CN110982831B - Cold-adaptive uses of gene AlgL23 - Google Patents

Abstract

The invention discloses the use of the gene AlgL23 with cold adaptability. The enzyme activity still has 48% of the maximum enzyme activity at 4°C, and it has relatively good cold adaptability.

Description

Cold-adaptive uses of gene AlgL23

technical field

The present invention relates to the field of gene use, in particular to the use of the gene AlgL23 with cold adaptability.

Background technique

Algin is one of the most abundant fucoidan, consisting of two kinds of L-guluronic acid (α-L-guluronic acid, G for short) and D-mannuronic acid (β-D-mannuronic acid, M for short). The units are linked by 1,4-glycosidic bonds. There are three kinds of combinations, namely: homopolymeric gulutonate fragment (Poly-gulutonate, Poly-G), homopolymeric mannuronate fragment (Poly-mannuronate, Poly-M) and mannuronic acid- Guluronic acid mixed chimeric fragment (Poly-MG). At present, the methods of algin degradation mainly include chemical degradation, physical degradation and enzymatic hydrolysis. In comparison, enzymatic degradation of algin has more advantages because of its high efficiency and non-toxic by-products, and is more suitable for algin oligosaccharides. preparation. Various oligosaccharides derived from alginate obtained by enzymatic degradation have physiological functions and biological activities such as promoting plant growth, antioxidant, anticoagulant, antitumor, antiallergic, antiproliferative and antiallergic activities. Because of their small molecular weight, oligosaccharides and oligosaccharides are easy to be utilized, and their functional activities are often higher than those of sugars, so they are more widely used. The third-generation bioenergy using microalgae and macroalgae as substrates has attracted widespread attention at home and abroad. Seaweed is an ideal feedstock for biofuel production and has great potential. The biotectonic characteristics of seaweed endow it with an advantage over lignocellulosic biomass, which is prone to high yields and avoids the energy-intensive pretreatment and hydrolysis processes before fermentation. However, the full potential of ethanol production from seaweed has not yet been realized, mainly because industrial microorganisms cannot metabolize seaweed polysaccharide components and must first be degraded into monosaccharides or oligosaccharides before they can be utilized by microorganisms. Therefore, the mild degradation of algin by alginate lyase becomes the key point for the utilization of macroalgae.

Algin lyases exist widely in nature, microorganisms, mollusks and algae in water systems, microorganisms and viruses in terrestrial systems all have algin lyases. At present, many fucoidan lyase genes have been cloned and sequenced, and a variety of recombinant fucoidan lyase genetically engineered bacteria have been constructed. However, the expression level of recombinant fucoidan lyase genetic engineering is low, which limits the application of the enzyme. Therefore, it is still an urgent task in the technical field to further obtain fucoidan lyase with excellent characteristics by means of genetic engineering.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a cold-adaptive use of the gene AlgL23.

In order to achieve the above object, the present invention provides the use of a gene AlgL23 and/or a vector containing the gene AlgL23 with cold adaptability.

Further, the cold adaptability refers to the ability to have a larger enzyme activity at 4-55°C.

Further, the cold adaptability refers to the ability to have a larger enzyme activity at 4-50°C.

The present invention also provides the use of the gene AlgL23 and/or the vector containing the gene AlgL23 to act on kelp to produce reducing sugar.

The present invention obtains the DNA sequence of AlgL23 of alginate lyase by designing specific primers. The coding region of the gene is 2223bp and encodes 740 amino acids, of which 1-28 amino acids are signal peptides, and the theoretical molecular weight is 82.59kDa. AlgL23 obtained by recombinant expression in Escherichia coli has high enzymatic activity and cold adaptability. The enzyme can still maintain good enzymatic activity at low temperature. When reacted at 4°C, the enzymatic activity can retain nearly 48.94% of the maximum enzymatic activity. This is undoubtedly very advantageous compared to enzymes from other sources.

As a preferred mode of the embodiment, the temperature range of the hydrolysis catalyzed by the alginate lyase is 4-50°C, and the optimum temperature is 35°C, and the enzyme can still retain 55% of the remaining activity after being treated at 40°C for 60 minutes. The pH range of hydrolysis is 5-9, the optimum pH is 6, and more than 55% of the maximum enzymatic activity can still be retained under the condition of pH 5-7 for 2 hours. Mn ²⁺ has obvious promoting effect on the enzyme, while Cu ²⁺ has obvious inhibitory effect on the enzyme.

Description of drawings

Figure 1 is the SDS-PAGE map of the recombinant expression and purification of the alginate lyase gene AlgL23.

Fig. 2 is a graph showing the effect of temperature on alginate lyase AlgL23 activity of the present invention;

3 is a graph showing the influence of temperature on the stability of alginate lyase AlgL23 of the present invention;

Fig. 4 is a graph showing the influence of pH of the present invention on the activity of alginate lyase AlgL23;

Fig. 5 is a graph showing the effect of pH on the stability of alginate lyase AlgL23;

6 is a graph showing the influence of metal ions of the present invention on the activity of alginate lyase AlgL23;

Figure 7 is a table showing the effects of inhibitors and detergents on enzyme activity;

Figure 8 is a graph showing the action of the alginate lyase AlgL23 of the present invention on kelp meal.

Detailed ways

The following describes in detail the embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals refer to the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary, and are intended to explain the present invention and should not be construed as limiting the present invention. If no specific technology or condition is indicated in the examples, the technology or condition described in the literature in the field or the product specification is used. The reagents or instruments used without the manufacturer's indication are conventional products that can be obtained from the market.

source of biological material

Pseudoalteromonas carrageenovora ASY5, Chinese name is Pseudoalteromonas carrageensis, isolated from Xiamen mangrove soil rot leaf samples, from China Industrial Microorganism Culture Collection and Management Center (CICC), deposit number 23819.

Example 1: Acquisition of cold-adapted alginate lyase AlgL23 gene

Inoculate Pseudoalteromonas carrageenovora ASY5 into artificial seawater medium, under the condition of 25 ℃, 180r/min, shake and cultivate to OD ₆₀₀ of 1-1.5, take 1 mL of cultured bacterial solution, use Dongsheng Bio-Bacterial Genome Rapid Extraction Kit ( The genomic DNA of Pseudoalteromonas carrageenovora ASY5 strain was extracted by silica membrane spin column method.

The above-mentioned artificial seawater culture medium configuration method is as follows:

Artificial seawater culture medium: beef extract 10g, tryptone 10g, distilled water 250mL, artificial seawater 750mL (NaCl 37.51g, KCl 1.03g, CaCl ₂ 1.61g, MgCl ₂ ·6H ₂ O 6.4g, NaHCO ₃ 0.15g, MgSO ₄ 7H ₂ O 4.67 g, distilled water 1000 mL). Beef extract and tryptone were dissolved in distilled water, adjusted to pH 7.8 with NaOH, heated and boiled for 10 min, cooled and adjusted to pH 7.3 with NaOH again, then mixed with artificial seawater, sterilized at 121 °C for 20 min. The solid medium was supplemented with 20 g of agar.

Using the genomic DNA of Pseudoalteromonas carrageenovora ASY5 strain as a template, primers were designed for PCR amplification. The primer sequences are as follows:

Forward primer AlgL23-F:

5'-CGC GGATCC ATGATGAATTTATCTCGAAG-3'; SEQ ID NO: 3;

Reverse primer AlgL23-R:

5'-CCG GAATTC CTCCTGAGTATTCTTCAACG-3'; SEQ ID NO:4.

The forward primer AlgL23-F is underlined the restriction endonuclease site BamHI, and the reverse primer AlgL23-R is underlined the restriction endonuclease EcoR I site. The used high-fidelity DNA polymerase PrimeSTAR HS was purchased from TaKaRa Biological Company, Dalian, China, and the PCR reaction reagents used were operated according to the product instructions provided by the company.

The PCR reaction system is:

PCR conditions were: 95°C, 5 min; 94°C, 15s; 55°C, 15s, 72°C, 1 min, 30 cycles, and finally 72°C, 10 min.

The PCR product obtained is the alginate lyase AlgL23 gene.

Example 2: Sequence Analysis

The BLAST program of NCBI was used to search the homology of nucleic acid sequence and amino acid sequence in GenBank database, the signal peptide prediction tool SignalP4.0 of SMART database was used to predict whether there is a signal peptide in alginate lyase, and the protparam tool of ExPASy was used to predict the protein physicochemical properties.

The results of analysis with the above biological software show that the coding region of the gene AlgL23 is 2223 bp long, and the nucleotide sequence is shown in SEQ ID NO: 1. The alginate lyase AlgL23 gene contains a total of 740 amino acids, and its amino acid sequence is shown in SEQ ID NO: 2. Analysis with the protparam tool of ExPASy showed that the theoretical molecular weight of the protein AlgL23 was about 82.59 kDa, and the theoretical isoelectric point was 6.55. The analysis results of signal peptide showed that 1-28 amino acids at the N-terminal were signal peptide sequences.

SEQ ID NO: 1 is shown below:

ATGATGAATTTATCTCGAAGCAAAACATATTTTAAAACAGCCGGCGTAACTGCAGCCTTGTTATTATCTTTAAATGCACATGCTGTGCATCCTAATTTGGTAATCACTAACGACGATGTACAACACATGCGTCAAGCTATTAGCACTAACAGCGAGAGCCAATTTGCTACAGCGTTTGAGTCATTAAAAGCGCAGGTAGATGAGCAAATAGCACAACCTATCACTGTACCAGTTCCAAAAGATGGTGGTGGTGGTTATACCCATGAACGCCATAAAAAAAATTACCAACTGATGTACAACGCGGGCGTTATTTATCAGCTAAGTAAAGATGAAAAATACGCAAATTATGTTCGCGATATGCTACTTGCATACGCACAGTTATACCCAACGCTTGATGTACATCCAAAGCGTAAAGTGAAATCGCAAAACCCAGGTAAACTTTTTTGGCAAAGCCTGAATGAAGCTATGTGGCTTGTATACACTATTCAAGCATATGACCTAGTACATGACACGCTAAGCGCTGCGAATATAAAAACTATCGAAGATGATTTATTGCGCCCGGTTTCATTATTTATGTCTGAAGGGCAACCTTCTACGTTTAATAAAGTACACAACCATGGGACATGGGCTACAGCTGGTGTTGGTATGGCTGGTTATGTATTAGACGAGCCAGAGTGGGTAGAGAAATCATTATTTGATTTAAAAAAGTCGGGTAAGGGTGGTTTTGTTAAGCAGCTCGAAATGCTGTTTTCTCCCCAAGGCTATTACAATGAAGGGCCTTACTATCAACGTTTTGCATTATTACCATTTGTAACGTTTGCTAAAGCGATTGAAAACAACGAACCTCAAAGAAAGATATTTGAATACCGCGATGGCATATTATTAAAAGCAATCGATACGACTATTCAGCTTAGTTATAACGGTTTGTTTTTTCCTATAAACGATGCCATAAAAAGTAAAGGTATTGACACTATAGAGCTTGTGCAAGGGGTTACTGCGG CTTACGGTTTAACAAATGATGCGGGCTATTTAGATGTAGCTAAAAAGCAAAATCAAATTGTTTTAACGGGTGATGGCTTAAAAGTTGCTCAAGCTTTGGATAAAAGCCAAGAAAAGCCATACGTATTTAAATCCGTTGCTTTTGGTGATGGTAACGATGGTAAGCAAGGCGCTTTGGTTGTTATGCGCAGTGACGTAGGTGGCGATCAAGCATTGCTATTTAAACCTGCCGCACAAGGTTTAGGCCATGGTCATTTTGATAAGCTTACATGGCAGTTTTACGATCATGGTAATGAAATCGTATCTGATTACGGCGCTGCACGCTTTTTAAATGTAGAAGCTAAATATGGTGGTCGTTACTTACCTGAAAACGAGACTTATGCAAAGCATACAGTTGCACACAACACCGTTGTTGTTGATGAAACCACGCACTTTAATGCAAATGTTGAAGTGGGTAATAATAACCACCCAACGCTTAATTTTTTCGAAACCAATCAATACGGTACAGTATCAAGTGGGCAAATAAAAACAGCTTATAAAGGTGTTGAGTTAGAGCGTACTTTAGCGCTTGTTAACCTGCCAGAGCTTGACAGTACCATTGCTGTAGATATGTTTAATGTAACTGCAAATAAGGCACATCAGCTTGATTTACCGCTACATTATAAAGGTCAGTTAATTGATACAAGTTTTGAATTAACAGGTAACGCTAAGCAGTTATCAGCATTGGGTGATAAAAACGGATACCAGCATTTATGGCTTAAGGCACAGGCAAAACCTGAGCAAGGGCTAGCTAAGGTAACGTGGTTAAATGATAACGGACGTTTTTATACACAAACTAGTTTAGTTAAAGGAGATGAGTCGTTCCTATTTACTCAAATAGGCGCAAACGACCCGCACTTTAATTTACGTAACGAAAACGGTTTTATTCGCCGAGTAGATAGCGCTAAACAGCATAAGTTTATATCTATTTTAGAGCCGCATGGCGAGTACAACCCAAGTAA AGAATATACGCTAGAAGCAAATAGCAGAGTAACGGCACTTAACTACAGCGAACAAGATACGCTTACACTTGTTAATGTTGATATTAAGGGCAAATCTTATTTAGTTGCGATCAATAAAGCTGCACAAGCAAACCCTAGCAAGCACACTTTCACATATCAAAATAAAGCATTCACCTTAAATGGCCGTCTTGGCGTTTATGCGTTGAAGAATACTCAGGAGTAA

SEQ ID NO: 2 is shown below:

MMNLSRSKTYFKTAGVTAALLLSLNAHAVHPNLVITNDDVQHMRQAISTNSESQFATAFESLKAQVDEQIAQPITVPVPKDGGGGYTHERHKKNYQLMYNAGVIYQLSKDEKYANYVRDMLLAYAQLYPTLDVHPKRKVKSQNPGKLFWQSLNEAMWLVYTIQAYDLVHDTLSAANIKTIEDDLLRPVSLFMSEGQPSTFNKVHNHGTWATAGVGMAGYVLDEPEWVEKSLFDLKKSGKGGFVKQLEMLFSPQGYYNEGPYYQRFALLPFVTFAKAIENNEPQRKIFEYRDGILLKAIDTTIQLSYNGLFFPINDAIKSKGIDTIELVQGVTAAYGLTNDAGYLDVAKKQNQIVLTGDGLKVAQALDKSQEKPYVFKSVAFGDGNDGKQGALVVMRSDVGGDQALLFKPAAQGLGHGHFDKLTWQFYDHGNEIVSDYGAARFLNVEAKYGGRYLPENETYAKHTVAHNTVVVDETTHFNANVEVGNNNHPTLNFFETNQYGTVSSGQIKTAYKGVELERTLALVNLPELDSTIAVDMFNVTANKAHQLDLPLHYKGQLIDTSFELTGNAKQLSALGDKNGYQHLWLKAQAKPEQGLAKVTWLNDNGRFYTQTSLVKGDESFLFTQIGANDPHFNLRNENGFIRRVDSAKQHKFISILEPHGEYNPSKEYTLEANSRVTALNYSEQDTLTLVNVDIKGKSYLVAINKAAQANPSKHTFTYQNKAFTLNGRLGVYALKNTQE。

Example 3: Recombinant expression and purification of gene AlgL23 in E. coli BL21(DE3) strain

The PCR products obtained in the examples and the pET-28a plasmid were double digested with the restriction enzymes EcoR I and BamHI, and the product fragments after the digestion were recovered. Restriction endonucleases EcoR I and BamHI were purchased from TaKaRa Biological Company, Dalian, China, and the reaction system, temperature and time between the enzyme and the substrate used for enzymatic cleavage were all operated in accordance with the product instructions provided by the company. The PCR product that has undergone double digestion with EcoRI and BamHI is connected with the pET-28a plasmid vector that has also undergone double digestion under the catalysis of T4 DNA ligase, and the ligation product is transformed into E. On the LB solid medium containing 0.1 mg/mL kanamycin, invert at 37 °C for 16 h, pick the positive transformants, and transfer the positive transformants into the liquid LB medium containing 0.1 mg/mL kanamycin at 37 °C. , 180r/min, cultured for 12h, using forward primer AlgL23-F and reverse primer AlgL23-R for bacterial liquid PCR verification. Correctly named the recombinant plasmid pET-28a-AlgL23.

Next, the recombinant plasmid pET-28a-AlgL23 was transformed into Escherichia coli BL21 (DE3), spread on LB solid medium containing 0.1 mg/mL kanamycin, and cultured upside down at 37°C for 16 h. The positive transformants were inserted into liquid LB medium containing 0.1mg/mL kanamycin, 37°C, 180r/min, cultured for 12h, and the bacterial liquid PCR was verified with forward primer AlgL23-F and reverse primer AlgL23-R , the amplified product with a size of about 2200bp was obtained, which preliminarily proved that the constructed recombinant plasmid was correct. The recombinant plasmid was sent to Xiamen Primus Biotechnology Co., Ltd. for sequencing. The results showed that the gene AlgL23 shown in SEQ ID NO: 1 was inserted into the EcoR I and BamHI restriction sites of pET-28a, and the insertion direction was correct, so further It was proved that the constructed recombinant plasmid was correct, and the recombinant plasmid was named pET-28a-AlgL23. Inducible expression of recombinant alginate lyase was performed using isopropylthiogalactosyl (IPTG). Isopropylthio-β-D-galactoside (IPTG) was added to a final concentration of 0.05 mmol/L, and the bacterial liquid was collected into a 200 mL centrifuge tube after induction at 16 °C for 20 h, and the bacterial cells were pelleted by centrifugation at 6500 rpm. The bacterial cells were resuspended in 20 mL of lysis buffer (dissolution buffer formula: 0.3mol/LNaCl, 15mmol/L imidazole, 50mmol/LNaH ₂ PO ₄ , pH 8.0), and ultrasonically disrupted until the bacterial liquid became translucent. (The parameters are set to 300w, ultrasonic time 5s, intermittent time 5s, total working time 15min), centrifuge at 11000rpm for 20min, mix the supernatant with Ni-NTA Agarose pre-equilibrated with lysis buffer, combine at 4°C for 1 hour, the purification process is as follows Purification kit (purchased from Qiagen) was performed according to the instructions. The purified protein was analyzed by SDS-PAGE electrophoresis, and its molecular weight was about 83kDa. The protein concentration was determined by Bradford method, and the recombinant algin lyase with a concentration of about 1.0mg/ml was obtained (see Figure 1 for the results). In Figure 1, M is the protein marker, lane 1 is the expression of the empty vector pET-28a in E.coliBL21, lane 2 is the induced expression of the recombinant vector pET-28a-AlgL23 in E.coli BL21, and lanes 3-6 are purified by Ni-NTA The obtained target protein AlgL23 (82.59kDa). It can be seen from Figure 1 that the target protein AlgL23 was induced, and the purity was higher after being purified by Ni-NTA.

Example 4: Analysis of the enzymatic properties of recombinant alginate lyase

1. Determination of recombinase activity:

Mix 200 μL of the purified recombinant seaweed polysaccharide lyase obtained in Example 3 with 800 μL of sodium alginate (0.5%, pH 8.0), react at 35° C. for 60 min and terminate the reaction in a boiling water bath, and then add 1 mL of DNS solution (DNS The solution formula is: weigh 10 g of 3,5-dinitrosalicylic acid, put it in 600 ml of water, gradually add 10 sodium hydroxide, stir and dissolve in a 50 ° C water bath, and then add 200 g of potassium sodium tartrate, 2 g of phenol and no Water sodium sulfite 5g, all dissolved and clarified, cooled to room temperature, volume was adjusted to 1000ml with water, filtered, stored in a brown reagent bottle, placed in the dark for 7 days before use.) and reacted in a boiling water bath for 10min, and the absorbance value was measured at 540nm. The enzyme activity is defined as: under the above conditions, the amount of enzyme required to catalyze the production of 1 μg of reducing sugar (such as alginate oligosaccharide) per minute is one enzyme activity unit (U).

2. The effect of temperature on enzyme activity and stability

The enzyme activity of recombinant alginate lyase was determined after the reaction at the reaction temperature of 4 °C, 10 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, and 60 °C. The highest enzyme activity is 100%. When measuring the temperature stability of the recombinant alginate lyase, the enzyme solution was treated at 30°C, 35°C, and 40°C for 0-180min, respectively, and the remaining enzyme activity was measured at the optimum reaction temperature, taking the untreated enzyme activity as 100% . The optimal temperature measurement results are shown in Figure 2: the alginate lyase AlgL23 reaches the maximum activity at 35 °C, indicating that the optimal reaction temperature of the algin lyase AlgL23 is 35 °C, and the enzyme activity still has the maximum enzyme activity at 4 °C. 48%, indicating that the enzyme can react at lower temperature and has better cold adaptability. The temperature stability results are shown in Figure 3. The enzyme is relatively stable at 30 °C and 35 °C, and 60% of the enzyme activity is still retained after 2 hours of incubation, and 50% of the original enzyme activity is still maintained at 40 °C for 1 hour.

3. The effect of pH value on enzyme activity and stability

The substrates were prepared with different pH buffer systems, and the enzyme activity was determined at the optimum reaction temperature, with the highest enzyme activity being 100%. The buffer system was 50mmol/L acetic acid-sodium acetate (pH 4-6), sodium dihydrogen phosphate-disodium hydrogen phosphate (pH 6-8), Tris-HCl (pH8-9) glycine-NaOH (pH9- 10). The pH stability was studied by mixing the enzyme solution with buffer solutions of different pH in equal proportions, and placing it at 4°C for 2 hours, and then measuring the remaining enzyme activity, taking the untreated enzyme activity as 100%. The results are shown in Figure 4. The activity of the recombinant algin lyase was the highest at pH 6.0, indicating that the optimum pH of the algin lyase AlgL23 was 6.0. The pH stability results are shown in Figure 5. The recombinant alginate lyase AlgL23 is relatively stable in the acid range (5-7), and can still maintain more than 55% of the enzyme activity after being treated at 4°C for 2 h.

4. The effect of metal ions on enzyme activity

Different metal ions (Mg ²⁺ , Sr ²⁺ , Mn ²⁺ , Fe ²⁺ , Ba ²⁺ , Cd ²⁺ , Fe ³⁺ , Ca ²⁺ , Cu ²⁺ ), placed at 37°C for 1 h, the remaining activity of the enzyme was determined, and the enzyme activity without metal ions was taken as 100% to study the effect of metal ions on the enzyme activity. As shown in Figure 6, the results show that the metal ion Mn ²⁺ has a significant promoting effect on AlgL23 at the concentration of 1 mmol/L and 10 mmol/L, and Fe ³⁺ slightly promotes the enzyme activity at the concentration of 1 mmol/L When the concentration increased to 10mmol/L, it had a great inhibitory effect on the enzyme activity. Cu ²⁺ , Mg ²⁺ , Sr ²⁺ , Ca ²⁺ , Ba ²⁺ all had different degrees of inhibition on AlgL23. When the concentration is 10mmol/L, Cu ²⁺ has a very strong inhibitory effect on the enzyme activity, almost completely inhibiting the enzyme activity.

5. Effects of inhibitors and detergents on enzyme activity

Add the final concentration of 1mmol/L or 10mmol/L inhibitor (EDTA, DTT, mercaptoethanol, CTAB, Urea) and 1% or 10% (V/V) detergent (SDS, Tween 20, Tween 80, TritonX 100), after being placed at 37°C for 1 h, the remaining activity of the enzyme was determined, and the enzyme activity without inhibitor or detergent was taken as 100%, and the effect of inhibitor and detergent on the enzyme activity was studied. The results are shown in Figure 7. The alginate lyase AlgL23 has good resistance to SDS, and CTAB has a strong inhibitory effect on AlgL23. At a concentration of 10 mM, the alginate lyase AlgL23 was substantially inactivated. EDTA and Tween 80 also had obvious inhibitory effect on AlgL23, which greatly inhibited the activity of the enzyme. The low concentration of urea has a slight inhibition on the enzyme activity, and the inhibitory effect is also significantly enhanced when the concentration is increased.

Example 5: Recombinant alginate lyase AlgL23 acts on kelp meal

After cleaning the kelp slices with distilled water, dry them outdoors for 1-2 days, then dry them in an oven, grind the dried samples into powder, and add the powder to 0.05mol/L Tris-HCl (pH8.0) buffer , 50 ℃ of stirring for 10min to make 5mg/ml substrate, 10U of recombinant algin lyase AlgL23 was added to 20ml of substrate, the enzymatic reaction was carried out at 35 ℃, and samples were taken every 1h. The samples were taken in a boiling water bath for 10 min to terminate the reaction, and then centrifuged at 4°C and 12000 rpm for 20 min, and the content of reducing sugar in the supernatant was determined by DNS method. After 6h of reaction, 5U of recombinant alginate lyase AlgL23 was added to the reaction mixture, and a sample was taken every 2h. After the reaction was carried out for 12h, the content of reducing sugar basically no longer increased and its amount reached about 12.5mg (as shown in Figure 8 ). shown), these results suggest that the recombinant alginate lyase AlgL23 can act on kelp to produce reducing sugars.

Although the embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and those of ordinary skill in the art will not depart from the principles and spirit of the present invention Variations, modifications, substitutions, and alterations to the above-described embodiments are possible within the scope of the present invention without departing from the scope of the present invention.

SEQUENCE LISTING

<110> Jimei University

<120> Use of the gene AlgL23 for cold adaptation

<130> JMDXL-19036-CNI

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<170> PatentIn version 3.5

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<213> Pseudoalteromonas carrageenovora ASY5

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Met Met Asn Leu Ser Arg Ser Lys Thr Tyr Phe Lys Thr Ala Gly Val

Thr Ala Ala Leu Leu Leu Ser Leu Asn Ala His Ala Val His Pro Asn

Leu Val Ile Thr Asn Asp Asp Val Gln His Met Arg Gln Ala Ile Ser

Thr Asn Ser Glu Ser Gln Phe Ala Thr Ala Phe Glu Ser Leu Lys Ala

Gln Val Asp Glu Gln Ile Ala Gln Pro Ile Thr Val Pro Val Pro Lys

Asp Gly Gly Gly Gly Tyr Thr His Glu Arg His Lys Lys Asn Tyr Gln

Leu Met Tyr Asn Ala Gly Val Ile Tyr Gln Leu Ser Lys Asp Glu Lys

Tyr Ala Asn Tyr Val Arg Asp Met Leu Leu Ala Tyr Ala Gln Leu Tyr

Pro Thr Leu Asp Val His Pro Lys Arg Lys Val Lys Ser Gln Asn Pro

Gly Lys Leu Phe Trp Gln Ser Leu Asn Glu Ala Met Trp Leu Val Tyr

Thr Ile Gln Ala Tyr Asp Leu Val His Asp Thr Leu Ser Ala Ala Asn

Ile Lys Thr Ile Glu Asp Asp Leu Leu Arg Pro Val Ser Leu Phe Met

Ser Glu Gly Gln Pro Ser Thr Phe Asn Lys Val His Asn His Gly Thr

Trp Ala Thr Ala Gly Val Gly Met Ala Gly Tyr Val Leu Asp Glu Pro

Glu Trp Val Glu Lys Ser Leu Phe Asp Leu Lys Lys Ser Gly Lys Gly

Gly Phe Val Lys Gln Leu Glu Met Leu Phe Ser Pro Gln Gly Tyr Tyr

Asn Glu Gly Pro Tyr Tyr Gln Arg Phe Ala Leu Leu Pro Phe Val Thr

Phe Ala Lys Ala Ile Glu Asn Asn Glu Pro Gln Arg Lys Ile Phe Glu

Tyr Arg Asp Gly Ile Leu Leu Lys Ala Ile Asp Thr Thr Ile Gln Leu

Ser Tyr Asn Gly Leu Phe Phe Pro Ile Asn Asp Ala Ile Lys Ser Lys

Gly Ile Asp Thr Ile Glu Leu Val Gln Gly Val Thr Ala Ala Tyr Gly

Leu Thr Asn Asp Ala Gly Tyr Leu Asp Val Ala Lys Lys Gln Asn Gln

Ile Val Leu Thr Gly Asp Gly Leu Lys Val Ala Gln Ala Leu Asp Lys

Ser Gln Glu Lys Pro Tyr Val Phe Lys Ser Val Ala Phe Gly Asp Gly

Asn Asp Gly Lys Gln Gly Ala Leu Val Val Met Arg Ser Asp Val Gly

Gly Asp Gln Ala Leu Leu Phe Lys Pro Ala Ala Gln Gly Leu Gly His

Gly His Phe Asp Lys Leu Thr Trp Gln Phe Tyr Asp His Gly Asn Glu

Ile Val Ser Asp Tyr Gly Ala Ala Arg Phe Leu Asn Val Glu Ala Lys

Tyr Gly Gly Arg Tyr Leu Pro Glu Asn Glu Thr Tyr Ala Lys His Thr

Val Ala His Asn Thr Val Val Val Asp Glu Thr Thr His Phe Asn Ala

Asn Val Glu Val Gly Asn Asn Asn His Pro Thr Leu Asn Phe Phe Glu

Thr Asn Gln Tyr Gly Thr Val Ser Ser Gly Gln Ile Lys Thr Ala Tyr

Lys Gly Val Glu Leu Glu Arg Thr Leu Ala Leu Val Asn Leu Pro Glu

Leu Asp Ser Thr Ile Ala Val Asp Met Phe Asn Val Thr Ala Asn Lys

Ala His Gln Leu Asp Leu Pro Leu His Tyr Lys Gly Gln Leu Ile Asp

Thr Ser Phe Glu Leu Thr Gly Asn Ala Lys Gln Leu Ser Ala Leu Gly

Asp Lys Asn Gly Tyr Gln His Leu Trp Leu Lys Ala Gln Ala Lys Pro

Glu Gln Gly Leu Ala Lys Val Thr Trp Leu Asn Asp Asn Gly Arg Phe

Tyr Thr Gln Thr Ser Leu Val Lys Gly Asp Glu Ser Phe Leu Phe Thr

Gln Ile Gly Ala Asn Asp Pro His Phe Asn Leu Arg Asn Glu Asn Gly

Phe Ile Arg Arg Val Asp Ser Ala Lys Gln His Lys Phe Ile Ser Ile

Leu Glu Pro His Gly Glu Tyr Asn Pro Ser Lys Glu Tyr Thr Leu Glu

Ala Asn Ser Arg Val Thr Ala Leu Asn Tyr Ser Glu Gln Asp Thr Leu

Thr Leu Val Asn Val Asp Ile Lys Gly Lys Ser Tyr Leu Val Ala Ile

Asn Lys Ala Ala Gln Ala Asn Pro Ser Lys His Thr Phe Thr Tyr Gln

Asn Lys Ala Phe Thr Leu Asn Gly Arg Leu Gly Val Tyr Ala Leu Lys

Asn Thr Gln Glu

<210> 3

<211> 29

<212> DNA

<213> Synthetic

<400> 3

cgcggatcca tgatgaattt atctcgaag 29

<210> 4

<211> 29

<212> DNA

<213> Synthetic

<400> 4

ccggaattcc tcctgagtat tcttcaacg 29

Claims (1)

1. alginate lyase gene AlgL23 and/or the carrier containing algin lyase gene AlgL23 acts on the purposes of kelp to produce reducing sugar; the nucleotide sequence of described algin lyase gene AlgL23 is as shown in SEQ ID NO:1 .

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