A fucanase based on ancestral sequence reconstruction and use thereof

Abstract

The application belongs to the technical field of bioengineering and its application, and particularly relates to a fucanase based on ancestor sequence reconstruction and application thereof. The application constructs a supposed artificial protein with endo-1,3-fucan activity through an ancestor sequence reconstruction strategy, thereby obtaining an artificial endo-1,3-fucanase which is completely different from the sequence of the fucanase of a natural source, and the amino acid sequence of the artificial endo-1,3-fucanase is SEQ ID NO. 1. The fucanase can degrade fucans of various sources in an endo manner, and under the condition of controlling the enzyme amount or the reaction time, the fucan can be rapidly degraded to generate fucosyl oligosaccharides with specific structures within 10 min.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and its application technology, specifically relating to a fucoidanase based on ancestral sequence reconstruction and its application. Background Technology

[0002] Fucoidan is an important class of marine food polysaccharides, primarily composed of sulfated L-fucose, with highly diverse distribution patterns of its sulfate ester groups along the sugar chains. According to current classification, fucoidan can be divided into Type I and Type II. Type I fucoidan consists of repeating unit structures composed of fucose linked by α-1,3 glycosidic bonds, and is mainly distributed in sea cucumbers, sea urchins, and brown algae of the Laminaria and Sylvales orders. Type II fucoidan consists of repeating unit structures composed of fucose linked by alternating α-1,3 and α-1,4 glycosidic bonds, and is mainly distributed in brown algae of the Fucus orders. Fucoidan possesses rich physiological regulatory functions and excellent biomaterial properties, and its application potential is widely recognized.

[0003] Low molecular weight fucoidan or fucoidan oligosaccharides obtained by fucoidanase degradation of fucoidan have the characteristics of low viscosity, good solubility, and high bioavailability, and have been proven to possess a variety of physiological activities. Fucoidan oligosaccharides are used to develop high value-added products such as functional foods. Pharmaceuticals and third-generation functional foods require their active ingredients to have a clearly defined structure; therefore, preparing high-purity oligosaccharide monomers rather than oligosaccharide mixtures has become a recognized trend in oligosaccharide preparation.

[0004] Targeted preparation of oligosaccharides refers to the preparation of high-purity oligosaccharide products with specific structures. Endo-1,3-fucosanase is the core enzyme for targeted preparation of fucoidan oligosaccharides. Currently, there are relatively few known fucases. A search revealed only two Chinese patents related to this topic: Chinese patent CN112654709A discloses the enzymatic hydrolysis of fucoidan, involving fucases from *Psychrophilus* species, including P5AFcnA and P19DFcnA fucases; Chinese patent CN111789235A discloses a combined enzymatic hydrolysis method for sea cucumber based on fucase and protease, involving an endo-1,3-fucosanase. However, all patented enzymes and fucases disclosed in databases are of naturally derived origin, not artificial enzymes. The ancestral sequences of enzyme families can be deduced through ancestral sequence reconstruction strategies. Compared with modern enzymes, ancestral enzymes usually have better thermal stability and have more significant advantages in the actual production of oligosaccharides. This not only has the potential to overcome the bottleneck of insufficient thermal stability of natural enzymes, but also to further optimize the enzymatic properties through subsequent molecular modification, making them more suitable for the process parameters of industrial production. At the same time, artificial synthesis can also achieve large-scale and standardized preparation, providing higher-quality enzyme preparation support for the efficient enzymatic hydrolysis and conversion of polysaccharides such as fucoidan and the industrial production of oligosaccharides. However, research on the ancestral sequence reconstruction of fucoidanase and the development of artificial enzymes is currently lacking, and relevant technological exploration is urgently needed to fill this technological gap. Summary of the Invention

[0005] The technical problem that this invention aims to solve is that there is currently a lack of research on the reconstruction of the ancestral sequence of fucoidanase and the development of artificial enzymes, and there is an urgent need to explore related technologies to fill this gap.

[0006] To address the aforementioned issues, this invention constructs a putative artificial protein with endoglucan activity using an ancestral sequence reconstruction strategy, thereby obtaining an artificial endoglucanase with a sequence completely different from that of naturally derived fucoidanase. This fucoidanase enables the large-scale production of fucoidan oligosaccharides.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: a fucoidanase based on ancestral sequence reconstruction, the amino acid sequence of which is SEQ ID NO. 1, and an enzyme derived from SEQ ID NO. 1 that has been substituted, deleted or added to one or more amino acids and still has continuous endoglucan activity for α-1,3-fucosan.

[0008] SEQ ID NO. 1:

[0009] MKFYSILMNKRLIVGSLVFIVSAGLPNVTLTQNSTIVDNEKISDFYISDGSKFVPKDFYPKFSWETTPMYCMFGDGTRLLTPKEVEFIAARTDFICIEKNHGR TTLGAAEVGAKHEAKAFKKIKPDIKVLFYFNSAYAWPFTSYNENFTRNKIDEYPELKKFLIVDKTTGELQHRNNVFFFDVLNPEFREWWSNTVAQGVKDSGADG VFIDQMHGFAWLRSDKKEEVEKAMGEMMANLKRKLGPDKILLGNNASSDIAKDVFPAIDAAMFEHYNNKKLSKENLLKDWDDMLKNAKAGKMSIFRIGVESEEE ASQDQGIRGSRRDALEKLAKERLEYYLACYLIGAQPYSYFQYGWGWRLDTGSLVDYPELQKPLGPPKGAYKRVHENGWEFTREFEHASVWVDTENKEAKITWK.

[0010] This sequence is the amino acid sequence of an artificial enzyme constructed using an ancestral sequence reconstruction strategy. Sequence alignment revealed that this hypothetical protein has no significant similarity to currently known enzymes. High-performance liquid chromatography with tandem differential detection technology confirmed that this enzyme possesses degradation activity against α-1,3-fucosan. Figure 1 This protein can specifically cleave the α-1,3 glycosidic bonds on the fucoidan backbone and produce oligosaccharides with specific structures, thus confirming that this hypothetical protein is a novel artificial endo-1,3-fucosanase.

[0011] Its optimal reaction temperature is 40℃, making it suitable for common medium-temperature reaction systems in industrial production. This eliminates the need for additional high- or low-temperature control equipment, reducing energy consumption and equipment costs. Furthermore, its excellent stability allows it to remain stable for at least one day under refrigeration at 4℃, room temperature at 25℃, and near the reaction temperature at 30℃, with residual enzyme activity remaining above 80%. This significantly extends the shelf life and reaction duration of the enzyme preparation, reducing the frequency of enzyme replenishment and improving production continuity. Regarding pH adaptability, this ancestral enzyme has a wide pH tolerance range, exhibiting catalytic activity between pH 6.5 and 10.5, with an optimal reaction pH of 8.0. This makes it compatible with fucoidan hydrolysis systems from various raw material sources (where pH fluctuations are common after pretreatment of some raw materials), eliminating the need for frequent pH adjustments, simplifying the production process, and reducing the cost of acid-base regulators. More importantly, its enzyme activity is largely unaffected by NaCl, while polysaccharide raw materials such as fucoidan often contain a certain amount of salt. Therefore, no additional desalting pretreatment step is needed during industrial enzymatic hydrolysis, further simplifying the process and reducing pretreatment costs. Based on these superior characteristics, this ancestral enzyme not only holds promise for overcoming the bottleneck of insufficient thermal stability of natural enzymes, but its enzymatic properties can also be further optimized through subsequent molecular modification to better suit the process parameters of industrial production. Furthermore, its artificial synthesis method enables large-scale, standardized preparation, providing higher-quality enzyme preparation support for the efficient enzymatic hydrolysis and conversion of polysaccharides such as fucoidan and the industrial production of oligosaccharides.

[0012] The nucleotide sequence encoding the above-mentioned fucoidanase SEQ ID NO. 1 corresponds to all genes that can be translated into the amino acid sequence of SEQ ID NO. 1.

[0013] This invention provides a method for preparing the above-mentioned fucoidanase, wherein the enzyme is heterologously expressed in systems such as Escherichia coli, Bacillus subtilis, and Pichia pastoris, and artificial endo-1,3-fucoidanase can be prepared in large quantities by inducing enzyme production.

[0014] The above-mentioned fucoidanase is used in the targeted preparation of fucoidan oligosaccharides with specific structures. By controlling the amount of enzyme added, reaction time, or substrate concentration, fucoidan and oligosaccharides with different molecular weights of 28-560 kDa can be prepared.

[0015] This invention also provides a method for the targeted preparation of fucoidan oligosaccharides with specific structures based on the above-mentioned fucoidanase. The amount of enzyme added is adjusted according to the amount of substrate added to the reaction system, and the reaction is carried out at 30-40℃ for 1-60 minutes to obtain fucoidan oligosaccharides.

[0016] The beneficial effects of this invention are as follows:

[0017] (1) The artificial endonuclease-1,3-fucognanase gene reconstructed based on the ancestral sequence of this invention can be overexpressed in common expression systems such as Escherichia coli, lactic acid bacteria, Bacillus subtilis, and Pichia pastoris through genetic engineering.

[0018] (2) The fucoidanase based on the ancestral sequence reconstruction of the present invention can degrade fucoidan from various sources in an endoglucan manner. Under the condition of controlling the amount of enzyme added or the reaction time, fucoidan can be rapidly degraded to generate fucoidan oligosaccharides with specific structures within 10 minutes. Attached Figure Description

[0019] Figure 1 The results of enzymatic hydrolysis of oligosaccharides by the fucoidanase of the present invention;

[0020] Figure 2 The results of biochemical property analysis of the fucoidanase of the present invention; wherein, A is the effect of temperature on activity; B is the effect of pH on activity; C is the effect of time on residual activity; D is the effect of pH on residue; E is the effect of concentration on activity;

[0021] Figure 3 Schematic diagram of the fucoidanase of the present invention producing fucoidan of different molecular weights by controlling the amount of enzyme added;

[0022] Figure 4 Schematic diagram of the fucoidanase of the present invention producing fucoidan of different molecular weights under controlled reaction time conditions;

[0023] Figure 5 : Schematic diagram of the fucoidanase of the present invention producing fucoidan of different molecular weights under controlled substrate concentration conditions. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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 some embodiments of the present invention, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, all raw materials used in the following embodiments were purchased from the market.

[0025] The ancestral sequence was reconstructed to obtain the amino acid sequence:

[0026] First, existing homologous protein sequences of the target enzyme family (GH168) are collected and multiple sequence alignment is performed to determine conserved and variant sites. Then, a phylogenetic tree is constructed based on the alignment results to infer the evolutionary relationship between the sequences. On this basis, the maximum likelihood method is used to statistically infer the amino acid state of the internal nodes in the phylogenetic tree according to the evolutionary model. Finally, the combination of amino acid residues with the highest probability is selected to obtain the inferred ancestral enzyme sequence, which is the amino acid sequence of the present invention.

[0027] Example 1: Heterologous expression of artificial endonuclease-1,3-fucosanase in Escherichia coli:

[0028] The nucleotide sequence that can be correctly translated from the ancestral amino acid sequence was obtained through codon optimization, as shown in SEQ ID NO. 2.

[0029] SEQ ID NO. 2:

[0030] .

[0031] The entire gene was synthesized using a gene synthesizer and ligated into the pET-28a(+) vector to form a recombinant plasmid. The recombinant plasmid was then introduced into BL21(DE3) competent cells to form a recombinant bacterial strain. Positive clones were screened and induced to express the recombinant strain using isopropyl thiogalactoside in LB medium containing suitable antibiotics at 17°C for 12 hours. The bacterial cells were collected by centrifugation, washed and resuspended in buffer, and then sonicated to obtain an intracellular enzyme solution containing endo-1,3-fucosanase (crude enzyme solution).

[0032] Example 2: Heterologous expression of artificial endonuclease-1,3-fucosanase in Bacillus subtilis:

[0033] A nucleotide sequence that could correctly translate the ancestral amino acid sequence was obtained through codon optimization, and the entire gene was synthesized using a gene synthesizer. The target gene was ligated into the pMA5 vector via seamless cloning, and the constructed expression vector was transformed into the Bacillus subtilis BS WB600 expression host. Positive recombinants were screened on kanamycin-resistant plates. The verified recombinant bacteria were inoculated into LB medium containing kanamycin resistance and cultured at 37°C for 12 h. Then, a 1% inoculum was transferred to LB medium containing kanamycin resistance and cultured at 37°C for 16 h. The bacterial cells were collected by centrifugation, washed and resuspended with buffer, and sonicated to obtain an intracellular enzyme solution, i.e., a crude enzyme solution containing endo-1,3-fucosanase.

[0034] Example 3: Heterologous expression of artificial endonuclease-1,3-fucosanase in Pichia pastoris:

[0035] A nucleotide sequence that could correctly translate the ancestral amino acid sequence was obtained through codon optimization, and the whole gene was synthesized using a gene synthesizer. The target gene was ligated into a linearized pPIC9K vector using seamless cloning. The constructed recombinant plasmid was electroporated into GS115 competent cells, and single colonies were extracted for PCR verification. Single colonies with correct sequencing and antibiotic selection were cultured in YPD liquid medium at 30°C and 220 rpm for 12 h. Then, they were inoculated into BMGY medium at pH 6.0 and cultured at 30°C and 220 rpm until the OD600 reached 5.0. After centrifugation, the culture was repeated in BMMY medium at pH 6.0, and expression was induced for 72 h at 29°C and 220 rpm with the addition of 0.5% methanol. The extracellular supernatant was collected by centrifugation, which was the crude enzyme solution.

[0036] Example 4: Comparison of the activity of artificial endonuclease-1,3-fucosanase in various expression systems:

[0037] 50 μL of appropriately diluted enzyme solution from Examples 1-3 was mixed with 50 μL of 2 mg / mL fucoidan solution and reacted at 35°C for 10 min, followed by inactivation at 100°C for 5 min. The expressed protein from the empty host bacteria used in Examples 1-3 was used as a negative control to demonstrate that its endogenous expressed protein did not interfere with activity. The reducing sugar content in the experimental and control systems was detected using the pHBH method to calculate the enzyme activity of endo-1,3-fucosanase. 1 U of activity was defined as the activity of generating 1 μmol of reducing sugar within 1 min. The activity of 1 mL of fermentation broth in different expression systems detected by the pHBH method is shown in the table below:

[0038] Table 1: Activity of 1 mL fermentation broth in different expression systems:

[0039] .

[0040] No enzyme activity was detected in the negative control group, while enzyme activity was detected in the recombinant bacteria containing this sequence (see Table 1), proving that the active protein is the one expressed by this sequence. Furthermore, the results show that the endonuclease-1,3-fucosanase of this invention can be successfully expressed in heterologous systems such as *Escherichia coli*, *Bacillus subtilis*, and *Pichia pastoris*, with the highest expression activity in *E. coli*, making it suitable for the production of chemical reagents. The *Pichia pastoris* expression system enables extracellular expression of the recombinant enzyme with low exogenous protein content, simplifying subsequent isolation and purification procedures and facilitating its application in the development and production of health products, food, and pharmaceuticals.

[0041] Example 5: Enzymatic properties of endo-1,3-fucosanase:

[0042] To obtain the optimal reaction conditions for endo-1,3-fucosinase, the effects of temperature and pH were investigated.

[0043] 1) Optimal reaction temperature and temperature stability:

[0044] The recombinant enzyme solution obtained from *E. coli* in Example 1 was appropriately diluted and mixed with a fucoidan substrate solution at pH 8.0 and 2 mg / mL. The mixture was reacted at 15, 20, 25, 30, 35, 40, 45, 50, 55, and 60 °C for 10 min. After inactivation, the enzyme activity was determined using the pHBH method (reducing sugar increment detection method), and the specific enzyme activity was calculated. The results are as follows: Figure 2 As shown in Figure A, the optimal reaction temperature is 40℃. After placing an appropriate amount of enzyme at 4℃, 25℃, 30℃, and 40℃ for 0, 1, 2, 4, 6, 16, and 24 hours, a certain amount of enzyme was mixed with the substrate solution to determine its activity. The activity at 4℃ for 0 hours was taken as 100%, and the result is expressed as residual enzyme activity. Figure 2 (B) The results showed that this enzyme could be stably stored for at least 1 day at 4℃, 25℃, and 30℃ (with residual enzyme activity >80%).

[0045] 2) Reaction pH and pH stability:

[0046] Using the recombinant enzyme solution obtained from *E. coli* in Example 1, an appropriate amount was mixed with a 2 mg / mL fucoidan substrate solution prepared with different pH buffers (citrate-disodium hydrogen phosphate buffer at pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0; sodium dihydrogen phosphate-disodium hydrogen phosphate buffer at pH 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0; and sodium carbonate-sodium bicarbonate buffer at pH 9.0, 9.5, 10.0, 10.5, and 11.0). The reaction was carried out at different pH environments for 10 min. After inactivation, the activity was determined using the pHBH method, and the specific enzyme activity was calculated. The results are as follows: Figure 2 As shown in Figure C, endo-1,3-fucosanase exhibits activity at pH values ​​ranging from 6.5 to 10.5; its optimal reaction pH is 8.0. An appropriate amount of enzyme was placed at the above pH values ​​for 1 hour, and then the pH was adjusted to 8.0 before mixing with the substrate for enzyme activity assay. The enzyme activity at the highest point was recorded as 100%, and all other values ​​were expressed as residual enzyme activity. Figure 2 The results showed that the enzyme remained stable at pH 6.5-10.0 (with residual enzyme activity >80%), indicating that the enzyme has a wide pH stability range.

[0047] 3) Effect of NaCl on artificial endo-1,3-fucosanase:

[0048] Different concentrations of NaCl were added to the enzymatic hydrolysis reaction, and then the relative residual enzyme activity was calculated. Figure 2 (E), the results showed that the enzyme activity was basically unaffected by NaCl.

[0049] Example 6: The degradation ability of artificial endo-1,3-fucosanase on various fucoidans:

[0050] The recombinant enzyme from the *E. coli* expression system, diluted to 50 μL, was mixed with 50 μL of fucoidan solutions derived from *Sea cucumber*, *Sea cucumber*, *Sea cucumber*, *Sea cucumber*, and *Sea cucumber* (2 mg / mL). The mixture was reacted at 30°C for 10 min, followed by inactivation at 100°C for 5 min. Similarly, 50 μL of the inactivated enzyme solution was mixed with each type of fucoidan solution under the same conditions as a control. The reducing sugars in the experimental and control systems were detected using the pHBH method, and the enzyme activity of endo-1,3-fucosanase was calculated. The results are shown in the table below, with the enzyme activity defined as in Example 4. The results indicate that the endo-1,3-fucosanase of this invention has the ability to degrade fucoidan from at least the three sources described in this example, and can produce various low-molecular-weight fucoidan and oligosaccharides with different structures. This can be used for the structural analysis and structure-activity relationship studies of fucoidan.

[0051] Table 2: Degradation capacity of the fucoidanase of the present invention for various fucoidans:

[0052] .

[0053] Example 7: Fucoidan of different molecular weights can be prepared by controlling the amount of enzyme added:

[0054] The recombinases obtained from the *E. coli* system in Example 1 (0.001 U, 0.05 U, 0.1 U, 0.15 U, 0.2 U, 0.4 U, 0.6 U, 0.8 U, and 1 U) were added to 100 mg (2 mg / mL) fucoidan solution at pH 8.0, reacted at 40°C for 1 h, and then 500 μL of each solution was inactivated. The molecular weight of fucoidan was monitored using a Shodex OHpak LB-806M gel column connected to a differential detector and a multi-angle laser light scattering detector (HPSEC-MALLS method). The mobile phase was 0.15 M NaCl containing 10 mM PBS at pH 7.4, and the flow rate was 0.5 mL / min. The molecular weight detection results are as follows: Figure 3 As shown, with the increase of enzyme dosage, fucoidan and oligosaccharides with different molecular weights of 28kDa-560kDa can be obtained within 1 hour.

[0055] Example 8: Fucoidan of different molecular weights can be prepared by controlling the reaction time:

[0056] The 0.1 U recombinant enzyme obtained from the *E. coli* system in Example 1 was added to a 100 mg (2 mg / mL) fucoidan solution at pH 8.0. The solutions were reacted at 40°C for 1 min, 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min, after which 500 μL was inactivated. The molecular weight of the fucoidan was monitored using the HPSEC-MALLS method described above, with the mobile phase conditions the same as in Example 7. The molecular weight determination results are as follows: Figure 4 As shown, with the increase of enzyme dosage, fucoidan and oligosaccharides with different molecular weights of 32kDa-560kDa can be obtained within 1 hour.

[0057] Example 9: Fucoidan of different molecular weights can be prepared by controlling the substrate concentration:

[0058] The 0.1 U recombinant enzyme obtained from the *E. coli* system in Example 1 was added to 100 mg (concentrations of 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, and 5.0 mg / mL) fucoidan solutions at pH 8.0. After reacting at 40°C for 1 h, 500 μL of each solution was inactivated. The molecular weight of fucoidan was monitored using the HPSEC-MALLS method described above, with the mobile phase conditions the same as in Example 7. The molecular weight detection results are as follows: Figure 5 As shown, with the increase of enzyme dosage, fucoidan and oligosaccharides with different molecular weights of 85kDa-216kDa can be obtained within 1 hour.

[0059] By combining Examples 7-9, fucoidan and oligosaccharides with different molecular weights of 28-560 kDa can be prepared by controlling the amount of enzyme added, reaction time, or substrate concentration.

[0060] Finally, it should be noted that although the above embodiments describe specific implementations of the present invention, they are not intended to limit the invention. Those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended claims. All modifications or equivalent substitutions should be included within the scope of protection of the present invention.

Claims

1. A fucoidanase based on ancestral sequence reconstruction, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. The fucoidanase according to claim 1, characterized in that: The optimal reaction temperature is 40℃. It can be stably stored for at least 1 day at reaction temperatures of 4℃, 25℃ or 30℃, and the residual enzyme activity remains above 80%. It has catalytic activity under pH conditions of 6.5-10.5, and the optimal reaction pH is 8.

0.

3. A method for preparing the fucoidanase of claim 1, characterized in that: The enzyme was heterologously expressed in Escherichia coli, Bacillus subtilis, and Pichia pastoris systems, and artificial endo-1,3-fucosanase could be prepared in large quantities by inducing enzyme production.

4. The application of the fucoidanase according to claim 1 or 2 in the targeted preparation of fucoidan oligosaccharides with specific structures.

5. The application as described in claim 4, characterized in that: Fucoidan and oligosaccharides with molecular weights ranging from 28 to 560 kDa can be prepared by controlling the amount of enzyme added, reaction time, or substrate concentration.

6. A method for the targeted preparation of fucoidan oligosaccharides with specific structures based on the fucoidanase described in claim 1 or 2, characterized in that: Adjust the amount of enzyme added according to the amount of substrate added to the reaction system, and react at 30-40℃ for 1 min-60 min to prepare fucoidan.

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