A specific carrageenase and its use

Abstract

The application belongs to the technical field of bioengineering and its application, and particularly relates to a specific gel seaweed enzyme and application thereof. The amino acid sequence of the enzyme is shown as SEQ ID NO. 1, and the enzyme can provide a key tool enzyme for fine structure analysis, degradation and preparation of specific polymerization degree functional oligosaccharide of gel seaweed. The application can stably and efficiently prepare gel seaweed oligosaccharide with clear chemical structure and specific polymerization degree by accurately controlling reaction conditions, and realizes customized production of product molecular weight, thereby providing effective technical support for high-value conversion and industrial application of gel seaweed resources.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering and its application technology, specifically relating to a specific seaweed gum enzyme and its application. Background Technology

[0002] Seaweed (Ilex chinensis) is a common economic seaweed belonging to the genus Ilex in the family Cladosaceae of the phylum Rhodophyta, widely distributed along the southeastern coast of my country. Seaweed gum is the main water-soluble polysaccharide in seaweed, accounting for approximately 30%–40% of the thallus's dry weight. It is a linear heteropolysaccharide composed of alternating β-1,4-linked d-galactose-6-sulfate (G6S) and α-1,3-linked 3,6-lactoether-l-galactose (LA). Seaweed gum possesses excellent gelling properties, stability, and biocompatibility, showing potential application value in the food, cosmetics, and pharmaceutical industries. However, the development and utilization of seaweed and its polysaccharides are currently in their early stages, with limited product forms and lagging deep-processing technologies. Achieving high-value transformation of seaweed gum is one of the key directions for increasing its industrial added value.

[0003] Enzymatic degradation technology has become an important method for polysaccharide structure analysis and functional oligosaccharide preparation due to its advantages such as mild conditions, high specificity, and controllable products. However, for the enzymatic degradation of sea cucumber gum, there has long been a lack of specific enzymes. Early studies reported that crude enzyme solutions from certain microorganisms had degrading activity against sea cucumber gum, but the corresponding gene sequences and catalytic mechanisms were unknown, hindering the efficient preparation and targeted modification of these enzymes, which severely limited the enzymatic research and application development of sea cucumber gum.

[0004] Furthermore, although β-agarosease Aga86A_Wa from the GH86 family has been reported to possess activity in degrading seaweed gum in recent years, it is a bifunctional enzyme for both agarose and seaweed gum. During degradation, it inevitably hydrolyzes other polysaccharides such as agarose, resulting in limited product purity, and its catalytic efficiency for seaweed gum is relatively low. Currently, only one endonuclease previously discovered by the inventors, namely the endonuclease disclosed in Chinese patent CN116536287A, has been used in the enzymatic hydrolysis of seaweed gum. The amino acid sequence of this endonuclease is SEQ ID NO.1. The endonuclease of this invention has typical structural features capable of degrading seaweed gum. By controlling conditions such as the amount of enzyme added or the reaction time, seaweed gum can be degraded to produce seaweed gum and oligosaccharides with different molecular weights ranging from 1kDa to 100kDa. Existing β-agarose enzymes have inherent limitations: their active sites are not specifically designed for seaweed gum, their recognition of the characteristic structural unit (G6S) is not very specific, resulting in limited catalytic efficiency, and they cannot avoid cross-reaction with agarose, which restricts the preparation of high-purity oligosaccharides. Summary of the Invention

[0005] The technical problem to be solved by this invention is that there are few specific endo-β-funoran enzymes in the current research field of funoran degrading enzymes. As a result, the available enzyme tools for the structural analysis, controlled degradation and large-scale preparation of funoran oligosaccharides are relatively limited, making it difficult to efficiently prepare funoran oligosaccharides with different degrees of polymerization.

[0006] To address the aforementioned problems, this invention provides a novel β-caryophyllease. This enzyme's catalytic domain possesses a highly conserved active site, specifically and efficiently hydrolyzing the β-1,4 glycosidic bonds in caryophylle via an endoglucosamine process, generating characteristic oligosaccharides with a G6S reducing end. It exhibits no activity towards agarose or laver polysaccharides. Based on this, this specific β-caryophyllease is used for efficient hydrolysis of caryophylle, and the degree of polymerization of the product can be precisely controlled according to requirements, thereby preparing caryophylle oligosaccharides within a specific molecular weight range. This enzyme possesses outstanding advantages such as high substrate specificity, high catalytic specificity, and controllable product structure, making it suitable for the high-purity, refined enzymatic preparation of caryophylle, providing a new, efficient, and specific enzymatic tool for the high-value conversion of caryophylle.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: a specific seaweed gum enzyme, 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 random bifunctional seaweed gum enzyme activity, as well as other sequences with greater than 80% homology to SEQ ID NO. 1.

[0008] SEQ ID NO. 1:

[0009] MKLIRLVLTLAVYFTLLSCSTDTKQIVVSPDPVDSVEDDTEETEEDEDESGNEEELYEPAVGKPSAALSSCSPKNNILYDAPVNNNVQKVNLSEFDMGDWQLIDELSDEFDYAAGKTASVFLSKWKFGFINNYTGPAPTEWSGDQITFETLEGDNRAIVLMPTETGAGANRTLKCGMISSKAKSSYPIFQEAKVKI SNSQLANAVWMLSGESGTTEEIDNLEAYGPRLRPDNTLCDYPYYADRIHLSHHTFKSVGGQRMDYQPKIQTWMSRKKTEGDCSRDNEVVWSEDYHYFG VKWVNEQRLEYFVDGKRVKVVTGLRVADGIDPESYTACNGLTREMHMIISQAAQAWRYGGATNFWNSSDIKTGPNTKMYVDWIRVYSPTGTVNKRSCN.

[0010] In the active cavity of this specific β-gumase, the -1 and +2 subsites strictly recognize and match G6S residues, thereby enabling specific and efficient hydrolysis of the β-1,4-glycosidic bond between G6S and LA in the gum molecule. It belongs to the 16th family of glycoside hydrolases (GH16). This enzyme acts on the gum backbone via an endo-gum reaction, controllably generating a series of characteristic oligosaccharides containing a tetrasaccharide (LA-G6S)2 with G6S as the reducing terminal and uniform degree of polymerization. The main end products have an even degree of polymerization.

[0011] The gene encoding the aforementioned specific β-gumase (corresponding to the catalytic domain sequence SEQ ID NO. 1) has the nucleotide sequence shown in SEQ ID NO. 2, and also includes all degenerate sequences that can be translated into SEQ ID NO. 1, as well as gene sequences that still express the enzyme activity after codon optimization.

[0012] SEQ ID NO. 2:

[0013]

[0014] This invention provides a method for preparing the aforementioned β-caryophyllase. The method involves cloning the gene sequence encoding the enzyme into an expression vector, heterologously expressing it in systems such as *Escherichia coli*, *Bacillus subtilis*, and *Pichia pastoris*, and then purifying it with high purity via IPTG induction, affinity chromatography, and gel filtration chromatography. The successful heterologous expression of the aforementioned β-caryophyllase in systems such as *Escherichia coli*, *Bacillus subtilis*, and *Pichia pastoris* allows for the large-scale production and preparation of the target enzyme, which can be effectively applied in fields such as chemical analysis and the food industry.

[0015] The application of the above-mentioned β-caryophyllum enzyme in the specific enzymatic hydrolysis of caryophyllum. The β-caryophyllum enzyme described in this invention can specifically degrade caryophyllum to obtain various caryophyllum oligosaccharides with different degrees of polymerization.

[0016] Furthermore, the enzyme exhibits an optimal reaction temperature of 35–45 °C and an optimal reaction pH of 7.0–8.0 when catalyzing the reaction of seaweed gum, and its activity is stable in a 20 mM phosphate buffer system. The optimal temperature of 35–45 °C eliminates the need for high-temperature equipment, resulting in low energy consumption and stable activity; the optimal pH of 7.0–8.0 is compatible with conventional systems and offers high tolerance for errors; and the stability in 20 mM phosphate buffer simplifies system construction. The β-seaweed gum enzyme of this invention provides mild overall reaction conditions, is easy to operate, adaptable to various scenarios, and aligns with the concept of green production.

[0017] The application of the aforementioned β-carotene enzyme in the targeted preparation of carotene oligosaccharides with specific structures. Based on this specific β-carotene enzyme, a controllable enzymatic hydrolysis model for carotene molecular weight was established; by adjusting the hydrolysis time and enzyme dosage, precise control over the molecular weight and oligosaccharide composition of the degradation products can be achieved.

[0018] The beneficial effects of this invention are as follows:

[0019] (1) The β-hesperidin gene of the present invention can be efficiently prepared by cloning and expression, and the expression system is stable and produces a high amount of enzyme.

[0020] (2) The β-gumase of the present invention can specifically degrade the β-1,4 glycosidic bond between G6S and LA in gum via endonucleolysis. The present invention also provides the encoding gene and preparation method of the enzyme.

[0021] (3) The enzyme provided by the present invention can prepare oligosaccharides of different degrees of polymerization by controlling the enzymatic hydrolysis conditions, providing a tool enzyme for the preparation and structure-function study of functional oligosaccharides.

[0022] (4) The β-gumase of the present invention has potential application value in the fields of food, medicine, cosmetics, etc., and can be used for the specific degradation of gum and the preparation of specific oligosaccharides.

[0023] In summary, by obtaining and applying the specific sea cucumber gum enzyme of the present invention, sea cucumber gum resources can be utilized in a more refined manner, while enriching the tool library of agar-type galactanases, thereby significantly promoting the application of marine polysaccharide enzymes in the fields of food, medicine and biomaterials, and creating important technical and economic value. Attached Figure Description

[0024] Figure 1 : SDS-PAGE results of the specific seaweed gum enzyme of the present invention; where the left band is the molecular weight standard, and the right band is the molecular weight of the crude enzyme solution of the target protein successfully expressed in the E. coli system;

[0025] Figure 2 : Graph showing the reaction process of the specific seaweed gum enzyme of this invention;

[0026] Figure 3 : Ion chromatogram of the specific seaweed gum enzyme extracted from seaweed gum according to the present invention;

[0027] Figure 4 The graph shows the change in molecular weight of the oligosaccharide product in the reaction system as a function of reaction time under controlled reaction time conditions using the specific seaweed gum enzyme of the present invention.

[0028] Figure 5 : The graph showing the change in oligosaccharide molecular weight with reaction time in the specific seaweed gum enzyme reaction system of the present invention. Detailed Implementation

[0029] 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.

[0030] Example 1: Heterologous expression of specific β-hesperidin in Escherichia coli:

[0031] Escherichia coli carrying the specific β-glucanase target gene (derived from the OF219_2522 gene sequence of strain Wenyingzhuangia aestuarii OF219) was passaged in LB liquid medium containing kanamycin and cultured at 37℃ and 170 rpm until the OD600 reached approximately 0.4. Isopropyl thiogalactoside was added to a final concentration of 0.5 mM for induction expression at 17℃ for 16 h. The bacterial cells were collected by centrifugation, resuspended in a certain amount of pH 7.5 NaH2PO4-Na2HPO4 buffer, and then sonicated in an ice-water bath. The supernatant was collected by centrifugation at 4℃ to obtain the crude enzyme solution containing the specific β-glucanase. To verify the expression of the target protein, the crude enzyme solution was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After mixing the sample with the loading buffer and boiling to denature it, electrophoresis was performed using a 12% separating gel, followed by Coomassie Brilliant Blue R-250 staining. Results are as follows: Figure 1 As shown, a distinct specific band appeared at approximately 55 kDa in the experimental group lane, consistent with the theoretical molecular weight of the target protein; while the empty vector control group lane did not show this band at the corresponding position. This result confirms the successful expression of specific β-hesperidinase in the *E. coli* system.

[0032] Example 2: Heterologous expression of specific seaweed gumase in Bacillus subtilis:

[0033] The upstream and downstream primers (5′-TCTAGAATGAAATTAATTAGATTGGTTTTAACCTTA-3′) were designed, and the target gene was amplified by PCR. The PCR reaction program was as follows: pre-denaturation at 95℃ for 3 min; followed by 24 cycles, each cycle consisting of denaturation at 95℃ for 20 s, annealing at 42℃ for 30 s, and extension at 72℃ for 60 s; after the cycles, a final extension at 72℃ for 5 min was performed to obtain the target gene fragment. This fragment was ligated into the pMA5 vector using seamless cloning technology to construct a recombinant expression vector. The constructed expression vector was transformed into Bacillus subtilis WB600 expression host, and positive transformants were obtained after screening. Positive clones were inoculated into LB medium and induced to express by adding isopropyl-β-D-thiogalactoside (IPTG) at 37℃ for 16 h. The bacterial cells were collected by centrifugation, resuspended in 20 mM phosphate-buffered saline (PBS), and the cells were sonicated in an ice-water bath to disrupt the cell structure. After centrifugation at 4°C, the supernatant was collected to obtain a crude enzyme solution containing specific seaweed gumase.

[0034] Example 3: Heterologous expression of specific seaweed gumase in Pichia pastoris:

[0035] The upstream and downstream primers (5′-TTAATTACAACTTCTTTTATTTACGGTACCTGTTGG-3′) were designed, and the target gene fragment was amplified by PCR. The PCR reaction procedure was the same as in Example 2. The obtained fragment was ligated into the Pichia pastoris expression vector pPIC9K through seamless cloning to construct a recombinant expression plasmid. The recombinant plasmid was electroporated into Pichia pastoris GS115 competent cells, and positive transformants were screened. The positive clone was inoculated into YPD medium and cultured at 30°C and 220 rpm for 16 h. Then it was transferred to BMGY medium at pH 6.0 and cultured under the same conditions until the OD600 was about 5.0. After centrifugation, the cells were collected and resuspended in fresh BMGY medium. 0.3% (v / v) methanol was added to induce expression at 29°C for 72 h. After induction, the supernatant was collected by centrifugation at 4°C to obtain the crude enzyme solution containing specific seaweed gumase.

[0036] Example 4: Enzyme activity verification of specific seaweed gumase:

[0037] To verify the catalytic activity of the specific β-gumase (Sequence 1) and eliminate interference from endogenous host proteins, the following experimental groups were set up for comparative analysis in this embodiment: The experimental group used the crude enzyme solution containing the target protein (Sequence 1) prepared in Example 1; Negative control group A (heat inactivation control) was pretreated at 100℃ for 10 minutes to completely inactivate the enzyme; Negative control group B (empty host control) used *E. coli* hosts transformed with an empty vector without the target gene, and prepared the control crude enzyme solution under the same culture, induction, and disruption conditions as in Example 1; The blank control group used an equal volume of phosphate buffer (pH 7.5) as a reference. During the assay, 375 μL of appropriately diluted test sample was mixed with an equal volume of 2 mg / mL gumase solution, reacted at 55℃ for 5 minutes, and immediately inactivated at 100℃. The amount of reducing sugar generated was determined using the pHBH method. Enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the generation of 1 μmol of reducing sugar per minute under the above conditions. The results showed that the enzyme activity in the experimental group was 107.44 ± 3.6 U / mL; the enzyme activity in the negative control group A (heat-inactivated) was undetectable; the enzyme activity in the negative control group B (empty host) was only 0.36 ± 0.10 U / mL, equivalent to 0.33% of the experimental group; the blank control group only showed background signal. These results clearly indicate that the heat-inactivated control verified the enzyme catalytic characteristics of the reaction; the residual activity of the empty host control was negligible, proving that the endogenous enzyme in the host contributed very little to the degradation activity of seaweed gum in this detection system. Therefore, the measured enzyme activity was entirely derived from the heterologously expressed specific β-seaweed gum enzyme (sequence 1), effectively excluding host background interference. Using the same control design, Examples 2 (Bacillus subtilis system) and Example 3 (Pichia pastoris system) were validated. The enzyme activity of the empty host control was lower than 1% of the experimental group, further demonstrating the specific expression and functional activity of this enzyme in different expression systems.

[0038] Example 5: Gel size exclusion chromatography monitoring of the enzymatic hydrolysis process:

[0039] To investigate the mechanism of action of β-gumase (e.g., OF219_2522), the reaction process was analyzed using both endo- and exo-excision modes. The enzyme from Example 1 was incubated with gumase substrate (final concentration 1 mg / mL) at 30 °C. Samples were taken at time points of 0, 5, 10, 30, and 60 min, and the reaction was immediately terminated by heating at 100 °C for 5 min. After filtration through a 0.22 μm aqueous filter, the samples were analyzed using high-performance gel permeation chromatography (HPLC) with a differential detector. The chromatographic column was a Superdex 30 Increase 10 / 300 GL, the mobile phase was 50 mM ammonium formate aqueous solution, the flow rate was 0.5 mL / min, the column temperature was 30 °C, and the sample loading volume was 30 μL. The results are as follows: Figure 2As shown, when 1 U of enzyme was added and incubated with seaweed gum substrate, rapid generation of oligosaccharides with different degrees of polymerization (retention time 20 min-30 min) could be observed in the early stage of the reaction (30 min), indicating that the enzyme acts on the seaweed gum substrate in an endo-cleavage manner.

[0040] Example 6: LC-MS analysis of the reaction products of specific seaweed gumase:

[0041] The final products of specific seaweed gumase acting on seaweed gum substrate were systematically monitored and analyzed using ultra-high performance size exclusion chromatography-high resolution mass spectrometry (UPSEC-HRMS). For example... Figure 3 As shown, two types of characteristic structural units were clearly identified in the mass spectra of the product: one type is the typical saturated oligosaccharides of seaweed gum, including the disaccharide LA-G6S, the tetrasaccharide (LA-G6S)2, and the hexasaccharide (LA-G6S)3, which is consistent with the characteristic of endo-β-seaweed gum enzymes hydrolyzing alternating polysaccharide chains to produce oligosaccharides with even degrees of polymerization. The other type is methylated saturated oligosaccharides, such as the disaccharide LAMe-G6S (i.e., (LA-G6S)Me), indicating that the enzyme's active cavity is receptive to methylation modifications (LAMe) in the seaweed gum chain. The above results confirm that this enzyme can specifically recognize and cleave the characteristic structural units in the seaweed gum backbone (LA and G6S are linked by β-1,4 glycosidic bonds), making it a novel β-seaweed gum enzyme.

[0042] Example 7: Low molecular weight polysaccharide of seaweed gum can be prepared by controlling the amount of enzyme added:

[0043] The enzyme solution obtained in Example 1 was added to the seaweed gum solution, and the reaction was carried out at a ratio of 1 g substrate (0.5 mg / mL) corresponding to 1 U, 2 U, 4 U, 6 U, 8 U, and 10 U seaweed gum enzyme. After reacting at 35°C for 1 h, 500 μL of each enzyme was inactivated. The molecular weight of seaweed gum was monitored using high performance size exclusion chromatography-multiangle laser light scattering-differential refractive index detection (HPSEC-MALLS-RI 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 4 As shown, with the increase of the amount of seaweed gum enzyme added, seaweed gum degradation products with a molecular weight range of about 1 kDa to about 100 kDa can be obtained within 1 h of reaction, including oligosaccharides with different degrees of polymerization and low molecular weight polysaccharide fragments, indicating that the molecular weight of degradation products can be precisely controlled by adjusting the amount of enzyme.

[0044] Example 8: Low molecular weight polysaccharide of seaweed gum can be prepared by controlling the reaction time:

[0045] One U of the enzyme solution obtained in Example 1 was added to 20 mL of a 5 mg / mL seaweed gum solution at pH 7.0. The reaction was carried out at 55 °C for 10, 20, 30, 40, 50, and 60 min, after which 600 μL of the solution was inactivated. After the reaction, the molecular weight of the seaweed gum was monitored using HPSEC-MALLS-RI. The molecular weight detection results are as follows: Figure 5 As shown, the average molecular weight of the product gradually decreases with the extension of reaction time. A series of degradation products with decreasing molecular weight can be obtained at different time points. Thus, by controlling the reaction time, degradation products with different target molecular weights can be obtained.

[0046] Example 9: Preparation of oligosaccharides of different degrees of polymerization from seaweed gum using specific seaweed gum enzyme:

[0047] Two U of the enzyme solution obtained in Example 1 was added to 20 mL of a 5 mg / mL seaweed gum solution at pH 7.0 and reacted at 55 °C for 4 h. After the reaction, the resulting enzymatic hydrolysate was lyophilized and concentrated. The hydrolysate was then purified using a GE HiLoad™ 26 / 600 Superdex™ 30 pg column with a 5 mm ammonium formate mobile phase and a flow rate of 2.6 mL / min. The fractions containing seaweed gum tetrasaccharide and seaweed gum disodium were collected separately, lyophilized, and the ammonium formate was volatilized to desalt the product. Finally, seaweed gum tetrasaccharide and seaweed gum disodium were obtained.

[0048] 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 specific seaweed gum enzyme, characterized in that: The amino acid sequence of this enzyme is shown in SEQ ID NO.

1.

2. The gene encoding the specific seaweed gumase of claim 1, characterized in that: The nucleotide sequence is shown in SEQ ID NO.

2.

3. A method for preparing the specific seaweed gum enzyme according to claim 1, characterized in that: The target enzyme was obtained by cloning the gene sequence encoding the enzyme into an expression vector and expressing it heterologously in Escherichia coli, Bacillus subtilis, and Pichia pastoris systems. After IPTG induction, the enzyme was purified by affinity chromatography and gel filtration chromatography to obtain high purity.

4. The application of the seaweed gum enzyme according to claim 1 in the specific enzymatic hydrolysis of seaweed gum.

5. The application as described in claim 4, characterized in that: The enzyme exhibits an optimal reaction temperature of 35–45 °C and an optimal reaction pH of 7.0–8.0 when catalyzing the reaction of seaweed gum, and its activity is stable in a 20 mM phosphate buffer system.

6. The application of the seaweed gum enzyme of claim 1 in the targeted preparation of seaweed gum oligosaccharides with specific structures.

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