Low-temperature alginate lyase and application thereof

Abstract

The invention provides low-temperature alginate lyase and application thereof, namely alginate lyase with low-temperature catalytic activity, which is separated from marine-derived cold monad bacteria, and the amino acid sequence of the alginate lyase is SEQ ID NO: 1; the amino acid sequence of the alginate lyase after the signal peptide is removed is SEQ ID NO: 3. The algin lyase Alg3889 provided by the invention has the optimal reaction temperature of about 10-16 DEG C, has good temperature stability, and still keeps more than 70% of enzyme activity after being incubated at 0-70 DEG C for 1 hour; the optimum pH value of the strain is 8.0, after the strain is incubated in a buffer system with the pH value of 3.0-10.6 for 12 h, about 90% of the highest enzyme activity can be kept, and the strain has high acid resistance and alkali resistance. The degradation product of the alginate oligosaccharide is alginate oligosaccharide with the polymerization degree of 2-6, and the alginate oligosaccharide has high application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of protease, and particularly relates to a low-temperature alginate lyase and application thereof. BACKGROUND

[0002] Alginate is an important polysaccharide in the ocean, and widely exists in the cell wall of kelp, giant kelp and other brown algae. The content of alginate can reach 30% to 60% of the dry weight of brown algae. Alginate is a linear polysaccharide composed of β-D-mannuronate (M) and α-L-guluronate (G). According to the arrangement of the two monomers, alginate can be divided into three forms: polyM composed of M monomers connected by β-1, 4 glycosidic bonds, polyG composed of G monomers connected by α-1, 4 glycosidic bonds, and polyMG composed of two monomers arranged randomly. Alginate oligosaccharide is a product obtained by biological enzymolysis of alginate lyase or by physical and chemical methods. Alginate oligosaccharide is widely used in various fields such as biomedical science, feed additives and food industry due to its low molecular weight, good biocompatibility and high water solubility. Alginate lyase belongs to the polysaccharide lyase family, and breaks 1, 4-O-glycosidic bonds by β-elimination to realize the biological enzymolysis of alginate. Alginate lyase has important economic and scientific research value in alginate structure research, alginate oligosaccharide preparation, protoplast preparation, biofuel preparation and drug development, and has attracted widespread attention from researchers. At present, most of the reported alginate lyases have low activity at low temperature, and must be heated to a certain temperature (40-50℃) to exert maximum effect, which greatly increases the energy consumption and production cost in the process of industrial application. Therefore, it is of great significance to find alginate lyase with catalytic activity at low temperature and to study its degradation conditions. SUMMARY

[0003] The application aims to provide a low-temperature alginate lyase and application thereof, i.e. an alginate lyase Alg3889 with low-temperature catalytic activity isolated from a psychrophilic bacterium of the genus psychrobacter from a marine source, so as to make up for the shortcomings of the prior art.

[0004] The alginate lyase provided by the application has an amino acid sequence of SEQ ID NO: 1. MYLNLTKTKISLCVSIALTPLLSPLAYSATFNMQKNGTEYSVDGNNGEIEGQQIYLWSTDTSNENQNWVQLDRDGYYSYKKEGTSLCWDGGDDGANGQAVTLETCDEDDYNQQWKKVKITSGTEIYRFEKRNDSDYSLDGDNGGENGQSMYLWESDSDNENQQWELTNLDDSSSTTTSSTDYDLDSSVAPSSNFDLTDWYLSVPTDTDDDGYADSIKEAELNDDYESDYFYTGSDGAMVFMCTVGGYKTSANTTYTRTELREMLRAGDTSIDTDDKENNWAFSSIDSDDQSDFGGIDGTLTATVAVNHVTTTSTDDEQRGRIIIGQIHAENNEPLRLYYHKYEGNTYGSIYFAHEASKDSGGEEKWYNLLGTMIDDDNDGYVDEDDGLTDDPSDGIALDEKFTYVVDVDGDDLTVTISDEDGNTLASKTVDMSDSGYDDSDNYMYFKAGLYLNDKTSDGDDYSTVSFYKLTNTHD; the nucleotide sequence of the coding gene is SEQ ID NO: 2; The brown algin lyase has an amino acid sequence of SEQ ID NO: 3 after removing the signal peptide. MATFNMQKNGTEYSVDGNNGEIEGQQIYLWSTDTSNENQNWVQLDRDGYYSYKKEGTSLCWDGGDDGANGQAVTLETCDEDDYNQQWKKVKITSGTEIYRFEKRNDSDYSLDGDNGGENGQSMYLWESDSDNENQQWELTNLDDSSSTTTSSTDYDLDSSVAPSSNFDLTDWYLSVPTDTDDDGYADSIKEAELNDDYESDYFYTGSDGAMVFMCTVGGYKTSANTTYTRTELREMLRAGDTSIDTDDKENNWAFSSIDSDDQSDFGGIDGTLTATVAVNHVTTTSTDDEQRGRIIIGQIHAENNEPLRLYYHKYEGNTYGSIYFAHEASKDSGGEEKWYNLLGTMIDDDNDGYVDEDDGLTDDPSDGIALDEKFTYVVDVDGDDLTVTISDEDGNTLASKTVDMSDSGYDDSDNYMYFKAGLYLNDKTSDGDDYSTVSFYKLTNTHD; Correspondingly, the nucleotide sequence of the brown algin lyase encoding the amino acid sequence of SEQ ID NO: 3 is SEQ ID NO: 4.

[0005]

[0006] The present application also provides a recombinant expression plasmid, wherein a nucleotide fragment for encoding the alginate lyase Alg3889 without signal peptide, i.e., SEQ ID NO: 4 without stop codon taa, is inserted into the recombinant expression plasmid pET-24a(+). alg3889 ; The present application also provides a recombinant expression strain, wherein the recombinant expression plasmid pET-24a(+) alg3889 is carried by the recombinant expression strain. E. coli BL21(DE3) / pET-24a(+) alg3889 ; The present application also provides a method for preparing alginate lyase Alg3889, which is prepared by fermenting the recombinant expression strain E. coli BL21(DE3) / pET-24a(+) alg3889 , and purifying the fermentation product.

[0007] The present application also provides an application of the alginate lyase, which is used in degrading alginate. The present application also provides a method for preparing alginate oligosaccharide, which is prepared by using the alginate lyase.

[0008] The alginate lyase Alg3889 provided by the present application has an optimal reaction temperature of about 10-16℃, and has good temperature stability, and can maintain more than 70% of enzyme activity after incubation at 0-70℃ for 1 h; the optimal pH thereof is 8.0, and after incubation in a buffer system with pH of 3.0-10.6 for 12 h, it can maintain about 90% of the highest enzyme activity, and has strong acid and alkali resistance; and the degradation product thereof is alginate oligosaccharide with a polymerization degree of 2-6, and has high application value. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 : Hydrolysis circle of original strain C. psychrerythraea ZH108 on alginate plate; wherein, in the figure: a is a hydrolysis circle at 16℃, and b is a hydrolysis circle at 4℃; Figure 2 : Agarose gel electrophoresis diagram of PCR amplification product of alginate lyase gene alg3889 under different annealing temperatures; Figure 3: Agarose gel electrophoresis map of double enzyme digestion verification of recombinant expression plasmid in recombinant expression strain, wherein lane 1: double enzyme digestion recombinant expression plasmid, lane 2: recombinant expression plasmid, lane 3: double enzyme digestion plasmid pET-24a(+), lane 4: plasmid pET-24a(+), lane M: DNA Marker; Figure 4 : Purity detection map of alginate lyase Alg3889, wherein lane 1: E. coli BL21(DE3) / pET-24a(+) / alg3889 : Lane 1-8: 10 mM, 20 mM, 50 mM, 75 mM, 100 mM, 250 mM and 500 mM imidazole elution protein effluent, lane M: 15-130 kDa protein Marker; Figure 5 : Optimum reaction temperature and temperature stability map of alginate lyase Alg3889; wherein, in the figure: a is optimum reaction temperature, b is temperature stability; Figure 6 : Optimum pH and pH stability map of alginate lyase Alg3889; wherein, in the figure: a is optimum pH, b is pH stability; Figure 7 : Substrate preference analysis map of alginate lyase Alg3889; Figure 8 : Degradation product analysis map of alginate lyase Alg3889. DETAILED DESCRIPTION

[0010] The strain of psychrobacter ZH108 used in the present application is isolated from a rotten seaweed sample collected at NCJW03 station (11°56'14.1191"E, 78°55'39.8765"N). As shown in FIG. 1, it has strong alginate lyase activity at low temperature (16℃, 4℃) through alginate enzyme plate detection, and then the potential alginate lyase gene in the above strain is screened and recombinantly expressed, and the obtained recombinant protein Alg3889 has obvious alginate lyase activity. Figure 1 alg3889

[0011] The present application will be described in detail below in combination with examples and drawings.

[0012] Example 1: Construction of recombinant expression strain E. coli BL21(DE3) / pET-24a(+) / alg3889 I. Amplification of alginate lyase gene alg3889 ​​​​By sequencing analysis and RAST annotation of the whole genome of the bacteria ZH108 (preserved in China Ocean Microbial Culture Collection Center, preservation number MCCO181-A5) of the genus Psychrobacillus, 5 potential alginate lyase genes and their sequences were obtained, and the gene encoding alginate lyase was obtained alg3889 The nucleotide sequence of the gene is SEQ ID NO: 2 (1428 bp), the amino acid sequence of the translated protein is SEQ ID NO: 1 (475 aa), the amino acid sequence after removing the signal peptide is SEQ ID NO: 3, and the nucleotide sequence of the corresponding coding gene is SEQ ID NO: 4.

[0013] According to the gene sequence and the expression vector pET-24a(+), appropriate enzyme cutting sites were added to the upstream and downstream of the target gene, and the enzyme cutting sites were Eco R I, Xho I, and SnapGene software was used to design primers.

[0014] The primer sequences are as follows: alg3889 -F: 5'-CCGGAATTCATGGCTACTTTTAATATGC-3' alg3889 -R: 5'-CCGCTCGAGATCATGGGTATTGGTTAG-3' The genomic DNA of the strain ZH108 was used as a template to amplify the alginate lyase gene at different annealing temperatures alg3889 The PCR amplification product was subjected to agarose gel electrophoresis, as shown in Figure 2 The optimal annealing temperature of the alginate lyase gene alg3889 was 50.0℃; the reaction system was scaled up to 200 μL, the PCR amplification product was subjected to agarose gel electrophoresis detection, and whether it met the length of the known alginate lyase gene alg3889 was verified, and the high-quality target gene cloning fragment was recovered and purified. The PCR reaction system is shown in Table 1, and the PCR reaction program is shown in Table 2.

[0015] Table 1: PCR reaction system table Reaction system Volume (µL) PrimeSTAR GXL DNA Polymerase 1 5×PrimeSTAR GXL Buffer 10 dNTP Mixture (2.5 mM) 4 -F (10 µM) 1 -R (10 µM) 1 Genomic DNA 2.5 Double distilled water 30.5 Total system 50 Table 2: PCR reaction program table

[0016] II. Construction of recombinant cloning strain A poly-A tail needs to be added to the 3' end of the gel recovery product to realize the base complementary pairing of the pUCm-T vector and the target gene, and the recombinant cloning plasmid pUCm-T alg3889The reaction systems with added poly-A are shown in Table 3, and the connection reaction systems are shown in Table 4.

[0017] Table 3: Reaction System with Poly-A Reaction system Volume (µL) Gel recovery product 14.5 10×PCR Buffer 2 rTaq enzyme 0.5 dNTP Mixture 3 Total system 20 Table 4: Connection Reaction System Table Ligation system Volume (µL) Target gene with poly-A tail 4 pUCm-T vector 1 Solution I 5 Total system 10 The recombinant cloning plasmid was transformed into the cloning host using the heat shock method. E. coli JM109 was then plated onto LB solid medium (containing 100 μg / mL Amp, 20 mg / mL X-gal, and 24 mg / mL IPTG) and incubated overnight at 37°C. A single white colony was picked and inoculated into 1 mL of LB liquid medium (containing 100 μg / mL Amp) and incubated at 37°C and 170 rpm for 2 h. Once the bacterial culture became turbid, PCR was performed to verify whether the recombinant cloning plasmid had been successfully transferred into the cloning host. E. coli If the band size of JM109 competent cells is the same as that of the target gene, 200 µL of the corresponding bacterial culture is aspirated for sequencing to ensure that no mismatch occurs during the amplification process; the returned sequencing results are then compared with those of the alginate lyase gene. alg3889 Sequence alignment was performed, and recombinant clones with completely correct sequencing were preserved and stored at -80℃ for later use. The bacterial culture PCR reaction system is shown in Table 5, and the bacterial culture PCR reaction procedure is shown in Table 6.

[0018] Table 5: Bacterial PCR Reaction System Reaction system Volume (µL) Premix Taq 5 Bacterial solution 1 -F (10 µM) 0.8 -R (10 µM) 0.8 Double distilled water 2.4 Total system 10 Table 6: PCR reaction procedure for bacterial culture

[0019] III. Construction of Recombinant Expression Strains Recombinant clone strains and E. coli BL21(DE3) / pET-24a(+) were inoculated onto LB solid medium (containing 100 µg / mL Amp and 50 µg / mL Kana, respectively) for activation and incubated at 37°C inverted for 24 h. Single colonies were then transferred to 5 mL LB liquid medium (containing 100 µg / mL Amp and 50 µg / mL Kana, respectively) and cultured at 37°C with shaking at 170 rpm for 12–16 h. Plasmids were then extracted. Eco RI XhoThe recombinant cloning plasmid and expression vector were digested with enzyme I to generate complementary sticky ends. The reaction temperature was 37℃, and the reaction time was 1-2 h. The enzyme digestion products were detected by agarose gel electrophoresis. The target gene and linear expression vector with the same sticky ends were recovered using a kit and ligated at a molar ratio of 10:1. The double enzyme digestion reaction system is shown in Table 7.

[0020] Table 7: Double Enzyme Digestion Reaction System Enzyme digestion system Volume (µL) Plasmid 8 Double distilled water 8 10×fast Buffer 2 R I 1 I 1 Total system 20 The recombinant expression plasmid after ligation was transformed into [the target cell] using the heat shock method. E. coli JM109 competent cells were used, and the strains were validated by culture PCR. The validated strains were preserved and cryopreserved at -80℃ for later use. The successfully constructed recombinant expression plasmid was extracted and transformed into the expression host cell using a heat shock method. E. coli BL21(DE3) was used to construct a recombinant expression strain, and its correctness was verified by bacterial culture PCR and double enzyme digestion. The verified recombinant expression strain was preserved and stored at -80℃ for later use. Figure 3 As shown, a recombinant expression strain was successfully constructed. E. coli BL21(DE3) / pET-24a(+) / alg3889 .

[0021] Example 2: Induction and purification of alginate lyase Alg3889 recombinant expression strain E. coli BL21(DE3) / pET-24a(+) / alg3889 The culture was activated by inoculation onto LB solid medium (containing Kana at a final concentration of 50 µg / mL) and cultured at 37°C for 24 h, while simultaneously inoculating the bacterial strain. E. coli BL21(DE3) / pET-24a(+) was used as a negative control, and all other treatments were consistent with those of the recombinant expression strain. Single colonies were picked and cultured in 5 ml of LB liquid medium (containing a final concentration of 50 µg / mL Kana) at 37°C with shaking at 170 rpm to prepare a seed culture. The seed culture was then inoculated at a rate of 1% into 300 ml of LB liquid medium (containing a final concentration of 50 µg / mL Kana) and cultured at 37°C with shaking at 170 rpm for 1–2 h to allow OD to develop. 600 The concentration should reach between 0.4 and 0.6; add IPTG (final concentration 0.1 mM) to the bacterial culture, and incubate at 16°C and 150 rpm with shaking for 12 to 16 h.

[0022] The bacteria were collected at low temperature (4°C, 6000 rpm, centrifugation for 10 min), and part of the supernatant was reserved for activity test; the bacteria were resuspended by adding Binding Buffer to the bacteria, and the bacteria were concentrated 10-20 times and mixed by blowing; the bacteria were ultrasonically broken, the breaking time was 5 s, the interval time was 10 s, the breaking was performed 200-300 times, and the whole process was operated on ice; after the breaking was completed, the supernatant (i.e., crude enzyme solution) was collected by centrifugation at 4°C, 12000 rpm for 15 min; the broken precipitate was resuspended with Binding Buffer and reserved for subsequent activity test.

[0023] After the supernatant was filtered by using a 0.22 µm filter, the supernatant was added to a nickel ion affinity chromatography column, and the column was loaded 3 times, and then different concentrations of imidazole (10 mM, 20 mM, 50 mM, 75 mM, 100 mM, 250 mM, 500 mM) were sequentially added to elute the nickel column, and the effluent was collected for SDS-PAGE electrophoresis for purity detection, as shown in Figure 4 The effluent eluted by 20 mM imidazole was loaded into a dialysis bag, placed in a beaker, and dialyzed by adding 4°C pre-cooled dialysis solution, and the whole process was performed at 4°C, and finally the pure enzyme was obtained.

[0024] Example 3: Enzymatic property research of alginate lyase Alg3889 I. Protein content determination The protein content of the recombinant alginate lyase Alg3889 was determined by the Coomassie brilliant blue method, and was 0.274 mg / ml.

[0025] II. Optimum reaction temperature and temperature stability determination Optimum reaction temperature: the substrate was incubated at different temperatures for 5 min, and 3 parallel tests were set for each temperature. 20 µL alginate lyase Alg3889 was added to 180 µL 0.3% sodium alginate substrate (dissolved in a 20 mM Tris-HCl buffer with a pH of 8.0 containing 500 mM NaCl), and the system was reacted at 0°C, 4°C, 10°C, 16°C, 28°C, 37°C, 45°C, 50°C, 60°C, 70°C, 80°C, 90°C and 100°C for 10 min, respectively; after the reaction was completed, the DNS method was used for detection (note: unless otherwise specified, the DNS method in the present application means that 200 µL DNS solution was added to the reaction system after the enzyme catalytic reaction was completed, mixed, color developed in a boiling water bath for 5 min, and the absorbance value of the reaction system at a wavelength of 540 nm was determined). As shown in Figure 5As shown in FIG. 2a, alginate lyase Alg3889 has high enzyme activity in the range of 4-45℃, and can maintain more than 90% of the highest enzyme activity, and the optimum reaction temperature is about 10-16℃, and has high enzyme activity at low temperature.

[0026] Temperature stability: a certain amount of alginate lyase Alg3889 was incubated at 0℃, 4℃, 10℃, 16℃, 28℃, 37℃, 45℃, 50℃, 60℃, 70℃, 80℃, and 90℃ for 1 h; 20 μL of enzyme solution incubated at different temperatures was taken and added to 180 μL of reaction system, and reacted for 10 min under the optimum conditions; after the reaction, the DNS method was used for detection, and the highest remaining enzyme activity was taken as 100%, so as to determine the stability of alginate lyase at different temperatures. As shown in FIG. 2b, alginate lyase Alg3889 has good temperature stability, and still maintains more than 70% of enzyme activity after incubation at 0-70℃ for 1 h, maintains about 30% of enzyme activity after incubation at 80℃ for 1 h, and basically loses enzyme activity after incubation at 90℃ for 1 h. Figure 5

[0027] III. Optimum reaction pH and pH stability determination Optimum reaction pH: the substrate was incubated at the optimum reaction temperature for 5 min, 20 μL of alginate lyase Alg3889 was added to 180 μL of substrate containing 0.3% (w / v) sodium alginate and 500 mM NaCl prepared in different pH buffer systems, and reacted for 10 min, and then the DNS method was used for detection; 3 parallel experiments were set for each pH buffer system, and different pH buffer systems are shown in Table 8. As shown in FIG. 3a, the optimum reaction pH of alginate lyase Alg3889 is 8.0. Figure 6

[0028] Table 8: Different pH buffer system table pH Buffer 3.0、4.0、5.0、6.0、7.0、7.2、7.8、8.0 50 mM Na2HPO4-citric acid buffer 8.0、8.2、8.6 50 mM Tris-HCl buffer 8.6、9.0、10.0、10.6 50 mM Glycine-NaOH buffer pH stability: a certain amount of alginate lyase Alg3889 was incubated at 4℃ for 12 h using different pH buffer systems (pH 3.0, 4.0, 5.0, 6.0, 7.0, 7.2, 7.8, 8.0, 8.2, 8.6, 9.0, 10.0, 10.6), and 20 μL of enzyme solution treated from the above systems was added to 180 μL of 0.3% sodium alginate substrate, mixed, and reacted for 10 min under the optimum conditions; after the reaction, the DNS method was used for detection, and the highest remaining enzyme activity was taken as 100%, so as to determine the stability of alginate lyase Alg3889 at different pH values. As shown in FIG. 2b, alginate lyase Alg3889 has good temperature stability, and still maintains more than 70% of enzyme activity after incubation at 0-70℃ for 1 h, maintains about 30% of enzyme activity after incubation at 80℃ for 1 h, and basically loses enzyme activity after incubation at 90℃ for 1 h. Figure 6 ​​As shown in FIG. 3, alginate lyase Alg3889 can maintain about 90% of the highest enzyme activity after incubation in a buffer system with pH ranging from 3.0 to 10.6 for 12 h, and has strong acid and alkali resistance.

[0029] IV. Substrate preference determination Three substrates (0.3% (w / v) sodium alginate, 0.3% (w / v) polyG, and 0.3% (w / v) polyM) were incubated at the optimal reaction temperature for 5 min, and then 20 μL alginate lyase Alg3889 was added to 180 μL of different substrates, and reacted for 0 min, 10 min, 30 min, 60 min, 150 min, and 180 min, respectively. After the reaction, the DNS method was used for detection, and the increase in absorbance at 540 nm was determined to obtain the corresponding degradation curve. Each reaction time was set in triplicate. As shown in FIG. 4, the substrate preference of alginate lyase Alg3889 was sodium alginate > polyG > polyM. Figure 7

[0030] V. Degradation product analysis 0.3% (w / v) sodium alginate (dissolved in a pH 8.0 Tris-HCl buffer containing 500 mM NaCl) was incubated at the optimal reaction temperature for 5 min, and then 100 μL alginate lyase was added to 900 μL of the substrate. After reaction at the optimal reaction temperature for 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, and 18 h, respectively, 125 μL of each sample was taken into a 2 mL EP tube, boiled to inactivate the alginate lyase, centrifuged at 12000 rpm for 10 min, and the supernatant was used as the subsequent chromatography sample and stored at -20°C.

[0031] ​The enzymatic product of each reaction time was spotted on a thin layer chromatography (TLC) plate in turn for detection of the enzymatic product, and the specific operation steps were as follows: the TLC plate was placed in an oven for drying, cut into a rectangle with a width of 5 cm and a length of 10 cm, a horizontal line was drawn at a distance of 1.5 cm from the top as the upper limit of the running plate, a horizontal line was drawn at a distance of 1 cm from the bottom, and a spotting point was marked, with a distance of about 1 cm between each spotting point; 0.3% (w / v) of the standard alginate oligosaccharide (a mixture of guluronic acid with a degree of polymerization of 3-6), the enzymatic product sample of each reaction time was spotted on the spotting point, a small amount at a time, about 2 μL of sample in total, and the next spotting was performed after the spotting point was dried to ensure the running plate effect; 10 mL of developing agent was taken in a chromatographic cylinder, and was placed in a closed state for 15-30 min, and after the developing agent reached saturation in the closed chromatographic cylinder, the TLC plate after spotting was placed vertically in the chromatographic liquid, avoiding the chromatographic liquid from touching the spotting point, and was developed for 40-60 min, and after the developing agent developed to the upper line drawn at a distance of 1.5 cm from the top, the plate was taken out and dried with a hair dryer; the dried chromatographic plate was placed in a chromogenic agent for about 3-5 s, immediately taken out, dried with a hair dryer, and placed in a baking plate device, and baked at 150°C until obvious bands appeared. As shown in FIG. 8, when the reaction time was 0.5 h, alginate oligosaccharides with a degree of polymerization of 2-6 were produced in the reaction system, and when the reaction time was 18 h, the degradation product was still alginate oligosaccharides with a degree of polymerization of 2-6, so the alginate lyase Alg3889 was an endo-enzyme, and the degradation product was alginate oligosaccharides with a degree of polymerization of 2-6. Figure 8 As shown in FIG. 8, when the reaction time was 0.5 h, alginate oligosaccharides with a degree of polymerization of 2-6 were produced in the reaction system, and when the reaction time was 18 h, the degradation product was still alginate oligosaccharides with a degree of polymerization of 2-6, so the alginate lyase Alg3889 was an endo-enzyme, and the degradation product was alginate oligosaccharides with a degree of polymerization of 2-6.

Claims

1. A low temperature phlorotidnase, characterized in that, The amino acid sequence of the low-temperature alginate lyase is SEQ ID NO:

1.

2. The low-temperature phycocolloid lyase according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the low-temperature alginate lyase is SEQ ID NO:

2.

3. The low-temperature phycocolloid lyase according to claim 1, wherein, The amino acid sequence of the alginate lyase after removing the signal peptide of the low-temperature alginate lyase is SEQ ID NO:

3.

4. The low-temperature phycocolloid lyase according to claim 1, wherein The nucleotide sequence of the gene encoding the alginate lyase after removing the signal peptide of the low-temperature alginate lyase is SEQ ID NO:

4.

5. A recombinant expression plasmid, characterized in that, The recombinant expression plasmid has inserted therein a nucleotide fragment for encoding the low-temperature alginate lyase or the alginate lyase after removing the signal peptide according to claim 1.

6. A recombinant expression strain, characterized in that, The recombinant expression strain carries the recombinant expression plasmid according to claim 5.

7. A method for preparing the low-temperature phlorotoinnase of claim 1, characterized by, The method is to use the recombinant expression strain according to claim 6 to carry out fermentation, and the fermentation product is prepared by purification.

8. Use of the low-temperature alginate lyase according to claim 1 in degrading alginate to prepare oligosaccharides.

9. A method of preparing phloro-oligosaccharides, characterized in that, The method is to use the low-temperature alginate lyase according to claim 1 to carry out enzymolysis to prepare oligosaccharides.

10. The method of claim 9, wherein, The method, wherein the oligosaccharides are alginate oligosaccharides with a polymerization degree of 2-6.

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