A glucanase mutant gene and its application

Abstract

The present application provides a mutant glucanase, which has a mutation at the amino acid position corresponding to position 121 of SEQ ID No. 2 compared to the parent glucanase. The enzyme activity of the mutant glucanase of the present application is increased by 68% compared to the wild-type enzyme, and has a wide application prospect in degrading materials containing glucan (for example, Aureobasidium pullulans residues produced in the production of pullulan).

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a mutated glucanase gene and its application, and more particularly to a mutated gene Glu16A-2 of β-1,3-1,4-glucanase and its application. Background Technology

[0002] β-glucan is a class of non-starch polysaccharides composed of glucose structural units linked by β-glycosidic bonds, widely found in higher plants, fungi, bacteria, and other organisms. β-glucanases catalyze the breakdown of β-glucan into soluble oligosaccharides with prebiotic value, which have important applications in industry, agriculture, and medicine. β-1,3-1,4-glucanase (EC3.2.1.6) belongs to the β-glucanase family and specifically hydrolyzes the β-1,3-1,4-glycosidic bonds in β-glucan. The green, high-value conversion of β-glucan-rich bacterial residue produced during the fermentation of pullulan by *Phyllostachys buddingus* has always been a challenge. Using β-glucanases for degradation and conversion into prebiotic oligosaccharides is an economical and efficient method.

[0003] *Thermomyces lanuginosus* can ferment *Brachystomia buddingae* residue to produce oligosaccharides, and the thermostable nature of its secreted enzymes makes it a promising candidate for industrial applications. During residue fermentation, the GH16 family β-1,3-1,4-glucanase GUL16A (gil301070474) was specifically induced to express, indicating its important role in degrading β-glucan and *Brachystomia buddingae* residue. However, the low activity of the β-glucanase provided by this fungus limits its application in the conversion of *Brachystomia buddingae* residue.

[0004] To enhance the activity of Glul6A, the applicant analyzed its key amino acid sites using active structure sequence profiling and conducted research on them. Summary of the Invention

[0005] This application obtained a glucanase mutant with enhanced activity through mutation of key amino acid sites.

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

[0007] On one hand, the present invention provides a mutant dextranase, which, compared with the parental 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 dextranase 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 with SEQ ID No. 2.

[0010] In one embodiment, the dextranase is 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 dextranase is shown in SEQ ID No. 3.

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

[0014] On the other hand, the present invention also provides a recombinant vector containing the encoding gene of the mutated dextranase; preferably, the recombinant vector is a recombinant expression vector; preferably, it is a vector of the pET series, such as pET-22b, pET-15b, pET-28a.

[0015] On the other hand, the present invention also provides a recombinant strain containing the above-mentioned 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 dextranase, the encoding gene, the recombinant vector, or the recombinant strain in the degradation of dextran or dextran-containing materials. It should be understood that dextran-containing materials include naturally occurring dextran-containing materials or processed dextran-containing materials.

[0017] In one embodiment, the material containing dextran is fungal residue from fungi, such as the residue of *Brevipedia buddingis*.

[0018] In one embodiment, the dextran is oat-derived dextran.

[0019] In one embodiment, the budding short-stem mold residue is the residue remaining after fermenting budding short-stem mold to produce a specific product (e.g., pullulan).

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

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

[0022] This invention obtains the mutant gene Glu16A-2 by mutating β-1,3-1,4-glucanase Glu16A. The mutation site of the gene is L121F. The mutant β-1,3-1,4-glucanase encoded by this gene has an activity of 163 IU / mg, which is 68% higher than that of the wild-type enzyme. It has wide applications in the degradation of budding short-stemmed mold residue produced in pullulan production. Attached Figure Description

[0023] Figure 1 This is an SDS-PAGE image of purified protein Glu16A-2.

[0024] Figure 2 The results show the comparison of enzyme activities between wild-type glucanase Glu16A (WT) and mutant Glu16A-2 (L121F).

[0025] Figure 3 The results show the comparison of enzyme activity of wild-type glucanase Glu16A (WT) and mutant Glu16A-2 (L121F) in degrading budding short-stemmed mold residue. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0027] Example 1: Mutation of β-1,3-1,4-glucanase Glu16A and construction of recombinant vector

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

[0029] SEQ ID No.1

[0030] Atgcgttcccttcccatccttttcgccggtttgacctctcaactggccgcggcgtatcatcttgttgacgactacggccggggcaatggcttcttcgacaagttcaacttcttcaccggcgacgatcccacccatgggtacgtcgactatgtgagccgggatgtggctgcaggcgccggcctcatcggtgagcgcgacggtcgcacatacatgggtgtcgacttcaccaatcccgcttcgggccgtggccggcggagcgtgcgattggagagcaagaacacgtatgagcacggcctgattgtgatcgatcttgctcatatgccaggctcggtctgcggcacctggccggccttctggaccctgggcaccggtgactggccgtacggcggggagattgacatcattgagggtgtcaacgacaataccttcaaccacatggtactccacaccagcgatggttgcaccatcgataacgacggcttcacgggcaatctgaagacgtccaactgctacgtgtacgcccccggccaggacgccaacgccggctgtggcattgaggctaccgacccgaattcctacggcaaaggtttcaacagcattggcggcggcatctacgccacggagatcacccccaacgggatcagcatctggttcttccctcgtggctccgagcccggtgacgtcctcggcgacaacccgaacccggcgaactgggacacgcccgctgccaagttcgcgggaggtggctgcgactgggagggcaagttcaacgcccagagactgatctttgacgtcaccttctgcggcgattgggccggcaatgtttggggcattggtggctgcgccagccgtgcggccaactgcgtggacttcgttcgcgataacccgtccgccttcgccgagtcttactggctggtgaactcgctccgcgtgtacgcaccctaa

[0031] SEQ ID No.2

[0032] MRSLPILFAGLTSQLAAAYHLVDDYGRGNGFFDKFNFFTGDDPTHGYVDYVSRDVAAGAGLIGERDGRTYMGVDFTNPASGRGRRSVRLESKNTYEHGLIVIDLAHMPGSVCGTWPAFWTLGTGDWPYGGEIDIIEGVNDNTFNHMSLHTSDGCTID NDGFTGNLKTSNCYVYAPGQDANAGCGIEATDPNSYGKGFNSIGGGIYATEITPNGISIWFFPRGSEPGDVLGDNPNPANWDTPAAKFAGGGCDWEGKFNAQRLIFDVTFCGDWAGNVWGIGGCASRAANCVDFVRDNPSAFAESYWLVNSLRVYAP

[0033] The applicant predicted the key amino acid site (L at position 121) for enzyme activity by analyzing the active structure sequence of Glu16A. The L at position 121 of SEQ ID No.2 was mutated to F. The mutated amino acid sequence is shown in SEQ ID No.3. It was named Glu16A-2.

[0034] SEQ ID No. 3

[0035] MRSLPILFAGLTSQLAAAYHLVDDYGRGNGFFDKFNFFTGDDPTHGYVDYVSRDVAAGAGLIGERDGRTYMGVDFTNPASGRGRRSVRLESKNTYEHGLIVIDLAHMPGSVCGTWPAFWTFGTGDWPYGGEIDIIEGVNDNTFNHMSLHTSDGCTID NDGFTGNLKTSNCYVYAPGQDANAGCGIEATDPNSYGKGFNSIGGGIYATEITPNGISIWFFPRGSEPGDVLGDNPNPANWDTPAAKFAGGGCDWEGKFNAQRLIFDVTFCGDWAGNVWGIGGCASRAANCVDFVRDNPSAFAESYWLVNSLRVYAP

[0036] The specific mutation method can be any method conventional in the art. In this embodiment, site-directed mutagenesis is performed using 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 ligated to plasmid pET22b(+) to obtain recombinant plasmids.

[0040] Example 2: Construction of recombinant engineered bacteria containing the above-mentioned recombinant plasmid

[0041] The recombinant engineered bacteria containing the Glu16A gene and the mutant gene Glu16A-2 were constructed as follows: The correctly sequenced recombinant plasmid was added to 50 μL of *E. coli* BL21(DE3) competent cells and incubated on ice for 30 min; followed by heat shock at 42℃ for 90 s; then on ice for 2 min. 1 mL of LB medium was added, and the cells were cultured in a shaker at 37℃ for 1-1.5 h; the cells were centrifuged at 8000 rpm for 2 min, and the supernatant was discarded (leaving a small amount of liquid at the bottom). The remaining solution was spread evenly onto LB agar plates containing 50 μg / mL kanamycin until dry, and incubated overnight at 37℃ inverted mode. The next day, single colonies were picked and inoculated into 5 mL of LB medium containing antibiotics, and cultured overnight at 37℃ and 200 rpm to obtain the recombinant engineered bacteria containing the recombinant plasmid.

[0042] Example 3: Recombinant Expression of Glu16A and Glu16A-2

[0043] The recombinant engineered bacteria containing the Glu16A or Glu16A-2 gene obtained above were fermented in LB medium (containing 50 μg / mL kanamycin) at 37°C and 200 rpm until OD reached... 600 =0.6-0.8; Add IPTG to a final concentration of 0.5 mM, and induce culture at 16℃ for 20 h; Centrifuge at 8000 rpm, 4℃ for 10 min to obtain bacterial pellet, resuspend, and sonicate to disrupt cells; Centrifuge to obtain supernatant (crude enzyme solution), and filter the supernatant through a 0.22 μm filter; Combine the affinity column packing material with the filtrate, and prepare for affinity purification. A GE Healthcare Ni Sepharose 6Fast Flow affinity column was used to purify the target protein containing the 6×His tag. The purified protein was added to pH 5.0 Na2HPO4-citrate buffer, and ultrafiltered at 4900 rpm, 4℃, until the pH of the filtered buffer was 5.0; the concentration of the purified protein was measured using Coomassie Brilliant Blue staining.

[0044] Figure 1This is an SDS-PAGE image of purified protein Glu16A-2. Markers were 10, 15, 25.0, 35.0, 45.0, 60, 75, and 100 kDa. Each lane represented a marker, crude enzyme (CE), sediment enzyme (SE), effluent enzyme (EE), 5 mM imidazole elution buffer, 5 mM imidazole elution buffer, 10 mM imidazole elution buffer, 10 mM imidazole elution buffer, and 20 mM imidazole elution buffer. Figure 1 It can be seen that the target protein band is located at 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) Dextranase activity assay

[0047] Mutant dextranase L121F (SEQ ID No. 3) and wild-type enzyme WT (SEQ ID No. 2) were diluted to 0.005 mg / mL. 35 μL of pH 5.0 Na₂HPO₄-citrate buffer was added to the control group tube, and 35 μL of diluted enzyme solution was added to the experimental group tube. Then, 35 μL of 10 mg / mL dextran (dissolved in 50 mM NaH₂PO₄-citrate buffer at pH 5.0) was added to each tube. The reaction was carried out at 60°C for 30 min. 230 μL of DNS was added to each tube, and the mixture was incubated in a boiling water bath for 10 min. The mixture was rapidly cooled, shaken well, centrifuged, and the supernatant was collected to determine the OD. 540 Each enzyme was tested in triplicate. The amount of reducing sugar was calculated from the standard curve, and the specific enzyme activity was calculated using the formula. The enzyme activities of the two proteins were compared. Enzyme activity unit (IU / mg) is defined as the amount of reducing sugar produced in 1 minute by hydrolyzing dextran substrate to produce the equivalent of 1 μmol of glucose under the corresponding temperature and pH conditions.

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

[0049] (2) Determination of enzyme activity in residue of degraded budding short-stalked fungus

[0050] Mutant dextranase L121F and wild-type enzyme WT were diluted to 0.005 mg / mL. 150 μL of the diluted enzyme solution was added to each experimental tube, followed by 0.04 mg of *Bacillus buddingus* residue dissolved in 600 μL buffer. The mixture was reacted at 60°C for 1 h. A 100 μL sample was taken, and 80 μL of DNS was added. The mixture was then incubated in a boiling water bath for 10 min, cooled to room temperature, and 820 μL of H2O was added. The mixture was shaken well, centrifuged, and the supernatant was collected for OD measurement. 540 Perform three replicates for each enzyme; calculate the amount of reducing sugar based on the standard curve, and then calculate the specific enzyme activity using the formula; compare the enzyme activities of the two proteins.

[0051] The enzyme activities of mutant dextranase L121F and wild-type dextranase WT in degrading budding short-stemmed mold residue are as follows: Figure 3 As shown, the enzyme activity of the mutant dextranase L121F is 1.18 times that of the wild-type dextranase WT, which proves that the mutant dextranase has strong enzyme activity for the residue of *Brachystomata buddingii* and has potential application value.

[0052] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A mutated glucanase, characterized in that, The amino acid sequence of the mutated glucanase is shown in SEQ ID No.

3.

2. A gene encoding the mutated glucanase of claim 1.

3. A recombinant vector containing the gene of claim 2.

4. A recombinant strain comprising the recombinant vector of claim 3.

5. Use of the mutated glucanase of claim 1, or the gene of claim 2, or the recombinant vector of claim 3, or the recombinant strain of claim 4, in degrading glucan or a material containing glucan.

6. A method of degrading glucan or a material containing glucan, the method comprising the step of treating the glucan or the material containing glucan with the mutated glucanase of claim 1, or the gene of claim 2, or the recombinant vector of claim 3, or the recombinant strain of claim 4.

7. Use according to claim 5 or method according to claim 6, characterized in that, The glucan is glucan derived from oats.

8. Use according to claim 5 or method according to claim 6, characterized in that, The material containing glucan is fungal residue from fungi.

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