Application of a polysaccharide hydrolase and its mutants in the hydrolysis of Enteromorpha

Abstract

The present invention provides an application of a polysaccharide hydrolase and its mutant in the hydrolysis of Enteromorpha prolifera. The amino acid sequence of the polysaccharide hydrolase is SEQ ID NO:1. The present invention also provides an optimized polysaccharide hydrolase, whose amino acid sequence is SEQ ID NO:3 and the nucleotide sequence of its encoding gene is SEQ ID NO:4. The polysaccharide hydrolase provided by the present invention can be used to degrade Enteromorpha prolifera to produce reducing oligosaccharides. Compared with the original enzyme, its mutant has higher catalytic efficiency and better application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of proteases, and specifically relates to the application of a polysaccharide hydrolase and its mutants in the hydrolysis of Enteromorpha prolifera. Background Art

[0002] Enteromorpha prolifera belongs to the family Ulvaceae and the genus Ulva, and is a eurythermal and euryhaline green alga with strong environmental adaptability. From 2008 to 2021, large-scale green tides mainly composed of Enteromorpha prolifera occurred in the Yellow Sea every year, and the rapid disposal and utilization of Enteromorpha prolifera have attracted attention from all aspects.

[0003] The dry components of Enteromorpha prolifera contain carbohydrates (43 - 51%), proteins (26 - 33%), fats (0.2 - 0.8%), total amino acids (20.26 - 23.32%), etc. Among them, the content of carbohydrates, that is, Enteromorpha prolifera polysaccharides, is the highest.

[0004] The main components of Enteromorpha prolifera polysaccharides are sulfated polysaccharides, including sulfated rhamnose, uronic acid, xylose and other components, which have unique biological activities and broad application prospects in the fields of food, biomedicine, cosmetics, etc.

[0005] The Enteromorpha prolifera thallus can be destroyed by various enzymes such as proteases, cellulases, hemicellulases, xylanases, pectinases, etc. during the hydrolysis process: Most of these enzymes are non-specific enzymes. For example, xylanase can hydrolyze xylose units. However, the hydrolysis efficiency of non-specific enzymes is limited, the hydrolysis speed is slow, and the hydrolysis products have complex structures and non-uniform molecular weights.

[0006] Currently, it has been reported that polysaccharide hydrolases capable of hydrolyzing sulfated polysaccharides are isolated from the intestines of marine organisms and environmental microorganisms, but natural polysaccharide hydrolases have problems such as few types and low degradation efficiency, making it difficult to be actually applied. Summary of the Invention

[0007] The purpose of the present invention is to provide the application of a polysaccharide hydrolase and its mutants in the hydrolysis of Enteromorpha prolifera, so as to make up for the deficiencies of the existing technology.

[0008] The present invention first provides a polysaccharide hydrolase, comprising:

[0009] 1) A protease with an amino acid sequence of SEQ ID NO:1,

[0010] 2) A protease derived from 1) by substituting, deleting, or adding one or several amino acids in the sequence of 1).

[0011] The gene encoding the above polysaccharide hydrolase has a nucleotide sequence of SEQ ID NO:2; another aspect of the present invention also provides an optimized polysaccharide hydrolase with an amino acid sequence of SEQ ID NO:3 and a nucleotide sequence of the encoding gene of SEQ ID NO:4.

[0012] Another aspect of the present invention also provides a recombinant expression vector into which the nucleic acid fragment encoding the polysaccharide hydrolase is inserted;

[0013] The present invention also provides a recombinant engineering strain transformed with the above recombinant expression vector.

[0014] Another aspect of the present invention provides a use of the polysaccharide hydrolase, which is an application in the degradation of Enteromorpha prolifera to prepare reducing oligosaccharides.

[0015] The polysaccharide hydrolase provided by the present invention can be used to degrade Enteromorpha prolifera to produce reducing oligosaccharides, and its mutant has a higher catalytic efficiency compared with the original enzyme and has a better application prospect. Description of the Drawings

[0016] Figure 1 : Photo of strain screening;

[0017] Figure 2 : Diagram of the effect of pH on enzyme activity;

[0018] Figure 3 : Diagram of the effect of temperature on enzyme activity;

[0019] Figure 4 : Diagram of the effect of NaCl concentration on enzyme activity. Detailed Embodiments

[0020] The present invention will be described in detail below with reference to the embodiments and the drawings.

[0021] Example 1: Amplification of the original polysaccharide hydrolase gene

[0022] I. Isolation and screening of polysaccharide hydrolase-producing bacteria

[0023] Samples of rotting Enteromorpha prolifera near the shore were collected and cultured in seawater medium. After preliminary isolation and culture by the gradient dilution plate method, plates containing Enteromorpha prolifera hydrolysate as the sole carbon source were used for directional screening. According to the growth of colonies on the plates, the degradation ability of the strains was initially judged.

[0024] Add the rotten Enteromorpha prolifera samples to an appropriate amount of seawater and culture them at 30 °C for 3 - 5 days until completely rotten. Take the supernatant, dilute it serially and spread it on the seawater nutrient medium, and culture it at 30 °C for one week. Pick single colonies for further purification. The rescreening is for directional screening. Inoculate the single colonies on the plate with Enteromorpha prolifera hydrolysate as the sole carbon source and culture it statically at 30 °C for one week, and observe the time and size of the transparent zones produced.

[0025] The composition of the primary screening nutrient medium is as follows:

[0026] Glucose 3%, yeast powder 1%, seawater crystal 2%, agar 2%, pH 6.5;

[0027] The composition of the rescreening solid medium is as follows:

[0028] Enteromorpha prolifera hydrolysate, ammonium chloride 0.3%, magnesium sulfate 0.05%, seawater crystal 1.5%, agar 1.5%, pH 6.5 - 7;

[0029] The preparation method of the Enteromorpha prolifera hydrolysate is as follows: Take 20 g of dried Enteromorpha prolifera, add it to 100 ml of dilute sulfuric acid solution with a concentration of 0.2 mol / L, and carry out acid hydrolysis at 115 °C for 30 - 50 min. Use sodium hydroxide to neutralize the excess sulfuric acid and adjust the pH to neutral to obtain the Enteromorpha prolifera hydrolysate.

[0030] Finally, a strain of bacillus was obtained and named strain HT02. This strain of bacteria can grow in the seawater medium, is Gram-negative, rod-shaped, does not produce spores, the colonies are yellow, and does not produce gas when cultured in the glucose peptone medium. Referring to "Bergey's Manual of Determinative Bacteriology", it belongs to the genus Flavobacterium.

[0031] Example 2: Screening for the enzyme gene with the function of hydrolyzing polysaccharides

[0032] Genome sequencing and sequence alignment were carried out on strain HT02, and it was found that there is a potential polysaccharide hydrolase, and the nucleotide sequence of its encoding gene is as follows:

[0033] ATGGTGTTCTTCAAGGACCTGTTCATCTTCAAGAGCCTGATCAAGGGCACCCTGTTCACCCTGTTCGTGAGCGGCCAGATCCTGAGGGCCCCCGGCGCCGGCGGCCCCGGCGGCGCCTACGTGGTGAGCGCCCCCGACGAGGACACCAACCCCGACATCAGCTGCCCCGCCAGCGACGAGTTCGAGAACCCCAACAGGAGCGCCGACCTGCCCAACGCCGTGAACGTGGGCACCATCGACGACAGGACCTGCTACAGCAACTACACCGAGAGCAACGTGTACGGCAAGACCTGGGGCGTGTACAACATCACCGCCGGCAGCAACCACTTCGGCGAGAGGCTGCAGCCCAGGATGGAGAGGAGCCTGAGCAGGAGCAGGGAGACCGGCATCGGCAGCTTCGCCAAGTTCACCGGCGTGTTCAGGATCCTGGAGGTGGGCGACGCCGGCAGCTTCGGCCAGGACGGCAGCTACATCATGCAGGCCAAGGGCAAGCACACCGGCGGCGGCGGCCCCAACGACCCCGCCATCTGCCTGTACCTGGCCAAGCCCGTGTACGGCGACGACGGCAACGGCAACCAGGTGAGCTTCGACATCTACGCCGAGAGGATCCTGTACAGGGGCGGCGAGGGCAGCGGCAGGGAGGTGGTGTTCCTGAAGAACGTGAACAAGAACGTGGAGACCAACTTCGAGCTGGAGGTGGGCTTCGGCGAGGACCCCAGCGACGCCACCAAGAAGATCCACTACTGCAACGCCATCATCGGCGGCGACGCCTTCAACTGGAACATCCCCGAGCCCGAGAAGGGCACCGAGAGCGGCATCAGGTACGGCGCCTACAGGGTGAAGGGCGGCAGGGCCCAGATCAGGTGGGCCAACACCGCCTACCAGAAGGAGGAGGTGGTGGACAACGGCAACCCCGGCCCCAGCGACGACGTGTTCAGGCTGAGGAACGTGGCCACCGGCCAGTTCCTGACCGACAGC(SEQ ID NO:2).

[0034] The encoded amino acid sequence is as follows:

[0035] MVFFKDLFIFKSLIKGTLFTLFVSGQILRAPGAGGPGGAYVVSAPDEDTNPDISCPASDEFENPNRSADLPNAVNVGTIDDRTCYSNYTESNVYGKTWGVYNITAGSNHFGERLQPRMERSLSRSRETGIGSFAKFTGVFRILEVGDAGSFGQDGSYIMQAKGKHTGGGGPNDPAICLYLAKPVYGDDGNGNQVSFDIYAERILYRGGEGSGREVVFLKNVNKNVETNFELEVGFGEDPSDATKKIHYCNAIIGGDAFNWNIPEPEKGTESGIRYGAYRVKGGRAQIRWANTAYQKEEVVDNGNPGPSDDVFRLRNVATGQFLTDS (SEQ ID NO:1).

[0036] II. Construction of an expression vector containing a polysaccharide hydrolase

[0037] After adding restriction enzyme cleavage sites for BamHI and HindIII to both ends of the nucleic acid fragment with the sequence of SEQ ID NO:1, it was synthesized by Sangon Biotech (Shanghai). The synthesized target gene and plasmid pET-28a were respectively double digested with BamHI and HindIII, and the target fragment was recovered and purified using a gel extraction kit ((Omega); the recovered plasmid was ligated with the purified target gene fragment to prepare a recombinant expression vector.

[0038] After the ligation, the recombinant expression vector was transferred into competent cells using the heat shock method. It was spread and cultured on an LB plate supplemented with ampicillin to screen for positive transformants. The bacterial solution of the positive transformants was sent for sequencing and comparison to determine that the polysaccharide hydrolase gene sequence had been cloned into the expression vector.

[0039] III. Construction of a recombinant engineering bacterium

[0040] The recombinant plasmid in the positive clone with correct sequencing was extracted and transformed into the host E. coli BL21 competent cells, and positive transformants resistant to ampicillin were screened and sequenced to determine the target engineering bacterium.

[0041] Inoculate the target engineering bacteria into LB medium containing 50 μg / mL ampicillin, and culture them overnight at 37 °C with 180 rpm for activation. After the bacterial liquid is well activated, add it to the induction medium (Enteromorpha prolifera hydrolysate, 0.3% ammonium chloride, 0.05% magnesium sulfate, 0.5% glycerol, 100 μg / mL ampicillin) at an inoculation amount of 2%, and induce expression at 35 °C for 48 h.

[0042] Centrifuge the fermentation broth to collect the cell precipitate, add 50 mM Tris-HCl buffer solution at pH 8.0 and mix well, and perform ultrasonic disruption. After disruption, centrifuge at 10000 rpm for 20 min, and take the supernatant, which is the crude enzyme solution of polysaccharide hydrolase.

[0043] IV. Determination of the hydrolysis of Enteromorpha prolifera polysaccharide by polysaccharide hydrolase

[0044] Make appropriate modifications according to the detection method provided by Melcher R, Neumann M, Werner J F, et al. Revised domain structure of ulvan lyase and characterization of the first ulvan binding domain[J]. Scientific Reports, 2017, 7: 44115.

[0045] Extraction of Enteromorpha prolifera polysaccharide: Take 100 g of dry Enteromorpha prolifera powder and add 1 L of a mixed solution of dichloromethane and acetone (v / v = 1:1) to remove most of the lipids and pigments. Dry the residual substances at low temperature, use 3 L of distilled water for hot water extraction for 7 h, centrifuge, and take the supernatant. Add proteinase K (1%) and hydrolyze at 37 °C for 24 h to remove proteins. After boiling for 10 min to terminate the reaction, centrifuge and take the supernatant. Add 4 volumes of 95% ethanol to the supernatant and precipitate the polysaccharide at 4 °C. Take the precipitate, wash it 2 - 3 times with cold acetone, then wash it once with ethanol, and dry it for standby.

[0046] Reducing sugars will be produced after the hydrolysis of Enteromorpha prolifera polysaccharide, and the reaction of reducing sugars with DNS can be quantified. Take 20 μL of the crude enzyme solution, add 100 mM Tris-HCl buffer solution (pH 8.5), 200 mM NaCl, and 1 g / L Enteromorpha prolifera polysaccharide substrate to a 200 μL reaction system, incubate at 30 °C for 5 min, then add 10 μL of the crude enzyme solution and react for 30 min, using the heat-inactivated crude enzyme solution as a blank control. After the reaction, measure the absorbance at 235 nm, and the amount of enzyme required for the OD235 value to increase by 0.1 is defined as one enzyme activity unit.

[0047] Take 20 μL of the enzyme solution and react it in a phosphate - acetate - boric acid buffer (pH 5 - 11). The system also contains 200 mM NaCl and 1 g / L Enteromorpha polysaccharide substrate. Select 35°C for the reaction. According to the activity of the polysaccharide hydrolase, the optimal pH of this enzyme is determined to be 9, and the activity is relatively high at pH 8 - 9.5( Figure 2 ).

[0048] Take 20 μL of the enzyme solution and react it in a 100 mM Tris - HCl buffer system (pH 8.5). The system also contains 200 mM NaCl and 1 g / L Enteromorpha polysaccharide substrate. Select 20°C, 30°C, 35°C, 40°C, 45°C, 50°C as the reaction temperatures of the enzyme. According to the activity of the polysaccharide hydrolase, the optimal temperature of this enzyme in this buffer system is determined to be 35°C( Figure 3 ).

[0049] Take 20 μL of the enzyme solution and react it respectively in a 100 mM Tris - HCl buffer system (pH 8.5). The system selects 50 mM, 100 mM, 200 mM, 300 mM, 400 mM and 500 mM NaCl and 1 g / L Enteromorpha polysaccharide substrate. Select 35°C for the reaction. According to the activity of the polysaccharide hydrolase, it is determined that the activity is the highest at a NaCl concentration of 200 mM( Figure 4 ).

[0050] Example 3: Modify the polysaccharide hydrolase

[0051] In order to further improve the catalytic efficiency of the polysaccharide hydrolase with the amino acid sequence SEQ ID NO: 2, it was modified. First, through alignment, the catalytic region and binding region of the polysaccharide hydrolase were determined; the C - terminal signal peptide of the type IX secretion system (T9SS) was removed, and 300 amino acids containing the catalytic domain at the front end were retained. Based on this template, mutant primers for site - directed mutagenesis were designed to construct the mutant plasmid pET - 28a - K162M.

[0052] Analyze the polysaccharide hydrolase with the amino acid sequence SEQ ID NO: 2, and it is speculated that the substrate - binding sites are located at positions 117 / 160 / 162 / 216 / 281. It was determined to mutate Lys162, which is located close to the proton side chain, into the non - polar hydrophobic amino acid Met to improve the entry process of the hydrophobic substrate.

[0053] Using the recombinant plasmid pET - 28a in Example 1 as a template and primers for whole - plasmid PCR mutagenesis, the sequence information of the mutant primers is as follows:

[0054] Mutant forward primer: 5'-GGTGTGCTTGCCCATGGCCTGCATGA - 3'

[0055] Mutant reverse primer: 5'-ACATCATGCAGGCCATGGGCAAGCA-3'.

[0056] The PCR reaction system is as follows:

[0057] 10×PCR Buffer 5 μL; dNTP (2 mmol / L) 4 μL; 1 μL each of forward and reverse primers; 1 μL of plasmid template; 0.5 μL of Taq enzyme; made up to 50 μL with water. The amplification conditions are: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 1 min, annealing at 56°C for 1 min, extension at 72°C for 1 min, 35 cycles, and incubation at 72°C for 10 min.

[0058] The PCR amplification product is the positive K162M mutation, named pET-28a-K162M.

[0059] The amino acid sequence after mutation is as follows:

[0060] MVFFKDLFIFKSLIKGTLFTLFVSGQILRAPGAGGPGGAYVVSAPDEDTNPDISCPASDEFENPNRSADLPNAVNVGTIDDRTCYSNYTESNVYGKTWGVYNITAGSNHFGERLQPRMERSLSRSRETGIGSFAKFTGVFRILEVGDAGSFGQDGSYIMQAMGKHTGGGGPNDPAICLYLAKPVYGDDGNGNQVSFDIYAERILYRGGEGSGREVVFLKNVNKNVETNFELEVGFGEDPSDATKKIHYCNAIIGGDAFNWNIPEPEKGTESGIRYGAYRVKGGRAQIRWANTAYQKEEVV (SEQ ID NO: 3);

[0061] The sequence of the coding gene is as follows:

[0062] ATGGTGTTCTTCAAGGACCTGTTCATCTTCAAGAGCCTGATCAAGGGCACCCTGTTCACCCTGTTCGTGAGCGGCCAGATCCTGAGGGCCCCCGGCGCCGGCGGCCCCGGCGGCGCCTACGTGGTGAGCGCCCCCGACGAGGACACCAACCCCGACATCAGCTGCCCCGCCAGCGACGAGTTCGAGAACCCCAACAGGAGCGCCGACCTGCCCAACGCCGTGAACGTGGGCACCATCGACGACAGGACCTGCTACAGCAACTACACCGAGAGCAACGTGTACGGCAAGACCTGGGGCGTGTACAACATCACCGCCGGCAGCAACCACTTCGGCGAGAGGCTGCAGCCCAGGATGGAGAGGAGCCTGAGCAGGAGCAGGGAGACCGGCATCGGCAGCTTCGCCAAGTTCACCGGCGTGTTCAGGATCCTGGAGGTGGGCGACGCCGGCAGCTTCGGCCAGGACGGCAGCTACATCATGCAGGCCATGGGCAAGCACACCGGCGGCGGCGGCCCCAACGACCCCGCCATCTGCCTGTACCTGGCCAAGCCCGTGTACGGCGACGACGGCAACGGCAACCAGGTGAGCTTCGACATCTACGCCGAGAGGATCCTGTACAGGGGCGGCGAGGGCAGCGGCAGGGAGGTGGTGTTCCTGAAGAACGTGAACAAGAACGTGGAGACCAACTTCGAGCTGGAGGTGGGCTTCGGCGAGGACCCCAGCGACGCCACCAAGAAGATCCACTACTGCAACGCCATCATCGGCGGCGACGCCTTCAACTGGAACATCCCCGAGCCCGAGAAGGGCACCGAGAGCGGCATCAGGTACGGCGCCTACAGGGTGAAGGGCGGCAGGGCCCAGATCAGGTGGGCCAACACCGCCTACCAGAAGGAGGAGGTGGTG(SEQ ID NO: 3).

[0063] The mutant plasmid pET-28a-K162M was transformed into the host E. coli BL21 competent cells. Positive transformants resistant to ampicillin were screened, and the target mutant bacteria were confirmed by sequencing. The target engineered bacteria were inoculated into LB medium containing 50 μg / mL ampicillin and cultured overnight at 37 °C with 180 rpm for activation. After the bacterial solution was well activated, it was added to the induction medium (Enteromorpha prolifera hydrolysate, 0.3% ammonium chloride, 0.05% magnesium sulfate, 0.5% glycerol, 100 μg / mL ampicillin) at an inoculation amount of 2% and induced at 35 °C for 48 h for expression.

[0064] The fermentation broth was centrifuged to collect the cell precipitate, and 50 mM Tris-HCl buffer (pH 8.0) was added and mixed evenly for ultrasonic disruption. After disruption, it was centrifuged at 10000 rpm for 20 min, and the supernatant was taken, which was the crude enzyme solution of polysaccharide hydrolase.

[0065] Take 20 μL of the crude enzyme solution of the unmutated protein and the K162M mutant protein respectively. Add 100 mM Tris-HCl buffer (pH 8.5), 200 mM NaCl, and 1 g / L Enteromorpha prolifera polysaccharide substrate to a 200 μL reaction system. After incubating at 30 °C for 5 min, add 10 μL of the crude enzyme solution and react for 30 min. The crude enzyme solution inactivated at high temperature was used as the blank control. The activity of the unmutated protein was 32.43 U / mg, while the activity of the mutated protein was 106.58 U / mg, which was increased by about 3 times.

Claims

1. A polysaccharide hydrolase, characterized in that, The amino acid sequence of the polysaccharide hydrolase described above is SEQ ID NO:

1.

2. A gene, characterized in that, The gene encodes the polysaccharide hydrolase described in claim 1.

3. The gene according to claim 2, wherein The nucleotide sequence of the gene described above is SEQ ID NO:

2.

4. A polysaccharide hydrolase, characterized in that, The amino acid sequence of the polysaccharide hydrolase described above is SEQ ID NO:

3.

5. The polysaccharide hydrolase according to claim 4, wherein The nucleotide sequence of the coding gene of the polysaccharide hydrolase described above is SEQ ID NO:

4.

6. A recombinant expression vector, characterized in that, The nucleic acid fragment encoding the polysaccharide hydrolase described in claim 1 or claim 4 is inserted into the recombinant expression vector described above.

7. A recombinant engineering strain, characterized in that, The recombinant expression vector described in claim 6 is transformed into the recombinant engineering strain described above.

8. Use of the polysaccharide hydrolase described in claim 1 or claim 4 in the preparation of oligosaccharides by degrading Enteromorpha polysaccharides.

9. A method for degrading Enteromorpha polysaccharide, characterized in that, The method described above is to use the polysaccharide hydrolase described in claim 1 or claim 4 to degrade Enteromorpha polysaccharides.

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