Glucanase mutant gene and application thereof

Abstract

The invention provides a mutant glucanase, and compared with a parent glucanase, the mutant glucanase has mutation at a 121st amino acid site corresponding to SEQ ID No.2 (Sequence Identifier Number 2). The enzyme activity of the mutant glucanase is improved by 68% compared with that of a wild type enzyme, and the mutant glucanase has a wide application prospect in degradation of glucan-containing materials (such as aureobasidium pullulans residues generated in pullulan production).

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering, and particularly relates to a dextranase mutant gene and its application, and more particularly to a mutant gene Glu16A-2 of β-1,3-1,4-dextranase and its application. Background Art

[0002] β-glucan is a class of non-starch polysaccharides formed by connecting glucose structural units with β-glycosidic bonds, and is widely present in organisms such as higher plants, fungi, and bacteria. β-glucanase can catalyze the decomposition of β-glucan into soluble oligosaccharides with prebiotic value, and has important application value in the industrial, agricultural, and medical fields. And β-1,3-1,4-glucanase (EC3.2.1.6) belongs to β-glucanase and can specifically hydrolyze the β-1,3-1,4-glycosidic bond in β-glucan. The green high-value conversion of the β-glucan-rich bacterial residue produced during the fermentation of Aureobasidium pullulans to produce pullulan polysaccharide has always been a difficult problem. Using β-glucanase for degradation and conversion into prebiotic oligosaccharides is an economical and efficient means.

[0003] Thermomyces lanuginosus can ferment the bacterial residue of Aureobasidium pullulans to produce oligosaccharides, and the high-temperature resistance of its secreted enzymes makes it have certain application prospects in industry. During the fermentation of the bacterial residue, the β-1,3-1,4-glucanase Glul6A (gil301070474) of the GH16 family is specifically induced to express, indicating its important role in degrading β-glucan and the bacterial residue of Aureobasidium pullulans. However, the enzyme activity of the β-glucanase provided by this bacterium is relatively low, which limits its application in the conversion of the bacterial residue of Aureobasidium pullulans.

[0004] In order to improve the activity of Glul6A, the applicant analyzed its key amino acid sites through an active architecture sequence spectrum and studied them. Summary of the Invention

[0005] In this application, a dextranase mutant with improved activity was obtained through mutation of key amino acid sites.

[0006] In the present invention, the mutant dextranase is named Glu16A-2 or referred to as L121F.

[0007] On the one hand, the present invention provides a mutant dextranase, and compared with the parental dextranase, the mutant dextranase has a mutation at the 121st amino acid site corresponding to SEQ ID No.2.

[0008] In one embodiment, the 121st amino acid is mutated to F (phenylalanine).

[0009] In one embodiment, the amino acid sequence of the parental glucanase has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity compared to SEQ ID No. 2.

[0010] In one embodiment, the glucanase is a β-1,3-1,4-glucanase.

[0011] In one embodiment, the parental glucanase is derived from Thermomyces lanuginosus.

[0012] In one embodiment, the amino acid sequence of the mutated glucanase is as shown in SEQ ID No. 3.

[0013] On the other hand, the present invention also provides the coding gene of the mutated glucanase.

[0014] On the other hand, the present invention also provides a recombinant vector containing the coding gene of the mutated glucanase; preferably, the recombinant vector is a recombinant expression vector; preferably a vector of the pET series, for example, pET-22b, pET-15b, pET-28a.

[0015] On the other hand, the present invention also provides a recombinant strain containing the above recombinant vector; preferably, the recombinant strain is Escherichia coli, such as Escherichia coli BL21.

[0016] On the other hand, the present invention also provides the use of the mutated glucanase, the coding gene, the recombinant vector or the recombinant strain in degrading glucan or materials containing glucan. It should be understood that materials containing glucan include materials naturally containing glucan, or processed materials containing glucan.

[0017] In one embodiment, the material containing glucan is fungal residue from fungi, for example, the residue of Aureobasidium pullulans.

[0018] In one embodiment, the glucan is glucan derived from oats.

[0019] In one embodiment, the Aureobasidium pullulans residue is the residue remaining after using Aureobasidium pullulans to ferment and produce a specific product (for example, pullulan).

[0020] On the other hand, the present invention also provides a method for degrading dextran or a material containing dextran, which method comprises the step of treating dextran or a material containing dextran with the mutated dextranase, the encoding gene, the recombinant vector or the recombinant strain.

[0021] In one embodiment, the dextran is β-dextran.

[0022] By mutating β-1,3-1,4-dextranase Glu16A, the present invention obtained a mutated gene Glu16A-2, the mutation site of which is L121F. The activity of the mutated β-1,3-1,4-dextranase encoded by this gene can reach 163 IU / mg, which is 68% higher than that of the wild-type enzyme, and it has a wide application in degrading Aureobasidium pullulans residues produced in the production of pullulan polysaccharide. Description of the Drawings

[0023] Figure 1 It is the SDS-PAGE diagram after purification of protein Glu16A-2.

[0024] Figure 2 It is the comparison result of the enzyme activities of wild-type dextranase Glu16A (WT) and mutant Glu16A-2 (L121F).

[0025] Figure 3 It is the comparison result of the enzyme activities of wild-type dextranase Glu16A (WT) and mutant Glu16A-2 (L121F) in degrading Aureobasidium pullulans residues. Detailed Embodiments

[0026] The present invention will be further described below in conjunction with the drawings and specific embodiments. The following description is only for the preferred embodiments of the present invention, and does not limit the present invention in other forms. Any person skilled in the art may use the disclosed technical content to make equivalent changes into equivalent embodiments. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention's solution falls within the protection scope of the present invention.

[0027] Example 1. Mutation of β-1,3-1,4-dextranase Glu16A and Construction of Recombinant Vector

[0028] The β-1,3-1,4-dextranase Glu16A gene was obtained from the NCBI database. Its nucleic acid sequence is as shown in SEQ ID No.1, and its amino acid sequence is as shown in SEQ ID No.2.

[0029] SEQ ID No.1

[0030] Atgcgttcccttcccatccttttcgccggtttgacctctcaactggccgcggcgtatcatcttgttgacgactacggccggggcaatggcttcttcgacaagttcaacttcttcaccggcgacgatcccacccatgggtacgtcgactatgtgagccgggatgtggctgcaggcgccggcctcatcggtgagcgcgacggtcgcacatacatgggtgtcgacttcaccaatcccgcttcgggccgtggccggcggagcgtgcgattggagagcaagaacacgtatgagcacggcctgattgtgatcgatcttgctcatatgccaggctcggtctgcggcacctggccggccttctggaccctgggcaccggtgactggccgtacggcggggagattgacatcattgagggtgtcaacgacaataccttcaaccacatggtactccacaccagcgatggttgcaccatcgataacgacggcttcacgggcaatctgaagacgtccaactgctacgtgtacgcccccggccaggacgccaacgccggctgtggcattgaggctaccgacccgaattcctacggcaaaggtttcaacagcattggcggcggcatctacgccacggagatcacccccaacgggatcagcatctggttcttccctcgtggctccgagcccggtgacgtcctcggcgacaacccgaacccggcgaactgggacacgcccgctgccaagttcgcgggaggtggctgcgactgggagggcaagttcaacgcccagagactgatctttgacgtcaccttctgcggcgattgggccggcaatgtttggggcattggtggctgcgccagccgtgcggccaactgcgtggacttcgttcgcgataacccgtccgccttcgccgagtcttactggctggtgaactcgctccgcgtgtacgcaccctaa

[0031] SEQ ID No.2

[0032] MRSLPILFAGLTSQLAAAYHLVDDYGRGNGFFDKFNFFTGDDPTHGYVDYVSRDVAAGAGLIGERDGRTYMGVDFTNPASGRGRRSVRLESKNTYEHGLIVIDLAHMPGSVCGTWPAFWTLGTGDWPYGGEIDIIEGVNDNTFNHMSLHTSDGCTIDNDGFTGNLKTSNCYVYAPGQDANAGCGIEATDPNSYGKGFNSIGGGIYATEITPNGISIWFFPRGSEPGDVLGDNPNPANWDTPAAKFAGGGCDWEGKFNAQRLIFDVTFCGDWAGNVWGIGGCASRAANCVDFVRDNPSAFAESYWLVNSLRVYAP

[0033] Through the sequence spectrum analysis of the Glu16A active architecture, the applicant predicted the key amino acid sites for its enzyme activity (L at position 121), mutated L at position 121 of SEQ ID No. 2 to F, and the mutated amino acid sequence is shown in SEQ ID No. 3, which is named Glu16A-2.

[0034] SEQ ID No.3

[0035] MRSLPILFAGLTSQLAAAYHLVDDYGRGNGFFDKFNFFTGDDPTHGYVDYVSRDVAAGAGLIGERDGRTYMGVDFTNPASGRGRRSVRLESKNTYEHGLIVIDLAHMPGSVCGTWPAFWTFGTGDWPYGGEIDIIEGVNDNTFNHMSLHTSDGCTIDNDGFTGNLKTSNCYVYAPGQDANAGCGIEATDPNSYGKGFNSIGGGIYATEITPNGISIWFFPRGSEPGDVLGDNPNPANWDTPAAKFAGGGCDWEGKFNAQRLIFDVTFCGDWAGNVWGIGGCASRAANCVDFVRDNPSAFAESYWLVNSLRVYAP

[0036] Specific mutagenesis methods can adopt the conventional methods in the art. In this example, site-directed mutagenesis was carried out by designing primers, and the primer sequences are as follows:

[0037] L121F-sense: tctggaccttcggcaccggtgactggcc

[0038] L121F-antisense: accggtgccgaaggtccagaaggcc

[0039] The Glu16A gene and the Glu16A-2 gene were respectively ligated to the plasmid pET22b(+), and recombinant plasmids were obtained.

[0040] Example 2. Construction of recombinant engineering bacteria containing the above recombinant plasmids

[0041] To construct recombinant engineering bacteria containing the above Glu16A gene and the mutant gene Glu16A-2, the specific steps are as follows: Add the above recombinant plasmid with correct sequencing to 50 μL of Escherichia coli BL21(DE3) competent cells, and incubate on ice for 30 min; heat shock at 42 °C for 90 s; incubate on ice for 2 min, add 1 mL of liquid LB, and culture in a shaker at 37 °C for 1 - 1.5 h; centrifuge at 8000 rpm for 2 min, and discard the supernatant (leave a little bottom liquid). Spread the remaining solution on an LB plate containing 50 μg / mL kanamycin, spread evenly until dry, and culture overnight at 37 °C in an inverted position; pick monoclonal colonies the next day, inoculate them into 5 mL of LB medium containing antibiotics, and culture overnight at 37 °C and 200 rpm to obtain recombinant engineering bacteria containing the recombinant plasmid.

[0042] Example 3. Recombinant expression of Glu16A and Glu16A-2

[0043] Take the obtained recombinant engineering bacteria containing the above Glu16A or Glu16A-2 gene, and ferment and culture them in an LB medium (containing 50 μg / mL kanamycin) at 37 °C and 200 rpm until OD 600 = 0.6 - 0.8; add IPTG with a final concentration of 0.5 mM, and induce culture at 16 °C for 20 h; centrifuge at 8000 rpm and 4 °C for 10 min to obtain cell pellets. After resuspension, ultrasonically disrupt the cells; centrifuge to obtain the supernatant (crude enzyme solution), and filter the supernatant with a 0.22 μm filter head; bind the packing of the affinity column to the filtrate to prepare for affinity purification. Use a GE Healthcare Ni Sepharose 6 Fast Flow affinity column to perform affinity purification on the target protein containing a 6×His tag. Add Na2HPO4-citrate buffer with pH 5.0 to the purified protein, and ultrafilter at 4900 rpm and 4 °C until the pH of the filtered buffer is 5.0; use the Coomassie Brilliant Blue staining method to measure the concentration of the purified protein.

[0044] Figure 1SDS-PAGE diagram of purified protein Glu16A-2. The markers are 10, 15, 25.0, 35.0, 45.0, 60, 75, 100 kDa respectively. Each lane is Marker, crude enzyme (CE), sediment enzyme (SE), effluent enzyme (EE), 5 mM imidazole eluent, 5 mM imidazole eluent, 10 mM imidazole eluent, 10 mM imidazole eluent, 20 mM imidazole eluent. From Figure 1 it can be seen that the target protein band is located around 35 kDa, which is consistent with the size of the target protein.

[0045] Example 4. Comparison of enzymatic properties between mutant dextranase Glu16A-2 and wild-type enzyme Glu16A

[0046] (1) Determination of dextranase activity

[0047] Dilute mutant dextranase L121F (SEQ ID No.3) and wild-type enzyme WT (SEQ ID No.2) to 0.005 mg / mL; add 35 μL of Na2HPO4-citrate buffer at pH 5.0 to the control tube, and add 35 μL of the diluted enzyme solution to the experimental group tube. Then add 35 μL of dextran with a concentration of 10 mg / mL (dissolved in 50 mM NaH2PO4-citrate buffer at pH 5.0) to each tube, and react at 60 °C for 30 min; add 230 μL of DNS to each tube, and boil in a water bath for 10 min; quickly cool, shake well, centrifuge, take the supernatant, and measure OD 540 ; Conduct three replicate experiments for each enzyme; calculate the amount of reducing sugar according to the standard curve, and then calculate the specific enzyme activity according to the formula; compare the enzyme activities of the two proteins. Definition of enzyme activity unit (IU / mg): Under the corresponding temperature and pH conditions, the amount of reducing sugar produced by hydrolyzing dextran substrate for 1 min equivalent to 1 μmol of glucose is one enzyme activity unit.

[0048] The enzyme activities of mutant dextranase L121F and wild-type dextranase WT under the optimal conditions are as Figure 2 shown. The enzyme activity of mutant dextranase L121F is 1.68 times that of wild-type dextranase WT, which proves that mutant dextranase has strong enzyme activity towards dextran and has potential application value.

[0049] (2) Determination of enzyme activity for degrading Aureobasidium pullulans residue

[0050] Dilute the mutant glucanase L121F and the wild-type enzyme WT to 0.005 mg / mL; add 150 μL of the diluted enzyme solution to the experimental group tubes, and then add 0.04 mg of Aureobasidium pullulans residue dissolved in 600 μL of buffer. Take 100 μL of the sample after reacting at 60 °C for 1 h, add 80 μL of DNS, boil in a water bath for 10 min, cool to room temperature, add 820 μL of H2O, shake well, centrifuge, take the supernatant, and measure the OD 540 ; Conduct three replicate experiments for each enzyme; calculate the amount of reducing sugar according to the standard curve, and then calculate the specific enzyme activity according to the formula; compare the enzyme activities of the two proteins.

[0051] The enzyme activities of the mutant glucanase L121F and the wild-type glucanase WT in degrading Aureobasidium pullulans residue are as Figure 3 shown. The enzyme activity of the mutant glucanase L121F is 1.18 times that of the wild-type glucanase WT, which proves that the mutant glucanase has strong enzyme activity towards Aureobasidium pullulans residue and has potential application value.

[0052] The above embodiments are only used to illustrate the technical concept and characteristics of the present invention, and their purpose is to enable those who are familiar with this technology to understand the content of the present invention and implement it accordingly, and cannot be used to limit the protection scope of the present invention. All equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A mutant glucanase, wherein compared with the parental glucanase, a mutation exists at the amino acid position 121 corresponding to SEQ ID No.

2.

2. The mutant glucanase according to claim 1, characterized in that, The amino acid mutation at position 121 is F.

3. The mutant glucanase according to claim 1, wherein, The parental glucanase is derived from Thermomyces lanuginosus.

4. A gene encoding the mutant glucanase according to any one of claims 1-3.

5. A recombinant vector containing the gene according to claim 4.

6. A recombinant strain containing the recombinant vector according to claim 5.

7. Use of the mutant glucanase according to any one of claims 1-3, or the gene according to claim 4, or the recombinant vector according to claim 5, or the recombinant strain according to claim 6 in degrading glucan or a material containing glucan.

8. A method for degrading glucan or a material containing glucan, the method comprising the step of treating glucan or a material containing glucan with the mutant glucanase according to any one of claims 1-3, or the gene according to claim 4, or the recombinant vector according to claim 5, or the recombinant strain according to claim 6.

9. The application according to claim 7 or the method according to claim 8, characterized in that The glucan is derived from oats.

10. The application according to claim 7 or the method according to claim 8, characterized in that The material containing glucan is the fungal residue from fungi.

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