A bifunctional enzyme with degrading glucan and chitosan, gene and application

Abstract

The application discloses a bifunctional enzyme with the functions of degrading glucan and chitosan, a gene and application, the amino acid sequence of the bifunctional enzyme is shown as SEQ ID NO.1, and the nucleotide sequence of the gene for encoding the bifunctional enzyme is shown as SEQ ID NO.2. The application screens a new enzyme gene for hydrolyzing cellulose and polysaccharide complex by using metagenomic sequencing data, and provides a new idea for mining enzyme gene resources in microorganisms. Since the bifunctional enzyme can efficiently degrade glucan and chitosan, the bifunctional enzyme has a more extensive application prospect in the fields of animal feed, livestock and poultry breeding and the like.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a bifunctional enzyme, gene, and application of degrading dextran and chitosan. Background Technology

[0002] β-glucan is a non-starch polysaccharide composed of D-glucan linked by β-1,3 and β-1,4 glycosidic bonds. It is a major component of water-soluble dietary fiber in cereals, but its high viscosity negatively impacts industrial production. β-glucanase can reduce viscosity by cleaving the 1,3-β-glycosidic and 1,4-β-glycosidic bonds in β-glucan molecules, converting them into oligosaccharides or monosaccharides. As an important glycosidic hydrolase, β-glucanase has been applied in various fields such as beer brewing, feed industry, biofuels, and biocontrol. Chitosan, also known as deacetylated chitin, is a linear polysaccharide composed of acetylglucosamine and aminodeoxyglucose linked by β-1,4 glycosidic bonds. Chitosan degradation by chitosanase yields chitosan oligosaccharides with a degree of polymerization between 2 and 20, also known as oligochitosan. Due to its high water solubility, high biocompatibility, and biodegradability, it is used in livestock farming, pharmaceuticals, and agriculture.

[0003] Microorganisms in nature are the main source of β-glucanase and chitosanase, but in industrial applications, it is still necessary to improve the catalytic activity of these enzymes. Therefore, it is of great significance to discover efficient bifunctional enzymes and their genes that can simultaneously degrade β-glucan and chitosan. Summary of the Invention

[0004] To address the aforementioned shortcomings of the prior art, the first objective of this invention is to provide a bifunctional enzyme, namely Glu7 enzyme, that degrades dextran and chitosan, the amino acid sequence of which is shown in SEQ ID NO.1.

[0005] A second object of the present invention is to provide a gene encoding the above-mentioned bifunctional enzyme, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0006] A third objective of this invention is to provide a recombinant vector comprising the gene fragment with the nucleotide sequence shown in SEQ ID NO.2.

[0007] A fourth objective of the present invention is to provide an engineered bacterium comprising the above-mentioned recombinant vector or gene fragment with nucleotide sequences as shown in SEQ ID NO.2.

[0008] The fifth objective of this invention is to provide the application of the above-mentioned bifunctional enzyme in the degradation of dextran and chitosan. The specific application process is as follows: using dextran and chitosan as substrates, adding the above-mentioned bifunctional enzyme, and carrying out enzymatic hydrolysis reaction at 40-60°C and pH 5-7.

[0009] Based on the above technical solution, the present invention can be further improved as follows:

[0010] Furthermore, the mass concentrations of both dextran and chitosan are 1-10%.

[0011] Furthermore, the dosage of the bifunctional enzyme is 1-5 U / mL.

[0012] Furthermore, the enzymatic hydrolysis conditions are 50-60℃ and pH 6-7.

[0013] Furthermore, Co is added to the reaction system during the enzymatic hydrolysis process. 2+ Cu 2+ Fe 2+ Zn 2+ .

[0014] Furthermore, Co is added to the reaction system during the enzymatic hydrolysis process. 2+ Cu 2+ Fe 2+ Zn 2+ The concentration is 5-500 mmol / L.

[0015] When the above-mentioned bifunctional enzymes are applied to the degradation of glucan and chitosan, in order to improve enzyme activity, recombinant bacteria containing bifunctional enzyme genes can be cultured in a fermenter, followed by fermentation induction and purification to improve the activity of the bifunctional enzymes. The specific culture medium and culture conditions are as follows: 8 g / L tryptone, 20 g / L yeast extract, 2 mL / L glycerol, 2.31 g KH2PO4, 12.54 g K2HPO4·3H2O, initial pH 7.0, culture medium volume of 70 / 250 mL, and inoculum size of 3%. After 6 h of induction of recombinant bacteria, the β-glucanase activity in the fermenter is 232.70 U / mL.

[0016] The present invention has the following beneficial effects:

[0017] This invention utilizes metagenomic sequencing data to screen a novel enzyme gene for hydrolyzing cellulose and polysaccharide complexes, providing a new approach for mining enzyme gene resources in microorganisms. The novel bifunctional enzyme screened in this invention is designated Glu7, and its amino acid sequence is shown in SEQ ID NO.1. The nucleotide sequence of the gene encoding this bifunctional enzyme is shown in SEQ ID NO.2. This bifunctional enzyme can efficiently degrade dextran and chitosan. Specifically, at the shake-flask level, the β-glucanase activity is 112.76 U / mL, with optimal operating conditions of 50℃ and pH 6.0, and it exhibits good stability at 50℃; at the shake-flask level, the chitosanase activity is 63.32 U / mL, with optimal operating conditions of 50℃ and pH 7.0. Furthermore, metal ions such as Co... 2+ Cu 2+ Fe 2+ Zn 2+ Both can activate β-glucanase and chitosanase activity. Furthermore, this invention also enhances the enzyme activity of Glu7 through fermenter-scale cultivation.

[0018] Existing enzymes hydrolyze dextran or chitosan under acidic conditions, while the bifunctional enzyme provided by this invention can hydrolyze dextran and chitosan under neutral conditions, thus broadening the conditions for hydrolyzing dextran and chitosan and making them more widely applicable in animal feed, livestock and poultry farming and other fields. Attached Figure Description

[0019] Figure 1 The results of double enzyme digestion electrophoresis during the construction of the recombinant expression vector are shown; where M: Marker; 1: double enzyme digestion result of pET-30a(+) plasmid; 2: pET-30a(+) plasmid.

[0020] Figure 2 The results of SDS-PAGE electrophoresis after induction of recombinant bacterial expression are shown; where M: Marker; 1: empty vector control bacteria; 2: supernatant; 3: supernatant after lysis.

[0021] Figure 3 To optimize the SDS-PAGE electrophoresis results for recombinase-induced expression conditions.

[0022] Figure 4 The results of SDS-PAGE electrophoresis of the purified recombinant enzyme are shown below; where M: Marker; 1: crude enzyme solution; 2: flow-through solution; 3: washing solution; 4-7: purification tubes 1, 2, 3 and 4.

[0023] Figure 5 The results show the optimal temperature for determining Glu7β-glucanase activity and chitosanase activity.

[0024] Figure 6The results show the optimal pH values ​​for Glu7β-glucanase and chitosanase activities.

[0025] Figure 7 The results show the thermostability of Glu7β-glucanase activity and chitosanase activity.

[0026] Figure 8 The results show the growth curves of the recombinant bacteria on four different basal culture media.

[0027] Figure 9 The results show the β-glucanase activity assays on four different basal culture media of the recombinant bacteria.

[0028] Figure 10 The results were determined to optimize the inoculum size for recombinant bacteria under shake-flask conditions.

[0029] Figure 11 The results show the initial pH optimization of the culture medium for recombinant bacteria under shake-flask conditions.

[0030] Figure 12 The results were determined to optimize the liquid volume of the recombinant bacteria under shake-flask conditions.

[0031] Figure 13 To measure β-glucanase activity and OD in the fermenter 600 Measurement results. Detailed Implementation

[0032] This invention utilizes metagenomic sequencing data to screen for a complete GH family-related gene sequence (Glu7) in a bacterial community capable of degrading natural cellulose. The full-length Glu7 is 1164 bp, and its novel gene was confirmed by BLAST alignment. Gene cloning was then performed, and the gene was expressed in *Escherichia coli* strain BL21. To improve the induced expression level of the bifunctional enzyme, the induction conditions were optimized. The optimal induction conditions for this bifunctional enzyme were a final IPTG concentration of 0.25 mmol / L and induction at 37°C for 6 h. Furthermore, the enzymatic properties of this bifunctional enzyme were studied. Specifically, the optimal temperature for β-glucanase activity was 50°C, and the optimal pH was 6.0. It maintained over 80% of its enzyme activity even after treatment at 50°C for 60 min, exhibiting good thermostability. The optimal temperature and pH for chitosanase activity of Glu7 were 50°C and pH 7.0, respectively. At the shake-flask level, the β-glucanase activity of the recombinant bacteria reached 112.76 U / mL, and the chitosanase activity reached 63.32 U / mL; Na + K + Ca 2+ Mg 2+ Zn 2+ Mn 2+ Fe 2+ Cu 2+ Co2+ Ni 2+ Both EDTA and Cu have an activating effect on chitosanase activity. 2+ Ca 2+ Ni 2+ Mn 2+ It has the strongest activating effect on chitosanase activity; Co 2+ Cu 2+ Fe 2+ Zn 2+ All of them have an activating effect on β-glucanase activity.

[0033] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0034] Example 1:

[0035] I. Cloning of the recombinase gene Glu7

[0036] 1. Screening for cellulose degradation-related genes

[0037] Metagenomic sequencing data analysis was performed on cellulose-degrading bacteria, and a complete β-glucan gene sequence (Glu7) of the GH8 family was screened from the sequencing data. The gene is 1164 bp in length and encodes 387 amino acids. Comparison with the NCBI database confirmed that this gene is 100% homologous to the GH8 family glucanase gene from Paenibacillus ihbetae IHBB 9852 (CP016809.1), but it lacks 22 amino acids at the N-terminus (WP_099477748).

[0038] The selected gene sequences are shown below:

[0039] ATGCTGCCGGCGGGACTGTCCATGGCGGAGAATCAGAGGCCGTTTCCG

[0040] CAGCATACGACATATACGAGCGGTTCGATCAAGCCCACCATGTGACG

[0041] CAGACGGCGATGGACGAAGCCGTCAAGAGTAAGTGGAACAGCTGGAA

[0042] GGCTTCGTTTCTCAAGCCTGCCGGCACGGGGCAATATTACGTAAAATAC

[0043] AACTCGGCCGGGGAAACCGTATCGGAGGCCCACGGCTACGGGATGATC

[0044] CTGACGGTCATGATGGCCGGAGCCGATGCCAACGCTCAGACATACTTC

[0045] GATGGACTGTACCGATATTATAAGGCTCATCCGAGCAACCAGAATCCAT

[0046] ACTTAATGGCCTGGAAACAGAACAGCAGCTTCCAGAACATTGAGGGG

[0047] GCCAACTCGGCGACGGACGGCGATATGGATATCGCATACGCCCTCCTGT

[0048] TGGCAGATAGACAGTGGGGCAGCAGCGGAAGCATTAACTATCTGCAAG

[0049] CGGCCAAGAACATCATAGGCGCAATCATGAGCCATGACGTGAATCAAT

[0050] CGCAATGGACTCTTCGTCTGGGAGATTGGGCGACGAGCGGCAGCTTCA

[0051] ATACAGCCACGCGCCCGTCCGATTTCATGCTGAACCATCTGAAGGCGTT

[0052] CCGGAAGGCTACCGGGGATGCCAGATGGGATCAGGTAATCGACAAAAC

[0053] ATACAGCATCATCAACTCCGTTCACGGCAGCTATAGCCCGAATACGGGG

[0054] CTGCTTCCGGATTTCGTCGTGCTGCAGGGGGGGAGCTATCAGCCTGCG

[0055] GCGGCCGGCTTCCTTGAAGGTGCGAACGACGGGAACTATTATTACAAT

[0056] TCGGCACGGACGCCTTGGCGAATCGCCACCGACTATTTGATGACCGGC

[0057] GATACGAGGGCCTTGGGACAGCTCGATTTGATGAACGCGTTCATCAAA

[0058] TCGAATACGAGCCAGAAGCCGGCAAATATCAAGGCAGGCTATACGCTC

[0059] GGCGGAAGCCCGCTTGTGTCGTATAACAGCGGCGCATTCTACGCCCCG

[0060] TTCGGCATCAGCGCGATGGTCTCCCAGAGCCACCAAAGCTGGCTGAAC

[0061] GTCGTGTGGACCTATACGGCGAATGCATCGGCCGAGGGATATTACGAA

[0062] GAAAGCATCAAGCTGTTCTCGATGCTGGTCATGTCCGGGAATTGGTGGAGCTATTAA(SEQ IDNO.5)

[0063] Based on the screened gene sequences, polymerase chain reaction primers were designed for PCR amplification. The primers, PCR amplification system, and PCR reaction procedure are as follows:

[0064] Upstream primer sequence: 5'-GCGGCCGCATGCTGCCGGCGGGACTG-3' (SEQ ID NO.3);

[0065] Downstream primer sequence: 5'-TTAGTAGGTCCACCAATTCCCGG GGTACC-3' (SEQ ID NO.4).

[0066] PCR reaction system: 10 μL of 2×San Taq PCR Mix, 1 μL of upstream primer (36 μL / 100 μm), 1 μL of downstream primer (47 μL / 100 μm), 1 μL of template DNA, 7 μL of ultrapure water, and a total volume of 20 μL.

[0067] PCR reaction conditions: 94℃ for 5 min, 94℃ for 30 s, 59℃ for 30 s, 72℃ for 90 s, 30 cycles, 72℃ for 5 min.

[0068] The PCR products were recovered using the AxyPrep DNA Gel Recovery Kit to extract the target gene fragments.

[0069] 2. Optimization of the Glu7 codon

[0070] When exogenous genes are expressed as heterologous proteins in E. coli, codon bias leads to very low expression levels of recombinant proteins. Codon optimization, which replaces low-frequency codons with high-frequency codons, can improve the synthesis efficiency of recombinant proteins, thereby increasing their expression levels.

[0071] The amino acid sequence is shown below:

[0072] MLPAGLMAWK TRPSDFMLNHHLKAFRKATGDARWDQVIDKTYSIINSVHGSYSPNTGLLPDFVVLQGGSYQPAAAGFLEGANDGNYYYNSARTPWRIATDYLMTGDTRALGQLDLMNAFIKSNTSQKPANIKAGYTLGGSPLVSYNSGAFYAPFGISAMVSQSHQSWLNVVWTYTANASAEGYYEESIKLFSMLVMSGNWWSY(SEQ ID NO.1)

[0073] The complete sequence information of the optimized gene codon is shown below:

[0074]

[0075] II. Construction and Transformation of Prokaryotic Expression Vectors

[0076] The target gene and pET-30a(+) plasmid were double-digested with KpnI and XhoI, and ligated with T4 DNA ligase at 16℃ for 16 h. The ligation product was transformed into competent *E. coli* DH5α cells using the heat shock method. Positive clones were screened on LB agar plates containing 50 μg / mL kanamycin and identified by PCR and sequencing. Recombinant plasmids were extracted from the pET-30a(+)-DH5α clone strain, purified, and transformed into the expression strain *E. coli* BL21(DE3) using the heat shock method. Positive clones were screened on LB agar plates containing 50 μg / mL kanamycin and verified by PCR, double digestion, and sequencing before induction of expression. The results are shown in the figure. Figure 1 .

[0077] Depend on Figure 1 It can be seen that the pET-30a(+) plasmid is 5422bp in size and the Glu7 gene fragment is 1164bp in size, both of which are consistent with the sizes of the two band fragments in lane 1, indicating that the whole gene synthesis and prokaryotic expression vector construction were successful.

[0078] III. Induction and Optimization of Recombinase Glu7 Expression

[0079] Single colonies of the transformed recombinant bacteria were picked and cultured overnight at 37°C and 200 rpm with shaking in LB medium containing 50 μg / mL kanamycin. A 5% inoculum was then transferred to 100 mL of the same medium and cultured with shaking for 2–3 hours until the A600 reached 0.5–0.8. One mL of the pre-induction sample was collected and stored at -20°C. IPTG was added to the remaining samples to a final concentration of 0.5 mmol / L, and the samples were cultured at 37°C with shaking. After 6 hours of culture, one mL of the post-induction sample was collected and stored at -20°C. The pre-induction and post-induction samples were centrifuged at 12000 rpm for 1 minute, and the bacterial pellets were collected separately. 75 μL of PBS (pH 7.4) buffer and 25 μL of 4×SDS loading buffer were added to each pellet, and the pellets were shaken vigorously for 1 minute to ensure complete lysis. SDS-PAGE analysis confirmed the induced expression of the recombinant protein. The results are shown in the figure below. Figure 2 .Depend on Figure 2 It can be seen that the protein expressed by the Glu7 gene is an intracellular protein, and the molecular weight of the protein is consistent with the molecular weight of 42.57 kD calculated based on the gene fragment length.

[0080] Furthermore, the induction conditions for recombinant protein expression were optimized in terms of IPTG concentration, induction temperature, and induction time. IPTG was added at final concentrations of 0.25 mmol / L, 0.5 mmol / L, 0.75 mmol / L, and 1 mmol / L for induction. The optimal IPTG concentration was determined by SDS-PAGE electrophoresis. Under the optimal IPTG concentration, expression was induced at three temperature gradients of 28℃, 30℃, and 37℃. The optimal culture temperature was determined by SDS-PAGE electrophoresis. The optimal induction time was determined by setting time gradients of 2 h, 4 h, 6 h, 8 h, and 10 h at the optimal IPTG concentration and temperature.

[0081] The results of optimizing recombinase-induced expression conditions are shown in Figure 3 ;in, Figure 3 A represents the effect of IPTG concentration on the expression level of recombinant protein; Figure 3 B represents the optimized results of temperature-induced expression conditions; Figure 3 C represents the result of optimizing time-induced expression conditions.

[0082] Figure 3 In A, M is the standard molecular weight of the protein; 1 to 7 represent the values ​​before induction, 0 mM, 0.25 mM, 0.50 mM, 0.75 mM, 1 mM, and empty control bacteria, respectively.

[0083] Figure 3 In B, M represents the standard molecular weight of the protein; 1: 28℃; 2: 30℃; 3: 37℃; 4: before induction.

[0084] Figure 3 In C, M represents the standard molecular weight of the protein; 1 to 6 represent induction times of 2h, 4h, 6h, 8h, 10h, and before induction, respectively.

[0085] Depend on Figure 3 It can be seen that the optimal induction conditions after optimization are: the optimal IPTG induction concentration is 0.25 mmol / L. After induction with 0.25 mmol / L IPTG, the expression level of recombinant protein reaches its maximum after 6 hours of induction at 37℃.

[0086] IV. Purification of recombinase Glu7

[0087] Recombinant enzyme Glu7 was induced at 37°C for 6 h with 0.25 mmol / L IPTG, and bacterial cells were collected after centrifugation at 4000 r / min for 10 min at 4°C. The cells were washed twice with PBS (pH 7.4) buffer, sonicated, and centrifuged at 12000 r / min for 30 min at 4°C to obtain crude enzyme solution.

[0088] Recombinant protein was purified using a Ni-NTA column. The column was washed with ultrapure water to remove 20% ethanol and air from the matrix. The column was equilibrated with binding buffer, and the crude enzyme solution was loaded and incubated at 4°C for 1 hour. Impurities were washed with washing buffer, and the target protein was eluted with buffers containing 20 mmol / L, 50 mmol / L, 100 mmol / L, 150 mmol / L, 200 mmol / L, 250 mmol / L, 500 mmol / L, and 1000 mmol / L imidazole, respectively. The eluent was collected, and the optimal imidazole concentration was determined. The column was washed with ultrapure water and then rinsed with 20% ethanol. The column was stored at 4°C. The purified enzyme solution was then verified by SDS-PAGE electrophoresis to determine band size and purity. The results are shown below. Figure 4 The recovery rate and purification fold of the target protein during the purification process are shown in Table 1.

[0089] Depend on Figure 4 It can be seen that there is a single band at 43kD, which is consistent with the predicted protein molecular weight of 42.57kD.

[0090] Table 1 shows that the specific activity of β-glucanase in the purified recombinant enzyme Glu7 increased from 49.03 U / mg to 155.53 U / mg, with a purification fold of 3.17 and a recovery rate of 51.86%. The specific activity of chitosanase increased from 27.53 U / mg to 76.89 U / mg, with a purification fold of 2.79. The enzyme activity recovery rate was 47.44%.

[0091] Table 1 Purification of Recombinant β-glucanase

[0092]

[0093]

[0094] The enzyme activity and protein concentration were determined using the following methods:

[0095] 1. The β-glucanase activity was determined using the 3,5-dinitrosalicylic acid (DNS) method: 1% barley glucan was used as the substrate, and 0.1 mol / L phosphate buffer was used as the solvent. 200 μL of the substrate was placed in a centrifuge tube and preheated for 10 min. 100 μL of appropriately diluted enzyme solution was added, mixed, and reacted in a 50℃ water bath for 10 min. 700 μL of DNS was added, mixed, and then boiled in a water bath for 5 min before being brought to a final volume of 5 mL. After cooling, the absorbance was measured at 540 nm. The amount of reducing sugar produced was obtained according to the glucose standard curve, and the enzyme activity was calculated. The enzyme solution added to the blank tube was inactivated.

[0096] 2. Chitosanase activity was determined using the 3,5-dinitrosalicylic acid (DNS) method: 1% colloidal chitosan was used as the substrate, and 0.1 mol / L phosphate buffer was used as the solvent. 350 μL of the substrate was placed in a centrifuge tube and preheated for 10 min. 50 μL of appropriately diluted enzyme solution was added, mixed, and reacted in a 50℃ water bath for 10 min. 600 μL of DNS was added, mixed, and then in a boiling water bath for 10 min. After cooling, the absorbance was measured at 540 nm. The amount of reducing sugar produced was obtained according to the glucosamine hydrochloride standard curve, and the enzyme activity was calculated. The enzyme solution added to the blank tube was inactivated.

[0097] 3. Definition of β-glucanase activity: Under the above reaction conditions, the amount of enzyme required to release 1 μmol of reducing sugar per minute by hydrolyzing the substrate is defined as 1 unit of enzyme activity (U).

[0098] 4. Definition of chitosanase activity: Under the above reaction conditions, the amount of enzyme required to release 1 μmol of reducing sugar per minute by hydrolyzing the substrate is defined as 1 unit of enzyme activity (U).

[0099] 5. The specific activity of an enzyme is defined as the number of enzyme activity units per milligram of protein, expressed in U / mg.

[0100] 6. Protein concentration was determined using the Bradford method. A concentration gradient standard solution was prepared using bovine serum albumin (BSA) as the standard. The absorbance was measured at a wavelength of 595 nm, and a BSA standard curve was plotted. The protein concentration of the enzyme solution was determined from the standard curve.

[0101] V. Determination of the enzymatic properties of recombinase Glu7

[0102] 1. Determination of optimal temperature

[0103] 1% barley glucan and 1% colloidal chitosan were prepared using a pH 5 buffer solution. Enzyme activity was measured at 30℃, 40℃, 50℃, 60℃, and 70℃, with the highest enzyme activity defined as 100%. Relative enzyme activity was calculated at each temperature. A graph was plotted with temperature on the x-axis and relative enzyme activity on the y-axis. The results are shown below. Figure 5 .

[0104] 2. Determination of optimal pH

[0105] At the optimal reaction temperature, 1% barley glucan and 1% colloidal chitosan were prepared using buffer solutions of different pH values. The highest enzyme activity was recorded as 100%, and the relative enzyme activity at different reaction pH values ​​was calculated. A graph was plotted with pH on the x-axis and relative enzyme activity on the y-axis. The results are shown below. Figure 6 .

[0106] 3. Thermal stability test

[0107] Glu7 enzyme solution was incubated in water baths at 50℃ and 60℃, with samples taken every 10 minutes to determine residual enzyme activity. The activity of untreated enzyme was taken as 100%, and relative enzyme activity was calculated. A graph was plotted with treatment time on the x-axis and relative enzyme activity on the y-axis. The results are shown below. Figure 7 .

[0108] 4. Determination of the effects of metal ions and compounds on enzyme activity

[0109] The enzyme solution was diluted with 0.1 mol / L phosphate buffer (pH 7.4), and 5 mmol / L of metal ions and compounds were added respectively. After being incubated at room temperature for 30 min, the residual enzyme activity was measured. The enzyme activity without the addition of metal ions and compounds was taken as 100%. The results are shown in Table 2.

[0110] Depend on Figure 5-7 It is known that the optimal temperature for both β-glucanase and chitosanase activities of Glu7 is 50℃, and the optimal pH for β-glucanase and chitosanase activities of Glu7 is pH 6.0 and pH 7.0, respectively. The β-glucanase activity of Glu7 can still maintain more than 80% of its enzyme activity after treatment at 50℃ for 60 min, indicating a certain degree of thermal stability.

[0111] In the above enzymatic hydrolysis process, the amount of bifunctional enzyme Glu7 used was 1 U / mL.

[0112] As shown in Table 2, Na + K + Ca 2+ Mg 2+ Zn 2+ Mn 2+ Fe 2+ Cu 2+ Co 2+ Ni 2+ Both EDTA and Cu have an activating effect on chitosanase activity. 2+ Ca 2+ Ni 2+ Mn 2+ It exhibited the strongest activating effect on chitosanase activity, increasing it by 80.7%, 66.23%, 43.41%, and 42.26%, respectively. + K + Ca 2+ Mg 2+ EDTA, Ni 2+ Mn 2+ All enzymes inhibited β-glucanase activity, reducing it by 25.21%, 23.11%, 13.04%, 25.05%, 27.93%, 29.40%, and 46.27%, respectively; Co2+ Cu 2+ Fe 2+ Zn 2+ All of them activated β-glucanase activity, increasing it by 134.03%, 18.18%, 10.92%, and 9.89%, respectively.

[0113] Table 2. Effects of different metal ions and compounds on Glu7 enzyme activity.

[0114]

[0115] VI. Optimization of Culture Conditions and Small-Scale Fermentation of Recombinant Bacteria

[0116] 1. Growth curves and enzyme activity determination of recombinant bacteria in different basal culture media

[0117] Growth curves of recombinant bacteria in different culture media were determined: Single colonies of recombinant bacteria were picked and placed in 100 mL LB medium (250 mL Erlenmeyer flask) containing kanamycin (final concentration 50 μg / mL) and cultured at 37℃ and 200 rpm for 12 h to obtain seed culture. 5 mL of seed culture was then transferred to four different culture media: LB, TB, SB, and SOB (100 mL / 250 mL Erlenmeyer flask, kanamycin 50 μg / mL). 5 mL of seed culture was added to each medium and cultured at 37℃ and 200 rpm. Samples were taken every 2 h to measure OD. 600 The results are shown Figure 8 .

[0118] Recombinant bacteria were induced to express their enzymes in four different culture media. After induction, the bacterial suspension was centrifuged at 4000 rpm for 10 min at 4°C, and the bacterial cells were collected. The cells were washed twice with PBS (pH 7.4) buffer, sonicated (300W, 4 s on, 4 s off, 10 min), and then centrifuged at 12000 rpm for 30 min at 4°C. The supernatant, i.e., the crude enzyme solution, was collected. The β-glucanase activity of the recombinant bacteria in different culture media was measured. The results are shown in the table below. Figure 9 .

[0119] Depend on Figure 8 and Figure 9 It can be seen that the recombinant bacteria grow best and have the highest β-glucanase activity in TB medium. Therefore, TB medium is used as the base medium for optimizing the composition of the culture medium.

[0120] 2. Optimization of culture medium composition

[0121] An orthogonal experiment was designed to investigate the effects of three factors—tryptone, yeast extract, and glycerol—on β-glucanase activity in TB medium. Each of these three factors had four levels. The orthogonal experiment was conducted as shown in Table 3 to determine the optimal medium. The results are shown in Tables 4 and 5.

[0122] Table 3 Orthogonal Experimental Design

[0123]

[0124] Table 4. Results of orthogonal experiments for optimizing the culture medium for Glu7.

[0125]

[0126]

[0127] Table 5. Range Analysis Results of Orthogonal Experiments

[0128]

[0129] As shown in Table 5, the R values ​​of the three factors are ranked in the order of A > B > C. Among them, the range of tryptone factor is the largest. The optimal combination of culture medium is A1B2C1, that is, 8 g / L tryptone, 20 g / L yeast extract, and 2 mL / L glycerol. Based on the optimal culture medium, the β-glucanase activity of Glu7 reaches 171.75 U / mL.

[0130] 3. Optimization of recombinant bacterial culture conditions

[0131] Using enzyme activity as an indicator of enzyme production culture conditions, the effects of factors such as inoculum size, initial pH, and liquid volume on enzyme activity were investigated to determine the optimal enzyme production culture conditions.

[0132] (1) Inoculation volume

[0133] The culture medium volume was 100 / 250 mL, pH 7.0, and the inoculum volume was adjusted to 1%, 3%, 5%, 7%, and 10% (V / V), with three replicates for each level. The culture was incubated overnight at 37°C with shaking at 200 rpm until OD reached. 600 When the concentration reaches 0.5–0.8, add IPTG to a final concentration of 0.25 mmol / L, incubate at 37°C with shaking for 6 hours, then take samples to measure enzyme activity. Results are shown below. Figure 10 .

[0134] (2) Initial pH

[0135] The culture medium volume was 100 / 250 mL, the inoculum size was 3%, and the initial pH of the medium was adjusted to pH 6.0, pH 6.5, pH 7.0, pH 7.5, and pH 8.0, with three replicates for each level. The medium was incubated overnight at 37°C with shaking at 200 rpm until OD reached.600 When the concentration reaches 0.5–0.8, add IPTG to a final concentration of 0.25 mmol / L, incubate at 37°C with shaking for 6 hours, then take samples to measure enzyme activity. Results are shown below. Figure 11 .

[0136] (3) Liquid volume

[0137] The initial pH of the culture medium was 7.0, the inoculum size was 3%, and the liquid volumes were adjusted to 30 / 250 mL, 50 / 250 mL, 70 / 250 mL, and 100 / 250 mL, with three replicates for each level. The culture was incubated overnight at 37°C with shaking at 200 rpm until OD reached. 600 When the concentration reaches 0.5–0.8, add IPTG to a final concentration of 0.25 mmol / L, incubate at 37°C with shaking for 6 hours, then take samples to measure enzyme activity. Results are shown below. Figure 12 .

[0138] Depend on Figures 10-12 It was found that the highest enzyme activity of Glu7 was observed when the culture medium volume was 70 / 250 mL, the initial pH was 7.0, and the inoculum size was 3%. Through optimization of the culture medium and conditions for recombinant Glu7, the β-glucanase activity of Glu7 increased from 112.76 U / mL to 207.99 U / mL, an increase of 84.30%, and the chitosanase activity increased from 63.32 U / mL to 75.48 U / mL, an increase of 19.20%.

[0139] 4. Small-scale test in a 3L fermenter

[0140] The culture medium formulation optimized in the above experiments was used to observe the growth and enzyme production of the recombinant bacteria in the fermenter. The initial fermentation conditions were: 1.8 L of culture medium, 37℃, 200 rpm, and pH 7.0. Dissolved oxygen was maintained at a constant 20%–30% by adjusting the stirring speed and aeration rate. The inoculum size was 5% (w / v), and kanamycin was added to achieve a final concentration of 50 μg / mL. OD was measured after inoculation. 600 When the OD value was approximately 0.6, IPTG was added to a final concentration of 0.25 mmol / L for induction at 37°C for 6 hours. Samples were taken every 1 hour to measure the OD value. 600 and β-glucanase activity, results are shown in Figure 13 .

[0141] Depend on Figure 13 To date, the β-glucanase activity in the fermenter is 232.70 U / mL, representing an increase of 34.13% in enzyme activity.

[0142] In summary, through metagenomic sequencing data analysis of cellulase-producing bacteria, a complete β-glucan and chitosan bifunctional enzyme gene sequence (Glu7) from the GH8 family was screened. The full-length sequence is 1164 bp, and the recombinant enzyme Glu7 has a size of approximately 42.6 kDa in SDS-PAGE. At the shake-flask level, the β-glucanase activity of the recombinant bacteria was 112.76 U / mL, with optimal reaction conditions of 50℃ and pH 6.0, and it exhibited good stability at 50℃. At the shake-flask level, the chitosanase activity was 63.32 U / mL, with optimal reaction conditions of 50℃ and pH 7.0. Furthermore, Na… + K + Ca 2+ Mg 2+ Zn 2+ Mn 2+ Fe 2+ Cu 2+ Co 2+ Ni 2+ Both EDTA and Co have an activating effect on the chitosanase activity of Glu7. 2+ Cu 2+ Fe 2+ Zn 2+ The β-glucanase activity of Glu7 was activated. Under optimized culture medium and conditions (8 g / L tryptone, 20 g / L yeast extract, 2 mL / L glycerol, 2.31 g KH2PO4, 12.54 g K2HPO4·3H2O, initial pH 7.0, culture medium volume 70 / 250 mL, inoculum size 3%), after 6 h of induction with recombinant bacteria, the β-glucanase activity in shake flasks increased to 207.99 U / mL, an increase of 84.30%, and the chitosanase activity of Glu7 increased from 63.32 U / mL to 75.48 U / mL, an increase of 11.88%. Because of the bifunctional enzyme activity of Glu7 in β-glucan and chitosan, it has a wider range of application prospects in animal feed, livestock and poultry farming, and other fields.

[0143] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of bifunctional enzymes capable of degrading dextran and chitosan in the degradation of dextran and chitosan; among which, The amino acid sequence of the bifunctional enzyme is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The specific application process is as follows: using dextran and chitosan as substrates, adding a bifunctional enzyme, and carrying out an enzymatic hydrolysis reaction at 40-60℃ and pH 5-7.

3. The application according to claim 2, characterized in that, The mass concentrations of both dextran and chitosan are 1-10%.

4. The application according to claim 2, characterized in that, The amount of the added bifunctional enzyme is 1-5 U / mL.

5. The application according to claim 2, characterized in that, The enzymatic hydrolysis conditions are 50-60℃ and pH 6-7.

6. The application according to claim 2, characterized in that, Co is added to the reaction system during enzymatic hydrolysis. 2+ Cu 2+ Fe 2+ Zn 2+ .

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