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After the SS-bgly gene sequence is optimized by codon，GC content decreased from 39.7% to 37.3%，the Codon Adaptation Index number (CAI) increased from 0.74 to 0.93. And then activate the successfully constructed strains and extract pPIC9K-SS-bgly plasmid. After Sal Ⅰ enzyme is linearized, it is electroporated into competent cells and spread on the MD plate. For single colonies appearing on the plate, repeated freezing and thawing methods can be used to release genomic genes. Take the supernatant for PCR verification，the result is shown in Figure 2.4. There is a band of interest with the theoretical size at about 2000 bp, while the blank control GS115-pPIC9K only has a band of about 500 bp, which proves that the recombinant strain was successfully constructed.
Figure.Screening transformants by MD plate
Figure. PCR analysis of partial P. pastoris transformantsLane M: DNA marker; Lanes 1-7: PCR products of transformants; Lane 8: Control
Different transformants have slightly different enzyme production activities. In order to obtain transformants with high enzyme activity, 6 transformants verified by colony PCR were selected for shake flask fermentation screening, and their enzyme activity was about 32-70 U/mL. Among them, the 6# transformant has the highest enzyme activity:70 U/mL.
Figure.The standard curve of pNP concentration and absorbance value
Activate the GS115-pPIC9K-SS-bgly 6# transformant on the YPD plate. After the single bacteria grow, proceed to fermentation.Sampling is taken every 24 hours. The sampled bacterial liquid is diluted with ultrapure water and measured for OD600, and then centrifuged to take the supernatant to determine its β-glycosidase activity and protein content. The result is shown in the figure below. In the first 72 hours, the recombinant strain grew rapidly, after which the growth rate slowed down and reached a stable level in 120 hours. The activity of β-glycosidase kept increasing from 0-120 h, reaching the highest value of 70 U/mL at 120 h of fermentation, and then gradually decreasing.The protein content has been increasing steadily, reaching 1.5 mg/mL at 120 h of induction. The methanol continued to induce, and the protein content continued to increase, but the enzyme activity decreased. It is speculated that it may be caused by the accumulation of protease.
Figure. Fermentation process curves
Figure.Standard curve between standard protein concentration and absorbance value
After 120 hours of methanol induction, 10 μL supernatant was taken for SDS-PAGE electrophoresis. A clear band was observed near the target molecular weight of 57 kDa (Figure 2.9), indicating that the SS-bgly gene was successfully expressed in P. pastoris .
Figure.SDS-PAGE electrophoresis
This chapter is mainly to construct GS115-pPIC9K-SS-bgly Pichia pastoris recombinant engineering strain. First, after codon optimization of the SS-bgly gene, it was sent to Shanghai Shenggong for synthesis, and the E.coli TOP 10 strain containing the pPIC9K-SS-bgly plasmid was obtained. After the strain is activated, the plasmid is extracted and linearized by Sal Ⅰ enzyme, and then transferred to Pichia pastoris GS115 by electroporation. Use MD plate and colony PCR to initially screen positive transformants. After the successfully verified transformants are screened by shake flask fermentation, a 6# transformant with high enzyme activity is obtained. During the fermentation process, the recombinant strain grows normally, and the enzyme activity and protein content can reach 70 U/mL and 1.5 mg/mL respectively after 120 h of methanol induction. Take 10 μL supernatant for SDS-PAGE detection, and the band of interest appears at 57 kDa, which proves that the SS-bgly gene was successfully expressed in Pichia pastoris.
The plasmid pPIC9K-SS-bgly was extracted and preserved in the previous step as a template, and the SS-bgly gene fragment containing the restriction sites Kpn Ⅰ and Not Ⅰ was amplified by primers. The size is about 1500 bp. And the electrophoresis results showed that it was successfully amplified.
Figure.Agarose gel electropheresis of the SS-bgly gene Lane M: DNA marker; Lane 1: SS-bgly
The PCR-amplified gene SS-bgly introduced restriction sites Kpn Ⅰ and Not Ⅰ at both ends, and double-enzyme digestion with the expression vector pPICZαA. After purification and recovery by electrophoresis, it is connected and transformed into competent large intestine cells.Then evenly spread on the low-salt LB plate with corresponding resistance. The colony PCR result (Figure 3.2) shows that the band size is 2000 bp, which is consistent with theory. The positive clones that have been successfully verified by PCR are selected for further restriction enzyme digestion verification. The results are shown in Figure 3.3. After double digestion of the recombinant vector, pPICZαA and SS-bgly bands consistent with the theoretical size appeared. Combined with the sequencing results of Shanghai Shenggong, it was confirmed that the plasmid pPICZαA-SS-bgly was successfully constructed.
Figure.Screening transformants by MD plate
Figure. PCR analysis of partial P. pastoris transformantsLane M: DNA marker; Lanes 1-7: PCR products of transformants; Lane 8: Control
Figure .Restriction identification of recombinant plasmid Lane M: DNA marker; Lane 1: recombinant plasmid; Lane 2: double-digested product of recombinant plasmid
The strains with successful sequencing verification results were cultured overnight and the plasmid pPICZA-SS-bgly was extracted. After linearization with Sac Ⅰ enzyme and purification and recovery, electrotransfer to the GS115-pPIC9K-SS-bgly 6# strain selected in the last process. Spread on YPD resistant plate (containing 100 μg/mL Zeocin) and collect the single colonies grown on the plates with sterile water and spread them on YPD plates with different resistances in a gradient. For the finally screened transformants, the genomic genes are released by repeated freezing and thawing operations, and the supernatant is used as a template, and the primers are used for PCR verification. The result is shown in Figure 3.5, there is a clear band around 3000 bp, which is consistent with the theoretical value, indicating that the gene has been successfully inserted into the target strain.
Figure.Identification of positive transformants by colony PCR Lane M: DNA marker; Lane 1-7: PCR products of transformants; Lane 8: Control
Figure 3.5 PCR identification of recombinant P. pastoris strainsLane M: DNA marker; Lanes 1-7: PCR products of transformants
Different transformants are subjected to shake flask experiments, and their enzyme activities are slightly different. In order to screen high vigorous strains, select transformants that have been successfully screened by high resistance plates and verified by colony PCR for shaking flask fermentation. Its enzyme activity is between 102-152 U/mL, and the 4# transformant has the highest activity, which is 152 U/mL.
Figure.Enzyme activity of transformants
Ferment the recombinant strain GS115/9K-ZαA-SS-bgly 4# to determine the growth curve, enzyme activity and protein content. As shown in Figure 3.7, there is no difference in the growth of recombinant bacteria GS115/9K-SS-bgly 6# and GS115/9K-ZαA-SS-bgly 4#. The enzyme activity and protein content of GS115/9K-ZαA-SS-bgly 4# are higher than that of GS115/9K SS-bgly 6# at all stages. The enzyme activity of GS115/9K-SS-bgly 6# kept increasing from 0-120 h, and then gradually decreased. The enzyme activity reached the highest value of 152 U/mL at 120 h, which was an increase of 117%. The protein content has been increasing steadily, reaching 1.8 mg/mL after 120 hours of methanol induction, which is a 20% increase compared to 20%. The methanol continued to induce, but the protein content still increased, but the enzyme activity decreased. It may be due to the accumulation of proteases, which leads to the degradation of the target protein.
The glycerol phosphate dehydrogenase gene GAP is used as an internal reference gene basis：△CT=(CT, bgly -CT,GAP)Test-(CT, bgly - CT,GAP)Calibrator. Calculate the average CT value of the target gene and the internal reference gene and the expression level of the target gene SS-bgly in the transformant GS115/9K-SS-bgly 6# and the transformant GS115/9K-ZαA-SS-bgly 4#. Using the plasmid pPIC9K SS-bgly as a single copy, it was calculated that the chromosome copy numbers of the SS-bgly gene in the GS115/9K-SS-bgly 6# and GS115/9K-ZαA SS-bgly 4# strains were 1.89 and 4.03, rounded to 2 and 4.
Table. Calculation of gene copy number
Use the plasmid pPIC9K-SS-bgly extracted in Chapter 2 as a template to amplify the SS-bgly gene sequence and connect it to the pPICZαA vector.The E.coli TOP 10 strain containing pPICZαA-SS-bgly plasmid was successfully constructed. After the strain was activated on a low-salt LB resistant plate, the plasmid was extracted, linearized by the restriction enzyme Sac 1, and electrotransferred to the GS115-pPIC9K-SS-bgly 6# strain constructed in Chapter 2. After initial screening of the transformants with concentration gradient resistance plates and colony PCR, the high vigor strain GS115/9K-ZA-SS-bgly 4# was obtained by shaking flask fermentation. After 120 h of methanol induction, its enzyme activity was as high as 152 U/mL, which is an increase of 117%, and the protein content is 1.8 mg/mL, which is an increase of 20%. The fluorescence quantitative PCR technology determines that its gene copy number is 4.
Temperature and pH are both important parameters that affect enzymatic reactions. Under the condition that other conditions remain the same, we studied the effect of SS-bgly on the conversion of ginsenoside substrate to CK activity in the range of 40~90 ℃ . As shown in Figure 4.2, when the temperature is 40~80 ℃, the activity of SS-bgly transforming ginsenoside substrate to produce CK increases with the increase of temperature, and when the temperature increases to 90 ℃, the activity is reduced, therefore, 80 ℃ is the best reaction temperature for SS-bgly to hydrolyze the ginsenoside substrate. SS-bgly transforms ginsenoside substrate to produce CK at a higher temperature than most reported ginsenoside hydrolases, and its high temperature resistance makes it more suitable for industrial conversion of ginsenoside substrate to produce CK.
Figure. Effect of temperature on the production of CK
Similarly, when other conditions were consistent, we studied the effect of SS-bgly on the conversion of ginsenoside substrate to CK in the range of pH 4.0~8.0. The results are shown in Figure 4.3. When the pH is 4.0-6.0, the activity of SS-bgly transforming ginsenoside substrate to produce CK increases with the increase of pH, and when the pH increases to 7.0, its activity to produce CK decreases. Therefore, the optimal reaction pH for SS-bgly hydrolysis of ginsenoside substrate is 6.0. The SS-bgly gene is expressed in E. coli, and the optimal pH for its transformation of ginsenoside substrate is also 6.0.
Figure. Effect of pH on the production of CK
Some metal ions have been found to promote the conversion of ginsenosides. For example, Ca2+ regulates the external calcium signal of microorganisms and participates in the regulation of β-glucosidase activity, thereby promoting the biotransformation of ginsenoside Rd. Mg2+ and Fe3+ also promoted the production of ginsenoside CK, but the mechanism is still unclear. CaCO3 CaCl2 and MnCl2 metal ions have been used to increase the β-glucosidase activity of T. purpureogenus in Microbacterium esteraromaticum， Thermotoga petrophila and T. purpureogenus. This study explored the effect of some metal ions on the activity of SS-bgly transformed ginsenoside substrate to produce CK. As shown in Figure 4.4, the addition of ZnCl2, KCl, CaCl2, FeCl3 metal ions in the enzyme reaction system has no effect on the activity of transforming ginsenoside substrates to produce CK, while the addition of CuCl2 metal ions reduces the activity of CK production to 57.2%. The addition of LiCl and MgSO4 metal ions can increase the activity of CK production by 25% and 15%. In order to determine the optimal type and concentration of metal ions, we designed further experiments. The result is shown in Figure 4.5. When LiCl and MgSO4 metal ions are added to the enzyme reaction system at a final concentration of 3 mM, the CK production activity is the highest. Under the optimal metal ion concentration, the activity of LiCl metal ion for CK production is 1.15 times that of MgSO4. Therefore, in the subsequent enzymatic reactions, LiCl metal ions with a final concentration of 3 mM were added to promote the production of CK. In previous reports, LiCl has not been found to promote SS-bgly conversion of ginsenoside substrate to produce CK. We speculated that the addition of LiCl metal ions changed the structure of the enzyme and increased the affinity of the enzyme for the ginsenoside substrate, thereby promoting the production of CK.Its specific mechanism needs further analysis.
Figure.Effects of metal ions on CK producing activity
Figure.Effects of LiCl and MgSO4 concentration on CK producing activity
When the temperature of the enzymatic reaction system is set to 80 ℃ and the pH is 6.0, the final concentration of LiCl metal ions is 3 mM. We have studied the influence of the substrate concentration on the conversion of saponin, and the results are shown in Table 4.4. When the ginsenoside substrate concentration is 10 mg/mL and 20 mg/mL, it can be completely converted into 3.79 mg/mL and 7.58 mg/mL CK within 12 h and 30 h, respectively. When the substrate concentration is 30 mg/mL, At mL, the output of CK reaches the maximum value of 9.39 mg/mL, and the conversion rate is 82.5%. When the substrate concentration continued to increase to 40 mg/mL, the CK yield decreased to 9.08 mg/mL, and the conversion rate decreased to 59.8%. According to the production of CK, it is determined that 30 mg/mL is the optimal substrate concentration. Regarding SS-bgly, the highest CK yield previously reported is that when combined with Cs-abf, 4.2 mg/mL CK can be obtained. However, in this study, the yield of CK was as high as 9.39 mg/mL, which is the highest concentration of CK currently reported by SS-bgly, which is higher than the yield of CK reported in most of the literature. At present, there are many reports about glycosidase conversion of ginsenoside Rb1 to CK. In this topic, the concentration of substrate Rb1 is 15 mg/mL, which is higher than most of the substrate concentration for glycosidase conversion of ginsenoside Rb1.
Figure.Effect of crude enzyme concentration on the production of CK
Figure.Time course for CK production from ginsenoside substrate
When the temperature of the enzymatic reaction system was set to 80 ℃, the pH was 6.0, the final concentration of LiCl metal ions was 3 mM, and the substrate concentration was 30 mg/mL, the relationship between CK production and enzyme concentration was studied. The results are shown in the figure. As shown in 4.6, when the enzyme amount is increased from 0 mg/mL to 9 mg/mL, the CK output increases as the enzyme amount increases, and when the enzyme amount increases to 12 mg/mL, the CK output increases. Compared with the previous slowdown, 9 mg/mL was confirmed to be the optimal enzyme concentration.
Under the above optimized enzymatic reaction conditions, 9 mg/mL SS-bgly can convert 30 mg/mL ginsenoside substrate into 9.39 mg/mL CK within 48 hours, with a productivity of 195.6 mg/L/h, molar conversion Rate 82.5%.
According to the measured initial reaction velocity of different substrate concentrations, take 1/[S] as the abscissa and 1/V as the ordinate. We can obtain the double reciprocal form of the Michaelis equation, and obtain the maximum reaction rate Vmax and Michaelis constant Km according to the Linweaver-Burk mapping method, and calculate the conversion number kcat. Among them, the Michaelis constant represents the affinity of the enzyme to the substrate. The smaller the Km, the higher the affinity of the enzyme to the substrate. Conversion number kcat is the number of micromoles of substrate converted per micromol of enzyme molecule per second of enzymatic reaction. The larger the kcat, the higher the efficiency of enzyme catalysis.
The kinetic parameters are shown in Table 4.6. It can be seen that the values of Km, kcat and kcat/Km follow the order of Rd> Rb1> F2, F2> Rb1> Rd and F2> Rb1> Rd, respectively. This indicates that the catalytic efficiency of SS-bgly on ginsenoside F2 is much higher than that of Rb1 and Rd. F2 is converted to CK as soon as it appears, so the presence of F2 cannot be detected in HPLC. On the contrary, SS-bgly has the lowest catalytic efficiency for ginsenoside Rd, which indicates that the conversion of ginsenoside Rd to F2 is a limiting step.
Table. Kinetic parameters of enzymatic reaction
The intermediate product of SS-bgly conversion of ginsenoside substrate to CK was analyzed by HPLC. In the HPLC chromatogram, the ginsenoside Rb1, Rd, F2, and CK standard products showed obvious peaks at about 6 min, 14.5 min, and 17 min. It can be seen from Figure 4.8 that SS-bgly converted 5 mg/mL ginsenoside substrate (including 2.5 mg/mL Rb1 and 0.75 mg/mL Rd). After 3 hours, Rb1 disappeared completely and converted to 1.52 mg/mL Rd and 0.87. mg/mL CK, until after 6 h of reaction, Rd disappears completely and all is converted to CK. Therefore, the hydrolysis path of SS-bgly to convert ginsenoside substrate to CK is Rb1→ Rd→ F2→ CK. This shows that SS-bgly firstly hydrolyzes the 20-O-glycosidic bond to convert Rb1 to Rd, then hydrolyzes the 3-O-glycosidic bond of Rd to become F2, and finally hydrolyzes the 3-O-glycosidic bond of F2 to become CK (Figure 4.9). Ginsenoside F2 cannot be detected in HPLC due to its high substrate specificity. There are many different ways to convert ginsenoside Rb1 into CK using glycosidase. For example, Rb1→Rd→CK,Rb1→XVII→F2→CK, Rb1 → gypenoside XVII → gypenoside LXXV → CK. This is mainly determined by the specificity of the enzyme for the aglycone at the C-3 or C-20 position of ginsenoside Rb1.
Figure.Transformation pathway of ginsenoside Rb1 to CK
In order to improve the catalytic efficiency of SS-bgly on ginsenoside Rd, AutoDock version 4.2 was used to dock the structure of ginsenoside Rd on the active center of SS-bgly (PDB code 1GOW). The coordination files of protein and ligand required for docking calculation are prepared by AutoDock-Tools, and the docking conformation has been analyzed using Pymol software and LigPlot. The results are shown in Figure 4.9. If you want to improve the catalytic efficiency of SS-bgly on ginsenoside Rd, you can choose Tyr322, Ala263, Val209, Leu213, Phe359, Trp361, His342 as possible mutation sites. For example, Kyung-Chul et al. [156] reported that by mutating the Val209 protein, the activity of SS-bgly on ginsenoside Rd was increased by 4.2 times, and the catalytic efficiency was increased by 3.7 times.
The constructed strains were fermented, and concentrated crude enzymes were obtained through operations such as salting out, dialysis and ultrafiltration. The enzymatic reaction was carried out in sodium acetate buffer, and we carried out a series of optimizations on the enzymatic reaction conditions. We found that when the temperature of the enzyme reaction system is 80 ℃, the pH is 6.0, and the final concentration of LiCl metal ions is 3 mM, 9 mg/mL SS-bgly can completely convert 10 mg/mL and 20 mg /mL of ginsenoside substrate within 12 h and 30 h. Finally, 3.79 mg/mL and 7.58 mg/mL of CK were obtained. When the substrate concentration reached 30 mg/mL, after 48 hours of reaction, the CK yield reached 9.39 mg/mL, the conversion rate reached 82.5%, and when the substrate concentration further increased to 40 mg/mL, the CK yield dropped to 9.08 mg/mL mL, the conversion rate is 59.8%.The conversion pathway of ginsenoside Rb1 analyzed by HPLC is Rb1→ Rd→ F2→ CK. We studied the kinetic parameters of SS-bgly catalyzed hydrolysis of ginsenosides and found that the Michaelis constants Km of SS-bgly to the substrates Rb1, Rd, F2 are 3.64, 5.08, and 1.86 mM. Through analysis, the biological conversion of ginsenoside Rd to F2 is the rate-limiting step. For this reason, the structure of ginsenoside Rd is docked to the active center of SS-bgly, and hydrophobin sites that may improve its catalytic efficiency are given.