A mutant alginate lyase

Abstract

The present application relates to the technical field of genetic engineering and protein engineering, and particularly relates to a mutant of alginate lyase. The mutant provided by the present application comprises a mutation site selected from D9V, D10V, S12F, P15G, S18T and T140Q. After being treated at 45 DEG C for 20 min, the residual rate of enzyme activity of the mutant is generally higher than that of wild-type alginate lyase, the stability is significantly improved, and an unexpected technical effect is achieved. The mutant can be widely used in the field of seaweed processing, effectively improves the enzymatic hydrolysis efficiency of Sargassum thunbergii, Ascophyllum nodosum and other seaweeds, and has a wide market prospect.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and protein engineering technology, and specifically relates to an alginate lyase mutant. Background Technology

[0002] Brown algae are mainly composed of alginate, laminarin, mannitol, and sulfated alginate. Alginate, an anionic polysaccharide derived from the cell wall of brown algae, is the main structural component of brown algae and is also known as sodium alginate. Utilizing brown algae requires first addressing the issue of utilizing alginate.

[0003] The main methods for degrading alginate are: (1) Chemical degradation. Acid, hydrothermal or alkali pretreatment has been used for the hydrolysis of alginate. Acid hydrolysis is more commonly used in chemical degradation, but alginate is relatively acid-resistant and it is difficult to control the production of uronic acid. In addition, a high concentration of acid is required to obtain a high yield of uronic acid. (2) Water preheating treatment. Alginate can produce alginate monomers (mannuronate and guluronate) after hydrothermal treatment (180℃-240℃), and lactic acid and glycolic acid are produced at the same time. (3) Enzymatic degradation. Enzymatic degradation of alginate is mild, the process is controllable, the yield is high, it is green and safe, environmentally friendly, the mechanism of action is clear, and the products are certain. Different substrate-specific enzyme preparations can be selected to be used alone or in combination according to the specific product requirements. Endo-alginates produce alginate oligosaccharides with different DP, while exo-alginates degrade alginate or alginate oligosaccharides to produce monosaccharides.

[0004] The production of alginate lyase largely relies on alginate-degrading bacteria. Although wild-type alginate-degrading bacteria can effectively produce a quantitative amount of enzyme protein, the yield is low and the cost is high, making it difficult to meet practical application requirements. Therefore, heterologous expression of alginate lyase genes using genetic engineering is the most effective way to increase alginate lyase yield. Research mainly focuses on cloning alginate lyase genes from alginate-degrading bacteria and overexpressing them in Bacillus subtilis. Currently, alginate lyase genes from more than twenty alginate-degrading bacteria have been cloned, and most of these genes have been successfully heterologously expressed. The expression levels of recombinant alginate lyase are all higher than those of wild-type strains. According to the CAZY database classification, alginate lyase belongs to polysaccharide degrading enzymes (PL), and is specifically divided into seven families: PL5, PL6, PL7, PL14, PL15, PL17, and PL18.

[0005] In recent years, with the widespread application of protein engineering technology in the field of enzyme preparations, the development of novel alginate lyases with high enzyme activity and excellent properties has become a research hotspot in this field. This is of great significance for reducing the production cost of alginate lyases and promoting their industrialization. Summary of the Invention

[0006] The purpose of this invention is to provide an alginate lyase mutant. The mutant exhibits significantly improved stability compared to the wild type, which is beneficial for its widespread application in industrial fields such as seaweed processing.

[0007] One aspect of the present invention relates to an alginate lyase mutant comprising an amino acid sequence having at least 90% identity with SEQ ID NO:1, and comprising, compared with SEQ ID NO:1, an amino acid substitution at at least one position selected from the group consisting of: 9, 10, 12, 15, 18, 140.

[0008] In some embodiments of the present invention, the amino acid sequence of the mutant has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity with SEQ ID NO:1.

[0009] In some more specific embodiments, the amino acid sequence of the mutant has at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9% identity with SEQ ID NO:1.

[0010] In some embodiments of the present invention, the mutant comprises a substitution of at least one amino acid from the group consisting of: D9V, D10V, S12F, P15G, S18T, and T140Q.

[0011] In some embodiments of the present invention, the mutant comprises substitutions or combinations of substitutions for the following amino acids: D9V; D10V; S12F; P15G; S18T; T140Q; D9V / D10V; D9V / S12F; D9V / P15G; D9V / A17S; D9V / S18T; D9V / T140R; D9V / T140Q; D10V / S12F; D10V / P15G; D10V / A17S; D10V / S18T; D10V / T140R; D10V / T140Q; S12F / P15G; S12F / A17S; S12F / S18T; S12F / T140R; S12F / T140Q; P15G / A17S; P15G / S18T; P15G / T140R; P15G / T140Q; A17S / S18T; A17S / T140R; A17S / T140Q; S18T / T140R; S18T / T140Q; D9V / D10V / S12F; D9V / D10V / P15G; D9V / D10V / A17S; D9V / D10V / S18T; D9V / D10V / T140R; D9V / D10V / T140Q; D9V / S12F / P15G; D9V / S12F / A17S; D9V / S12F / S18T; D9V / S12F / T140R; D9V / S12F / T140Q; D9V / P15G / A17S; D9V / P15G / S18T; D9V / P15G / T140R; D9V / P15G / T140Q; D10V / S12F / P15G; D10V / S12F / A17S; D10V / S12F / S18T; D10V / S12F / T140R; D10V / S12F / T140Q; D10V / P15G / A17S; D10V / P15G / S18T; D10V / P15G / T140R; D10V / P15G / T140Q; D10V / A17S / S18T; D10V / A17S / T140R; D10V / A17S / T140Q; D10V / S18T / T140R; D10V / S18T / T140Q; S12F / A17S / S18T; S12F / A17S / T140R; S12F / A17S / T140Q; S12F / S18T / T140R; S12F / S18T / T140Q; P15G / A17S / S18T; P15G / A17S / T140R; P15G / A17S / T140Q; P15G / S18T / T140R; P15G / S18T / T140Q; A17S / S18T / T140R; A17S / S18T / T140Q; D9V / D10V / S12F / P15G; D9V / D10V / S12F / A17S; D9V / D10V / S12F / S18T; D9V / D10V / S12F / T140R; D9V / D10V / S12F / T140Q; D9V / D10V / P15G / A17S; D9V / D10V / P15G / S18T; D9V / D10V / P15G / T140R; D9V / D10V / P15G / T140Q; D9V / A17S / S18T / T140Q; D9V / S12F / P15G / S18T; D9V / S12F / P15G / T140R; D9V / S12F / P15G / T140Q; D9V / S12F / A17S / S18T; D9V / S12F / A17S / T140R; D9V / S12F / A17S / T140Q; D9V / S12F / S18T / T140R; D9V / S12F / S18T / T140Q; D10V / S12F / P15G / T140Q; D10V / S12F / A17S / T140Q; D10V / S12F / S18T / T140R; D10V / S12F / P15G / T140R; D10V / S12F / A17S / T140R; D10V / S12F / S18T / T140R; S12F / P15G / A17S / S18T; S12F / P15G / A17S / T140R; S12F / P15G / A17S / T140Q; S12F / P15G / S18T / T140R; S12F / P15G / S18T / T140Q; P15G / A17S / S18T / T140R; P15G / A17S / S18T / T140Q; D9V / D10V / S12F / P15G / T140Q; D9V / D10V / S12F / A17S / T140Q; D9V / D10V / S12F / S18T / T140R; D9V / D10V / S12F / P15G / T140R; D9V / D10V / S12F / A17S / T140R; D9V / D10V / S12F / S18T / T140R; D9V / S12F / P15G / A17S / S18T; D9V / S12F / P15G / A17S / T140R; D9V / S12F / P15G / A17S / T140Q; D9V / S12F / P15G / S18T / T140R; D9V / S12F / P15G / S18T / T140Q; D9V / P15G / A17S / S18T / T140R; D9V / P15G / A17S / S18T / T140Q; D10V / S12F / P15G / A17S / S18T; D10V / S12F / P15G / A17S / T140R; D10V / S12F / P15G / A17S / T140Q; D10V / S12F / P15G / S18T / T140R; D10V / S12F / P15G / S18T / T140Q; D10V / P15G / A17S / S18T / T140R; D10V / P15G / A17S / S18T / T140Q; S12F / P15G / A17S / S18T / T140R; S12F / P15G / A17S / S18T / T140Q; D9V / D10V / S12F / P15G / A17S / S18T; D9V / D10V / S12F / P15G / A17S / T140R; D9V / D10V / S12F / P15G / A17S / T140Q; D9V / D10V / S12F / P15G / S18T / T140R; D9V / D10V / S12F / P15G / S18T / T140Q; D9V / D10V / P15G / A17S / S18T / T140R; D9V / D10V / P15G / A17S / S18T / T140Q; D9V / S12F / P15G / A17S / S18T / T140R; D9V / S12F / P15G / A17S / S18T / T140Q; D10V / S12F / P15G / A17S / S18T / T140R; D10V / S12F / P15G / A17S / S18T / T140Q; D9V / D10V / S12F / P15G / A17S / S18T / T140R; D9V / D10V / S12F / P15G / A17S / S18T / T140Q.

[0012] The present invention also relates to a DNA molecule encoding the above-mentioned alginate lyase mutant.

[0013] The present invention also relates to recombinant expression vectors comprising the above-described DNA molecules.

[0014] The present invention also relates to a host cell comprising the above-described recombinant expression vector.

[0015] When the above plasmids were transferred into host cells, the stability of the recombinantly expressed alginate lyase mutant was significantly improved.

[0016] In some embodiments of the present invention, the host cell is Bacillus subtilis (Bacillus subtilis). Bacillus subtilis ).

[0017] Compared to wild-type alginate lyase AH1, the mutants provided by this invention, containing any one of the mutation sites D9V, D10V, S12F, P15G, S18T, and T140Q, showed a generally increased enzyme activity residual rate of 15.8%-32.9% and significantly improved stability after treatment at 45℃ for 20 min. Among them, the T140Q single-point mutant exhibited the highest stability, with an enzyme activity residual rate as high as 60.1%. Furthermore, the mutants provided by this invention, containing two or more mutation sites selected from D9V, D10V, S12F, P15G, A17S, S18T, and T140R / Q, showed a generally increased enzyme activity residual rate of 27.3%-78.1% after treatment at 45℃ for 20 min, achieving unexpected technical effects.

[0018] The alginate lyase mutant provided by this invention has significantly improved stability and can be widely used in the seaweed processing field, effectively improving the enzymatic hydrolysis efficiency of seaweeds such as Sargassum and Paecilomyces, with broad market prospects. Detailed Implementation

[0019] The method of the present invention will be further illustrated below with reference to examples. Experimental methods not specified in the examples can generally be performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual* by J. Sambrook et al., or according to the manufacturer's recommendations. Those skilled in the art can better understand and master the present invention with the help of these examples. However, the methods for implementing the present invention should not be limited to the specific method steps described in the embodiments of the present invention.

[0020] The present invention will now be described in detail with reference to specific embodiments.

[0021] Example 1: Screening of alginate lyase mutants The amino acid sequence of wild-type alginate lyase AH1 is SEQ ID NO: 1, and the encoding nucleotide sequence is SEQ ID NO: 2.

[0022] To further improve the stability of alginate lyase AH1, the applicant analyzed its protein structure. This protein is a PL7 family alginate lyase with a β-jelly roll structure. Both the protein surface and the active site are exposed to the external environment, so changes in the external environment can directly affect the stability of the enzyme's active site. The applicant further mutated this gene without disrupting the protein's secondary structure and active site.

[0023] Primer AH1-ss-F: CGG GGTACC ATGGATCCGAATCTGGAGATTT; Primer AH1-ss-R: CG ACGCGT TTAATCATGTGTATGTTCCAGGC.

[0024] Using the AH1 gene as a template, PCR amplification was performed using the aforementioned primers and the GeneMorph II random mutation PCR kit (Bomais). The PCR products were recovered from the gel, double-digested with KpnI and MluI, and ligated into the similarly digested pSZX101 vector. The ligation was then performed into *E. coli* DH5α, plated on LB+Amp plates, and incubated upside down at 37°C. After transformants appeared, plasmids were extracted and transformed into *Bacillus subtilis*. Once transformants had grown, they were individually transferred to 48-well plates using toothpicks. 20 μg / ml kanamycin was added to each well, and the plates were incubated at 37°C and 500 rpm for approximately 48 hours. The supernatant was centrifuged and subjected to high-throughput assays. The alginate lyase activity was measured by incubating at 45°C for 20 minutes, and the residual enzyme activity of different mutants was calculated.

[0025] Experimental results showed that different mutants maintained varying degrees of stability. Some mutations still exhibited high enzyme activity under the same temperature treatment conditions, while others even worsened their tolerance; additionally, some mutations resulted in significant changes in enzymatic properties, all of which did not meet the requirements. Ultimately, the applicant screened and obtained mutation sites that significantly improved the stability of alginate lyase without significantly affecting its original enzymatic properties, namely: D9V, D10V, S12F, P15G, S18T, and T140Q.

[0026] Based on the truncated alginate lyase AH1, this invention provides mutants containing the aforementioned single mutation sites. Referring to the amino acid sequences of the mutants, the coding nucleotide sequences of the alginate lyase mutants were obtained.

[0027] This invention also provides cellulase mutants containing at least two, three, four, five, or six mutation sites from D9V, D10V, S12F, P15G, A17S, S18T, and T140R / Q. Examples include: two-point mutants such as P15G / S18T, S18T / T140Q, P15G / T140R, D9V / P15G, D10V / T140Q, and S12F / S18T; three-point mutants such as P15G / S18T / T140Q, P15F / S18T / T140R, D9V / S12F / P15G, D9V / P15G / T140Q, and D10V / S18T / T140Q; and S12F... Four-point mutants such as / P15G / S18T / T140Q, S12F / P15G / S18T / T140R, and D9V / S12F / P15G / T140Q; five-point mutants such as D9V / S12F / P15G / S18T / T140Q and D10V / P15G / A17S / S18T / T140Q; and six-point mutants such as D9V / S12F / P15G / A17S / S18T / T140Q.

[0028] Example 2 Expression of alginate lyase mutant in Bacillus subtilis Based on the codon preference of Bacillus, the gene sequences of alginate lyase AH1 and its mutants were optimized and synthesized, and two restriction sites, KpnI and MluI, were added to the 5' and 3' ends of the synthesized sequences, respectively.

[0029] 2.1 Carrier Construction The synthesized alginate lyase gene fragment was obtained by PCR. The obtained gene fragment and expression vector pSZX101 were double-digested with KpnI and MluI, respectively. The target fragment was recovered by gel electrophoresis and ligated overnight with T4 ligase. The ligation product was transformed into E. coli DH5α competent cells, plated on LB+Amp plates, and incubated overnight at 37°C. Single colonies grew. Colony PCR was used to verify the correct ligation of transformants. Plasmids were extracted from the transformed cells and sent to the Beijing BGI Genomics Research Center for sequencing analysis.

[0030] Plasmids were purified from correctly sequenced E. coli clones using a plasmid medium-quantity preparation kit (Axygen).

[0031] 2.2 Preparation and transformation of Bacillus subtilis competent cells The conversion solution was prepared as follows: 1× Minimum salt solution: K2HPO4 7g, KH2PO4 3g, (NH4)2SO4 1g, trisodium citrate Na2C6H5O7·2H2O 0.5g, MgSO4·7H2O 0.1g, add water to 500 mL.

[0032] GMⅠ solution: 95.6 mL of 1× minimum salt solution, 2.5 mL of 20% glucose, 0.4 mL of 5% hydrolyzed casein, and 1 mL of 10% yeast extract.

[0033] GMⅡ solution: 96.98 mL of 1× minimum salt solution, 2.5 mL of 20% glucose, 0.08 mL of 5% hydrolyzed casein, 0.04 mL of 10% yeast extract, 0.25 mL of 1M MgCl2, and 0.05 mL of 1M CaCl2.

[0034] Transformation procedure: Inoculate the bacteria onto LB agar plates and incubate overnight at 37°C. Inoculate a loopful of bacterial growth into 5 mL of GMⅠ solution and incubate overnight at 30°C with gentle shaking (125 rpm). The next day, transfer 2 mL of the culture medium to 18 mL of GMⅠ and incubate at 37°C with rapid shaking (220 rpm) for 3.5 h. Then, transfer 5 mL of the culture medium from the previous step to 45 mL of GMⅡ and incubate at 37°C with gentle shaking (125 rpm) for 90 min. Centrifuge at 8000g for 10 min to collect the bacterial cells. Gently resuspend the bacterial cells in 5 mL of the original culture supernatant. The resuspended cells are competent cells and can be used for transformation. Preservation of competent cells: Add 30% sterile glycerol to a final concentration of 10%, mix well, aliquot into centrifuge tubes, and store immediately at -70°C.

[0035] Mix 1 μg of recombinant plasmid with 200 μL of the above competent cells thoroughly, incubate at 37°C with shaking (200 rpm) for 30 min, then spread on the appropriate resistant medium and incubate overnight at 37°C. The single colony that grows overnight is the engineered strain containing alginate lyase and the mutant.

[0036] Example 3 Fermentation Verification The engineered strain was inoculated into 5 mL of LB medium (0.5% yeast extract, 1% tryptone, 1% sodium chloride) and cultured at 37°C and 220 rpm for about 6-8 hours. It was then transferred to 50 mL of liquid fermentation medium (0.5% yeast extract, 0.5% tryptone, 1% glucose, 1.8% K2HPO4) and fermented in shake flasks at 37°C and 220 rpm for 72 hours. The supernatant was collected by centrifugation at 5000g for 10 minutes, which yielded the fermentation supernatant containing alginate lyase and its mutant.

[0037] 3.1 Assay of Alginate Lyase Activity The principle of alginate lyase activity assay: Alginate lyase can cleave the glycosidic bond in alginate molecules through β-elimination reaction, producing a non-reducing end with an unsaturated double bond. The double bond is located between C4 and C5 of the non-reducing end of the product and produces maximum ultraviolet absorption at 235 nm.

[0038] (1) Definition of enzyme activity unit: Under the conditions of 40°C and pH 7.0, and in the reaction system specified in this method, the degradation of the substrate sodium alginate produces unsaturated double bonds per minute. At 235 nm, each increase of 0.1 oz absorbance corresponds to 1 unit of enzyme activity (U).

[0039] (2) Measurement method Dilute with buffer to the appropriate concentration and control the absorbance value (OD). 235 = Between 0.22 and 0.35, the enzyme activity is approximately 0.5 U / mL.

[0040] Enzyme reaction: Take three 15mm*150mm test tubes, add 1.8mL of substrate, preheat in a 40℃ water bath for 5min, add 0.2mL of diluted enzyme solution, time accurately, vortex, incubate at 40℃ for 10min, remove the test tubes from the water bath and immediately add 2mL of phosphate stop solution, vortex, and place the test tubes on a test tube rack outside the water bath.

[0041] Blank: Take a 15mm*150mm test tube, add 1.8mL of substrate, preheat in a 40℃ water bath for 5min, add 0.2ml of buffer solution, time accurately, vortex, keep warm at 40℃ for 10min, remove the test tube from the water bath and immediately add 2mL of phosphate stop solution, vortex, and place the test tube on a test tube rack outside the water bath.

[0042] 3.2 Stability analysis of alginate lyase The fermentation supernatant of the recombinant Bacillus subtilis engineered bacteria expressing alginate lyase AH1 and its mutants obtained above was incubated at 45℃ for 20 min, and the alginate lyase activity was measured. The enzyme activity residual rate was calculated with the initial enzyme activity as 100%. The results are shown in Table 1.

[0043] Enzyme activity residual rate: refers to the catalytic ability that an enzyme retains after treatment under specific conditions, usually expressed as a percentage. Generally speaking, the higher the enzyme activity residual rate, the more stable the enzyme.

[0044] Calculation formula: Residual enzyme activity (%) = Enzyme activity after treatment / Initial enzyme activity × 100%.

[0045] Table 1. Residual enzyme activity of alginate lyase and its mutants Alginate lyase enzyme activity residual rate Wild-type AH1 27.2% D9V single-point mutant 43.6% D10V single-point mutant 43.1% S12F single-point mutant 45.8% P15G single-point mutant 43.0% S18T single-point mutant 47.2% T140Q single-point mutant 60.1% As shown in Table 1, compared with wild-type alginate lyase AH1, the single-point mutants provided in this invention, after treatment at 45℃ for 20 min, generally showed a 15.8%-32.9% increase in enzyme activity residue and a significant improvement in stability. Among them, the T140Q single-point mutant exhibited the highest stability, with an enzyme activity residue as high as 60.1%, achieving unexpected technical results.

[0046] Furthermore, the present invention provides two-point mutants such as P15G / S18T, S18T / T140Q, P15G / T140R, D9V / P15G, D10V / T140Q, and S12F / S18T; three-point mutants such as P15G / S18T / T140Q, P15F / S18T / T140R, D9V / S12F / P15G, D9V / P15G / T140Q, and D10V / S18T / T140Q; and S12F / P15G / S18T / T140Q and S12F / P18T / T140Q. Four-point mutants such as 15G / S18T / T140R and D9V / S12F / P15G / T140Q; five-point mutants such as D9V / S12F / P15G / S18T / T140Q and D10V / P15G / A17S / S18T / T140Q; and six-point mutants such as D9V / S12F / P15G / A17S / S18T / T140Q, after being treated at 45℃ for 20 min, generally showed an increase in enzyme activity residual rate of 27.3%-78.1%, achieving unexpected technical results.

[0047] In summary, the stability of the alginate lyase mutant provided by this invention is significantly improved, and it can be widely used in the seaweed processing field to effectively improve the enzymatic hydrolysis efficiency of seaweeds such as Sargassum and Laminaria japonica, with broad market prospects.

Claims

1. An alginate lyase mutant, characterized in that, The mutant comprises an amino acid sequence having at least 90% identity with SEQ ID NO:1, and contains an amino acid substitution at at least one position selected from the group consisting of: 10, 12, 15, 18, 140, compared to SEQ ID NO:

1.

2. The alginate lyase mutant of claim 1, wherein, The amino acid sequence of the mutant has at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identity with SEQ ID NO:

1.

3. The alginate lyase mutant of claim 2, wherein, The amino acid sequence of the mutant has at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or at least 99.9% identity with SEQ ID NO:

1.

4. The alginate lyase mutant of claim 1, wherein, The mutant contains a substitution of at least one amino acid from the following group: D10V, S12F, P15G, S18T, T140Q.

5. The alginate lyase mutant as described in claim 4, characterized in that, The mutant contains substitutions or combinations of the following amino acids: D10V; S12F; P15G; S18T; D9V / D10V; D9V / S12F; D9V / P15G; D9V / A17S; D9V / S18T; D9V / T140R; D9V / T140Q; D10V / S12F; D10V / P15G; D10V / A17S; D10V / S18T; D10V / T140R; D10V / T140Q; S12F / P15G; S12F / A17S; S12F / S18T; S12F / T140R; S12F / T140Q; P15G / A17S; P15G / S18T; P15G / T140R; P15G / T140Q; A17S / S18T; A17S / T140R; A17S / T140Q; S18T / T140R; S18T / T140Q; D9V / D10V / S12F; D9V / D10V / P15G; D9V / D10V / A17S; D9V / D10V / S18T; D9V / D10V / T140R; D9V / D10V / T140Q; D9V / S12F / P15G; D9V / S12F / A17S; D9V / S12F / S18T; D9V / S12F / T140R; D9V / S12F / T140Q; D9V / P15G / A17S; D9V / P15G / S18T; D9V / P15G / T140R; D9V / P15G / T140Q; D10V / S12F / P15G; D10V / S12F / A17S; D10V / S12F / S18T; D10V / S12F / T140R; D10V / S12F / T140Q; D10V / P15G / A17S; D10V / P15G / S18T; D10V / P15G / T140R; D10V / P15G / T140Q; D10V / A17S / S18T; D10V / A17S / T140R; D10V / A17S / T140Q; D10V / S18T / T140R; D10V / S18T / T140Q; S12F / A17S / S18T; S12F / A17S / T140R; S12F / A17S / T140Q; S12F / S18T / T140R; S12F / S18T / T140Q; P15G / A17S / S18T; P15G / A17S / T140R; P15G / A17S / T140Q; P15G / S18T / T140R; P15G / S18T / T140Q; A17S / S18T / T140R; A17S / S18T / T140Q; D9V / D10V / S12F / P15G; D9V / D10V / S12F / A17S; D9V / D10V / S12F / S18T; D9V / D10V / S12F / T140R; D9V / D10V / S12F / T140Q; D9V / D10V / P15G / A17S; D9V / D10V / P15G / S18T; D9V / D10V / P15G / T140R; D9V / D10V / P15G / T140Q; D9V / A17S / S18T / T140Q; D9V / S12F / P15G / S18T; D9V / S12F / P15G / T140R; D9V / S12F / P15G / T140Q; D9V / S12F / A17S / S18T; D9V / S12F / A17S / T140R; D9V / S12F / A17S / T140Q; D9V / S12F / S18T / T140R; D9V / S12F / S18T / T140Q; D10V / S12F / P15G / T140Q; D10V / S12F / A17S / T140Q; D10V / S12F / S18T / T140R; D10V / S12F / P15G / T140R; D10V / S12F / A17S / T140R; D10V / S12F / S18T / T140R; S12F / P15G / A17S / S18T; S12F / P15G / A17S / T140R; S12F / P15G / A17S / T140Q; S12F / P15G / S18T / T140R; S12F / P15G / S18T / T140Q; P15G / A17S / S18T / T140R; P15G / A17S / S18T / T140Q; D9V / D10V / S12F / P15G / T140Q; D9V / D10V / S12F / A17S / T140Q; D9V / D10V / S12F / S18T / T140R; D9V / D10V / S12F / P15G / T140R; D9V / D10V / S12F / A17S / T140R; D9V / D10V / S12F / S18T / T140R; D9V / S12F / P15G / A17S / S18T; D9V / S12F / P15G / A17S / T140R; D9V / S12F / P15G / A17S / T140Q; D9V / S12F / P15G / S18T / T140R; D9V / S12F / P15G / S18T / T140Q; D9V / P15G / A17S / S18T / T140R; D9V / P15G / A17S / S18T / T140Q; D10V / S12F / P15G / A17S / S18T; D10V / S12F / P15G / A17S / T140R; D10V / S12F / P15G / A17S / T140Q; D10V / S12F / P15G / S18T / T140R; D10V / S12F / P15G / S18T / T140Q; D10V / P15G / A17S / S18T / T140R; D10V / P15G / A17S / S18T / T140Q; S12F / P15G / A17S / S18T / T140R; S12F / P15G / A17S / S18T / T140Q; D9V / D10V / S12F / P15G / A17S / S18T; D9V / D10V / S12F / P15G / A17S / T140R; D9V / D10V / S12F / P15G / A17S / T140Q; D9V / D10V / S12F / P15G / S18T / T140R; D9V / D10V / S12F / P15G / S18T / T140Q; D9V / D10V / P15G / A17S / S18T / T140R; D9V / D10V / P15G / A17S / S18T / T140Q; D9V / S12F / P15G / A17S / S18T / T140R; D9V / S12F / P15G / A17S / S18T / T140Q; D10V / S12F / P15G / A17S / S18T / T140R; D10V / S12F / P15G / A17S / S18T / T140Q; D9V / D10V / S12F / P15G / A17S / S18T / T140R; D9V / D10V / S12F / P15G / A17S / S18T / T140Q.

6. A DNA molecule encoding any of the alginate lyase mutants of claims 1-5.

7. A recombinant expression plasmid comprising the DNA molecule of claim 6.

8. A host cell, characterized in that, The host cell comprises the recombinant expression plasmid as described in claim 7.

9. The host cell as described in claim 8, characterized in that, The host cell is Bacillus subtilis (B. subtilis) Bacillus subtilis ).

10. The application of the alginate lyase mutant according to any one of claims 1-5 in seaweed processing.