Agarase Mutant with Improved Thermal Stability and Application Thereof

Abstract

Disclosed are an agarase mutant with improved thermal stability and application thereof, belonging to the fields of genetic engineering technology and enzyme engineering. The present disclosure provides an agarase mutant, which is obtained by mutating the amino acid at the 86.sup.th site, the 373.sup.rd site, the 374.sup.th site, the 496.sup.th site, the 507.sup.th site, or the 747.sup.th site of agarase with an amino acid sequence as shown in SEQ ID NO.1. The agarase mutant provided by the present disclosure improves the thermal stability and the hydrolytic activity of the agarase. Compared with the wild type enzyme, the mutant enzyme shows excellent heat resistance and can be industrially used at a relatively high temperature, so that the utilization rate of agar raw materials and the yield of oligosaccharides from agar are improved, and the mutant enzyme has a good industrial application prospect.

Claims

1. An agarase mutant, wherein the agarase mutant comprises mutation of the amino acid at the 86.sup.th site, the 373.sup.rd site, the 374.sup.th site, the 496.sup.th site, the 507.sup.th site, or the 747.sup.th site of agarase, with an amino acid sequence as set forth in SEQ ID NO.1; the agarase mutant is obtained by mutating the alanine at the 86.sup.th site of the agarase with the amino acid sequence as set forth in SEQ ID NO.1 to aspartic acid; or the agarase mutant is obtained by mutating the serine at the 373.sup.rd site of the agarase with the amino acid sequence as set forth in SEQ ID NO.1 to alanine; or the agarase mutant is obtained by mutating the phenylalanine at the 374.sup.th site of the agarase with the amino acid sequence as set forth in SEQ ID NO.1 to tryptophan; or the agarase mutant is obtained by mutating the alanine at the 496.sup.th site of the agarase with the amino acid sequence as set forth in SEQ ID NO.1 to proline; or the agarase mutant is obtained by mutating the valine at the 507.sup.th site of the agarase with the amino acid sequence as set forth in SEQ ID NO.1 to lysine; or the agarase mutant is obtained by mutating the serine at the 747.sup.th site of the agarase with the amino acid sequence as set forth in SEQ ID NO.1 to glutamine.

2. A recombinant cell expressing the agarase mutant according to claim 1, or containing a gene encoding the agarase mutant, or containing a recombinant vector carrying a gene encoding the agarase mutant.

3. The recombinant cell according to claim 2, wherein the recombinant cell takes a prokaryotic cell or a eukaryotic cell as an expression host.

4. A method for preparing neoagarobiose, wherein the neoagarobiose is prepared by hydrolyzing agarose with the agarase mutant according to claim 1.

Description

BRIEF DESCRIPTION OF FIGURES

[0045] FIG. 1: Schematic diagram of plasmid construction.

[0046] FIG. 2: Enzyme activity characterization of a wild enzyme Aga50D and its mutants.

[0047] FIG. 3: Tm characterization of a wild enzyme Aga50D and its mutants.

[0048] FIG. 4: SDS-PAGE images of a wild enzyme Aga50D and its mutants after purification; and in the figure, M represents protein molecular weight standard, 1 represents the purified wild type Aga50D, and 2-15 represent the purified mutants, which are PD1, A86D, V172N, K259P, S286D, S373A, F374W, N400R, A496P, V507K, P677H, S705M, Y706F, and S747Q in sequence.

[0049] FIG. 5: Thermal stability characterization of a wild enzyme Aga50D and its mutants at 50 C.

[0050] FIG. 6: Thermal stability characterization of a wild enzyme Aga50D and its mutants at 60 C.

[0051] FIG. 7: Characterization of optimal reaction temperature for a wild enzyme Aga50D and its mutants.

[0052] FIG. 8: Optimal reaction pH characterization of a wild enzyme Aga50D and its mutants.

[0053] FIG. 9: Characterization of kinetic parameters of a wild enzyme Aga50D and its mutants.

[0054] FIG. 10: Analysis on products of a wild enzyme Aga50D and its mutant at 50 C.

[0055] FIG. 11: Analysis on products of a wild enzyme Aga50D and its mutant at 60 C.

DETAILED DESCRIPTION

[0056] The pET28a-Aga50D and its mutants involved in the following examples were synthesized by Suzhou GENEWIZ Biotechnology Co., Ltd. The main reagents involved in the following examples: a BCA concentration determination kit was purchased from Beyotime Biotechnology Co., Ltd., gene synthesis was completed by GENEWIZ Biotechnology Co., Ltd., and other commonly used reagents were domestic analytically pure reagents.

[0057] The media involved in the following examples are as follows: [0058] LB liquid medium: 1 g of peptone, 0.5 g of a yeast extract and 1 g of NaCl were weighed. The weighed materials were dissolved in deionized water and diluted to 100 mL, and then sterilized at 121 C. for 20 min in an autoclave. [0059] LB solid medium: 1.8% agar powder was added to the LB liquid medium. [0060] LB liquid-resistant medium: kanamycin was added to the LB liquid medium at a final concentration of 50 g.Math.mL.sup.1. [0061] LB solid medium: kanamycin was added to the LB solid medium at a final concentration of 50 g.Math.mL.sup.1.

[0062] The purification method of the enzymes involved in the following examples is as follows: [0063] (1) Equilibration: a nickel column was equilibrated with a buffer solution which contained 50 mM of Tris-HCl and 500 mM of NaCl, and had a pH of 7.5; [0064] (2) Sample loading: a pretreated sample was loaded at a flow rate of 1 mL/min; [0065] (3) Washing: mixed proteins were washed with a buffer solution which contained 50 mM of Tris-HCl, 500 mM of NaCl and 20 mM of imidazole, and had a pH of 7.5; and [0066] (4) Elution: a buffer solution which contained 50 mM of Tris-HCl, 500 mM of NaCl and 300 mM of imidazole, and had a pH of 7.5 was used for elution, and a target protein was collected to obtain the purified enzyme.

[0067] The detection methods involved in the following examples are described below:

[0068] Mutant enzyme activity assay:

[0069] The enzyme activity was determined by using a 3,5-dinitrosalicylic acid method (DNS). Agarase catalyzed the hydrolysis of agarose under certain conditions to generate reducing sugar, and 3,5-dinitrosalicylic acid and the reducing sugar were reduced under thermal conditions so as to form a brownish red amino complex. Within a certain range, the color depth was proportional to the amount of the reducing sugar, which could be measured at a wavelength of 520 nm to calculate the enzyme activity. Definition of enzyme activity unit: the amount of enzyme required to catalyze the production of 1 mol of D-galactose per minute at 30 C. under the condition that pH is 7.0 is defined as one activity unit.

[0070] Enzyme activity assay steps: [0071] (1) Preheating: 2 mL of a 1 mg.Math.mL.sup.1 agarose solution (with pH of 7.0) was taken and placed in a colorimetric tube. [0072] (2) Reaction: 0.1 mL of an enzyme solution was added, and evenly mixed by shaking for reacting for 20 min; and 1.5 mL of DNS was added to terminate the reaction, and the product was boiled in a water bath for 5 min, and then immediately cooled. [0073] (3) Measurement: the absorbance was measured at a wavelength of 520 nm, and the enzyme activity was calculated.

[0074] Determination of Tm Value

[0075] Differential scanning fluoremetry (DSF) was used. The natural protein was in a folded state, with the hydrophobic part hidden inside. As the temperature rose, the protein structure gradually disintegrated, exposing the hydrophobic part. At this time, dyes with affinity for the hydrophobic part bound to the protein, showing an increase in the fluorescence signal intensity of the system; when the temperature reached a certain point, the unfolded protein chains aggregated, and the fluorescent dyes could not bind, and returned to the environment or were subjected to fluorescence quenching at a high temperature, resulting in a decrease in the fluorescence signal intensity; and by tracking and detecting changes in fluorescence signals, the Tm of the protein could be determined. The SYPRO Orange dye was diluted 100 times, and 5 L of the dye was mixed with 20 L of protein and placed in a 96-well thin-walled PCR plate. Then, in an ABI StepOnePlus real-time fluorescence quantitative PCR instrument system, the obtained mixture was heated from 25 C. to 99 C. to monitor the fluorescence change.

[0076] Steps for Determining Thermal Stability of Enzymes at 50 C. or 60 C. [0077] (1) Preheating: 2 mL of an agarose solution (1 mg.Math.mL.sup.1, pH 7.0) was taken and placed in a colorimetric tube, the colorimetric tube was placed in a water bath at 50 C. or 60 C., and heat preservation was carried out for 30 min, 60 min, 90 min, 120 min, 180 min and 6 h, respectively. [0078] (2) Reaction: 0.1 mL of an enzyme solution was added, and evenly mixed by shaking for reacting for 20 min; and 1.5 mL of DNS was added to terminate the reaction, and the product was boiled in a water bath for 5 min, and then immediately cooled. [0079] (3) Measurement: the absorbance was measured at a wavelength of 520 nm, and the enzyme activity was calculated.

[0080] Detection Method for Neoagarobiose (NA2)

[0081] An enzymatically hydrolyzed sample and a neoagaro-oligosaccharide standard (purchased from Qingdao BZ Oligo Biotech Co., Ltd., with a purity greater than 98%, diluted to 1 mg.Math.mL.sup.1) were tested by ion chromatography (ICS-5000, Thermo Fisher Scientific, USA) after passing through a 0.22 m filter membrane. The detection conditions were as follows: chromatographic column: a Dionex CarboPac PA-200 anion exchange column, including an analytical column (4 mm250 mm) and a guard column (4 mm50 mm); a mobile phase: a 100 mmol.Math.L.sup.1 NaOH solution and a 150 mmol.Math.L.sup.1 NaAc solution, with a flow rate being 0.5 mL.Math.min.sup.1; an amperometric detector adopted four-potential pulse amperometry for detection; and the column temperature was 30 C., and the injection volume was 2 L.

[0082] The present disclosure will be described in detail below in conjunction with the accompanying drawings and examples.

EXAMPLE 1

Construction of Mutants

[0083] The specific steps are described below.

[0084] (1) Construction of a Recombinant Vector pET28a-Aga50D Containing a Wild Type Agarase Aga50D

[0085] According to S. degradans 2-40 published by NCBI, the nucleotide sequence encoding a parental enzyme agarase was as shown in SEQ ID NO.2 and sent to GENEWIZ Biotechnology Co., Ltd. for gene synthesis and plasmid recombination. The vector was pET28a, and the recombinant plasmid was named pET28a-Aga50D.

[0086] (2) Construction of Recombinant Vectors Containing Mutants (As Shown in FIG. 1)

[0087] Site-directed mutagenesis primers were designed to perform site-directed mutagenesis by using the recombinant plasmid pET28a-Aga50D obtained in step (1) as a template so as to obtain recombinant plasmids pET28a-A86D, pET28a-V172N, pET28a-K259P, pET28a-S286D, pET28a-S373A, pET28a-F374W, pET28a-N400R, pET28a-A496P, pET28a-V507K, pET28a-P677H, pET28a-S705M, pET28a-Y706F, and pET28a-S747Q which contain mutants A86D, V172N, K259P, S286D, S373A, F374W, N400R, A496P, V507K, P677H, S705M, Y706F, and S747Q, respectively.

[0088] The primer sequences involved are described below.

[0089] The site-directed mutagenesis primers for introducing A86D mutation:

TABLE-US-00001 A86D-F: 5-GGTTAAAGTTGGATATGCAGTC-3; and A86D-R: 5-CTTGGACTGCATATCCAAC-3.

[0090] The site-directed mutagenesis primers for introducing V172N mutation:

TABLE-US-00002 V172N-F: 5-CCCGATAGTGGAGACAACAACG-3; and V172N-R: 5-GGCGAGGTTTAAATCGTTGTTGTC-3.

[0091] The site-directed mutagenesis primers for introducing K259P mutation:

TABLE-US-00003 K259P-F: 5-CCAAAGTTGATTACCCGGGTAAAATC-3; and K259P-R: 5-CTAAACTATGGATTTTACCCGGGTAATC-3.

[0092] The site-directed mutagenesis primers for introducing S286D mutation:

TABLE-US-00004 S286D-F: 5-CAAGCCAATGCCTGATCGCTC-3; and S286D-R: 5-CGCCAAACTTAGAGCGATCAGG-3.

[0093] The site-directed mutagenesis primers for introducing S373A mutation:

TABLE-US-00005 S373A-F: 5-GCAGTGAGCGAAAAAGCTTTTG-3; and S373A-R: 5-GCGCGTAGCAAAAGCTTTTTC-3.

[0094] The site-directed mutagenesis primers for introducing F374W mutation:

TABLE-US-00006 F374W-F: 5-GAGCGAAAAATCATGGGCTAC-3; and F374W-R: 5-GCGCGTAGCCCATGATT-3.

[0095] The site-directed mutagenesis primers for introducing N400R mutation:

TABLE-US-00007 N400R-F: 5-CCCTCTCGCACGCCATTATAAC-3; and N400R-R: 5-CGACGGTAGTTATAATGGCGTGC-3.

[0096] The site-directed mutagenesis primers for introducing A496P mutation:

TABLE-US-00008 A496P-F: 5-GGATTTTTGGGGCCCAATGCC-3; and A496P-R: 5-CGAATACATCTGGCATTGGGCC-3.

[0097] The site-directed mutagenesis primers for introducing V507K mutation:

TABLE-US-00009 V507K-F: 5-CGACCCAGAATTTAAAAAGCGC-3; and V507K-R: 5-CGTTTCCATAGCGCGCTTTTTAA-3.

[0098] The site-directed mutagenesis primers for introducing P677H mutation:

TABLE-US-00010 P677H-F: 5-CTACAAAGAGGGCTTGCACAAGC-3; and P677H-R: 5-GCCCACTTCTGCTTGTGCAAG-3.

[0099] The site-directed mutagenesis primers for introducing S705M mutation:

TABLE-US-00011 S705M-F: 5-GGTGCTATGGATCACGGTATGTATC-3; and S705M-R: 5-CCGGGGTGATACATACCGTG-3.

[0100] The site-directed mutagenesis primers for introducing Y706F mutation:

TABLE-US-00012 Y706F-F: 5-CTATGGATCACGGTTCGTTTCACC-3; and Y706F-R: 5-GTGAATTAAACCGGGGTGAAACGAAC-3.

[0101] The site-directed mutagenesis primers for introducing S747Q mutation:

TABLE-US-00013 S747Q-F: 5-CTGGTTCCAGTATATGGATCAACCATTAAC-3; and S747Q-R: 5-CTCTGCCCGTTAATGGTTGATCC-3.

[0102] The PCR reaction system is as follows:

TABLE-US-00014 TABLE 1 Mutation PCR reaction system Reagent Dosage (L) PrimerSTAR MAX 25 Upstream primer (10 M) 1 Downstream primer (10 M) 1 Template (pET28a-Aga50D) 0.5 ddH.sub.2O 22.5 PCR reaction conditions: pre-denaturation at 95 C. for 3 min, denaturation at 95 C. for 3 min, denaturation at 95 C. for 30 s, annealing at 56 C. for 30 s, and extension at 72 C. for 3 min and 48 s, with a total of 30 cycles.

[0103] A target fragment obtained by gel recovery was transformed into E. coli BL21 (DE3), a kanamycin (50 g/mL) LB plate was coated with a transformant, and the transformant was statically cultured overnight at 37 C. After the bacterial colonies grew out, the single colonies were picked and inoculated into an LB liquid medium containing kanamycin (50 g/mL), and cultured overnight at 37 C. under the condition of 200 rpm; and the bacterial liquid was sent to GENEWIZ Biotechnology Co., Ltd. for determination. The mutant engineered strains containing the correct mutants were respectively obtained:

[0104] E. coli BL21 (DE3)/pET28a-A86D, E. coli BL21 (DE3)/pET28a-V172N, E. coli BL21 (DE3)/pET28a-K259P, E. coli BL21 (DE3)/pET28a-S286D, E. coli BL21 (DE3)/pET28a-S373A, E. coli BL21 (DE3)/pET28a-F374W, E. coli BL21 (DE3)/pET28a-N400R, E. coli BL21 (DE3)/pET28a-A496P, E. coli BL21 (DE3)/-V507K, E. coli BL21 (DE3)/pET28a-P677H, E. coli BL21 (DE3)/pET28a-S705M, E. coli BL21 (DE3)/pET28a-Y706F, and E. coli BL21 (DE3)/pET28a-S747Q; and

[0105] according to the above method, the engineered strain E. coli BL21 (DE3)/pET28a-Aga50D containing the original enzyme was prepared.

[0106] The combination mutant PD1 was obtained by a combination mutation of 20 sites including A86D, V172N, K259P, V274K, S286D, A351P, A355P, S373A, F374W, A386V, N400R, A496P, V507K, S619E, H635K, P677H, H703R, S705M, Y706F and S747Q based on the pET28a-Aga50D; the primer sequence was the same as above, and the method was the same as above; and the engineered strain E. coli BL21 (DE3)/pET28a-A86D/V172N/K259P/V274K/S286D/A351P/A355P/S373A/F374W/A386V/N400R/A496P/V507K/S619E/H635K/P677H/H703R/S705M/Y706F/S747Q was obtained, and was named E. coli BL21 (DE3)/PD1.

EXAMPLE 2

Purification of Enzymes and Determination of Enzymatic Properties

[0107] (1) Production of Enzymes by Shake-Flask Fermentation

[0108] The genetically engineered strains obtained in Example 1 were respectively streaked on the kanamycin (50 g/mL) LB plate, and cultured at 37 C. for 12 h; then, single colonies were picked and inoculated into an LB liquid medium containing kanamycin (50 g/mL) for shake-flask fermentation; and the product was cultured at 37 C. for 12 h under the condition of 200 rpm so as to obtain seed liquid.

[0109] 1 mL of the seed liquid was absorbed and transferred to an LB liquid medium containing 100 mL of kanamycin (50 g/mL) for shake-flask fermentation, and then was cultured at 37 C. until OD.sub.600 reached 0.6-0.8; and IPTG with a final concentration of 0.5 mM was added, the mixture was cooled to 16 C., enzyme was then induced, the product was centrifuged after being cultured for 12 h, and bacterial cells were collected.

[0110] (2) Purification of Enzymes

[0111] An appropriate amount (2-3 times the volume) of a lysis buffer solution (containing 50 mmol/L Tris HCl and 100 mmol/L NaCl, with a pH of 7.5) was added into the centrifugally collected bacterial cells for resuspending the bacterial cells. The product was subjected to ultrasonication on ice for 15-20 min at a power of 30%; then, the ultrasonication was performed for 2 s, and stopped for 3 s; after the ultrasonication was finished, the product was centrifuged at 4 C. for 10 min under the condition of 8000 rpm to collect supernatant, and the supernatant was filtered through a 0.22 m filter membrane. Ni.sup.2+ column affinity chromatography purification was performed to obtain mutant enzymes. After enzyme activity assay and SDS-PAGE electrophoresis detection, the results are as shown in FIG. 4.

[0112] Pure enzyme solutions containing wild type agarase and its mutant enzymes A86D, V172N, K259P, S286D, S373A, F374W, N400R, A496P, V507K, P677H, S705M, Y706F, S747Q, and PD1 were prepared, respectively.

[0113] (3) Enzyme Activity Assay

[0114] The enzyme activities of the above wild type agarase and its mutant enzymes A86D, V172N, K259P, S286D, S373A, F374W, N400R, A496P, V507K, P677H, S705M, Y706F, S747Q and PD1 were determined. The enzyme activity of the wild type agarase was defined as 100%, and the relative enzyme activities of the mutant enzymes was detected. The results are as shown in Table 2 and FIG. 2.

TABLE-US-00015 TABLE 2 Relative enzyme activities of a wild type enzyme and its mutants Relative enzyme Enzyme Tm Tm activity (%) WT 41.974 100 PD1 48.802 6.828 129.1 9.9 A86D 65.176 23.202 132.6 2.5 V172N 42.768 0.793 84.6 4.1 K259P 42.487 0.513 66.3 1.8 S286D 43.196 1.221 92.4 9.8 S373A 41.987 0.013 160.7 6.3 F374W 42.677 0.703 137.3 10.6 N400R 43.202 1.227 61.9 9.7 A496P 42.210 0.236 115.5 1.1 V507K 42.615 0.641 107.0 3.8 P677H 44.043 2.069 86.0 7.4 S705M 44.137 2.163 23.8 7.0 Y706F 43.638 1.664 55.4 10.4 S747Q 47.651 5.677 141.1 4.5

[0115] The results show that the Tm values of the wild type agarase and its mutant enzymes PD1, A86D, V172N, K259P, S286D, S373A, F374W, N400R, A496P, V507K, P677H, S705M, Y706F, and S747Q are determined by differential scanning fluoremetry, with PD1 (Tm=6.828 C.), S747Q (Tm=5.677 C.), S705M (Tm=2.163 C.), and P677H (Tm=2.069 C.) being significant in effect. It is worth noting that the thermal melting temperature (Tm=65.176 C., Tm=23.202 C.) of A86D is much higher than that of the wild type agarase (Tm=41.974 C.), so that the heat resistance is greatly improved, as shown in Table 2 and FIG. 3.

[0116] Although the enzyme activities of S373A, F374W and A496P have been improved, the increase of their thermal melting temperature is not significant.

[0117] Based on the relative enzyme activities, the mutants PD1, A86D, and S747Q with superior overall performance were selected for subsequent experiments.

[0118] (4) Enzyme Thermal Stability at 50 C.

[0119] The results of an enzyme thermal stability experiment at 50 C. are as shown in FIG. 5.

[0120] The results showed that the heat resistance of the mutant A86D was significantly enhanced.

[0121] At 50 C., the experimental results showed that the wild enzyme Aga50D only retained 13.65% of the original enzyme activity after being subjected to incubation for 30 min, only retained 10.79% of the original enzyme activity after being subjected to incubation for 60 min, and almost lost all enzyme activity after being subjected to incubation for 180 min.

[0122] At 50 C., the mutant S747Q retained 72.17% of the original enzyme activity after being subjected to incubation for 15 min, retained 23.71% of the original enzyme activity after being subjected to incubation for 30 min, and was completely inactivated after being subjected to incubation for 180 min.

[0123] At 50 C., the mutant A86D retained 81.39% of the original enzyme activity after being subjected to incubation for 30 min, retained 63.68% of the original enzyme activity after being subjected to incubation for 60 min, and still retained 42.15% of the original enzyme activity after being subjected to incubation for 6 h.

[0124] At 50 C., the mutant PD1 retained 95.51% of the original enzyme activity after being subjected to incubation for 30 min, retained 83.37% of the original enzyme activity after being subjected to incubation for 60 min, and still retained 55.28% of the original enzyme activity after being subjected to incubation for 6 h.

[0125] Therefore, a heat resistance test at 60 C. was additionally performed to further explore the superior heat resistance performance of PD1 and A86D.

[0126] (5) Enzyme Thermal Stability at 60 C.

[0127] The results of an enzyme thermal stability experiment at 60 C. are as shown in FIG. 6.

[0128] The results showed that the heat resistance of the mutant A86D was significantly enhanced.

[0129] At 60 C., the experimental results showed that the wild enzyme Aga50D had poor heat resistance, and was completely inactivated after being subjected to incubation at 60 C. for 30 min.

[0130] At 60 C., the mutant S747Q retained 12.26% of the original enzyme activity after being subjected to incubation for 30 min, only retained 0.94% of the original enzyme activity after being subjected to incubation for 60 min, and was completely inactivated after being subjected to incubation for 90 min.

[0131] At 60 C., the mutant PD1 retained 8.50% of the original enzyme activity after being subjected to incubation for 30 min, only retained 4.50% of the original enzyme activity after being subjected to incubation for 60 min, and was completely inactivated after being subjected to incubation for 90 min.

[0132] At 60 C., the mutant A86D retained 98.75% of the original enzyme activity after being subjected to incubation for 30 min, retained 82.46% of the original enzyme activity after being subjected to incubation for 60 min, retained 72.18% of the original enzyme activity after being subjected to incubation for 180 min, and still retained 30.83% of the original enzyme activity after being subjected to incubation for 6 h, thus showing extremely superior heat resistance and having excellent industrial application potential.

EXAMPLE 3

Determination of Enzymatic Properties

[0133] (1) Optimal Reaction Temperature

[0134] In order to further explore the characteristics of mutant enzymes, the enzymatic properties of PD1, A86D and S747Q were further studied. A 0.1% agarose substrate was prepared by using a buffer solution which contained Tris-HCl and had a pH of 7, and an enzyme solution with a final concentration of 0.2 mg.Math.mL.sup.1 was added to react for 20 min at different temperatures so as to measure the enzyme activity. It can be seen from FIG. 7 that the optimal reaction temperature for both a wild enzyme and its mutants is 30 C.

[0135] (2) Optimal Reaction pH

[0136] A 1 mg.Math.mL.sup.1 agarose substrate having a pH within a range of 5-6 was prepared by using a buffer solution containing 50 mM of citric acid, a 0.1% agarose substrate having a pH within a range of 7-8 was prepared by using a buffer solution containing 50 mM of Tris-HCl, and a 0.1% agarose substrate having a pH within a range of 9-10 was prepared by using a glycine-NaOH buffer solution. An enzyme solution with a final concentration of 0.2 mg.Math.mL.sup.1 was added to react for 20 min at different temperatures so as to measure the enzyme activity. It can be seen from FIG. 8 that the optimal reaction pH for both a wild enzyme and its mutants is 7, and their properties are similar.

[0137] (3) Kinetic Parameters

[0138] Agarose substrates with concentrations of 1 mg.Math.mL.sup.1, 2 mg.Math.mL.sup.1, 3 mg.Math.mL.sup.1, 4 mg.Math.mL.sup.1, 5 mg.Math.mL.sup.1, 8 mg.Math.mL.sup.1 and 10 mg.Math.mL.sup.1 were respectively prepared by using a buffer solution which contained Tris-HCl and had a pH of 7. An enzyme solution with a final concentration of 0.2 mg.Math.mL.sup.1 was added to react under the optimal reaction conditions, and then the amount of reducing sugar produced was determined and plotted according to a Lineweaver-Burk method (FIG. 9). The linear fitting equations for the 4 curves in the figure are =589.92x+15.37 (WT), =621.46x+31.568 (PD1), =506.45x+22.215 (A86D), and =436.23x+58.247 (S747Q), respectively. Thus, various kinetic parameters of a wild enzyme and its mutants can be deduced (Table 3).

[0139] The kinetic parameters of a wild enzyme and its mutants under optimal reaction conditions are listed in Table 3.

TABLE-US-00016 TABLE 3 Kinetic parameters of wild type Aga50D and its mutants Kcat/Km Km (mg/ml) (mL/mg.Math.s) WT 38.381 0.139 PD1 19.686 0.154 A86D 22.794 0.162 S747Q 7.489 0.181

[0140] It can be seen from the data in the table that the Km values of mutants are lower than that of WT, indicating that the binding ability of the mutants to an agarose substrate is significantly increased, which is consistent with the previous conclusion about the relative enzyme activity. The Kcat/Km values of the mutants are greater than that of the WT, indicating that the catalytic ability of the mutants is improved.

EXAMPLE 4

Application of Enzymes

[0141] After a 1 mg.Math.mL.sup.1 agarose substrate solution was prepared by using ultrapure water, the mutant and wild type pure enzyme solutions (0.2 mg.Math.mL.sup.1 enzyme solution (9.528 U.Math.mg.sup.1)) prepared in step (2) of Example 2 were respectively added to react in a water bath shaker at 50 C., with a rotational speed of 200 rpm and a reaction time of 1-3 h. After the reaction was completed, a boiling water bath was used to terminate the reaction.

[0142] Part of the reaction solution was taken out every 1 h, 2 h and 3 h, unreacted polysaccharides in the reaction solution was removed by centrifugation, and supernatant was taken to obtain enzymatic hydrolysate samples: neoagarobiose (NA2). The contents of the neoagarobiose (NA2) in the above-obtained products were detected, respectively.

[0143] The results show (as shown in FIG. 10) that at 50 C., neoagarobiose produced by hydrolyzing agarose with a wild type enzyme is low in yield, which is presumed to be due to heat instability, and rapid heat inactivation; while mutant enzymes PD1, A86D, and S747Q have certain heat resistance, and can still play a catalytic role under high temperature conditions; and an agarose substrate is still in a solution state at 50 C., which promotes the binding of the substrate to the enzyme, thus further improving the yield of the neoagarobiose.

[0144] The hydrolysis reaction lasted for 3 h, and the yield of the product neoagarobiose (NA2) obtained by hydrolyzing agarose with the wild type enzyme was 278.678 mg/mL (859.3 M);

[0145] the yield of the product neoagarobiose (NA2) obtained by hydrolyzing agarose with the mutant enzyme PD1 was 2036.281 mg/mL (6279.3 M);

[0146] the yield of the product neoagarobiose (NA2) obtained by hydrolyzing agarose with the mutant enzyme A86D was 1670.887 mg/mL (5152.6 M); and

[0147] the yield of the product neoagarobiose (NA2) obtained by hydrolyzing agarose with the mutant enzyme S747Q was 1965.719 mg/mL (6061.7 M).

[0148] Under the condition of 50 C., the mutant enzymes show excellent hydrolysis efficiency due to its superior heat resistance, thus greatly improving the yield of neoagarobiose, and having good industrial application value.

EXAMPLE 5

Application of Enzymes

[0149] After a 1 mg.Math.mL.sup.1 agarose substrate solution was prepared by using ultrapure water, the mutant and wild type pure enzyme solutions (0.2 mg.Math.mL.sup.1 enzyme solution (9.528 U.Math.mg.sup.1)) prepared in step (2) of Example 2 were respectively added to react in a water bath shaker at 60 C., with a rotational speed of 200 rpm and a reaction time of 1-3 h. After the reaction was completed, a boiling water bath was used to terminate the reaction.

[0150] Part of the reaction solution was taken out every 1 h, 2 h and 3 h, unreacted polysaccharides in the reaction solution was removed by centrifugation, and supernatant was taken to obtain enzymatic hydrolysate samples: neoagarobiose (NA2). The contents of the neoagarobiose (NA2) in the above-obtained products were detected, respectively.

[0151] The results show (as shown in FIG. 11) that under the condition of 60 C., due to the heat instability and rapid inactivation of the wild type enzyme, no product neoagarobiose (NA2) is detected; and the mutants PD1 and S747Q fail to withstand the high temperature of 60 C., resulting in a large loss of enzyme activity. However, A86D can withstand a high temperature of 60 C., and still retains better enzyme activity at 60 C.; and an agarose substrate is still in a solution state at 60 C., which promotes the binding of the substrate to the enzyme, thus further improving the yield of the neoagarobiose.

[0152] The hydrolysis reaction lasted for 3 h, and the yield of the product neoagarobiose (NA2) obtained by hydrolyzing agarose with the wild type enzyme was 0 M;

[0153] the yield of the product neoagarobiose (NA2) obtained by hydrolyzing agarose with the mutant enzyme PD1 was 147.6 M;

[0154] the yield of the product neoagarobiose (NA2) obtained by hydrolyzing agarose with the mutant enzyme S747Q was 254.9 M; and

[0155] the yield of the product neoagarobiose (NA2) obtained by hydrolyzing agarose with the mutant enzyme A86D was 5513.6 M.

[0156] Under the condition of 60 C., the mutant enzyme A86D shows excellent hydrolysis efficiency due to its superior heat resistance, thus greatly improving the yield of neoagarobiose, and having good industrial application value.

[0157] Although the present disclosure has been disclosed as above in exemplary embodiments, it is not intended to limit the present disclosure. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present disclosure. Therefore, the protection scope of the present disclosure shall be as defined in the Claims.