α-L-Rhamnosidase Mutant and Its Application

Abstract

The present invention belongs to the fields of genetic engineering and biocatalysis technology, and specifically relates to an α-L-rhamnosidase mutant and its application. The α-L-rhamnosidase mutant is obtained by mutating glycine at the 227th position of the α-L-rhamnosidase with the amino acid sequence shown in SEQ ID NO.2 into glutamic acid, and the amino acid sequence of the α-L-rhamnosidase mutant is shown in SEQ ID NO.1. The α-L-rhamnosidase gene is derived from Aspergillus nidulans. By using the random mutation technology, an α-L-rhamnosidase mutant expressed in Escherichia coli is successfully screened out, and its enzyme activity is increased by 1.52 times. Using naringin as a substrate, the catalytic reaction is carried out under the conditions of pH = 6.0-7.0 and temperature of 37-38 °C, and the conversion rate of prunin reaches 95%.

Description

Technical Field

[0001] The present invention belongs to the technical fields of genetic engineering and biocatalysis, and specifically relates to an α-L-rhamnosidase mutant and its application. Background Art

[0002] α-L-rhamnosidase (EC 3.2.1.40) is a type of glycoside hydrolase, mainly belonging to the GH13, GH28, GH78, and GH106 families. It can specifically hydrolyze the α-1,2, α-1,3, α-1,4, α-1,5, and α-1,6 glycosidic bonds at the ends of polysaccharides or glycoside compounds, releasing L-rhamnose and generating new polysaccharides or glycoside compounds, including hydrolysis of natural flavonoid glycoside compounds such as hesperidin, neohesperidin, naringin, rutin, and quercitrin. The hydrolysis products are respectively hesperetin monoglucoside, prunin, isoquercitrin, and quercetin, etc. α-L-rhamnosidase can be applied to the debittering of citrus juices in the food industry, the improvement of beverage flavors, and the production of sweeteners, etc.; α-L-rhamnosidase can also be applied to the preparation of various drugs and drug precursors in the pharmaceutical industry; in the chemical industry, α-L-rhamnosidase can be used as a biocatalyst.

[0003] Naringin (4'-5,7'-trihydroxyflavanone-7-rhamnoglucoside) is a type of flavanone compound and is the main active ingredient of traditional Chinese medicines such as Drynaria rhizome, Fructus Aurantii Immaturus, and Exocarpium Citri Grandis. However, due to the poor water solubility of naringin, its utilization rate is very low, which greatly limits its pharmaceutical activity and its application in the development of functional foods. α-L-rhamnosidase can specifically hydrolyze naringin to generate prunin (naringenin-7-O-glucoside), which has one less rhamnoside than naringin, but has good solubility and has good biological activities such as antiviral, antitumor, antioxidant, cholesterol-lowering, and blood sugar balance regulation. The abundance of prunin in nature is relatively low, and it is difficult to extract and prepare from biological tissues. Therefore, the method of using α-L-rhamnosidase for biotransformation with naringin as the substrate is an effective means for preparing prunin. Currently, there are few reports on the molecular modification of α-L-rhamnosidase. Screening α-L-rhamnosidase based on random mutation to improve its enzyme activity is a key method in the field of genetic engineering. Therefore, it is of great significance to screen and modify α-L-rhamnosidase by random mutation to improve its activity to meet the large-scale production of prunin. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide an α-L-rhamnosidase mutant and its application in the transformation of naringin to prepare prunin. The enzyme activity of the α-L-rhamnosidase mutant is significantly higher than that of the wild type, and the conversion rate of preparing prunin is relatively high.

[0005] The technical solution of the present invention is as follows:

[0006] The α-L-rhamnosidase mutant of the present invention is a mutant in which glycine at the 227th position of the α-L-rhamnosidase with the amino acid sequence shown in SEQ ID NO.2 is mutated to glutamic acid. The amino acid sequence of the α-L-rhamnosidase mutant is shown in SEQ ID NO.1 and is named G227E.

[0007] The amino acid sequence of the α-L-rhamnosidase derived from Aspergillus nidulans after codon optimization is shown in SEQ ID NO.2.

[0008] The α-L-rhamnosidase mutant is a mutant obtained by random mutagenesis technology and screening. The screening method includes the following steps:

[0009] (1) Construct a random mutation fragment using a random mutagenesis kit and ligate it to a plasmid vector;

[0010] (2) Transform the plasmid containing the random mutation fragment into a host cell;

[0011] (3) Select monoclonal colonies for culture and induction. After cell disruption, a crude enzyme solution of the mutant is obtained, and mutants with high enzyme activity are screened by enzyme activity detection to obtain the α-L-rhamnosidase mutant.

[0012] The plasmid vector is a pGEX series vector.

[0013] The host cell is a bacterium, preferably Escherichia coli.

[0014] The present invention also provides the application of the α-L-rhamnosidase mutant in the preparation of prunin by transforming naringin.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] The present invention has screened an α-L-rhamnosidase mutant G227E by random mutagenesis, and its enzyme activity is 1.52 times that of the wild-type enzyme. The α-L-rhamnosidase mutant G227E can catalyze naringin to obtain prunin, and the conversion rate reaches 95%. The α-L-rhamnosidase mutant G227E proposed by the present invention improves the application potential of this enzyme in the fields of food, medicine, and chemical industry. DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 It is the HPLC chart of naringin standard;

[0018] Figure 2 It is the HPLC chart of prunin standard;

[0019] Figure 3It is the HPLC chart of the catalytic result of wild-type enzyme solution;

[0020] Figure 4 It is the HPLC chart of the catalytic result of mutant G227E enzyme solution. Specific implementation manners

[0021] The embodiments of the present invention are only for further illustration of the content of the present invention and cannot be used as the limiting content or scope of the present invention. For the molecular biology experimental methods not specifically described in this embodiment, reference can be made to "Molecular Cloning: A Laboratory Manual".

[0022] (1) The culture media involved in the embodiments are as follows:

[0023] LB liquid culture medium: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride;

[0024] TB liquid culture medium: 12 g / L of tryptone, 24 g / L of yeast extract, 5 mL / L of glycerol, 2.31 g / L of KH2PO4, 16.43 g / L of K2HPO4·3H2O.

[0025] The solid culture medium is added with 1.5% agar powder on the basis of the formula.

[0026] (2) Activity detection of the enzyme

[0027] Enzyme activity determination method: The activity of recombinant α-L-rhamnosidase was determined using pNPR (p-nitrophenyl-α-L-rhamnoside) as the substrate. 400 μL of Tris-HCl buffer (0.1 M, pH = 7.0) was mixed with 20 μL of pNPR with a concentration of 5 mM, incubated at 50 °C for 2 min, then immediately added with 80 μL of crude enzyme solution, and the reaction continued at 50 °C for 5 min. Then, 500 μL of sodium carbonate solution with a concentration of 1 M was quickly added to terminate the reaction and develop color. After centrifugation at 12000×g for 2 min, the absorbance was immediately measured at 405 nm. The enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1 μmol of p-nitrophenol per minute.

[0028] (3) Determination of the p-nitrophenol standard curve

[0029] Weigh p-nitrophenol, dissolve it with deionized water and make up the volume to prepare a stock solution of 1 mM, and dilute it in gradients to p-nitrophenol standard solutions of 0.01 mM, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM. Using the p-nitrophenol concentration as the abscissa and the absorbance value as the ordinate, the standard curve of p-nitrophenol was plotted.

[0030] (4) HPLC determination method for naringin and prunin

[0031] HPLC was performed using a C18 reversed-phase bonded silica separation column (4.6 mm × 250 mm, 5 μm). The mobile phase was methanol: ultrapure water = 1:1, the flow rate was 1 mL / min, the column temperature was 40 °C, a VWD ultraviolet detector was used, the wavelength of the VWD detector was 282 nm, and the injection volume was 20 μL.

[0032] Example 1 Construction of the wild-type strain of α-L-rhamnosidase

[0033] The sequence of α-L-rhamnosidase (GenBank: FR873475.1) was submitted to a gene company for codon optimization and synthesis to construct the wild-type strain pGEX4T1-AnRha plasmid. Its amino acid sequence is shown in SEQ ID NO.1. The strain was inoculated into LB liquid medium (containing 100 μg / mL sodium ampicillin at the final concentration) at a ratio of 1:100 to prepare a seed solution. After culturing overnight, the seed solution was inoculated into TB liquid medium at an inoculation amount of 1%. When the bacterial solution was cultured to an OD 600 of 0.6 - 0.8, it was cooled in a refrigerator for 10 min, IPTG with a final concentration of 0.08 mM was added, and it was induced to culture on a shaker at 20 °C for 16 h. Then, the fermentation broth was centrifuged at 4 °C and 4000 × g for 20 min. The supernatant was discarded, the precipitate was taken, and the cells were thoroughly resuspended with 5 mL of Tris-HCl buffer (0.1 mM, pH = 7.0). After ultrasonic disruption, the supernatant was obtained by centrifugation, which was the crude enzyme solution of the wild-type enzyme.

[0034] Example 2 Screening, preparation, and expression of α-L-rhamnosidase mutants

[0035] According to the gene sequence of α-L-rhamnosidase, a primer pair for amplifying the target gene AnRha sequence was designed, and the nucleotide sequences were as follows:

[0036] Forward primer: 5’- CCC GGG TCG ACT CGA GAT GAG TCT ATC TAT ATC AGG AGT A-3’;

[0037] Reverse primer: 5’-AGT CAC GAT GCG GCC GCT TAG CCG AGG GTG GAC TCG AAA C-3’.

[0038] Using the recombinant plasmid pGEX4T1-AnRha as a template, a gene random mutagenesis kit was used to introduce random mutations into the target gene AnRha. The primer mixture was a 1:1 mixture of the upstream and downstream primers. The specific PCR reaction system is shown in Table 1. The PCR program was as follows: pre-denaturation at 94°C for 3 min; then 30 cycles (94°C for 30 s, 55°C for 30 s, 72°C for 2.5 min); extension at 72°C for 10 min; and finally incubation at 4°C.

[0039] Table 1 PCR reaction system

[0040]

[0041] After the PCR reaction was completed, 10 μL of 6×DNA LOADING Dye was added and mixed evenly. After electrophoresis on a 1% agarose gel, the product was recovered and purified to obtain the randomly mutated product. Then, the vector pGEX-4T-1 was ligated to the randomly mutated product in a seamless cloning manner, and then transformed into Escherichia coli Rosetta (DE3) competent cells. After culturing for 1 h, it was spread on an LB solid plate containing sodium ampicillin and cultured overnight at 37°C to obtain a mutant library of α-L-rhamnosidase.

[0042] 500 μL of LB liquid medium containing sodium ampicillin was added to each well of a sterile 96-well deep-well plate. Single colonies with uniform size and consistent morphology on the LB solid plate cultured overnight were picked and inoculated into each well of the 96-well deep-well plate, with one single colony inoculated into each well; the plate was sealed with a sealing film and placed in a constant temperature shaker at 37°C and 200 rpm for 16 h. After the culture was completed, each well was inoculated into a sterile 96-well deep-well plate containing 600 μL of TB liquid medium with sodium ampicillin at an inoculation amount of 5% per well, sealed with a sealing film, and placed in a constant temperature shaker at 37°C and 200 rpm for 4 h; after the culture was completed, it was cooled in a 4°C refrigerator for 10 min, and then IPTG with a final concentration of 0.08 mM was added to each well. After sealing the plate with a sealing film, it was placed in a constant temperature shaker at 20°C and 200 rpm for induction culture for 16 h.

[0043] After the induction culture was completed, centrifugation was carried out at 4000×g for 20 min, the supernatant was discarded, and it was placed in an ultra-low temperature refrigerator and frozen and thawed three times at -80°C. 200 μL of 1 mg / mL lysozyme solution was added, the cells were thoroughly pipetted and resuspended, and the reaction was carried out in a 37°C incubator for 30 min. After the reaction was completed, centrifugation was carried out at 4000×g for 20 min, and the supernatant was the crude enzyme solution. Mutants with the enzyme activity of the crude enzyme solution increased by more than 30% compared with the wild-type non-mutated strain were screened out, inoculated into a 250 mL shake flask for re-screening, and the mutants with increased enzyme activity after re-screening were sent for sequencing.

[0044] The results are shown in Table 2. Finally, the α-L-rhamnosidase mutant G227E of the present invention was obtained by screening. Its enzyme activity was the highest among all mutants in this example, and its enzyme activity was 1.52 times that of the wild-type enzyme.

[0045] Table 2 Enzyme Activity of α-L-Rhamnosidase

[0046]

[0047] Example 3 Conversion of Naringin to Prunin by α-L-Rhamnosidase Mutant

[0048] The crude enzyme solution of the α-L-rhamnosidase mutant and the crude enzyme solution of the wild-type enzyme were used to catalyze naringin respectively. The concentration of naringin was 20 g / L, and the reaction conditions were 37 °C and pH = 6. After reacting for 24 h, 1 mL of the reaction solution was taken into a centrifuge tube. After terminating the catalytic reaction in a 100 °C water bath and cooling to room temperature, an equal volume of methanol was added for extraction. The reaction solution was centrifuged at 12000×g for 10 min, and the supernatant was taken and diluted with methanol, and then filtered through a 0.22 μm organic filter membrane for HPLC analysis. Figure 1 This is the HPLC chromatogram of the naringin standard product. Figure 2 This is the HPLC chromatogram of the prunin standard product. Figure 3 This is the HPLC chromatogram of the catalytic result of the wild-type enzyme solution. Figure 4 This is the HPLC chromatogram of the catalytic result of the mutant G227E enzyme solution. The conversion rate of wild-type α-L-rhamnosidase to naringin was 80%, and the conversion rate of α-L-rhamnosidase mutant G227E to naringin was 95%.

[0049] SEQ ID NO.1

[0050] 1 MSLSISGVTF EHHRSALGIG EPSPRISWRF DGTVSNWTQS AYEIEINRAG QANTFRVNSS

[0051] 61 DSVLVPWPSD PLQSGEEATV RVRSFGRANQ PDAPWSDPVT VEPGLLDEDD WQSAVAIVSD

[0052] 121 RETEVNATHR PIYFRKDFDV DEEILSARLY ITALGVYEAE INGQPVGDHV LAPGWQAYSH

[0053] 181 RHEYNTYDVT DLLQTGDNTI GVTVGEGWYA GALTWSMTRN IYGDTLELLS LLSIATADGK

[0054] 241 TIYVPSDETW QSSTGPIIAS EIYNGETYDS TQAIEGWSQP GFDASGWLGT HEVTFDKSVL

[0055] 301 AAPDAPAVRR VEERRLESVF KSASGKTVLD FGQNLVGWLR VRVKGPRGST ISFVHTEVME

[0056] 361 NGEVATRPLR NAKATDNLTL SGEEQEWEPS FTFHGFRYVQ VTGWPEETEL NADSVTAIVI

[0057] 421 NSDMEQTGFF SCSNPLLNKL HENIIWSMRG NFLSIPTDCP QRDERLGWTG DIHAFARTAN

[0058] 481 FIYDTSGFLR GWLRDAYSEQ LENNYAPPYV IPNVLGPGSP TSIWGDAIVS VPWDLFQTYG

[0059] 541 DKAMLSEQYA GATAWLDKGI LRNEAGLWNR STFQYADWLD PLAPPDDPGA ATTNKYLVSD

[0060] 601 AYLIHSTELV ANISAYLDRP DDAERYAADR ADLTRAFQKA WISANGTVAN ETQTGLTLPL

[0061] 661 YFKLFERPEH YTDAVSRLVD IIKENEYKVG TGFAGTHLLG HTLSAYNASS TFYNTLLQED

[0062] 721 VPGWLFQVLM NGTTTWERWD SMLANGSVNP GEMTSFNHYA VGSVGAWMHE NIGGLRPIEP

[0063] 781 GWRRFAVDVK VGGGLSSAQE RFLSPYGSAE SSWEVRDGKF MLGVKVPPNS EAVVSLPGAP

[0064] 841 TRGKKEVIVG SGMHRFESTL G

[0065] SEQ ID NO.2

[0066] 1 MSLSISGVTF EHHRSALGIG EPSPRISWRF DGTVSNWTQS AYEIEINRAG QANTFRVNSS

[0067] 61 DSVLVPWPSD PLQSGEEATV RVRSFGRANQ PDAPWSDPVT VEPGLLDEDD WQSAVAIVSD

[0068] 121 RETEVNATHR PIYFRKDFDV DEEILSARLY ITALGVYEAE INGQPVGDHV LAPGWQAYSH

[0069] 181 RHEYNTYDVT DLLQTGDNTI GVTVGEGWYA GALTWSMTRN IYGDTLGLLS LLSIATADGK

[0070] 241 TIYVPSDETW QSSTGPIIAS EIYNGETYDS TQAIEGWSQP GFDASGWLGT HEVTFDKSVL

[0071] 301 AAPDAPAVRR VEERRLESVF KSASGKTVLD FGQNLVGWLR VRVKGPRGST ISFVHTEVME

[0072] 361 NGEVATRPLR NAKATDNLTL SGEEQEWEPS FTFHGFRYVQ VTGWPEETEL NADSVTAIVI

[0073] 421 NSDMEQTGFF SCSNPLLNKL HENIIWSMRG NFLSIPTDCP QRDERLGWTG DIHAFARTAN

[0074] 481 FIYDTSGFLR GWLRDAYSEQ LENNYAPPYV IPNVLGPGSP TSIWGDAIVS VPWDLFQTYG

[0075] 541 DKAMLSEQYA GATAWLDKGI LRNEAGLWNR STFQYADWLD PLAPPDDPGA ATTNKYLVSD

[0076] 601 AYLIHSTELV ANISAYLDRP DDAERYAADR ADLTRAFQKA WISANGTVAN ETQTGLTLPL

[0077] 661 YFKLFERPEH YTDAVSRLVD IIKENEYKVG TGFAGTHLLG HTLSAYNASS TFYNTLLQED

[0078] 721 VPGWLFQVLM NGTTTWERWD SMLANGSVNP GEMTSFNHYA VGSVGAWMHE NIGGLRPIEP

[0079] 781 GWRRFAVDVK VGGGLSSAQE RFLSPYGSAE SSWEVRDGKF MLGVKVPPNS EAVVSLPGAP

[0080] 841 TRGKKEVIVG SGMHRFESTL G

Claims

1. An α-L-rhamnosidase mutant, characterized in that: The α-L-rhamnosidase mutant is an α-L-rhamnosidase whose amino acid sequence is shown in SEQ ID NO.2, wherein the glycine at position 227 is mutated to glutamic acid, and the amino acid sequence of the α-L-rhamnosidase mutant is shown in SEQ ID NO.

1.

2. The α-L-rhamnosidase mutant according to claim 1, characterized in that: The α-L-rhamnosidase mutant is a mutant obtained through random mutation technology and screened.

3. The α-L-rhamnosidase mutant according to claim 2, characterized in that: The method for screening α-L-rhamnosidase mutants comprises the following steps: (1) Use a random mutagenesis kit to construct a random mutagenesis fragment and connect it to a plasmid vector; (2) Transforming the plasmid containing the random mutation fragment into the host cell; (3) Selecting a monoclonal colony for culture and induction, and obtaining a crude enzyme solution of the mutant after cell disruption, and screening a mutant with high enzyme activity by enzyme activity detection, thereby obtaining the α-L-rhamnosidase mutant.

4. The α-L-rhamnosidase mutant according to claim 3, characterized in that: The plasmid vector is a pGEX series vector.

5. The α-L-rhamnosidase mutant according to claim 4, characterized in that: The plasmid vector is pGEX-4T-1.

6. The α-L-rhamnosidase mutant according to claim 3, characterized in that: The host cell is a bacterium.

7. The α-L-rhamnosidase mutant according to claim 6, characterized in that: The host cell is Escherichia coli.

8. Use of the α-L-rhamnosidase mutant according to claim 1 in converting naringin to prepare prunin.

9. The use according to claim 8, characterized in that: When the α-L-rhamnosidase mutant converts naringin to prepare prunin, the pH is 6-7 and the temperature is 37-38°C.

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