Alpha-L-rhamnosidase mutant and application thereof

Abstract

The invention discloses an alpha-L-rhamnosidase mutant and application thereof, and belongs to the technical field of gene engineering and enzyme engineering. The amino acid sequence of the alpha-L-rhamnosidase mutant is shown as SEQ ID NO.13, the relative enzyme activity of the alpha-L-rhamnosidase mutant is 1.46 times that of a wild type, and the problems that existing alpha-L-rhamnosidase is low in rutin catalysis efficiency and many in by-product are effectively solved. The mutant can efficiently and specifically hydrolyze rutin to generate isoquercetin, does not have other byproducts, has excellent enzymatic characteristics, and provides an efficient catalyst for green biological preparation of isoquercetin. Meanwhile, by constructing a pichia pastoris surface display engineering bacterium containing the mutant gene, preparation of the whole-cell catalyst is achieved, and the production efficiency and industrial application potential of the isoquercetin are remarkably improved through the whole-cell catalyst.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genetic engineering and enzyme engineering, and particularly relates to an alpha-L-rhamnosidase mutant and application thereof. BACKGROUND

[0002] Isoquercitrin, also known as quercetin-3-O-glucoside, is a natural flavonoid compound, which is widely distributed in nature but has a very low content, has multiple pharmacological effects such as anti-oxidative stress, anti-tumor, cardiovascular protection, blood sugar reduction and anti-allergy, and can be used for synthesizing food additive EMIQ (enzyme modified isoquercitrin). Due to the very low natural content of isoquercitrin in plants, the traditional extraction method has low yield, large workload, and high reagent consumption, and cannot realize large-scale preparation. Rutin and isoquercitrin have the same parent nucleus structure, and only one rhamnose is connected to the C6 position of the glucose group of isoquercitrin, and the content of rutin in plants is higher. There are two methods for hydrolyzing rutin to generate isoquercitrin, namely chemical conversion method and biological conversion method. The chemical conversion method uses acid cleavage method, which can cause excessive hydrolysis of rutin to generate quercetin. The biological conversion method uses alpha-L-rhamnosidase to specifically cleave rutin to generate isoquercitrin. Compared with the chemical method, the biological conversion method has the advantages of mild conditions, high efficiency, environmental protection, less by-products, higher yield and purity, and has become a more feasible way for large-scale preparation of isoquercitrin.

[0003] Free enzymes are easy to be inactivated and difficult to be reused, and need to be purified, which greatly increases the production cost, so the application in industry is limited to a certain extent. In order to make the industrial application of enzymes more feasible, immobilization technology appears. However, the method for immobilizing alpha-L-rhamnosidase needs to purify the enzyme and use additional immobilization carriers, and the immobilization operation is relatively complex, the production cost is increased, and the current catalytic rutin concentration is low, and the catalytic efficiency is low.

[0004] Therefore, how to improve the catalytic efficiency of alpha-L-rhamnosidase for rutin and the application of alpha-L-rhamnosidase are of great significance for biological catalysis of rutin and generation of isoquercitrin. SUMMARY

[0005] The present application provides an alpha-L-rhamnosidase mutant and application thereof, which solves the problems of the prior art. The relative enzyme activity of the alpha-L-rhamnosidase mutant provided by the present application reaches 1.46 times that of the wild type, effectively solving the problems of low catalytic efficiency of the existing alpha-L-rhamnosidase for rutin and multiple by-products. The mutant can efficiently and specifically hydrolyze rutin to generate isoquercitrin without other by-products, has excellent enzymatic properties, and provides a high-efficiency catalyst for green biological preparation of isoquercitrin.

[0006] In order to achieve the above object, the present application provides the following scheme.

[0007] The present application provides an alpha-L-rhamnosidase mutant, the amino acid sequence of which is shown in SEQ ID NO. 13.

[0008] The present application also provides a gene encoding the alpha-L-rhamnosidase mutant.

[0009] The present application improves the relative enzyme activity of alpha-L-rhamnosidase by mutant screening of wild-type alpha-L-rhamnosidase, the enzyme activity of the obtained alpha-L-rhamnosidase mutant is increased by 1.46 times, thereby improving the efficiency of converting rutin to isoquercitrin, and the mutant has excellent enzymatic properties and can specifically convert the substrate to isoquercitrin without generating other by-products when catalyzing rutin.

[0010] The present application also provides a recombinant vector comprising the gene, and the recombinant vector is a Pichia pastoris cell surface display vector.

[0011] The recombinant vector is a DNA molecule used for introducing an exogenous gene into a host cell for expression. Based on the gene encoding the alpha-L-rhamnosidase mutant, the gene nucleotide sequence of the alpha-L-rhamnosidase mutant of the present application can be connected to various vectors to construct the above-mentioned recombinant vector by conventional methods in the art. Various vectors in the art, such as various plasmids, bacteriophages or viral vectors, etc., connected to the alpha-L-rhamnosidase mutant gene sequence of the present application, should all belong to the protection scope of the present application.

[0012] Optionally, in the recombinant vector, the gene encoding the alpha-L-rhamnosidase mutant contains a Flag tag.

[0013] Optionally, the recombinant vector comprises an anchor protein gene Gcw61.

[0014] The present application also provides an engineered bacterium comprising the recombinant vector.

[0015] Optionally, the engineered bacterium is a Pichia pastoris engineered bacterium.

[0016] The present application also provides the use of the alpha-L-rhamnosidase mutant, the gene, the recombinant vector or the recombinant bacterium in improving the catalytic efficiency of alpha-L-rhamnosidase.

[0017] The present application also provides the use of the alpha-L-rhamnosidase mutant, the gene, the recombinant vector or the recombinant bacterium in improving the production efficiency of isoquercitrin.

[0018] The application also provides a method for improving the production efficiency of isoquercitrin, comprising using the whole-cell catalyst produced by the engineering bacteria to catalyze the process of generating isoquercitrin from rutin.

[0019] The application discloses the following technical effects:

[0020] The application screens an alpha-L-rhamnosidase mutant by rational design and site-directed mutagenesis, significantly improves the catalytic efficiency of the alpha-L-rhamnosidase, and the relative enzyme activity reaches 1.46 times of that of the wild type, effectively solves the problems of low rutin catalytic efficiency and many by-products of the existing alpha-L-rhamnosidase, and the mutant can efficiently and specifically hydrolyze rutin to generate isoquercitrin without other by-products, and has excellent enzymatic properties, and provides a high-efficiency catalyst for green biological preparation of isoquercitrin.

[0021] The application realizes the preparation of the whole-cell catalyst by constructing the Pichia pastoris surface display engineering bacteria containing the mutant gene, avoids the complex process of traditional free enzyme purification and immobilization, and reduces the production cost. The whole-cell catalyst can realize efficient conversion under the condition of high-concentration rutin substrate (up to 800 g / L), and the conversion rate of 500 g / L rutin is more than 99% within 24 hours, and the production efficiency and industrial application potential of isoquercitrin are significantly improved. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description only constitute some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor.

[0023] Figure 1 Figure 1 is a graph of relative enzyme activity of wild-type alpha-L-rhamnosidase and alpha-L-rhamnosidase mutant;

[0024] Figure 2 Figure 4 is a determination of the optimum reaction pH of the whole-cell catalyst;

[0025] Figure 3 Figure 5 is a determination of the optimum reaction temperature of the whole-cell catalyst;

[0026] Figure 4 Figure 6 is a hydrolysis reaction of 500 g / L rutin by whole-cell catalysts with different enzyme activities. DETAILED DESCRIPTION

[0027] The following will describe in detail various exemplary embodiments of the application, and the detailed description should not be considered as limiting the application, but should be understood as a more detailed description of some aspects, properties and embodiments of the application.

[0028] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of, an understanding exists that each intervening value, to the upper or lower limit, is also specifically included within the scope of the present application. Each smaller range between any stated value or intervening value in a stated range, and any other stated or intervening value or range in stated values are all included within the present application. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, as values.

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the present specification will control.

[0030] Various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the application. Other embodiments will be apparent to those of ordinary skill in the art from consideration of the description and practice of the application. The description and examples are illustrative only.

[0031] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended terms that mean including, but not limited to.

[0032] The experimental methods used in the experiments involved in the present application are conventional methods unless otherwise specified; the materials and reagents used in the examples of the present application are commercially available biological experimental materials unless otherwise specified.

[0033] Example 1 Construction of α-L-rhamnosidase mutants

[0034] In this example, based on the rational design strategy of conservation and stability analysis, the amino acid sites near the active center of α-L-rhamnosidase were systematically modified. This strategy starts from the in-depth analysis of its molecular structure, and then specific single or multiple amino acid sites are subjected to site-directed mutagenesis or local sequence regions are subjected to combinatorial mutagenesis, and then a mutant library is constructed, and the function of the mutants is evaluated by specific screening and selection methods.

[0035] The present application takes the α-L-rhamnosidase with the amino acid sequence shown in SEQ ID NO. 1 as the starting template, and performs the following (a)-(e) single-point or multi-point combination mutations on the key sites to obtain enzyme variants with optimized performance:

[0036] (a) the amino acid corresponding to position 95 on SEQ ID NO. 1 is mutated from aspartic acid (D) to tryptophan (W) or phenylalanine (F);

[0037] (b) the amino acid corresponding to position 192 on SEQ ID NO. 1 is mutated from leucine (L) to methionine (M), tyrosine (Y) or tryptophan (W);

[0038] (c) the amino acid corresponding to position 195 on SEQ ID NO. 1 is mutated from leucine (L) to arginine (R) or phenylalanine (F);

[0039] (d) the amino acid corresponding to position 234 on SEQ ID NO. 1 is mutated from arginine (R) to leucine (L) or methionine (M);

[0040] (e) the amino acid corresponding to position 282 on SEQ ID NO. 1 is mutated from tyrosine (Y) to glutamic acid (E), isoleucine (I) or tryptophan (W).

[0041] The sequence shown in SEQ ID NO. 1 is the amino acid sequence of wild-type a-L-rhamnosidase Rha, which is as follows:

[0042] MWSSWLLSALLATEALAVPYEEYILAPSSRDLAPASVRQVNGSVTNAAALTGAGGQATFNGVSSVTYDFGINVAGIVSVDVASASSESAFIGVTFTESSMWISNEACDATQDAGLDTPLWFAVGQGAGVYSVGKKYTRGAFRYMTVVSNTTATVSLNSVKINYTASPIQDLRAYTGYFHSSDELLNRIWYAGAYTLQLCSIDPTTGDALVGLGAITSSETITLPQTDKWWTNYTITNGSSTLTDGAKRDRLVWPGDMSIALESVAVSTEDLYSVRTALESLYALQKADGQLPYAGKPFYDTVSFTYHLHSLVGAASYYQYTGDRAWLTRYWGQYKKGVQWALSGVDSTGLANITASADWLRFGMGAHNIEANAILYYVLNDAISLAQSLNDNAPIRNWTATAARIKTVANELLWDDKNGLYTDNETTTLHPQDGNSWAVKANLTLSANQSAIISESLAARWGPYGAPAPEAGATVSPFIGGFELQAHYQAGQPDRALDLLRLQWGFMLDDPRMTNSTFIEGYSTDGSLVYAPYTNRPRVSHAHGWSTGPTSALTIYTAGLRVTGPAGATWLYKPQPGNLTQVEAGFSTRLGSFASSFSRSGGRYQELSFTTPNGTTGSVELGDVSGQLVSEGGVKVQLVGGKASGLQGGKWRLNV.

[0043] The gene sequence encoding wild type a-L-rhamnosidase Rha is shown as SEQ ID NO. 2.

[0044] SEQ ID NO. 2:

[0045]

[0046] The gene sequence encoding wild-type a-L-rhamnosidase Rha was inserted into plasmid pPIC9K to construct recombinant plasmid pPIC9K-Rha. The recombinant plasmid pPIC9K-Rha was used as a template to construct a mutant enzyme recombinant expression vector using a site-directed mutagenesis kit, and the specific steps were as follows:

[0047] 1. PCR amplification

[0048] According to the gene sequence of wild-type a-L-rhamnosidase and the mutation of the target site, corresponding site-directed mutagenesis primers were designed, as shown in Table 1. Taking the site-directed mutagenesis of site 95 as an example, the corresponding site-directed mutagenesis primers were D95W-F and D95W-R. The recombinant plasmid pPIC9K-Rha was used as a template for PCR amplification to obtain a linear DNA fragment of the full plasmid length.

[0049] Table 1 Site-directed mutagenesis primers

[0050] Primer name Sequence (5' - 3') SEQ ID NO. D95W-F TGGGCTGGTTTGGATACTCCA 3 D95W-R TTGAGTAGCATCACAAGCTTCGTTG 4 L192M-F ATGGTTGGTTTGGGTGCTATTACTT 5 L192M-R AGCATCACCAGTGGTTGGG 6 L195R-F CGCGGTGCTATTACTTCTTCT 7 L195R-R ACCAACCAAAGCATCACCAG 8 R234L-F CGTGTTTGGCCAGGTGATATGT 9 R234L-R TCTATCTCTCTTAGCACCATCGGTC 10 Y282I-F TGGTACGATACTGTTTCTTTTACCTACCAC 11 Y282I-R TGGCTTACCAGCGTATGGC 12

[0051] 2. Digestion of template plasmid DNA with Dpn I

[0052] 1 μL of Dpn I was added to 5-10 μL of PCR amplification product, mixed gently, and reacted at 37°C for 1 hour to specifically degrade the original template plasmid with methylation.

[0053] 3. Self-circularization of PCR product

[0054] After purification of the digestion product, in vitro self-ligation reaction was performed to circularize the complete plasmid. The ligation product was transformed into E. coli JM109 to obtain the corresponding recombinant E. coli, which was plated on a plate containing ampicillin sodium and cultured at 37°C overnight. Randomly selected clones were subjected to colony PCR identification and sequencing verification. The results showed that the recombinant expression vector containing the a-L-rhamnosidase mutant gene was successfully transformed into the expression host E. coli JM109. The bacterial solution with successful mutation verified by sequencing was added with glycerol and preserved in a -80°C refrigerator.

[0055] 4. Electroporation of mutant recombinant plasmid into Pichia pastoris

[0056] High-purity and high-concentration mutant plasmid was extracted from the verified correct strain and subjected to linearization treatment. The linearized DNA was transformed into Pichia pastoris GS115 competent cells by electroporation. The transformed cells were plated on a selection plate and cultured at 30°C for 2-4 days until single colonies appeared. Colony PCR was used to identify whether the target gene was successfully integrated into the Pichia pastoris genome, and a positive recombinant yeast engineering strain was screened.

[0057] Example 2 Expression and purification of wild type and mutant α-L-rhamnosidases and enzyme activity determination

[0058] 1. Expression and purification of wild type and mutant α-L-rhamnosidases

[0059] The Pichia pastoris recombinant strains containing wild type and mutant α-L-rhamnosidases were respectively subjected to high-density fermentation under optimal conditions, and the promoter AOX1 was strongly induced by methanol to drive the efficient expression of the target genes (wild type or mutant α-L-rhamnosidase genes). After the expression was completed, the supernatant was collected by centrifugation, and the crude enzyme solutions of wild type and mutant α-L-rhamnosidases were obtained, respectively.

[0060] After the crude enzyme was filtered by a 0.45 μm filter membrane, it was purified by a nickel column and eluted by a buffer solution containing 200 mM imidazole, and the pure enzymes of wild type and mutant α-L-rhamnosidases were obtained, respectively.

[0061] 2. Enzyme activity determination of wild type and mutant α-L-rhamnosidases

[0062] Using rutin as the substrate: 10 g / L rutin solution was prepared by using 950 μL sodium phosphate buffer (50 mM, pH 5.0), 50 μL pure enzyme solution was added, and the reaction was carried out at 60°C and 150 rpm for 30 min, and then the enzyme was inactivated by boiling in water bath at 100°C for 5 min. The rhamnose content was determined by DNS reagent. Under the optimal reaction conditions, the amount of enzyme required to produce 1 μmol of rhamnose per minute was defined as one enzyme activity unit (U).

[0063] The results of relative enzyme activity determination of wild type and mutant α-L-rhamnosidases are shown in Table 1. Figure 1 As can be seen from the results of relative enzyme activity determination, the enzyme activity of mutant Y282I is 1.46 times that of the wild type, and the relative enzyme activity is greatly improved. The relative enzyme activities of other mutants are not improved or are reduced to different degrees. Therefore, mutant Y282I can efficiently convert rutin to isoquercitrin.

[0064] The amino acid sequence of mutant Y282I is shown in SEQ ID NO. 13.

[0065] MWSSWLLSALLATEALAVPYEEYILAPSSRDLAPASVRQVNGSVTNAAALTGAGGQATFNGVSSVTYDFGINVAGIVSVDVASASSESAFIGVTFTESSMWISNEACDATQDAGLDTPLWFAVGQGAGVYSVGKKYTRGAFRYMTVVSNTTATVSLNSVKINYTASPIQDLRAYTGYFHSSDELLNRIWYAGAYTLQLCSIDPTTGDALVGLGAITSSETITLPQTDKWWTNYTITNGSSTLTDGAKRDRLVWPGDMSIALESVAVSTEDLYSVRTALESLIALQKADGQLPYAGKPFYDTVSFTYHLHSLVGAASYYQYTGDRAWLTRYWGQYKKGVQWALSGVDSTGLANITASADWLRFGMGAHNIEANAILYYVLNDAISLAQSLNDNAPIRNWTATAARIKTVANELLWDDKNGLYTDNETTTLHPQDGNSWAVKANLTLSANQSAIISESLAARWGPYGAPAPEAGATVSPFIGGFELQAHYQAGQPDRALDLLRLQWGFMLDDPRMTNSTFIEGYSTDGSLVYAPYTNRPRVSHAHGWSTGPTSALTIYTAGLRVTGPAGATWLYKPQPGNLTQVEAGFSTRLGSFASSFSRSGGRYQELSFTTPNGTTGSVELGDVSGQLVSEGGVKVQLVGGKASGLQGGKWRLNV.

[0066] Example 3 Construction of Pichia pastoris whole-cell catalyst engineering strain

[0067] The specific operation steps are as follows:

[0068] Step S1. The coding gene of Rha mutant Y282I is amplified by PCR using appropriate primers, and a Flag tag is introduced at the 5' end.

[0069] Step S2. The Pichia pastoris GS115 genome DNA is extracted using a yeast genome extraction kit, appropriate primers are designed, and the anchor protein gene Gcw61 is amplified by PCR using the same as a template, while a linker sequence is introduced at the 5' end of the amplified fragment. The gene is homologously recombined with the vector pPIC9K, the ligation product is obtained to obtain the recombinant plasmid pPIC9K-linker-Gcw61, which is transformed into E. coli JM109.

[0070] Step S3. The alpha-L-rhamnosidase mutant gene used for homologous recombination was amplified by PCR, reverse PCR was performed on the plasmid pPIC9K-linker-Gcw61 in step S2 to obtain a vector, the gene was subjected to homologous recombination with the vector, the ligation product was used to obtain a surface display recombination vector, which was transformed into E. coli JM109. Then the surface display recombination vector was electroporated into Pichia pastoris GS115 to form transformants, PCR was used to identify the correct integration of the target gene in the transformants to obtain recombinants, and the positive recombinants were induced for expression by methanol. After the expression was completed, the supernatant was removed by centrifugation to obtain the Pichia pastoris engineering bacteria for surface display of the alpha-L-rhamnosidase Rha mutant, i.e., the Pichia pastoris whole-cell catalyst engineering bacteria.

[0071] The specific primers used in PCR amplification in steps S1, S2 and S3 are shown in Table 2.

[0072] Table 2 Primers used in the construction of a surface display recombination vector

[0073]

[0074] Example 4 Determination of the enzymatic properties of the whole-cell catalyst

[0075] The Pichia pastoris whole-cell catalyst engineering bacteria obtained by screening a third generation on a G418 plate were inoculated into 5 mL YPD medium, and cultured at 30°C and a rotation speed of 190 rpm for 24 h. The overnight culture was inoculated into 50 mL BMGY liquid medium at an inoculation amount of 2%, and cultured at 30°C and a rotation speed of 190 rpm until the OD 600 was about 1. The bacterial liquid was transferred to a 50 mL centrifuge tube, centrifuged at 5000 rpm for 10 min to collect the bacterial cells, and the bacterial cells were resuspended and washed by adding BMMY liquid medium, which was washed three times. The resuspended bacterial cells were transferred to BMMY medium, and cultured at 30°C and a rotation speed of 190 rpm for 5 days. Methanol was added every 24 h, and the amount of methanol added was 1% (v / v) of the total volume of the BMMY liquid medium. The bacterial cells were collected after centrifugation to obtain the whole-cell catalyst.

[0076] 1. Determination of the optimum pH of the whole-cell catalyst

[0077] 12-48 U of the whole-cell catalyst was resuspended with citric acid-phosphate buffer, and the bacterial suspension was used as the reaction catalyst. Rutin was used as the substrate, and the enzyme activity of the whole-cell catalyst in pH 3-8 buffer was determined at 60°C to determine the optimum pH. The results are shown in Table 3. Figure 2 The optimum pH of the whole-cell catalyst for catalyzing rutin was 5.

[0078] 2. Determination of the optimal temperature for whole-cell catalysts

[0079] 12-48 U of whole-cell catalyst was resuspended in 50 mM citrate-phosphate buffer (pH 5), and the bacterial suspension was used as the reaction catalyst. Using rutin as a substrate, the enzyme activity of the whole-cell catalyst was measured at 30-80℃ to determine the optimal temperature. Results are as follows: Figure 3 As shown, the optimal temperature for whole-cell catalysts to catalyze rutin is 60℃.

[0080] Example 5: Hydrolysis of Rutin using a Whole-Cell Catalyst

[0081] 1. Hydrolysis of rutin at different high concentrations using whole-cell catalysts

[0082] Whole-cell catalysts of 12-48 U were resuspended in citrate-phosphate buffer, and the bacterial suspension was used as the reaction catalyst. Rutin concentrations ranged from 150-800 g / L. Rutin was dissolved using 8% DMSO. The reaction temperature was 60℃, and the reaction time was 1-48 h. The reaction was terminated with 10 times the volume of DMSO. The supernatant was collected by centrifugation, filtered through a 0.22 μm organic membrane, and the product was analyzed by HPLC. The results are shown in Table 3. When the rutin concentration was 150-500 g / L, the conversion rate was >99% within 24 h.

[0083] Table 3. Hydrolysis reaction of rutin at different high concentrations using whole-cell catalysts.

[0084] Reaction concentration (g / L) Reaction time (h) Conversion rate 150-500 24h >99％ 550 24h 99.82％ 600 24h 98.80％ 650 24h 95.20％ 700 48h 98.79％ 750 48h 97.77％ 800 48h 95.35％

[0085] 2. Hydrolysis of 500 g / L rutin by whole-cell catalysts with different enzyme activities

[0086] Whole-cell catalysts of 12-48 U were resuspended in citrate-phosphate buffer, and the bacterial suspension was used as the reaction catalyst. Rutin concentration was 500 g / L. Rutin was dissolved using 8% DMSO. The reaction temperature was 60℃, and the reaction time was 1-48 h. The reaction was terminated with 10 times the volume of DMSO. The supernatant was collected by centrifugation, filtered through a 0.22 μm organic membrane, and the product was detected by HPLC. At 36 U and 48 U, the rutin conversion rate was >99%. Figure 4 ).

[0087] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An α-L-rhamnosidase mutant, characterized in that, The amino acid sequence is shown as SEQ ID NO.

13.

2. A gene encoding the α-L-rhamnosidase mutant of claim 1.

3. A recombinant vector comprising the gene of claim 2, characterized in that, The recombinant vector is a Pichia pastoris cell surface display vector.

4. The recombinant vector of claim 3, wherein, In the recombinant vector, the gene encoding the α-L-rhamnosidase mutant contains a Flag tag.

5. The recombinant vector of claim 4, wherein, The recombinant vector includes an anchor protein gene Gcw61.

6. An engineered bacterium comprising the recombinant vector of any one of claims 3-5.

7. The engineered bacterium of claim 6, wherein, The engineered bacterium is a Pichia pastoris engineered bacterium.

8. Use of the α-L-rhamnosidase mutant of claim 1, the gene of claim 2, the recombinant vector of any one of claims 3-5, or the engineered bacterium of claim 6 or 7 in improving the catalytic efficiency of α-L-rhamnosidase.

9. Use of the α-L-rhamnosidase mutant of claim 1, the gene of claim 2, the recombinant vector of any one of claims 3-5, or the engineered bacterium of claim 6 or 7 in improving the production efficiency of isoquercitrin.

10. A method for improving the efficiency of production of isoquercitrin, characterized by, The process comprises using the engineered bacterium of claim 6 or 7 to produce a whole-cell catalyst for catalyzing the production of isoquercitrin from rutin.

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