A truncated alginate lyase with wide temperature adaptability and application thereof

Abstract

A truncated alginate lyase with wide temperature adaptability and its applications. This invention belongs to the fields of marine microbial genetic engineering and enzyme engineering, specifically disclosing a truncated alginate lyase, Aly7YΔCBM16ΔCBM32, and its applications. The amino acid sequence of this truncated form is shown in SEQ ID NO.4, and the nucleotide sequence is shown in SEQ ID NO.3. Compared with the wild type, this truncated form exhibits approximately 2-fold increased specific enzyme activity, more than 3-fold extended half-life at 40°C, and a significantly wider temperature and pH adaptability range. Furthermore, after normalization to 100% of the maximum activity of each enzyme, this truncated form retains approximately 78% activity in 1% SDS, demonstrates enhanced catalytic activity for Poly G, and achieves approximately 90% relative enzyme activity even under sodium-free conditions. This truncated form exhibits excellent environmental tolerance and can be used for the efficient preparation of low-polymerization alginate oligosaccharides primarily composed of ΔDP2–ΔDP4.

Description

Technical Field

[0001] This invention belongs to the fields of marine microbial genetic engineering and enzyme engineering, and provides a truncated form of alginate lyase Aly7YΔCBM16ΔCBM32, its encoding gene, recombinant expression vector, engineered bacteria, and application method. Background Technology

[0002] Alginate lyases degrade alginate through β-elimination reactions, producing alginate oligosaccharides with various biological activities. These enzymes have significant applications in food, pharmaceuticals, cosmetics, and the resource utilization of marine waste. Based on substrate preference, they are mainly classified into Poly M lyases (which preferentially degrade polymannuronic acid), Poly G lyases (which preferentially degrade polyguluronic acid), and bifunctional lyases capable of simultaneously degrading both components.

[0003] Most reported alginate lyases are derived from culturable microorganisms, and these natural enzymes typically possess inherent limitations that restrict their industrial applications. For example, many wild-type alginate lyases have low optimal reaction temperatures, generally 30°C–35°C, and narrow temperature tolerance ranges. Their catalytic activity rapidly declines under fluctuating ambient temperatures or conditions outside their optimal range, i.e., below 20°C or above 40°C. Furthermore, these natural enzymes typically exhibit extremely low activity at low temperatures, such as in the 4°C–10°C range, and are easily inactivated during storage, limiting their application in scenarios requiring cryogenic processing or cold chain transportation.

[0004] Another common limiting factor is ion dependence. The catalytic activity of some alginate lyases depends on the activation by metal ions such as sodium ions, which poses a significant obstacle in applications where the ionic strength of the reaction system fluctuates or low-salt conditions are required. Furthermore, these natural enzymes are generally sensitive to and have poor tolerance to surfactants in the reaction system, such as sodium dodecyl sulfate (SDS), further limiting their application in complex industrial environments.

[0005] To improve enzyme performance, researchers have developed protein engineering strategies such as site-directed mutagenesis and directed evolution. However, these methods involve long experimental cycles, extensive screening, or require in-depth understanding of the enzyme's three-dimensional structure and catalytic mechanism, and the results are often uncertain. Modifying enzymes by deleting or recombinating functional domains is another approach. For example, the carbohydrate-binding module (CBM) is thought to enhance the enzyme's ability to bind to insoluble substrates. Existing research shows that CBM deletion has vastly different effects on different enzymes, and most are negative, such as significantly reducing catalytic activity or worsening thermal stability. Although some reports show that CBM deletion can lead to certain performance changes, such as increased activity or altered product profiles, the structural background, modification strategies, and effects obtained in these reports are significantly different from those of this invention. Currently, there are no reports of simultaneously achieving a wider temperature tolerance range, enhanced thermal stability, a wider pH tolerance range, enhanced catalytic activity against Poly G, enhanced surfactant tolerance, and elimination of ion dependence by deleting CBM.

[0006] The alginate lyase Aly7Y of this invention is derived from the metagenomic genome of uncultured marine Shewanella. It is a multi-domain enzyme containing two carbohydrate-binding modules, CBM16 and CBM32, and a catalytic domain, CD. Currently, there are no studies on the structure-function relationship of Aly7Y, nor are there any reports on the systematic construction and enzymatic evaluation of its truncated mutants. The functional division of Aly7Y's domains, the influence of the CBM modules on catalytic efficiency and ion dependence, and the feasibility of releasing catalytic potential through truncation optimization are all unknown. This invention is the first to conduct a truncated mutant study on Aly7Y, constructing a mutant that retains only the catalytic domain CD, resulting in a novel alginate lyase with a wide temperature tolerance range, wide pH tolerance range, high surfactant tolerance, high thermal stability, enhanced catalytic activity towards Poly G, and ion-independent enzyme activity. Summary of the Invention

[0007] The purpose of this invention is to provide a truncated alginate lyase, Aly7YΔCBM16ΔCBM32, which, compared with the wild-type alginate lyase Aly7Y, exhibits enhanced enzyme activity, thermal stability, temperature adaptability, pH tolerance, surfactant tolerance, catalytic activity towards Poly G, and ion independence, thus demonstrating superior application prospects in the preparation of alginate oligosaccharides.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A wild-type alginate lyase, Aly7Y, has the amino acid sequence shown in SEQ ID NO. 2.

[0010] The nucleotide sequence of the gene encoding the wild-type alginate lyase Aly7Y is shown in SEQ ID NO.1.

[0011] A truncated form of alginate lyase with wide temperature adaptability, Aly7YΔCBM16ΔCBM32, has the amino acid sequence shown in SEQ ID NO.4.

[0012] Furthermore, compared to the wild type, the truncated form Aly7YΔCBM16ΔCBM32 exhibits approximately 2 times higher specific enzyme activity; a more than 3 times longer half-life at 40°C; a significantly wider temperature and pH tolerance range; maintains approximately 78% activity in 1% SDS; enhanced catalytic activity for Poly G compared to the wild type; and still achieves approximately 90% relative enzyme activity under sodium-free conditions.

[0013] The nucleotide sequence of the gene encoding the truncated alginate lyase Aly7YΔCBM16ΔCBM32 is shown in SEQ ID NO.3.

[0014] The present invention also provides a recombinant vector containing the gene of the above-mentioned alginate lyase truncated form Aly7YΔCBM16ΔCBM32, wherein the vector is pET28a(+).

[0015] The present invention also provides engineered bacteria containing the above-mentioned alginate lyase truncated form Aly7YΔCBM16ΔCBM32, wherein the engineered bacteria is Escherichia coli BL21(DE3).

[0016] Application of the truncated alginate lyase Aly7YΔCBM16ΔCBM32 in the preparation of alginate oligosaccharides, wherein the truncated alginate is used to enzymatically hydrolyze alginate into low-polymerization alginate oligosaccharides mainly composed of ΔDP2–ΔDP4.

[0017] Furthermore, the enzymatic hydrolysis conditions are: substrate concentration 0.5% (w / v), temperature 10℃-40℃, and pH 6.0-10.0.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] The truncated form Aly7YΔCBM16ΔCBM32 exhibits enhanced enzyme activity and thermostability compared to the wild-type alginate lyase Aly7Y. The enzyme activity of Aly7YΔCBM16ΔCBM32 is approximately twice that of Aly7Y. Thermostability is also significantly enhanced, with a half-life at 40°C more than three times longer than Aly7Y, and a significantly wider temperature tolerance range and pH tolerance range. After normalization to 100% of the enzyme's maximum activity, Aly7YΔCBM16ΔCBM32 retains approximately 78% activity in 1% SDS, exhibits enhanced catalytic activity for Poly G, and achieves approximately 90% relative enzyme activity without sodium ion dependence. It demonstrates good environmental tolerance and can be used for the efficient preparation of alginate oligosaccharides, with the main products being alginate oligosaccharides with a degree of polymerization of 2-4. Attached Figure Description

[0020] Figure 1 Conservative structural domain analysis of Aly7Y and truncated bodies.

[0021] Figure 2 SDS-PAGE analysis of Aly7Y and its truncated form, M: Marker, 1: Aly7Y before purification, 2: Aly7Y after purification, 3: Aly7Y ΔCBM16 after purification, 4: Aly7Y ΔCBM16 ΔCBM32 after purification, 5: Aly7Y ΔCBM32 after purification.

[0022] Figure 3 The relative enzyme activity trends of Aly7Y and its truncated form at different reaction temperatures.

[0023] Figure 4 Temperature stability curves of Aly7Y and its truncated body.

[0024] Figure 5 Half-life analysis of Aly7Y and its truncated form at 40°C.

[0025] Figure 6 The relative enzyme activity differences of Aly7Y and its truncated form in different pH environments.

[0026] Figure 7 Effect of Na⁺ concentration on the relative enzyme activity of Aly7Y and its truncated form.

[0027] Figure 8 Relative enzyme activity analysis of Aly7Y and its truncated form on different substrates.

[0028] Figure 9 The degree of influence of chemical reagents on the relative enzyme activity of Aly7Y and its truncated form.

[0029] Figure 10 TLC analysis of Aly7Y and its truncated enzymatic digestion end products ( Figure 10 ESI-MS analysis of the final enzymatic hydrolysis product of Aly7Y (A), Aly7Y (Aly7Y). Figure 10 (Middle B), lanes 1-4: enzymatic hydrolysis final products of Aly7Y, Aly7YΔCBM16, Aly7YΔCBM16ΔCBM32 and Aly7YΔCBM32.

[0030] Figure 11 Molecular docking analysis of Aly7Y and Aly7YΔCBM16ΔCBM32. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1: Source and Sequence Analysis of Alginate Lyase

[0033] Wild-type enzymes are from the NCBI database and are uncultured. Shewanella The uncharacterized PL7 family protein sp. was named Aly7Y after removing the signal peptide, and its amino acid sequence is shown in SEQ ID NO:2. After codon optimization, its nucleotide sequence is shown in SEQ ID NO:1. Aly7Y contains three domains: from the N-terminus to the C-terminus, the carbohydrate-binding domain CBM16, the carbohydrate-binding domain CBM32, and the catalytic domain CD. To study the function of each domain, the CBM domain of Aly7Y was truncated, and the truncated alginate lysins Aly7YΔCBM16, Aly7YΔCBM16ΔCBM32, and Aly7YΔCBM32 were obtained. Domain analysis is shown below. Figure 1 As shown.

[0034] Example 2: Construction and expression of alginate lyase Aly7Y and its truncated form

[0035] 1. Construction of the truncated form of alginate lyase Aly7Y

[0036] The Aly7Y gene was synthesized by Kangwei Century Biotechnology Co., Ltd. (Jiangsu, China). The synthesized gene was processed... Eco RI / HinAfter double digestion with dIII, the recombinant plasmid was cloned into the pET28a(+) vector. The validated recombinant plasmid was transformed into E. coli BL21(DE3) for protein expression. Using pET28a(+)-Aly7Y as a template, the gene fragments Aly7YΔCBM16 and Aly7YΔCBM16ΔCBM32 were amplified using the specific primers listed in Table 1. Among them, primers Aly7YΔCBM16-R and Aly7YΔCBM16ΔCBM32-R were complementary to pET28a(+) and were used to amplify the corresponding inserted fragments in the wild-type plasmid pET28a(+)-Aly7Y. Then, the fragments were inserted into the pET28a(+) vector via homologous recombination to obtain the recombinant plasmids pET28a(+)-Aly7YΔCBM16 and pET28a(+)-Aly7YΔCBM16ΔCBM32. The pET28a(+) vector was linearized by inverse PCR using primers pET28a(+)-F and pET28a(+)-R.

[0037] The pET28a(+)-Aly7YΔCBM32 recombinant plasmid was constructed using an enzyme digestion and ligation method. The CBM16 gene was amplified using primers Aly7YΔCBM32-F and Aly7YΔCBM32-R listed in Table 1. The successfully constructed pET28a(+)-Aly7YΔCBM16ΔCBM32 recombinant plasmid and the amplified CBM16 gene were then used separately... Eco The plasmid pET28a(+)-Aly7YΔCBM32 was obtained by digestion with RI (recognition site: GAATTC, cleavage site: between G and A) and then ligation reaction.

[0038] After all the constructed recombinant plasmids were transformed into E. coli DH5α competent cells and verified by sequencing, the correct plasmids were transformed into E. coli BL21(DE3) for subsequent experiments.

[0039] Table 1. Primers for constructing truncated enzyme plasmids

[0040]

[0041] 2. Induction and expression of recombinase and preparation of crude enzyme solution

[0042] Recombinant Escherichia coli BL21(DE3) was inoculated into LB medium containing 50 μg / mL kanamycin sulfate and cultured overnight. The inoculum was then transferred at a 1% inoculum to LB medium with the same antibiotic resistance and cultured at 37°C with shaking at 150 r / min until OD500. 600Approximately 0.5. Add IPTG to a final concentration of 0.2 mM, and induce expression at 16℃ and 150 r / min for 20 h. Collect bacterial cells by centrifugation, resuspend in lysis buffer (20 mM Tris-HCl, 800 mM NaCl, pH 8.0), sonicate (230 W, working / interval 3 s / 5 s, 20 min), centrifuge at 4℃ and collect the supernatant, which is the crude enzyme solution.

[0043] 3. Enzyme purification based on affinity chromatography

[0044] The crude enzyme solution was loaded onto BeyoGold™ His-tag purification resin and bound at 4°C for 2 h. After washing four times with washing buffer (containing 2 mM imidazole), it was eluted three times with elution buffer (containing 50 mM imidazole), and the eluent was collected. Purity was determined by SDS-PAGE. Figure 2 High-purity alginate lyase Aly7Y and its truncated form were successfully obtained.

[0045] Example 3: Enzymatic Characterization of Aly7Y and its Truncated Form

[0046] 1. Detection method for enzyme activity of alginate lyase Aly7Y and its truncated form

[0047] The DNS method was used to determine the activity of alginate lyase. The reaction system consisted of 400 μL of 0.5% (w / v) substrate (sodium alginate, Poly M, or Poly G) and 100 μL of enzyme solution (0.1 mg / mL). The mixture was incubated at the optimal temperature for each enzyme for 20 min. The reaction was terminated by adding 0.5 mL of DNS reagent, followed by color development in a 100°C boiling water bath for 5 min. After cooling to room temperature (25℃±3℃), the volume was adjusted to 2.5 mL, and the absorbance was measured at 540 nm. A heat-inactivated enzyme solution was used as a blank control. One unit of enzyme activity (U) is defined as the amount of enzyme required to release 1 μg of reducing sugar (in glucose equivalents) per minute under the above conditions. The highest specific enzyme activities of Aly7YΔCBM16, Aly7YΔCBM16ΔCBM32, and Aly7YΔCBM32 were approximately 1.4, 2, and 0.9 times that of Aly7Y, respectively.

[0048] 2. Screening of optimal temperature conditions for alginate lyase Aly7Y and its truncated form

[0049] The optimal temperature range for Aly7Y and its truncated form is 10°C–65°C, with a gradient of 5°C. Enzyme activity was determined using the DNS method described above. After normalization to 100% of the maximum activity of each enzyme, the relative activity was calculated using the highest enzyme activity as 100% for the optimal temperature determination.

[0050] The optimal temperature for Aly7Y is 30°C, with poor activity at both 10°C and 40°C. However, the optimal temperature for Aly7YΔCBM16ΔCBM32 rises to 35°C, maintaining approximately 87% and 91% of its activity at 10°C and 40°C, respectively. After normalization to 100% of the enzyme's maximum activity, the temperature adaptation range is significantly broadened. Aly7YΔCBM16 also shows improved low-temperature activity, reaching approximately 84% at 10°C, suggesting that CBM16 may limit conformational flexibility at low temperatures. Conversely, the activity of Aly7YΔCBM32 decreases at the mesophilic 35°C, indicating that CBM32 contributes to maintaining mesophilic activity. The two CBMs exhibit modular differential regulatory effects on the temperature adaptability of Aly7Y, and their functions are not simply additive. Figure 3 ).

[0051] 3. Temperature tolerance characterization of alginate lyase Aly7Y and its truncated form

[0052] Thermostability was assessed by measuring the residual activity of the enzyme solutions after incubation at 4, 25, 30, 35, and 40°C for 12 h, and their half-life at 40°C was also tested. For the thermostability determination, the enzyme activity before incubation was used as a control (100%), and the relative activity was calculated.

[0053] After incubation at 4–40°C for 12 h, the residual activity of Aly7YΔCBM16ΔCBM32 remained the highest, and at 30°C it was approximately 84% higher than that of Aly7Y. Figure 4 At 40°C, its half-life is more than 3 times longer than that of Aly7Y. Figure 5 The stability of Aly7YΔCBM16 also improved, but Aly7YΔCBM32 performed the worst. This indicates that the functionality of CBMs depends on the specific context.

[0054] 4. Determination of the optimal reaction pH for alginate lyase Aly7Y and its truncated form.

[0055] The optimal pH was determined using 50 mM citrate buffer (pH 3.0–6.0), 50 mM Tris-HCl buffer (pH 6.0–8.0), and 50 mM glycine-NaOH buffer (pH 8.0–10.0). Enzyme activity was measured using the DNS method described above, and relative activities were calculated by normalizing each enzyme to its maximum activity as 100%.

[0056] The optimal pH for both Aly7Y and its truncated form is 8.0, but Aly7YΔCBM16ΔCBM32 exhibits a significantly broadened pH tolerance range: at pH 10, the residual enzyme activity is approximately 59% higher than that of Aly7Y, and it retains approximately 24% activity under strongly acidic conditions at pH 4, while Aly7Y almost completely loses its activity under the same conditions. Figure 6 ).

[0057] 5. Sodium tolerance test of alginate lyase Aly7Y and its truncated form

[0058] Different concentrations of NaCl (0–2M) were added to the reaction system to investigate the effect of sodium ions on enzyme activity. The results were compared after normalization to 100% with the maximum activity of each enzyme as the baseline.

[0059] Aly7Y and Aly7YΔCBM16 exhibited extremely low activity in the absence of Na⁺, approximately 8% and 14%, respectively, with optimal Na⁺ concentrations of 800 mM and 200 mM, respectively. Aly7YΔCBM16ΔCBM32 showed the lowest Na⁺ dependence, retaining approximately 90% activity in the absence of Na⁺, with an optimal Na⁺ concentration of 500 mM. All three maintained greater than 40% activity at 2 M Na⁺, demonstrating outstanding high-salt tolerance. In contrast, Aly7YΔCBM32 exhibited a unique low-salt preference, with an optimal Na⁺ concentration of 0 mM; the presence of Na⁺ actually inhibited its activity. Figure 7 ).

[0060] 6. Determination of substrate specificity of alginate lyase Aly7Y and its truncated form

[0061] Substrate specificity was determined using sodium alginate, Poly M, and Poly G as substrates (0.5%, w / v), respectively. Enzyme activity was determined using the DNS method described above, and relative activities were calculated by normalizing to 100% of the maximum activity of each enzyme.

[0062] The relative enzyme activities of Aly7Y for sodium alginate, Poly G, and Poly M were approximately 100%, 7%, and 19%, respectively (Figure 8). This indicates that Aly7Y prefers the natural sodium alginate substrate and exhibits weaker catalytic activity for the two homopolymer substrates. However, the truncated forms Aly7YΔCBM16 and ΔCBM32 showed a significant increase in relative enzyme activity for Poly G to approximately 48%, while the relative enzyme activities for Poly M and sodium alginate remained essentially unchanged.

[0063] 7. Chemical tolerance test of alginate lyase Aly7Y and its truncated form

[0064] Different metal ions (final concentration of 1 mM) or chemical reagents (final concentration of 1% (w / v)) were added to the reaction system to investigate their effects on enzyme activity. The enzyme activity was determined by the DNS method described above. The reaction systems without metal ions or chemical reagents were used as controls (100%) to calculate the relative activity.

[0065] Most reagents showed no significant inhibition of these four enzymes. All four enzymes exhibited SDS tolerance, with Aly7YΔCBM16ΔCBM32 retaining approximately 78% activity in 1% SDS. The high proportion of acidic amino acids (low pI) may have weakened the effect of anionic surfactants to some extent through electrostatic repulsion, but the tolerance mechanism of the β-jelly roll structure of the PL7 family to denaturants requires further investigation. Figure 9 ).

[0066] 8. Identification of enzymatic hydrolysis products of alginate lyase Aly7Y and its truncated form

[0067] The purified enzyme and sodium alginate were mixed at a ratio of 1:9 (v / v) and incubated under optimal reaction conditions for 24 h. The reaction was terminated by boiling in a water bath for 10 min. The enzymatic hydrolysis product was separated and purified by ethanol precipitation: first, the supernatant was centrifuged at 10,000 r / min for 10 min to remove denatured proteins and residual substrates; the supernatant was collected, and anhydrous ethanol was added to a final volume fraction of 70%, and the mixture was allowed to stand at 4 ℃ for 24 h, then centrifuged again at 10,000 r / min for 10 min to discard the precipitate. The secondary supernatant was collected and concentrated by rotary evaporation under reduced pressure at 60 ℃ to obtain alginate oligosaccharide (AOS) sample. The AOS was reconstituted with ultrapure water to prepare a 10 mg / mL stock solution, which was then filtered through a 0.22 μm microporous membrane for sterilization and stored at 4 ℃ in the dark. Product distribution analysis was performed using thin-layer chromatography (TLC): 5 μL sample was applied, the developing solvent was n-butanol–formic acid–water (4:6:1, V / V / V), and the colorimetric reagent was 10% sulfuric acid in ethanol. The mixture was baked at 105 °C for 5 min for color development. Degree of polymerization was determined using negative ion mode electrospray ionization mass spectrometry (ESI-MS).

[0068] The degradation products were analyzed using TLC and ESI-MS. Figure 10 As shown in Figure A, after 24 h of degradation of sodium alginate by Aly7Y and its truncated form, TLC showed three main bands, with migration positions corresponding to unsaturated disaccharides, trisaccharides, and tetrasaccharides, respectively, indicating that the removal of CBM did not alter the product distribution spectrum. Since saturated sugars (glucose, sucrose, raffinose, and stachyose) were used as markers, a slight migration difference between the markers and the products was observed. ESI-MS further confirmed the molecular weight of the Aly7Y product, as shown in Figure A. Figure 10 As shown in Figure B, the unsaturated disaccharide (m / z 351.06), unsaturated trisaccharide (m / z 527.09), and unsaturated tetrasaccharide (m / z 703.13) are consistent with the TLC results. The products are mainly disaccharides, trisaccharides, and tetrasaccharides, indicating that Aly7Y and its truncated form are endoglucan lyases.

[0069] 9. Molecular docking of alginate lyase Aly7Y and its truncated form

[0070] Hydrogen bond analysis showed that ( Figure 11 The number of hydrogen bonds formed between Aly7YΔCBM16ΔCBM32 and the three substrates was greater than that of Aly7Y: for the H4 substrate, the number of hydrogen bonds increased from 8 to 14; for the M4 substrate, from 13 to 17; and for the G4 substrate, from 11 to 17. The specific residues involved in hydrogen bonding interactions are shown in Table 2. LigPlus analysis revealed that the hydrophobic interactions between Aly7YΔCBM16ΔCBM32 and the substrates were also increased compared to Aly7Y. This indicates that the interaction between the substrate and the catalytic domain is enhanced after removing CBM. Furthermore, the active pocket volume of Aly7YΔCBM16ΔCBM32 (579 ų) was significantly larger than that of Aly7Y (351 ų), and the distances between the Brønsted base His172 and the Brønsted acid Tyr279 and the substrate in Aly7YΔCBM16ΔCBM32 (4.5 Å and 5.1 Å, respectively) were shorter than the corresponding distances in Aly7Y (5.9 Å and 6.4 Å, respectively). Figure 11 (G, H). This indicates that the removal of CBM opens the catalytic crack by eliminating steric hindrance, making it easier for the substrate to be tuned to the catalytic conformation.

[0071] Table 2. List of residues involved in hydrogen bond interactions

[0072]

[0073] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and not restrictive.

Claims

1. A truncated alginate lyase Aly7YΔCBM16ΔCBM32 with wide temperature adaptability, characterized in that, The amino acid sequence of the truncated alginate lyase Aly7YΔCBM16ΔCBM32 is shown in SEQ ID NO.

4.

2. The truncated alginate lyase Aly7YΔCBM16ΔCBM32 according to claim 1, characterized in that, Compared with the wild type, the truncated alginate lyase has approximately 2 times higher specific enzyme activity; its half-life is extended by more than 3 times at 40°C; and its temperature and pH adaptation range is significantly broadened.

3. The truncated alginate lyase Aly7YΔCBM16ΔCBM32 according to claim 1, characterized in that, The truncated alginate lyase retains approximately 78% activity in 1% SDS; its catalytic ability for Poly G is enhanced compared to the wild type; and it can still achieve approximately 90% relative enzyme activity under sodium-free conditions.

4. The gene encoding the truncated alginate lyase Aly7YΔCBM16ΔCBM32 according to claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

3.

5. The application of the truncated alginate lyase Aly7YΔCBM16ΔCBM32 as described in claim 1 in the preparation of alginate oligosaccharides, characterized in that, The application involves using the truncated alginate lyase to degrade alginate into low-polymerization alginate oligosaccharides mainly composed of ΔDP2–ΔDP4.

6. A recombinant expression vector, characterized in that, The recombinant expression vector is a pET28a(+) vector containing the encoding gene of claim 4.

7. The application according to claim 5, characterized in that, The degradation conditions are as follows: substrate concentration 0.5% (w / v), temperature 10℃-40℃, and pH 6.0-10.

0.

8. An engineered bacterium, characterized in that, The engineered bacteria is Escherichia coli BL21(DE3) containing the recombinant expression vector of claim 6.

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