2) Spot the sample

The DNA sample solution was mixed with 3 μL of 10 × Loading Buffer, and 50 μL was taken with a micropipettor and added to the sample well of the agarose gel. Attention should be paid to avoid damaging the gel surface around the sample well and penetrating the bottom of the gel when adding the sample. Add 5 μL of 250 bp DNA Ladder to the hole on the left side of the sample well (record the order and amount of sampling).

(3) Electrophoresis

Electrophoresis was performed at a voltage of 200V. When the front of the bromophenol blue dye moved to the bottom edge of 1 ~ 2 cm, the electrophoresis was completed.

(4) Results observation

After electrophoresis, the gel was taken out and observed under the ultraviolet detector, and the electrophoresis results were recorded.

1. Inoculate Komagataella pastoris GS115 into 5 mL YPD medium, and culture at 30 °C with 200 rpm for 24 h.
2. Inoculate 50 μL of the above bacterial solution into 50 mL YPD medium, and culture at 30 °C with 200 rpm until the OD₆₀₀ reaches 1.3~1.5.
3. Centrifuge at 4 °C with 4000 rpm for 5 min to collect cells, resuspend with 20 mL ice water, and repeat this step 1~2 times.
4. Centrifuge at 4 °C with 4000 rpm for 5 min to collect cells, resuspend with 20 mL 1 M sorbitol, and repeat this step 3~4 times.
5. Centrifuge at 4 °C with 4000 rpm for 5 min to collect cells, resuspend with 1 mL 1 M sorbitol, place on ice, and use immediately.

1. Mix 80 μL of competent cells with 50 μg of linearized plasmid.
2. Incubate the competent cells on ice for 30 min.
3. Use the 1652100 MicroPulser electroporation instrument (BIO-RAD, USA) to set the Komagataella pastoris mode, place the electroporation cup in the electroporation instrument, perform one pulse.
4. Immediately after the pulse, add 900 μL of 1 M sorbitol and transfer to a 1.5 mL centrifuge tube.
5. Incubate the competent cells at 30 °C with 200 rpm for 1 h.
6. Spread 100 μL of competent cells on SD histidine-deficient solid medium.
7. Incubate at 30 °C for 3 days until colonies form, then store at 4 °C.

For electroporation transformation, two control groups must be set simultaneously: a negative control of non-electroporated competent cells, and a negative control of electroporated competent cells without linearized plasmid. Compare with positive results to ensure the reliability of the results.

1. Prepare SD histidine-deficient solid medium containing 5 μg/mL G418, and pour into plates.
2. Pick positive colonies and inoculate them onto SD histidine-deficient solid plates containing 5 μg/mL G418, then culture at 30 °C for 48 h.

Pick single colonies with large size, plump shape, and good growth status from the plates, inoculate them into 5 mL YPD medium, and culture overnight at 30 °C with 200 rpm to prepare the primary seed culture. Inoculate the primary seed culture into 20 mL fresh Basal Salt Medium（BSM） at a 10% ratio, and culture at 30 °C with 200 rpm for 72 h.

In a 500 mL reaction flask, add 150 mL ethanol, 48.36 g (0.3 mol) chitosan, 30 g (0.2 mol) xylose, and 26 g (0.24 mol) acetylacetone in sequence. React at 80 °C for 12 h, then adjust the pH to 7 with HCl.

Weigh the Pro-xylane standard, dissolve it in methanol, and prepare gradient solutions with concentrations of 0.001 μg/mL, 0.01 μg/mL, and 0.05 μg/mL. Store at 4 °C in a refrigerator.

Prepare the reaction mixture according to Table 2.8. Among the components, 50 mM sodium phosphate buffer (pH=7.4) is the reaction buffer; β-acetone xyloside is the substrate (the product obtained in Step 2.4.10 was used in this experiment); NAD⁺ is the coenzyme; sodium phosphite pentahydrate and isopropanol are hydrogen donors; RDH and PTDH are Pro-xylane Synthases (The Pro-Xylane synthase in the system is derived from the fermentation broth supernatant obtained in Step 2.4.9. PTDH was added in excess to ensure sufficient hydrogen supply).

Place the prepared reaction system in a constant-temperature shaking reactor, and react with gentle shaking at 30 °C for 6 h.

Filter the reaction solution and Pro-xylane standard solution through a 0.22 μm microporous filter membrane respectively. Place HPLC-grade methanol and up water in an ultrasonic instrument for ultrasonic treatment for 20 min.

High-performance liquid chromatography (HPLC) was performed using a Kromasil® 100-5-C₁₈ column (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of methanol (A) and up water (B) in an isocratic elution mode (methanol : water = 5 : 95, v/v). The flow rate was 1.0 mL·min⁻¹, the injection volume was 20 μL, the column temperature was maintained at 35 °C, and detection was carried out at 220 nm.

Prepare a 4 mL reaction system, dilute it 2-fold, 10-fold, 20-fold, 100-fold, and 1000-fold with methanol respectively, and perform liquid chromatographic detection sequentially.

Perform liquid chromatographic analysis on the sample containing only the product from Step 2.4.10 and the Pro-xylane standard to evaluate the separation effect of the chromatographic conditions.

In this study, a xylose operon-based gene expression regulation system was constructed using molecular cloning techniques. Functional recombination of the xylR regulatory gene, xylO operator sequence, and the reporter gene gfp was achieved in E. coli. The target gene was amplified by PCR, and the recombinant plasmid was constructed through Gibson assembly and double-enzyme digestion followed by ligation. Stable transformants were obtained after transformation and optimization. Ultimately, a standardized experimental procedure was established, and a recombinant plasmid capable of characterizing regulatory activity via green fluorescent protein (GFP) expression was successfully constructed.

1. Take a 1.5 mL centrifuge tube, take 1 mL of E. coli culture, and centrifuged at 11000 rpm for 5 min to collect the bacteria.
2. The supernatant was discarded, resuspended in 1 mL up water, and the bacteria were collected by centrifugation at 11000 rpm for 5 min. This step was repeated once.
3. The bacteria were lysed in a boiling water bath for 10 min.
4. The cells were centrifuged at 11000 rpm for 10 min and the supernatant was aspirated.
5. The bacterial cells were added to the lysis buffer and incubated in a boiling water bath for 10 minutes.

(1) Four PCR reaction tubes were used to prepare 50 μL reaction system in each tube:

The templates were the supernatant of lysed bacterial solution and pET-28a plasmid solution, respectively. The primer sequences used are shown in Table 3.2:

(2) After the preparation is completed, gently centrifuge in a handheld centrifuge to concentrate the reaction solution at the bottom of the test tube and eliminate bubbles that may be generated during the sample addition process.

(3) Press the following table to set up the PCR program, insert the sample, and start running:

(1) To prepare 100 mL of 50 × TAE Buffer: Weigh the reagents in Scale 3.4, mix thoroughly in a beaker, and add water to 1000 mL.

(2) Dilute 50×TAE Buffer at a ratio of 20 mL/1000 mL into 1×TAE buffer solution.

(3) Gel preparation

1. 1.0 % agarose gel was prepared by melting 0.3 g of agarose with 30 mL of 1×TAE buffer solution in a microwave oven.
2. Select a comb with suitable pore size and stand it vertically at one end of the plexiglass inner tank.
3. When the agarose gel solution is cooled to 50 ~ 60°C, add 3 μL of nucleic acid dye and mix thoroughly, pour the gel into the glue bed without stopping, 3 ~ 4 mm high, avoid bubbles, and solidify at room temperature.
4. After the solution is completely solidified, gently remove the comb, place the plexglass inner tank in the electrophoresis tank, and add about 700 mL of 1×TAE as electrophoresis buffer, 1~2 mm above the gel surface.

(4) Spot the sample

Mix the DNA sample solution with 3 μL of Loading Buffer (10× loading buffer), take 20 μL with a micropipettor and add to the sample well of the agarose gel (100 μL per well). Attention should be paid to avoid damaging the gel surface around the sample wells and penetrating the bottom of the gel when adding the sample. Add 5 μL of 250 bp DNA Ladder to the hole on the left side of the sample well (record the order and amount of sampling).

(5) Electrophoresis

Electrophoresis was performed at a voltage of 80 V. When the front of the bromophenol blue dye moved to the bottom edge of 1 ~ 2 cm, the electrophoresis was completed.

(6) Observation of results

After electrophoresis, the gel was taken out and observed under the UV detector. The pinkish purple band should be displayed at the site of DNA (protective glasses should be worn when observing under the UV lamp), and the electrophoresis results were recorded. The DNA band area gel was cut out with a knife and placed in two 10 mL centrifuge tubes. The weight of the two gels was 0.46 g and 0.76 g, respectively.

(1) Sol

Equal volumes of XP2 Binding Buffer (460 μL and 760 μL) were added to the above centrifuge tubes, respectively, and the mixture was heated in a water bath at 50~60 °C for 7 min or until the gel was completely melted, shaking or vortexing the mixture every 2~3 min.

After the gel is completely dissolved, note the pH value of the gel-XP2 Binding Buffer mixture. If the pH is higher than 8, the yield of DNA will be greatly reduced. Look at the color of the mixture. If it is orange or red, add 5 μl of sodium acetate (5 M, pH 5.2) to lower the pH. After this adjustment, the color of the mixture will return to its normal pale yellow color.

(2) DNA binding

1. Two HiBind® DNA Mini binding columns were loaded into a 2 mL collection tube, respectively. The two tubes of DNA gel solution obtained previously were all transferred to the HiBind® DNA Mini binding column. The samples were centrifuged at 10000 ×g for 1 min at room temperature. Discard the filtrate in the collection tube and sleeve the post back into the collection tube inside the 2 mL collection tube. If the volume of the DNA gel solution exceeds 700 μL, only 700 μL can be transferred to the HiBind® DNA Mini Binding column at a time, and the rest can continue to repeat this step until all the solution has passed through the HiBind® DNA Mini binding column. Each HiBind® DNA Mini binding column has a limited adsorption capacity of 25 μg DNA. If a large yield is expected, the samples are added to the appropriate number of HiBind® DNA Mini binding columns.
2. Discard the filtrate in the collection tube and sleeve the HiBind® DNA Mini binding column back into the 2 mL collection tube. Then 300 μL XP2 Binding Buffer was added to the column, centrifuged at the maximum speed (≥13000×g) for 1 min at room temperature, and the filtrate was discarded.

(3) Rinse

1. Slip the HiBind® DNA Mini binding column back into the collection tube in the 2 mL collection tube. Add 700 μL SPW Buffer (which has been correctly diluted with absolute ethanol) to the HiBind® DNA Mini binding column. The samples were centrifuged at 10000×g for 1 min at room temperature and the filtrate was discarded.
2. Slip the HiBind® DNA Mini binding column back into the 2 mL collection tube. Centrifugation at ≥13000×g for 2 min at room temperature was used to dry the residual liquid of the HiBind® DNA Mini binding column.

(4) Elution

Load each of the two HiBind® DNA Mini binding columns onto a clean 1.5 mL centrifuge tube, add 50 μL (depending on the expected end-product concentration) of Elution Buffer to the substrate of the HiBind DNA Mini binding column, The column was left at room temperature for 1 min and centrifuged at 13000×g for 1 min to eluting the DNA. The first elution can wash out 70~80% of the bound DNA. The previous eluate was added to the column for a secondary elution.

(5) Measure and store

The obtained DNA fragments were stored in a -20 °C refrigerator or directly used for subsequent experiments. The DNA concentration and A₂₆₀/A₂₈₀ ratio were determined by ultramicro ultraviolet spectrophotometer (Elution Buffer served as a blank control).

(1) Prepare 20 μL reaction mixture:

(2) The reaction was carried out in a water bath at 50 °C for 20 min. Immediately after the reaction, the centrifuge tube was placed on ice and cooled for 2 min until it was transformed.

(3) Add 10 μL reaction solution to DH5α competent cells, flick for a number of times, and then incubated on ice for 30 min.

(4) The cells were ed in a water bath at 42 °C for 75 s and then quickly placed on ice for 5 min.

(5) The bacteria were collected by centrifugation at 4000 rpm for 3 min and resuspended in 500 μL up water.

(6) After adding 50 μL LB liquid medium, the cells were incubated for 45 min at 37 °C in a shaker.

(7) The bacteria were collected by centrifugation at 4000 rpm for 3 min. The filtrate was poured out and resuspended in 500 μL up water

(8) 100 μL of bacterial solution was evenly coated on LB plate containing kanamycin sulfate and incubated at 37 °C incubator.

(1) Add 2 μL, 50 mg/L kanamycin sulfate and 2998 μL LB liquid medium to 15 mL centrifuge tube, respectively.

(2) Three monoclonal colonies were picked from each plate into the centrifuge tube and sealed with a sealing membrane.

(3) Centrifuge tubes labeled 1, 2, and 3 were cultured on a shaker overnight and sequenced by Tsingke Biotechnology Co., Ltd (Xi’an, China).

(4) The sequenced plasmid sample solution was added to E. coli DH5α competent cells (10 μL), and then incubated on ice for 30 minutes.

(5) Thermal stimulation was placed in a 42°C water bath for 75s and then quickly placed on ice for 5 min.

(6) 500 μL LB liquid medium was added and incubated at 37 °C for 45 min with shaking.

(7) The bacteria were collected by centrifugation at 4000 rpm for 3 min, the filtrate was poured out and resuspended in 500 μL up water.

(8) 100 μL of bacterial solution was evenly coated on LB plate containing kanamycin sulfate and incubated at 37 °C incubator.

Single colonies were picked from the plate and added to 100 mL LB liquid medium containing 50 μg/mL kanamycin sulfate, and incubated at 37 °C with 200 rpm shaking overnight.

(1) Add 800 μL bacterial solution and 200 μL 50 % glycerol at a ratio of 4:1, mix thoroughly, seal the centrifuge tube with a sealing membrane, and store in the refrigerator at -20 °C.

(2) Extract plasmids using the E.Z.N.A.® Plasmid DNA Mini Kit I (Omega Bio-Tek, USA)

(1) Four PCR reaction tubes were used to prepare 50 μL reaction mixture in each tube:

The templates were the supernatant of lysed bacterial solution and pET-28a-xylR plasmid solution, respectively. The primer sequences used are shown in Table 3.7:

(2) After the preparation is completed, gently centrifuge in a handheld centrifuge to concentrate the reaction solution at the bottom of the test tube and eliminate bubbles that may be generated during the sample addition process.

(3) Set the PCR program according to the following table, insert the sample, and start running:

The steps of Agarose Gel Electrophoresis refer to the step 2.4.5. The DNA band region gels were cut with a knife and placed in two 10 mL centrifuge tubes. The weights of the two gels were 0.53 g and 0.66 g, respectively.

(1) Melt the gel

Equal volumes of XP2 Binding Buffer (530 μL and 660 μL) were added to the above centrifuge tubes, respectively, and the mixture was heated in a water bath at 50~60 °C for 7 min or until the gel was completely melted. The mixture was shaken or vortexed every 2~3 min.

(2) DNA binding

1. Two HiBind® DNA Mini binding columns were each loaded in a 2 mL collection tube. The two tubes of DNA gel solution obtained previously were all transferred to the HiBind® DNA Mini binding column. The samples were centrifuged at 10000×g for 1 min at room temperature.
2. Discard the filtrate in the collection tube and sleeve the HiBind® DNA Mini binding column back into the 2 mL collection tube. Then 300 μL XP2 Binding Buffer was added to the column, centrifuged at room temperature for 1min at the maximum speed (≥ 13000×g), and the filtrate was discarded.

(3) Rinse

1. Slip the HiBind® DNA Mini binding column back into the collection tube in the 2 mL collection tube. Add 700 μL SPW Buffer (which has been correctly diluted with absolute ethanol) to the HiBind® DNA Mini binding column. The samples were centrifuged at 10000×g for 1 min at room temperature and the filtrate was discarded.
2. Slip the HiBind® DNA Mini binding column back into the 2 mL collection tube. Centrifugation at ≥ 13000×g for 2 min at room temperature was used to dry the residual liquid of the HiBind® DNA Mini binding column.

(4) Elution

Load each of the two HiBind® DNA Mini binding columns onto a clean 1.5 mL centrifuge tube, add 50 μL (depending on the expected end-product concentration) of Elution Buffer to the substrate of the HiBind DNA Mini binding column, The column was left at room temperature for 1 min and centrifuged at 13000×g for 1 min to eluting the DNA. The first elution can wash out 70~80 % of the bound DNA. The previous eluate was added to the column for a secondary elution.

(5) Measure and store

The DNA fragments were stored in a -20 °C refrigerator or directly used for subsequent experiments. The Elution Buffer was used as a blank control.

(1) Prepare 40 μL reaction mixture:

(2) After the reaction in a water bath at 50 °C for 20 min, immediately cool the centrifuge tube on ice for 2 min.

(3) Add 20 μL reaction solution to E. coli DH5α cells, flick for a few times, and incubated on ice for 30 min.

(4) Thermal stimulation in a 42 °C water bath for 75 s and then quickly put it on ice for 5 min.

(5) 500 μL LB liquid medium was added and incubated at 37 °C for 45 min.

(6) The bacteria were collected by centrifugation at 4000 rpm for 3 min. The filtrate was poured out and resuspended in 500 μL up water.

(7) 100 μL of bacterial solution was evenly coated on LB plate containing kanamycin sulfate.

(8) The plate was placed in a 37 °C incubator for culture.

(1) Add 2 μL, 50 mg/L kanamycin sulfate and 2998 μL LB liquid medium to 15 mL centrifuge tube, respectively.

(2) Three monoclonal colonies were selected on the plate into the centrifuge tube and sealed with the sealing membrane.

(3) Centrifuge tubes labeled 1, 2, and 3 were cultured on a shaker and sequenced by Tsingke Biotechnology Co., LTD. (Xi'an, China).

(4) The sequenced plasmid sample solution was added to E. coli DH5α competent cells (1 μL), and then incubated on ice for 30 minutes.

(2) Thermal stimulation was placed in a 42 °C water bath for 75 s and then quickly placed on ice for 5 min.

(3) 500 μL LB liquid medium was added and incubated at 37 °C for 45 min with shaking.

(4) The bacteria were collected by centrifugation at 4000 rpm for 3 min, then the filtrate was removed and resuspended in 500 μL up water.

(5) 100 μL of bacterial solution was evenly coated on LB plate containing kanamycin sulfate and incubated at 37 °C.

Single colonies were picked from the plate and added to 100 mL LB liquid medium containing 50 μg/mL kanamycin sulfate for overnight culture at 37 °C and 200 rpm.

(1) Add 800 μL bacterial solution and 200 μL 50 % glycerol at a ratio of 4:1, mix thoroughly, seal the centrifuge tube with a sealing membrane, and store in the refrigerator at -20 °C.

(2) Extract plasmids using the E.Z.N.A.® Plasmid DNA Mini Kit I (Omega Bio-Tek, USA)

(1) Add 100 μL kanamycin sulfate (50 mg/L) to 100 mL LB liquid medium and mix well.

(2) 10 μL of glycerol-stored bacteria with pET-28a-GFP recombinant plasmid was added to the medium and mixed evenly.

(3) The cells were cultured overnight at 37 °C and 200 rpm with shaking.

(4) Extract plasmid:

Extract plasmids using the E.Z.N.A.® Plasmid DNA Mini Kit I (Omega Bio-Tek, USA)

Double enzyme digestion of target vector and target gene to be inserted

Double enzyme digestion mixture was made by adding the following reagents in proportion to two 1.5 ml sterile centrifuge tubes:

After mixing well, the mixture was kept in a water bath at 30 °C for 2 h.

Agarose gel electrophoresis and gel recovery of double enzyme digestion products

The steps of Agarose Gel Electrophoresis refer to the step 2.4.5.

Linearize the connection between the target vector and the target gene to be inserted

(1) The following reagents were added into the sterile PCR tube according to the proportion of Table 11 to make the connection system, so that the number of molecules of target gene: number of molecules of vector = 10:1.

After mixing, the reaction was passed through a gene amplicon for 1 h at 16 °C.

Transformation of E. coli TOP 10

(1) The reaction solution was added to E. coli TOP10 competent cells, and the cells were incubated on ice for 30 min. After that, the cells were heated in a water bath at 42 °C for 90 s, and then quickly put back on ice for 2 min.

(2) Add 500 µL LB liquid medium into the centrifuge tube, mark the groups, and culture for 45 min at 37°C with 200 rpm shaking table.

(3) The above bacteria solution was centrifuged at 4000 rpm/min for 1min to collect the bacteria. The supernatant was discarded, and then the cells were blown out into cell suspension with up water, and 100 µL of the cell suspension was spread on the screening plate containing kanamycin sulfate.

The experiment was carried out in 2 groups:

In the positive control group, 100 µL of competent cells transformed with pET-28a-GFP recombinant plasmid were uniformly coated on the plate of LB medium containing kanamycin sulfate.

In the experimental group, 100 µL of competent cells transformed with pET-28a-xylR-xylO-GFP recombinant plasmid were uniformly coated on LB medium containing kanamycin sulfate.

(4) The plates were placed in a 37 °C constant temperature incubator for 12~16 hours, and then the results were observed.

Recombinant E. coli inoculation

Single colonies from the plates of the positive control group and the experimental group were selected and cultured in two bottles of 100 mL LB liquid medium containing 50 mg/L kanamycin sulfate at 37 °C and 200 rpm overnight.

Plasmid extraction by alkaline lysis

(1) Add 800 μL bacterial solution and 200 μL 50 % glycerol at a ratio of 4:1, mix thoroughly, seal the centrifuge tube with a sealing membrane, and store in the refrigerator at -20 °C.

(2) Extract plasmids using the E.Z.N.A.® Plasmid DNA Mini Kit I (Omega Bio-Tek, USA)

(1) 100 μL BL21 (DE3) competent cells were taken out of the -80 °C refrigerator and quickly inserted into ice. After 5 minutes, when the bacterial block was melted, 2 μL of the identified recombinant plasmid was added to the competent cells, and the bottom of the centrifuge tube was gently mixed by hand, and the cells were left on ice for 30 minutes.

(2) The centrifuge tube was heated in a water bath at 42 °C for 90 seconds, quickly transferred to ice, and left on ice for 2 minutes.

(3) 700 μL of fresh liquid LB medium without antibiotics was added to the centrifuge tube, mixed and then resuscitated in a shaker at 37 °C and 200 rpm for 60 min.

(4) The above bacterial solution was centrifuged at 4000 rpm/min for 1 min. Then, 50 μL up water was added and the cells were blown out to form cell suspension. Then, 50 μL bacterial solution was evenly spread on LB agar plate containing 50 mg/L kanamycin sulfate.

(5) The plates were placed upside down in an incubator at 37 °C for overnight culture.

(1) Inoculation of recombinant E. coli BL21(DE3) -pET-28a-GFP and BL21(DE3) -pET-28a-xylR-xylO-GFP:

Single colonies with good shape and uniform size were selected from the transformed plates and inoculated into 50 mL newly prepared LB liquid medium, respectively. Then, 50 μL filtered and sterilized kanamycin sulfate solution (50 mg/mL) was added, and incubated at 37 °C at 200 rpm overnight to prepare the seed bacterial solution.

5 mL of the seed bacteria solution prepared in the previous step was inoculated into 500 mL of LB liquid medium, and 500 μL of 50 mg/mL kanamycin sulfate was added to each of them. The bacteria were cultured at 37 °C and 200 rpm on a shaker. The OD₆₀₀ value of the culture was detected by a spectrophotometer.

(1) When the OD₆₀₀ value of the culture reached 0.6~0.8 detected by spectrophotometer, 1 mL bacterial solution was absorbed into a 1.5 mL centrifuge tube to keep the sample, and the sample was frozen in a -20 °C refrigerator.

(2) 500 μL IPTG mother solution (100 mmol/L) was added to the bacterial solution containing pET-28a-GFP plasmid. 500 μL D-xylose mother solution with a concentration of 100 mmol/L was added to the bacterial solution containing pET-28a-xylR-xylO-GFP plasmid, and the final concentration was 0.1 mmol/L. The bacteria were incubated overnight at 30 °C and 200 rpm on a shaking table.

The bacteria in the positive control group and the experimental group were dropped onto the slide, and the green fluorescence of the bacteria was observed under a fluorescence microscope.

The standard samples were sent to the microplate reader for testing, and a standard curve graph was produced.

XynC-CBM was constructed by adding a Carbohydrate-Binding Module (CBM) to the carboxyl terminus of XynC. As shown in Figure A of Figure 4-2, XynC-CBM retains higher residual activity than XynC after the same heat treatment. It can be seen that the affinity of XynC-CBM for the substrate is higher than that of XynC. However, when residual activity is defined as the percentage relative to the enzyme activity of the control group, there is no significant difference between the two. Thus, CBM has no significant effect on the thermal stability of xylanase; As can be seen from the figure, the activities of XynC-CBM and XynC decreased after heat treatment at 80°C. Therefore, the thermal stability of these two proteins will be modified in this sudy.

By comparing Figure A and Figure B in Figure 1-2, it can be concluded that: whether it was XynC or XynC-CBM, the enzyme activity of xylanase in the recombinant E. coli expression vector was much lower than that in the recombinant Komagataella pastoris expression vector. Therefore, it was finally necessary to construct a recombinant Komagataella pastoris expression vector in this study.

According to the Chinese National Standards for xylanase activity, the activity of xylanase at room temperature and 80 ℃ water bath was determined. The xylanase enzyme from the 37 °C treatment group was used as the control group. The xylanase activity measured under standard conditions was defined as 100%. The residual enzyme activity in the experiment was recorded as the percentage of the xylanase activity after treatment at 80 °C compared to the activity of the control group. (The protein encoded by xynC is abbreviated as XynC)

We then constructed the D57R/N88W double mutant protein and tested its thermal stability in accordance with the Chinese National Standards for xylanase activity (As shown in Figure A of Figure 1-4).

At the same time, we also measured the enzymatic activity of xylanase when it was heated in an 80-degree water bath for different durations, in order to explore the relationship between the decrease in xylanase activity and the duration of the water bath (As shown in Figure B of Figure 1-4).

Finally, through experimental measurements, we determined the initial reaction rate of the enzyme under different substrate concentrations. Eventually, we calculated that the Vmax of the D57R/N88W double-mutant protein was 1.007 and the Km was 0.5896 (As shown in Figure C of Figure 1-4).

The recombinant plasmid was introduced into the expression strain Komagataella pastoris and cultured 2-3 days.The results are shown in Figure A of Figure 1-5.

Theoretically, the greater the number of copies transferred to the vector, the faster the positive clones will grow on high-concentration Zeocin® resistance plates. At this point, after high-concentration antibiotic screening, the probability that the positive clones carry high-copy expression vectors will significantly increase (As shown in Figure B of Figure 1-5).

The electrophoresis results are shown in Figure 1-6. There is a band of approximately 29 kDa in the 25~35 kDa range. The theoretical molecular weight of the fusion protein XynC-CBM is 29.2 kDa. It can be basically concluded that the target protein indicated by the arrow is the fusion protein XynC-CBM.

High-copy-number strains were screened by assaying xylanase activity according to the Chinese National Standard method for xylanase assay.

Finally, we selected the 12 bacterial strains with the highest enzyme activity and transferred them to shake flasks for fermentation verification. At the same time, their thermal stability was also measured.

To elucidate the molecular basis for enhanced thermostability resulting from the mutations, in silico analyses, including interaction analysis and molecular dynamics (MD) simulations, were performed on both the wild-type XynC and its mutant. These studies aimed to determine the mutational impact on the protein's local and global structure.

Based on the 3D structure of XynC (PDB ID: 3wp4), we performed an interaction analysis for residues at positions 57 and 88 with PyMOL. As shown in Figure 1-8, in the wild-type XynC, the aspartic acid residue at position 57 forms two hydrogen bonds with the aspartic acid residue at position 34. Concurrently, the asparagine residue at position 88 is engaged in a hydrogen bond with the threonine residue at position 213.

Additionally, we generated the three-dimensional structures of mutant proteins XynC-D57R and XynC-N88W by introducing corresponding mutations at positions 57 and 88 using PyMOL, followed by interaction analysis. As shown in Figure 1-9, in the XynC-D57R mutant, the substitution of aspartic acid with arginine at position 57 increased the number of hydrogen bonds formed with aspartic acid at position 34 from two to three. Meanwhile, a new hydrogen bond was established with tyrosine at position 94, thereby enhancing the structural stability of the protein.

As shown in Figure 1-10, the interaction analysis of the XynC-N88W mutant reveals that the substitution of asparagine with tryptophan at position 88 does not lead to the formation of additional hydrogen bonds. However, the aromatic ring of the tryptophan side chain significantly enhances hydrophobic interactions at this site, thereby contributing to improved structural stability of the protein.

Molecular dynamics simulations of the wild-type XynC and its mutants (XynC-D57R and XynC-N88W) were conducted for 50 ns at 333 K using the GROMACS software package. Subsequent trajectory analysis included the calculation of root-mean-square fluctuation (RMSF), with the results presented in Figure 1-11.

Panel A compares the RMSF profiles of XynC and XynC-D57R at 333 K. The D57R mutant exhibits a general reduction in flexibility starting from residue 25 compared to the wild-type. Notably, a significant decrease in RMSF is observed within the region of residues 145~165, indicating enhanced structural rigidity that correlates well with the experimentally observed improvement in thermostability.

Panel B displays the RMSF comparison between XynC and XynC-N88W. Similar to the D57R mutant, the N88W substitution leads to a reduction in RMSF within the loop region of residues 151-155, indicating enhanced structural rigidity in this area. This structural stabilization contributes to the improved thermostability observed in the mutant.

Plasmids were extracted using the Plasmid Mini Kit I. The plasmid concentrations are shown in Table 2.1.

Chemical Transformation of E. coli BL21(DE3): The recombinant vectors were introduced into the expression strain E. coli BL21(DE3) and cultured overnight: Add 1 μL of plasmid to 100 μL of BL21(DE3) competent cells, and inoculate onto the plate containing kanamycin sulfate. The results are shown in Figure 2-1.

SDS-PAGE Detection: The results are shown in Figure 2-2 (the target protein is indicated by the red box). As shown in Figure 2-2, the target protein exhibits good expression.

M: Prestained Protein Marker (15~180 kDa); 1: Bacterial solution before induction; 2: Bacterial solution after induction; 3: Lysate supernatant; 4: Lysate precipitate; 5: Q1 (flow-through); 6: Q2 (impurity eluate); 7: Affinity chromatography eluate; 8: Desalting eluate

Determination of Protein Content by Bradford Method: The protein standard was detected using a microplate reader, and the standard curve was drawn as shown in Figure 2-3 (R² > 0.9).

The samples obtained in the experiment were the desalting eluates of RDH and PTDH. By comparing with the standard protein curve, the protein concentration results are shown in Table 2.2.

The enzyme activity and specific enzyme activity of the desalting eluates of PTDH and RDH were determined respectively. The results are shown in Table 2.3.

Plasmids were extracted using the Plasmid Mini Kit I. The plasmid concentration is shown in Table 2.4.

The agarose gel electrophoresis results of the plasmids before and after enzyme digestion are shown in Figure 2-4.

Electroporation Transformation: After transformation, the bacterial solution was spread on SD histidine-deficient medium to screen positive colonies. The results are shown in Figure 2-5.

High-Copy Screening: Positive colonies were picked and inoculated onto SD histidine-deficient solid plates containing 5 mg/mL G418, then cultured at 30 °C for 48 h. The screening results are shown in Figure 2-6.

Liquid Chromatographic Results of Pro-Xylane Standards with Gradient Concentrations: The liquid chromatographic results of pro-xylane standards with different concentrations were overlaid, as shown in Figure 2-7: All standards with different concentrations showed absorption peaks at around 2.8 minutes, indicating that the absorption peak of the pro-xylane standard appears at approximately 2.8 minutes.

Determination of the Reaction System: The liquid chromatographic results of the reaction systems with different dilutions were overlaid with the standard results, as shown in Figure 2-8: The reaction system showed an absorption peak consistent with the standard, indicating that Pro-xylane was produced in the reaction system.

Evaluation of Chromatographic Conditions: Liquid chromatographic analysis was performed on the sample containing the product from Step 2.4.10 and the Pro-xylane standard. The results are shown in Figure 2-9: The established chromatographic conditions can effectively separate the absorption peaks of the substrate and product, with good separation efficiency.

Amplification of target gene and vector: The target gene was amplified by PCR and the gene bands were detected by agarose gel electrophoresis.

The electrophoresis results were obtained in Figure 3-1. Lane 1 is the xylR target gene fragment, and it can be clearly seen that the band is between 1000 bp and 1500 bp, the corresponding size is about 1100 bp, while the size of xylR gene fragment is 1179 bp. Lane 2 was the pET-28a vector fragment, and the size of the band was about 5000 bp, while the size of the pET-28a vector fragment was 5372 bp. Therefore, we can judge that the target gene fragment was successfully amplified and the electrophoresis result was good.

Gel recovery of PCR products: After cutting the target band, the gel was recovered and detected by UV spectrophotometer to obtain the target gene fragment concentration as shown in Table 3.1:

According to the numerical results in the table, the DNA product extracted after gum recovery is relatively pure. At the same time, the DNA concentration of xylR sample was lower, presumably due to the shallow gel band, which resulted in less DNA after recovery, or due to repeated DNA passing through the adsorption column in the secondary elution step, more DNA remained on the adsorption column and was not eluted.

Transformation of recombinant plasmid: 10 µL of the recombinant plasmid was added to 100 µL of E. coli DH5α competent cells and spread onto plates containing kanamycin with bacterial growth (Figure 3-2).

Plasmid extraction by alkaline lysis: The above cultured bacterial solution was evenly added to the centrifuge tube to break the cells for plasmid extraction. The concentration of extracted plasmid DNA was determined by ultraviolet spectrophotometer, as shown in Table 3.2. According to Table 3.2, the plasmid DNA concentration was low, and it was speculated that the long fragmentation time during the fragmentation process of the bacteria led to the degradation of part of the plasmid.

Amplification of target gene and vector: The target gene was amplified by PCR and the gene bands were detected by agarose gel electrophoresis.

The electrophoresis results were obtained in Figure 3-3. Lane 1 is the xylO target gene fragment, and it can be clearly seen that the band is between 200 bp and 300 bp, the corresponding size is about 250 bp, while the size of xylO gene fragment is 252 bp. Lane 2 was the pET-28a-xylR vector fragment, and the size of the band was about 6000 bp, while the size of the pET-28a-xylR vector fragment was 6551 bp. Therefore, we can judge that the target gene fragment was successfully amplified and the electrophoresis result was good.

Gel recovery of PCR products: After cutting the target band, the gel was recovered and detected by UV spectrophotometer to obtain the target gene fragment concentration as shown in Table 3.3:

According to the numerical results in the table, the DNA product extracted after gum recovery is relatively pure. At the same time, the DNA concentration of xylO sample was lower, presumably due to the shallow gel band, which resulted in less DNA after recovery, or due to repeated DNA passing through the adsorption column in the secondary elution step, more DNA remained on the adsorption column and was not eluted.

Transformation of recombinant plasmid: 10 µL of the recombinant plasmid was added to 100 µL of E. coli DH5α competent cells and spread onto plates containing kanamycin with bacterial growth (Figure 3-4).

Plasmid small extraction by alkaline lysis: The above cultured bacterial solution was evenly added to the centrifuge tube to break the cells for plasmid extraction. The concentration of extracted plasmid DNA was determined by ultraviolet spectrophotometer, as shown in Table 3.4.

Test gene template preparation: The bacterial solution of glycerol-stored bacteria cultured with GFP recombinant plasmid was uniformly added to the centrifuge tube to break the cells for plasmid extraction. The concentration of extracted plasmid DNA was determined by ultraviolet spectrophotometer, as shown in Table 3.5.

Agarose gel recovery of double digestion products: After cutting the GFP gene and pET-28a-xylR-xylO recombinant vector plasmid fragments, the concentration of target gene fragments was obtained after the gel was recovered and detected by ultraviolet spectrophotometer, as shown in Table 3.6. The data in Table 3.6 can further confirm the correct purification of gene bands.

Plasmid extraction by alkaline lysis: The bacterial solution cultured after the transformed pET-28a-GFP and pET-28a-xylR-xylO-GFP plasmids were uniformly added to the centrifuge tube to break the cells for plasmid extraction. The concentration of plasmid DNA extracted was determined by ultraviolet spectrophotometer, as shown in Table 3.7.

Observation of green fluorescent protein expression: Under fluorescence microscope, no green fluorescence was observed before adding xylose, but after adding xylose, the bacteria showed green fluorescence, which was consistent with the green fluorescence of the positive control group before and after adding IPTG.