CN108410839B - Beta-glucuronidase mutant with improved thermal stability - Google Patents

Beta-glucuronidase mutant with improved thermal stability Download PDF

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CN108410839B
CN108410839B CN201810071464.XA CN201810071464A CN108410839B CN 108410839 B CN108410839 B CN 108410839B CN 201810071464 A CN201810071464 A CN 201810071464A CN 108410839 B CN108410839 B CN 108410839B
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李春
贾锦彤
冯旭东
吕波
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Beijing Institute of Technology BIT
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Abstract

The invention discloses a beta-glucuronidase mutant with improved thermal stability, belonging to the field of bioengineering. The beta-glucuronidase derived from Talaromyces pinophilum Li-93 (the preservation number is CGMCC No.11765) is subjected to site-directed mutagenesis, valine 212, threonine 220, cysteine 238 and valine 348 coded by the beta-glucuronidase are mutated into arginine, 23 amino acids at the N end of the beta-glucuronidase are cut off, and recombinant plasmids of the mutant genes are transferred into Pichia pastoris GS115 to be expressed, so that the beta-glucuronidase mutant with improved thermal stability is obtained. The half-life period of the beta-glucuronidase mutant is prolonged by 81min and 25min compared with that of a wild type at the temperature of 55 ℃ and 70 ℃, and is 3 times and 2.3 times of that of the wild type. And a process for converting GL at high temperature is established by using the mutant. The beta-glucuronidase mutant obtained by the invention has wide industrial application prospect.

Description

Beta-glucuronidase mutant with improved thermal stability
Technical Field
The invention relates to a beta-glucuronidase mutant with improved thermal stability, belonging to the technical field of biological engineering.
Background
Beta-glucuronidase (EC: 3.2.1.31) is capable of recognizing and catalyzing various types of beta-glucuronic acid glycosidic bonds, while releasing beta-glucuronic acid and the corresponding ligands or aglycones. The enzyme belongs to mostly glycosidase family 1 (GH1) and glycosidase family 2(GH2), and the distribution of the enzyme is found in glycosidase family 79(GH79) in recent years. It has now been found that mammals, fungi and bacteria are able to secrete β -glucuronidase. Beta-glucuronidase has wide application in many fields, such as human body drug analysis, precursor drug enzyme guide treatment, tumor pathological research and the like. In addition, the beta-glucuronidase can generate color reaction after reacting with a substrate, so that the beta-glucuronidase is often used as a marker gene to locate the expression of other target genes. Recently, beta-glucuronidase is widely applied to modification of natural glucoside compounds to produce derivatives with high additional values, and the demand of the market for the enzyme is rapidly increased. However, most of the β -glucuronidases on the market are mesophilic ones, and in practical applications, the enzymes have poor heat resistance and are easy to inactivate, so that the reaction efficiency is reduced, and thus the β -glucuronidases become one of the bottlenecks in industrial application. Therefore, β -glucuronidase having high thermostability is urgently required in the market.
The inventor screens out a Talaromyces pinophilum Li-93 from Xinjiang in the earlier stage, beta-glucuronidase generated by induction can hydrolyze commercial monoammonium glycyrrhizinate to generate GAMG with high added value, the directionality is good, and the conversion rate of glycyrrhizic acid can reach 95%. The strain Talaromyces pinophilum Li-93 is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number: CGMCC No. 11765. The coding gene tpgus of beta-glucuronidase produced by Talaromyces pinophilum Li-93 has the length of 1554bp (GenBank accession number: KY609919), the gene totally codes 518 amino acids, and the theoretical single subunit molecular weight is 66.304 KDa. At the early stage, the TPGUS gene is successfully subjected to heterologous expression in pichia pastoris to obtain the recombinant beta-glucuronidase TPGUS-P. However, the enzyme has poor heat resistance, which limits its practical application.
Therefore, the invention improves the thermal stability of TPGUS-P by modifying the gene, and further effectively improves the industrial application value of TPGUS-P.
Disclosure of Invention
The invention aims to effectively improve the thermal stability of the beta-glucuronidase and increase the industrial value of the beta-glucuronidase by site-directed mutagenesis and overlapping extension PCR technology on the basis of the existing beta-glucuronidase.
In order to achieve the purpose, the invention mutates beta-glucuronidase coded by a gene sequence tpgus (GenBank accession number: KY609919), mutates valine 212, threonine 220, cysteine 238 and valine 348 into arginine, and cuts 23 amino acids at the N end of the beta-glucuronidase. The recombinant plasmid of the mutant gene is transferred into pichia pastoris GS115 for expression, and the beta-glucuronidase mutant with improved thermal stability is obtained.
The nucleotide sequence for coding the original beta-glucuronidase is shown as a sequence with GenBank accession number KY 609919.
The method for obtaining the mutant comprises the steps of taking pGAPz alpha-tpgus plasmid containing tpgus gene as a template, designing a primer, carrying out site-directed mutagenesis through PCR to obtain a recombinant plasmid pGAPz alpha-tpgusM 1 containing a mutant gene, and expressing the plasmid pGAPz alpha-tpgusM 1 in Pichia pastoris GS115 to produce the beta-glucuronidase mutant.
Drawings
FIG. 1 shows half-lives of the original enzyme and the constructed mutant at different temperatures.
FIG. 2 shows the reaction process for hydrolyzing substrate GL to generate GAMG at different temperatures using TPGUS-P mutant.
Detailed Description
The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1: construction of beta-glucuronidase mutant engineering bacteria
Through analyzing the three-dimensional structure of TPGUS-P and the homologous sequence alignment of TPGUS-P and thermophilic glycosidase of the same family, the valine at the 212 th position, the threonine at the 220 th position, the cysteine at the 238 th position and the valine at the 348 th position are all mutated into arginine, and 23 amino acids at the N end of the beta-glucuronidase are cut off. pGAPz alpha-tpgus plasmid containing tpgus gene is taken as a template, primers are designed, and the site-directed mutagenesis is carried out by PCR to obtain a recombinant plasmid pGAPz alpha-tpgusM 1 containing mutant gene (the PCR system is shown in Table 2), wherein the primers used for the site-directed mutagenesis are as follows:
TABLE 1 primers used for the mutations
Figure BDA0001557441400000021
TABLE 2 plasmid cloning System
Figure BDA0001557441400000022
Figure BDA0001557441400000031
And (3) PCR reaction conditions: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 55 deg.C for 30s, extension at 72 deg.C for 3.5min,30 cycles, extension at 72 deg.C for 5min, and storage at 16 deg.C.
The PCR product was digested with Fastdigest Dpn1 enzyme at 37 ℃ for 4h to remove the template strand (the digestion system is shown in Table 2).
TABLE 3 template Elimination System
Figure BDA0001557441400000032
Directly transforming the enzyme digestion product into an escherichia coli Top10 competent cell; screening positive clones, carrying out transformation expression on plasmid pGAPz alpha-tpgusM 1 in Pichia pastoris GS115 after correct sequencing, and screening the positive clones to obtain the engineering bacteria containing the beta-glucuronidase mutant gene.
Example 2: expression and purification of beta-glucuronidase mutant
The positive monoclonal colonies were picked and transferred to 30mL YPD medium (2% peptone, 1% yeast extract, 2% glucose) containing 50. mu.g/mL bleomycin, and cultured overnight at 30 ℃ and 200rpm to obtain a seed solution, the seed solution was transferred at 1% inoculum size to 300mL YPD medium (2% peptone, 1% yeast extract, 2% glucose) containing 50. mu.g/mL thiobleomycin, cultured at 30 ℃ and 200rpm, and then glucose was added every 12 hours to a final concentration of 2%, and the culture was continued for 48 hours.
Centrifuging at 4 deg.C 12000rpm for 10min, collecting supernatant, mixing with precooled acetone at-20 deg.C, and centrifuging at 4 deg.C 12000rpm for 10 min. After centrifugation, the supernatant was removed, and the pellet was resuspended in buffer H (50mM pH4.5 HAc-NaAc) to obtain a crude enzyme solution, which was placed in an ice bath for further use.
The protein of interest was purified using an AKTA Purifier 10(GE Healthcare) chromatography system. The solution A (30mM pH4.5 HAc-NaAc, 50mM NaCl), solution B (30mM pH4.5 HAc-NaAc) and 20% ethanol used in the protein purification process were vacuum filtered through a 0.22 μm filter before use, and then degassed by ultrasound for 10 min.
Balancing SpFF Sepharose affinity chromatographic column with A solution, loading the obtained crude enzyme solution TPGUS-P on the column, eluting with 100% A solution to remove impurity protein, and waiting for OD280And the temperature is reduced to the minimum. Eluting part of the non-specifically adsorbed hybrid protein with 80% solution A and 20% solution B to obtain OD280And (5) reducing the content of the protein to the minimum, eluting the protein by using 60% of solution A and 40% of solution B to obtain the target protein with higher purity. The flow-through peak, the 20% B-eluted peak and the 40% B-eluted peak were collected separately, and the enzyme activity thereof was preliminarily measured with glycyrrhizic acid (GL).
After obtaining the electrophoretically pure target protein, the concentration of the target protein was determined by using Nano Drop. 30mM protein purification A solution of HAc-NaAc, pH4.5, was used as a blank for zeroing, and then 2. mu.L of the protein sample was dropped onto the detector to measure the concentration.
Example 3: determination of beta-glucuronidase Activity and thermal stability
The beta-glucuronidase can catalyze the hydrolysis of the glycosidic bond of glycyrrhizic acid (GL) to generate 3-O-monoglucuronic acid Glycyrrhetinic Acid (GAMG), the reaction has the characteristics of simplicity, rapidness and easiness in detection, and the enzyme activity of the beta-glucuronidase and the mutant thereof is determined through the reaction in the research. Taking 50 μ L of the extract with a concentration of 0.01 g.L-1The enzyme solution was added to 50. mu.L of a solution containing GL at a concentration of 2.5 mmol. multidot.L-1Reacting in acetic acid-sodium acetate buffer solution with pH of 4.5 at 45 deg.C for 10min, adding 900 μ L and methanol to terminate the reaction, and detecting GAMG content with high performance liquid chromatograph. β -glucuronidase activity definition: under the conditions, the amount of beta-glucuronidase required for converting 1nmol of GAMG per minute is an activity unit.
Taking 100 μ L of the extract with a concentration of 0.01 g.L-1The pure enzyme solution is subjected to thermal denaturation in water baths at 55 ℃, 60 ℃, 65 ℃, 70 ℃ for 30min, 60min,90min, 120min, 150min, 180min, immediately ice-cooling for 20min, and measuring enzyme activity. The enzyme activity of the enzyme solution without thermal denaturation is taken as 100%, and the percentage of the corresponding residual enzyme activity is plotted by different temperatures. The half-life time of the enzyme activity of the beta-glucuronidase is defined as the half-life period, and the result is shown in figure 1, the half-life period of the original beta-glucuronidase is 43min under the condition of 55 ℃, 35min under 60 ℃, 25min under 65 ℃, 20min under 70 ℃, the half-life period of the mutant is 124min under the condition of 55 ℃, 57min under 60 ℃, 51min under 65 ℃ and 45min under 70 ℃, so that the thermal stability is obviously improved.
Example 4: application of enzyme in high-temperature conversion of glycyrrhizic acid
The fermentation process suitable for the TpGUS heat-resistant mutant is designed, a high-temperature conversion GAMG strategy is adopted, crude enzyme liquid is directly used as an enzyme preparation of a reaction system, and simultaneously used as a solvent to directly dissolve substrate glycyrrhizic acid, and only products are recovered in the whole system, and the glycyrrhizic acid in the reaction liquid has high concentration and is not easy to contaminate bacteria, so that the direct stirring reaction can be carried out without a sterile operation environment. The crude enzyme solution reacts directly with substrate GL at 50 ℃ and the optimal ratio of enzyme (10U/ml) to substrate (10g/L) during the "reaction" is 1: 3, controlling the pH value of the enzyme and the substrate to be 4.5 at the optimal rotation speed of 250rpm, supplementing GL every 20 hours from the 28 th hour to the final concentration of 10g/L, and supplementing 30g of the substrate for three times within 100 hours to finally obtain 20.2g of GAMG, wherein the conversion rate of the finally obtained glycyrrhizic acid is 87%. Compared with the traditional method which uses 30 ℃ for conversion, the GAMG 9g/L can be obtained under the same material condition, and the conversion rate is 43 percent. Compared with the process for obtaining equivalent products, the conversion process has the advantages of 150% GAMG yield and half of reaction time.
Sequence listing
<110> Beijing university of science and technology
<120> beta-glucuronidase mutant with improved thermal stability
<130> 24
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<170> SIPOSequenceListing 1.0
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Ala Cys Ser Ala Gln Asn His Lys Asn Ile Thr Arg Thr Ile Asp Leu
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Ser Pro Ala Ser Thr Pro Ala Pro Gly Lys Gln Val Val Asp Gly Ala
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Tyr Gln Ser Phe Ser Ile Glu Phe Cys Tyr Met Ala Asp Tyr Ala Gly
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Asn Asn Thr Asn Pro Asn Lys Phe Ser Arg Gln Val Val Gln Asn Leu
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Tyr Asp Ile Ser Gly Thr Tyr Pro Ile Phe Arg Val Gly Gly Ser Thr
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Gln Asn Ser Ala Val Tyr Phe Pro Asn Gln Thr Asp Val Ala Ile Ile
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Ala Pro Phe Gln Ser Glu Ala Ser Asp Gln Pro Ser His Ser Phe Ile
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Gly Pro Lys Phe Met Glu Ser Phe Gln Gln Phe Pro Glu Gly Thr Arg
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Tyr Ile Tyr Gly Leu Asn Phe Phe Gln Ser Glu Asn Glu Thr Leu Phe
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Asn Val Gly Asp Gly Leu Asp Gln Cys Val Leu Glu Ala Tyr Ala Ala
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Tyr Thr Ala Leu Gly Glu Ser Leu Tyr Gly Phe Glu Ile Gly Asn Glu
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Val Asn Ser Trp Pro Gly Gly Ser Arg Arg Pro Ala Asn Trp Thr Leu
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His Ser Asn Lys Ala Lys Thr Val Ala Asp His Asp Tyr Met Gly Ala
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Asn Arg Thr Asn Met Leu Ser Arg Ile Trp Tyr His Asp Tyr Leu Gly
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Asn Ala Thr Ala Asp Ser Gly Ile Glu Tyr Val Leu Gly Glu Thr Asn
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Ser Ile Ser Cys Gln Gly Ala Phe Asn Ile Ser Asp Val Met Ala Ser
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Ala Arg Trp Ala Val Asp Tyr Val Leu Tyr Leu Ser Ser Leu Lys Val
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Ser Arg Val His Phe His Met Gly Thr Arg Tyr Arg Tyr Ser Pro Trp
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Gln Pro Ile Tyr Tyr Asn Asp Thr Glu Ala His Val Lys Pro Ile Tyr
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Thr Glu Val Leu Val Asn Glu Thr Asn Phe Gly Ala Tyr Thr Val Tyr
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Thr Val Tyr Val Gly Ala Gly Glu Ala Val Leu Ile Ser Lys
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Claims (5)

1. A beta-glucuronidase mutant with improved thermal stability is characterized in that the mutant is obtained by carrying out the following mutation on a gene sequence such as beta-glucuronidase coded by a GenBank accession number KY 609919: the 195 th serine is mutated into isoleucine, the 212 th valine, the 220 th threonine, the 238 th cysteine and the 348 th valine are all mutated into arginine, and 23 amino acids at the N end of the beta-glucuronidase are cut off.
2. A gene encoding the mutant of claim 1.
3. A plasmid or cell comprising the gene of claim 2.
4. The method for obtaining the mutant as claimed in claim 1, wherein the mutant is obtained by using plasmid pGAPz alpha-tpgus as a template, designing a primer, obtaining a gene and a plasmid for encoding the mutant by PCR, and expressing beta-glucuronidase by using pichia pastoris as a host.
5. Use of the mutant β -glucuronidase according to claim 1 for the conversion of glycyrrhizic acid.
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CN109706091B (en) * 2018-12-20 2021-01-01 北京理工大学 Engineering bacterium GA108/PGAPZ alpha A-Atgusmix for industrial production of glycyrrhetinic acid and method
CN109735518B (en) * 2019-02-28 2020-08-25 北京理工大学 Beta-glucuronidase mutant with optimum reaction pH value increased and glycyrrhizic acid conversion process thereof
CN113122519B (en) * 2019-12-31 2023-05-26 中国科学院天津工业生物技术研究所 Heat-resistant glucosamine phosphate phosphatase mutant and application thereof
CN113652409B (en) * 2021-08-11 2024-05-07 河北维达康生物科技有限公司 Novel glycyrrhetinic acid glucuronyl transferase mutant and application thereof

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