Glucosidase truncated mutant with improved thermal activity and pH adaptability as well as construction method and application of glucosidase truncated mutant

Abstract

The invention relates to the technical field of gene engineering. The invention provides a glucosidase truncated mutant with improved thermal activity and pH adaptability. The amino acid sequence of the mutant is as shown in SEQ ID NO. 1. On the basis of a homologous modeling method, specific amino acid is truncated to optimize a glucosidase structure, the thermal activity and pH adaptability are improved, the optimum temperature is increased by 10 DEG C, and gt is reserved under the condition that the pH is 5.0; the activity is 50%, and the affinity of a substrate is improved (Km is equal to 0.8517 mM). The invention further provides application of the mutant in preparation of rare ginsenoside CK. The high-titer yield of the rare ginsenoside CK prepared from the mutant is remarkably increased compared with that of the rare ginsenoside CK prepared from a wild enzyme. The preparation process is simple and has the potential of being applied to large-scale industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering, and in particular to a glucose glycosidase truncated mutant with improved thermal activity and pH adaptability, and a construction method and application thereof. BACKGROUND

[0002] As the main active ingredient of traditional Chinese medicinal material ginseng, ginsenosides have a wide range of pharmacological activities. However, due to their large molecular weight and strong polarity, the oral bioavailability of these compounds is low. Studies have found that rare ginsenosides (such as Rh2 and CK) generated by deglycosylation of ginsenosides have better intestinal absorption characteristics and can more effectively enter the blood circulation to exert their pharmacological effects. These rare saponins not only exhibit significant anti-tumor activity, but also exhibit multiple pharmacological effects in immune regulation, neuroprotection, and cardiovascular protection, and have important drug development value. In terms of industrial production, the traditional acid-base hydrolysis method has inherent defects such as severe reaction conditions, complex by-products, and serious environmental pollution. In contrast, enzyme-catalyzed or microbial transformation-based biosynthesis technology has become the mainstream research direction and development trend for the preparation of rare ginsenosides due to its mild reaction conditions, high selectivity, and environmental friendliness.

[0003] Currently, β-glucosidase, β-xylosidase, and α-rhamnosidase have been successfully used to prepare rare ginsenosides. However, due to the poor solubility of ginsenosides, modern industrial production often requires high temperatures to improve reaction efficiency and reduce the risk of microbial contamination during long-term fermentation. Another key challenge in glycoside biotransformation is the limited site selectivity of glycosidases for specific glycosyl groups during multi-step hydrolysis, which makes it difficult for single-enzyme systems to efficiently cleave multiple glycosidic bonds simultaneously. However, natural glycosidases often exhibit limited thermal activity and pH adaptability, making it difficult to balance the improvement of substrate solubility and enzyme activity, and the construction of multi-enzyme cascade catalysis also faces the problem of incompatible conditions. Therefore, it is of great significance to develop methods for rational regulation of enzyme optimal working conditions. SUMMARY

[0004] The present application aims to provide a glucose glycosidase truncated mutant with improved thermal activity and pH adaptability, which significantly improves thermal activity and pH adaptability compared to wild-type enzymes without affecting catalytic activity. The preparation process is simple, which improves the complex process and heavy workload in existing genetic engineering enzyme modification, and has the potential to be applied to large-scale industrial production.

[0005] To achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The present application provides a heat activity and pH adaptability improved glucosidase truncated mutant, the amino acid sequence of which is shown as SEQ ID NO. 1.

[0007] As preferred, based on the glucosidase B6 amino acid sequence shown as SEQ ID NO. 3, the first to the 33rd amino acid is truncated by kinetic simulation analysis.

[0008] The present application provides a coding gene of the heat activity and pH adaptability improved glucosidase truncated mutant, the nucleotide sequence of which is shown as SEQ ID NO. 2.

[0009] The present application provides a recombinant expression vector containing the coding gene of the heat activity and pH adaptability improved glucosidase truncated mutant.

[0010] The present application provides a recombinant expression bacterium containing the coding gene of the heat activity and pH adaptability improved glucosidase truncated mutant.

[0011] The present application provides a construction method of the recombinant expression bacterium, comprising the following steps: performing PCR amplification with a plasmid containing the glucosidase B6 coding gene shown as SEQ ID NO: 4 as a template, connecting the amplification product with a linearized vector digested by NcoI / XhoI, and transforming into BL21 competent cells, thereby obtaining the recombinant expression bacterium.

[0012] As preferred, the PCR amplification primer comprises 13aa-F shown as SEQ ID NO: 5 and 13aa-R shown as SEQ ID NO: 6.

[0013] The present application provides a preparation method of rare ginsenoside CK, comprising the following steps:

[0014] (1) performing high-pressure homogenization crushing on the prepared recombinant expression bacterium containing the coding gene of the heat activity and pH adaptability improved glucosidase truncated mutant, thereby obtaining a crude enzyme solution;

[0015] (2) mixing the crude enzyme solution with a ginsenoside Rb1 solution, and performing catalytic reaction, thereby obtaining the product.

[0016] As preferred, the final concentration of the crude enzyme solution is 0.5-2 mg / mL, and the concentration of the ginsenoside Rb1 solution is 0.5-10 mg / mL.

[0017] As preferred, the temperature of the catalytic reaction is 30-55℃, and the time of the catalytic reaction is 3-24 h.

[0018] By adopting the above technical solution, the present application has the following beneficial effects:

[0019] 1. The technical solution of the present application analyzes the stability, hydrophobicity and three-dimensional structure of glucosidase B6 by biological methods such as kinetic simulation, and the solvent accessible surface area (SASA) and radius of gyration (Rg) after virtual mutation, finds that the 43 amino acids at the N-terminal of the enzyme are an unstable extension region. Then based on the homology modeling method, the structure of the glucosidase is optimized by truncating specific amino acids (1st to 33rd amino acids), and the obtained truncated mutant effectively improves the thermal activity and pH adaptability, the optimum temperature is increased by 10℃ (up to 50℃), the acid resistance is enhanced (more than 50% activity is retained under pH = 5.0 conditions), and the substrate affinity is improved (Km = 0.8517 mM).

[0020] 2. The rare ginsenoside CK prepared by using the mutant described in the present application has a significantly improved high-titer yield compared to the rare ginsenoside CK prepared by using the wild-type enzyme.

[0021] 3. The technical solution of the present application effectively improves the problems of complex process and heavy workload in the existing genetic engineering enzyme modification, and the preparation process is simple, which has the potential to be applied to large-scale industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The stability, hydrophobicity and three-dimensional structure of the amino acids of wild-type glucosidase B6 are shown in (A represents stability, B represents hydrophobicity, and C represents three-dimensional structure); Figure 1

[0023] The SASA and Rg of wild-type glucosidase B6 and each truncated enzyme are shown in (A represents SASA, and B represents Rg); Figure 2 Figure 2 The thermal activity, pH adaptability and specific enzyme activity of wild-type glucosidase B6 and each truncated enzyme are shown in (A represents thermal activity, B represents pH adaptability, and C represents specific enzyme activity);

[0024] Figure 3 Figure 3

[0025] Figure 4 The yield of ginsenoside CK prepared by using wild-type glucosidase B6 and truncated enzyme 33aa is shown. DETAILED DESCRIPTION

[0026] ​​​The present application provides a glucose glycosidase truncated mutant with improved thermal activity and pH adaptability, the amino acid sequence of the mutant is shown as SEQ ID NO. 1, and the specific sequence is MAGVMDPYEDPRLSADERAADLVARMSLEEKCGLMFQTVIEVGEEGELLEAPGRISKSPTTTVVRGKHLSHFNVHAIRSARQAAVWNNNLQALAAQTPHGVPVTVSTDPRHAFVENTGVGFAAGPFSQWPEGLGLAAIDDVETVRRFADVARQEYRAVGIRAALHPQIDLATEPRWGRQAQTLGQDAGRVAEFTAAYLQGFQGDALGPDSVACTTKHFPGGGPQKDGEDAHFPYGREQVYPGGMFEYHLEPFREAIRRGTAAMMPYYGMPIGLERNGVPIEEVGFGYNRQIVTDLLRGELGFDGVVVTDWELVNDNHVGDQVLPARAWGVEELSPSERMLKILDAGADQFGGEECVDLLIALVRAGRVDEARIDASALRLLRVKFQLGLFDDPFVDPDEAERIVGNAQFRAEGERAQARSLTVVQNRPTPGADHPVLPLSGAGRRVYVEGFRPEDVAELGEIVADPADADLALVRLGAPFEPRDDLFLEAWFHQGSLEFPPGRVYRMRSIAAHCPLVLVVNLDRPGILTPFAAFASAIVVDFGSSSRAVVDVLTGRIAPEGRLPIELPRSMDAVRSSREDVPSDTGDPLFPVHFGLELPMRVGGRADAHHHHHH.

[0027] In the present application, the stability, hydrophobicity and three-dimensional structure of glucosidase B6 are analyzed by biological methods such as kinetic simulation, and the solvent accessible surface area (SASA) and radius of gyration (Rg) after virtual mutation, and then based on the homology modeling method, the structure of glucosidase is optimized by truncating specific amino acids of glucosidase B6, to obtain the glucosidase truncated mutant with improved thermal activity and pH adaptability. The amino acid sequence of the glucosidase B6 in the present application is shown in SEQ ID NO. 3, and the specific sequence is MGSSHHHHHHSQDPTPTYLTAPDGTRFRDLNGNGVMDPYEDPRLSADERAADLVARMSLEEKCGLMFQTVIEVGEEGELLEAPGRISKSPTTTVVRGKHLSHFNVHAIRSARQAAVWNNNLQALAAQTPHGVPVTVSTDPRHAFVENTGVGFAAGPFSQWPEGLGLAAIDDVETVRRFADVARQEYRAVGIRAALHPQIDLATEPRWGRQAQTLGQDAGRVAEFTAAYLQGFQGDALGPDSVACTTKHFPGGGPQKDGEDAHFPYGREQVYPGGMFEYHLEPFREAIRRGTAAMMPYYGMPIGLERNGVPIEEVGFGYNRQIVTDLLRGELGFDGVVVTDWELVNDNHVGDQVLPARAWGVEELSPSERMLKILDAGADQFGGEECVDLLIALVRAGRVDEARIDASALRLLRVKFQLGLFDDPFVDPDEAERIVGNAQFRAEGERAQARSLTVVQNRPTPGADHPVLPLSGAGRRVYVEGFRPEDVAELGEIVADPADADLALVRLGAPFEPRDDLFLEAWFHQGSLEFPPGRVYRMRSIAAHCPLVLVVNLDRPGILTPFAAFASAIVVDFGSSSRAVVDVLTGRIAPEGRLPIELPRSMDAVRSSREDVPSDTGDPLFPVHFGLELPMRVGGRADA. The first to 33th amino acids of the glucosidase B6 are truncated in the present application.

[0028]

[0029] The present application provides a recombinant expression vector comprising a gene encoding the heat-active and pH-adaptive improved glucosidase truncated mutant.

[0030] The present application provides a recombinant expression bacterium comprising a gene encoding the heat-active and pH-adaptive improved glucosidase truncated mutant.

[0031] The present application provides a construction method of the recombinant expression bacterium, comprising the following steps: performing PCR amplification with a plasmid containing a glucosidase B6 encoding gene as shown in SEQ ID NO: 4 as a template, connecting the amplification product with a linearized vector digested by NcoI / XhoI, and transforming into BL21 competent cells, thereby obtaining the recombinant expression bacterium.

[0032]

[0033] In the present application, the PCR amplification primer comprises 13aa-F and 13aa-R, the sequence of 13aa-F is shown as SEQ ID NO: 5, and the specific sequence is ctttaagaaggagatataccatggctATGACCCCTACGTACC; the sequence of 13aa-R is shown as SEQ ID NO: 6, and the specific sequence is cagcggtttctttaccagactcgagttagtggtgatgatggtgatgCGCATCCGCGCGG.

[0034] In the present application, the PCR amplification system is preferably 1.2 μL of 13aa-F, 1.2 μL of 13aa-R, 0.6 μL of a plasmid containing the glucosidase B6 coding gene shown as SEQ ID NO: 4, 12 μL of ultrapure water and 15 μL of PrimerstarMax.

[0035] In the present application, the PCR amplification program is preferably 95℃ pre-denaturation for 3 min; 98℃ pre-denaturation for 3 min; 98℃ denaturation for 15 s, 60℃ annealing for 30 s, 72℃ extension for 20 s, 30 cycles.

[0036] The present application provides a preparation method of rare ginsenoside CK, comprising the following steps:

[0037] (1) The recombinant expression bacteria containing the coding gene of the heat activity and pH adaptability improved glucosidase truncated mutant prepared are high-pressure homogenized to obtain a crude enzyme solution;

[0038] (2) The crude enzyme solution is mixed with a ginsenoside Rb1 solution to catalyze a reaction, and the rare ginsenoside CK is obtained.

[0039] In the present application, the recombinant expression bacteria containing the prepared coding gene of the heat-active and pH-adaptive improved glucosidase truncated mutant are inoculated into a test tube containing 4 mL of LB medium, cultured at 37℃ overnight, then transferred into a 100 mL shake flask to induce expression of the protein; after the expression is completed, the bacterial bodies are collected, resuspended in a phosphate buffer, then broken by 800 MPa high pressure homogenization to obtain a crude enzyme solution. The temperature of the induced expression is preferably 14-18℃, further preferably 15-17℃, and more preferably 16℃; the time of the induced expression is preferably 20-25 h, further preferably 21-23 h, and more preferably 22 h. The pH value of the phosphate buffer is preferably 7.2-7.8, further preferably 7.3-7.6, and more preferably 7.5; the concentration of the phosphate buffer is preferably 15-25 mM, further preferably 18-22 mM, and more preferably 20 mM.

[0040] In the present application, the crude enzyme solution is mixed with a ginsenoside Rb1 solution to catalyze a reaction, and a dilute ginsenoside CK is obtained. The final concentration of the crude enzyme solution in the present application is preferably 0.5-2 mg / mL, further preferably 0.5-1 mg / mL, and more preferably 0.5 mg / mL; the concentration of the ginsenoside Rb1 solution is preferably 0.5-10 mg / mL, further preferably 0.5-5 mg / mL, and more preferably 1 mg / mL.

[0041] In the present application, the temperature of the catalyzed reaction is preferably 30-55℃, further preferably 40-52℃, and more preferably 50℃; the time of the catalyzed reaction is preferably 3-24 h, further preferably 5-20 h, and more preferably 12 h.

[0042] The technical solutions provided by the present application are described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0043] Example 1 (I)

[0045] Based on wild-type glucosidase B6 (the amino acid sequence is shown as SEQ ID NO. 3), 200 ns dynamics simulation is performed using gromacs, the RMSF in the trajectory file is analyzed, the stability, hydrophilic and hydrophobic properties, and three-dimensional structure of the amino acids of the glucosidase B6 (as shown in Figure 1 ), and the solvent accessible surface area (SASA) and radius of gyration (Rg) after virtual mutation are analyzed, and it is found that the first 43 amino acids at the N-terminal are unstable regions. The results of the stability, hydrophilic and hydrophobic properties, and three-dimensional structure of the amino acids of the glucosidase B6 are shown in Figure 1 .

[0046] The wild glucosidase B6 amino acid sequence is shown in SEQ ID NO. 3, and the specific sequence is MGSSHHHHHHSQDPTPTYLTAPDGTRFRDLNGNGVMDPYEDPRLSADERAADLVARMSLEEKCGLMFQTVIEVGEEGELLEAPGRISKSPTTTVVRGKHLSHFNVHAIRSARQAAVWNNNLQALAAQTPHGVPVTVSTDPRHAFVENTGVGFAAGPFSQWPEGLGLAAIDDVETVRRFADVARQEYRAVGIRAALHPQIDLATEPRWGRQAQTLGQDAGRVAEFTAAYLQGFQGDALGPDSVACTTKHFPGGGPQKDGEDAHFPYGREQVYPGGMFEYHLEPFREAIRRGTAAMMPYYGMPIGLERNGVPIEEVGFGYNRQIVTDLLRGELGFDGVVVTDWELVNDNHVGDQVLPARAWGVEELSPSERMLKILDAGADQFGGEECVDLLIALVRAGRVDEARIDASALRLLRVKFQLGLFDDPFVDPDEAERIVGNAQFRAEGERAQARSLTVVQNRPTPGADHPVLPLSGAGRRVYVEGFRPEDVAELGEIVADPADADLALVRLGAPFEPRDDLFLEAWFHQGSLEFPPGRVYRMRSIAAHCPLVLVVNLDRPGILTPFAAFASAIVVDFGSSSRAVVDVLTGRIAPEGRLPIELPRSMDAVRSSREDVPSDTGDPLFPVHFGLELPMRVGGRADA.

[0047] Based on the wild grape glucosidase B6, truncated mutant sequences of N-terminal truncation of the 1st-13th, 1st-23rd, 1st-33rd and 1st-43rd amino acids are designed respectively (referred to as truncated mutant 13aa, truncated mutant 23aa, truncated mutant 33aa and truncated mutant 43aa respectively), and the molecular dynamics analysis of each truncated mutant is carried out for 200ns, and the SASA and Rg results of the wild enzyme and each truncated enzyme are as follows Figure 2 The smallest value obtains the largest thermal activity promotion.

[0048] The amino acid sequence of the truncated mutant 13aa is shown as SEQ ID NO. 13, and the specific sequence is MATPTYLTAPDGTRFRDLNGNGVMDPYEDPRLSADERAADLVARMSLEEKCGLMFQTVIEVGEEGELLEAPGRISKSPTTTVVRGKHLSHFNVHAIRSARQAAVWNNNLQALAAQTPHGVPVTVSTDPRHAFVENTGVGFAAGPFSQWPEGLGLAAIDDVETVRRFADVARQEYRAVGIRAALHPQIDLATEPRWGRQAQTLGQDAGRVAEFTAAYLQGFQGDALGPDSVACTTKHFPGGGPQKDGEDAHFPYGREQVYPGGMFEYHLEPFREAIRRGTAAMMPYYGMPIGLERNGVPIEEVGFGYNRQIVTDLLRGELGFDGVVVTDWELVNDNHVGDQVLPARAWGVEELSPSERMLKILDAGADQFGGEECVDLLIALVRAGRVDEARIDASALRLLRVKFQLGLFDDPFVDPDEAERIVGNAQFRAEGERAQARSLTVVQNRPTPGADHPVLPLSGAGRRVYVEGFRPEDVAELGEIVADPADADLALVRLGAPFEPRDDLFLEAWFHQGSLEFPPGRVYRMRSIAAHCPLVLVVNLDRPGILTPFAAFASAIVVDFGSSSRAVVDVLTGRIAPEGRLPIELPRSMDAVRSSREDVPSDTGDPLFPVHFGLELPMRVGGRADAHHHHHH.

[0049] The amino acid sequence of the truncated mutant 23aa is shown as SEQ ID NO. 14, and the specific sequence is MAGTRFRDLNGNGVMDPYEDPRLSADERAADLVARMSLEEKCGLMFQTVIEVGEEGELLEAPGRISKSPTTTVVRGKHLSHFNVHAIRSARQAAVWNNNLQALAAQTPHGVPVTVSTDPRHAFVENTGVGFAAGPFSQWPEGLGLAAIDDVETVRRFADVARQEYRAVGIRAALHPQIDLATEPRWGRQAQTLGQDAGRVAEFTAAYLQGFQGDALGPDSVACTTKHFPGGGPQKDGEDAHFPYGREQVYPGGMFEYHLEPFREAIRRGTAAMMPYYGMPIGLERNGVPIEEVGFGYNRQIVTDLLRGELGFDGVVVTDWELVNDNHVGDQVLPARAWGVEELSPSERMLKILDAGADQFGGEECVDLLIALVRAGRVDEARIDASALRLLRVKFQLGLFDDPFVDPDEAERIVGNAQFRAEGERAQARSLTVVQNRPTPGADHPVLPLSGAGRRVYVEGFRPEDVAELGEIVADPADADLALVRLGAPFEPRDDLFLEAWFHQGSLEFPPGRVYRMRSIAAHCPLVLVVNLDRPGILTPFAAFASAIVVDFGSSSRAVVDVLTGRIAPEGRLPIELPRSMDAVRSSREDVPSDTGDPLFPVHFGLELPMRVGGRADAHHHHHH.

[0050] The amino acid sequence of the truncated mutant 33aa is shown as SEQ ID NO. 1, and the specific sequence is MAGVMDPYEDPRLSADERAADLVARMSLEEKCGLMFQTVIEVGEEGELLEAPGRISKSPTTTVVRGKHLSHFNVHAIRSARQAAVWNNNLQALAAQTPHGVPVTVSTDPRHAFVENTGVGFAAGPFSQWPEGLGLAAIDDVETVRRFADVARQEYRAVGIRAALHPQIDLATEPRWGRQAQTLGQDAGRVAEFTAAYLQGFQGDALGPDSVACTTKHFPGGGPQKDGEDAHFPYGREQVYPGGMFEYHLEPFREAIRRGTAAMMPYYGMPIGLERNGVPIEEVGFGYNRQIVTDLLRGELGFDGVVVTDWELVNDNHVGDQVLPARAWGVEELSPSERMLKILDAGADQFGGEECVDLLIALVRAGRVDEARIDASALRLLRVKFQLGLFDDPFVDPDEAERIVGNAQFRAEGERAQARSLTVVQNRPTPGADHPVLPLSGAGRRVYVEGFRPEDVAELGEIVADPADADLALVRLGAPFEPRDDLFLEAWFHQGSLEFPPGRVYRMRSIAAHCPLVLVVNLDRPGILTPFAAFASAIVVDFGSSSRAVVDVLTGRIAPEGRLPIELPRSMDAVRSSREDVPSDTGDPLFPVHFGLELPMRVGGRADAHHHHHH.

[0051] The amino acid sequence of the truncated mutant 43aa is shown in SEQ ID NO. 15, and the specific sequence is MALSADERAADLVARMSLEEKCGLMFQTVIEVGEEGELLEAPGRISKSPTTTVVRGKHLSHFNVHAIRSARQAAVWNNNLQALAAQTPHGVPVTVSTDPRHAFVENTGVGFAAGPFSQWPEGLGLAAIDDVETVRRFADVARQEYRAVGIRAALHPQIDLATEPRWGRQAQTLGQDAGRVAEFTAAYLQGFQGDALGPDSVACTTKHFPGGGPQKDGEDAHFPYGREQVYPGGMFEYHLEPFREAIRRGTAAMMPYYGMPIGLERNGVPIEEVGFGYNRQIVTDLLRGELGFDGVVVTDWELVNDNHVGDQVLPARAWGVEELSPSERMLKILDAGADQFGGEECVDLLIALVRAGRVDEARIDASALRLLRVKFQLGLFDDPFVDPDEAERIVGNAQFRAEGERAQARSLTVVQNRPTPGADHPVLPLSGAGRRVYVEGFRPEDVAELGEIVADPADADLALVRLGAPFEPRDDLFLEAWFHQGSLEFPPGRVYRMRSIAAHCPLVLVVNLDRPGILTPFAAFASAIVVDFGSSSRAVVDVLTGRIAPEGRLPIELPRSMDAVRSSREDVPSDTGDPLFPVHFGLELPMRVGGRADAHHHHHH. (II)

[0053] Based on wild grape glucosidase B6, the point mutation primers of N-terminal truncated mutants of 13, 23, 33 and 43 amino acids were designed respectively by using SnapGene software (the obtained truncated mutants are respectively recorded as 13aa, 23aa, 33aa and 43aa).

[0054] Table 1 Point mutation primers of each truncated mutant

[0055]

[0056] The homologous recombination strategy was used, and pETduet1 plasmid (purchased from Beijing Quanshi Gold Technology Co., Ltd.) containing wild-type glucosidase B6 encoding gene (as shown in SEQ ID NO: 4) was used as a DNA template, and PCR amplification was performed using the point mutation primers shown in Table 1. The PCR system was as follows: 1.2 μL of upstream primer F, 1.2 μL of downstream primer R, 0.6 μL of pETduet1 plasmid containing wild-type glucosidase B6 encoding gene, 12 μL of ultrapure water, and 15 μL of Primer starMax. The PCR program was as follows: 95 °C pre-denaturation for 3 min; 98 °C pre-denaturation for 3 min; 98 °C denaturation for 15 s, 60 °C annealing for 30 s, 72 °C extension for 20 s, 30 cycles. The PCR amplification product was recovered by gel. Then the PCR amplification product was purified and connected with the linearized vector pETduetI digested by NcoI / XhoI by homologous recombination to construct the recombinant expression vector plasmid 13aa, plasmid 23aa, plasmid 33aa, and plasmid 43aa. Each recombinant expression vector was transformed into BL21 (DE3) competent cells, and the recombinant strains expressing the target mutants were successfully obtained through resistance screening and sequencing verification.

[0057]

[0058] The constructed plasmids 13aa, 23aa, 33aa, and 43aa were introduced into *E. coli* BL21 (DE3, purchased from Beijing TransGen Technologies Co., Ltd.) via chemical transformation to obtain recombinant expression bacteria capable of expressing truncated mutants. The recombinant expression bacteria and wild-type enzyme B6 (constructed using the same method as the recombinant expression bacteria) were inoculated into test tubes containing 4 mL of LB medium and cultured overnight at 37°C. The culture was then transferred to 100 mL shake flasks and induced to express the protein at 16°C for 22 h. After expression, the bacterial weight was collected and resuspended in pH 7.5 phosphate buffer (20 mM), and the mixture was homogenized using a high-pressure homogenizer to obtain crude enzyme solution. The enzymes, namely wild-type glucosidase B6, truncated enzyme 13aa, truncated enzyme 23aa, truncated enzyme 33aa, and truncated enzyme 43aa, were then purified using a Ni-NTA column.

[0059] Example 2 (one)

[0061] Wild-type glucosidase B6 and the truncated enzymes prepared in Example 1 were incubated at 30, 35, 40, 45, and 55°C for 5 minutes, respectively. The activities of each enzyme group were then measured, with the highest activity value set as 100%. The results of the thermal activity measurement are as follows: Figure 3 As shown. From Figure 3 It can be seen that the optimal operating temperature of the truncated enzyme 33aa is 50℃, which is higher than that of wild glucosidase B6 at 10℃, indicating that the truncated enzyme prepared in this invention has better thermal activity than wild glucosidase B6. (two)

[0063] Wild-type glucosidase B6 was reacted with the truncated enzymes prepared in Example 1 in systems ranging from pH 6 to 8. Each reaction system contained 0.005 mg of enzyme (wild-type glucosidase B6 or truncated enzyme) and 0.3 g of substrate pNPG. The reactions were carried out at their respective optimal operating temperatures (B6: 40℃, 13aa: 45℃, 23aa: 45℃, 33aa: 50℃, 43aa: 45℃) for 1 min, and the absorbance of each group was measured. The activity at its highest value was set as 100%. The results of the glucose tolerance test are as follows: Figure 3 As shown. From Figure 3 It can be seen that the truncated enzyme prepared in this invention has significantly better activity than wild-type glucosidase B6 under acidic pH conditions. (three)

[0065] The pNPG 5 g was dissolved in 5 mL of deionized water to obtain a pNPG solution with a concentration of 5 mM. 0.3 mL of the pNPG solution was taken, and wild-type glucosidase B6 and each truncated enzyme crude enzyme solution prepared in Example 1 were added, wherein the wild-type glucosidase B6 contained 0.005 mg of glucosidase. The reaction was carried out at 40°C for 1 min, 0.2 mL of 1M Na2CO3 was added, the absorbance was measured at 405 nM, and the final specific enzyme activity was calculated.

[0066] 1 U was set as 1 μmol of product generated per minute; U = ΔA / t*V / m, wherein ΔA represents the luminosity value of the measured amount change, t represents the reaction time, V represents the reaction liquid volume, and m represents the mass of the reaction substance.

[0067] Specific enzyme activity = total enzyme activity U / mg of enzyme.

[0068] The results are shown in Table 1. Figure 3 The specific enzyme activities of B6, 13aa, 23aa, 33aa, and 43aa were 58.07, 64.98, 55.14, 68.50, and 45.58 U / mg, respectively.

[0069] Example 3

[0070] The ginsenoside Rb1 was weighed and dissolved in water to prepare a substrate solution with a concentration of 1 mg / mL. 0.25 mg / mL of wild-type glucosidase B6 and 0.5 mL of truncated enzyme 33aa crude enzyme solution were added, respectively. The wild-type glucosidase B6 system was reacted at 40°C, and the truncated enzyme 33aa system was reacted at 50°C for 12 h. The product ginsenoside CK yield was detected by liquid chromatography.

[0071] Table 2 Gradient conditions for mobile phase operation

[0072]

[0073]

[0074] Table 3 Gradient conditions for mobile phase operation-peak position

[0075] Marker Retention time Rb1 13.932 Rd 16.990 Gyp17 18.188 F2 22.423 Gyp75 25.85 C-K 32.684

[0076] Liquid chromatography detection method: sample treatment: an equal volume of methanol (chromatography grade) was added to the reaction system, and the sample was filtered with a 0.22 μm filter; chromatographic column: C18 column, 250 x 4.6 mm; column temperature: 30°C; mobile phase: A phase acetonitrile, B phase ultrapure water; detector: ultraviolet detector; detection wavelength: 203 nm; injection volume: 20 μL; elution conditions: the reaction of hydrolyzed ginsenoside Rb1 used the gradient elution method in Table 2.

[0077] The results are shown in Table 1. Figure 4The yield of ginsenoside Rb1 hydrolyzed by wild ginsenosidase B6 and truncated enzyme 33aa is 0.21 mg / mL and 0.31 mg / mL respectively at low substrate concentration (1 mg / mL).

[0078] As can be seen from the above, the heat activity and pH adaptability of the ginsenosidase truncated mutant (truncated enzyme 33aa) prepared by the technology of the present application are improved, i.e. the optimum temperature is increased by 10℃ (up to 50℃), the acid resistance is enhanced (more than 50% activity is retained at pH=5.0), and the substrate affinity is improved (Km=0.8517 mM); the high-titer yield of rare ginsenoside CK prepared by the wild-type enzyme is significantly improved.

[0079] The above only describes the preferred embodiments of the present application, and it should be noted that, for those skilled in the art, some improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A truncated mutant of glucosidase with improved thermal activity and pH adaptability, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO.

1.

2. The truncated mutant of glucosidase with improved thermal activity and pH adaptability according to claim 1, characterized in that, Based on kinetic simulation analysis, amino acids from position 1 to position 33 were truncated from the amino acid sequence of glucosidase B6 as shown in SEQ ID NO.

3.

3. The encoding gene of the truncated glucosidase mutant with enhanced thermal activity and pH adaptability as described in claim 1 or 2, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

4. A recombinant expression vector comprising the encoding gene of claim 3.

5. A recombinant expression bacterium comprising the encoding gene of claim 3.

6. The method for constructing the recombinant expression bacteria according to claim 5, characterized in that, Includes the following steps: The plasmid containing the glucosidase B6 encoding gene as shown in SEQ ID NO:4 was used as a template for PCR amplification. The amplification product was ligated into a linearized vector that had been double-digested with NcoI / XhoI and transformed into BL21 competent cells to obtain the product. The PCR amplification primers include 13aa-F as shown in SEQ ID NO:5 and 13aa-R as shown in SEQ ID NO:

6.

7. The application of the truncated glucosidase mutant with improved thermal activity and pH adaptability as described in claim 1 in the preparation of rare ginsenoside CK.

8. A method for preparing a rare ginsenoside CK, characterized in that, Includes the following steps: (1) The recombinant expression bacteria containing the encoding gene of the glucosidase truncated mutant with improved thermal activity and pH adaptability as described in claim 1 were homogenized under high pressure and crushed to obtain crude enzyme solution; (2) Mix the crude enzyme solution with the ginsenoside Rb1 solution and catalyze the reaction to obtain the final product.

9. The preparation method according to claim 8, characterized in that, The final concentration of the crude enzyme solution is 0.5-2 mg / mL, and the concentration of the ginsenoside Rb1 solution is 0.5-10 mg / mL.

10. The preparation method according to claim 8, characterized in that, The temperature of the catalytic reaction is 30-55℃, and the time of the catalytic reaction is 3-24h.

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