A disaccharide exo-type beta-agaro-oligosaccharide lyase having galactosidase activity and use thereof

Abstract

This invention relates to a disaccharide exoglycosidase β-agarase with both galactosidase and β-galactosidase activities and its applications, belonging to the field of biotechnology. This invention discloses for the first time a method for developing a β-agarase from *Pseudomonas alterniflora* (…). Pseudoalteromonas An enzyme, ORF0276, and its encoding gene, possessing both galactosidase and disaccharide exonuclease activities, were obtained from the genome of strain Q02 (sp.). This enzyme exhibits strong thermostability, high pH tolerance, and stable physicochemical properties, thus possessing potential for industrial applications. When degrading agar polysaccharide substrates, it can act on the β-1,4 glycosidic bonds of the sugar chains to produce a product primarily composed of neo-agarbiose, which can be used for the industrial preparation of neo-agarbiose (NA2). This research provides a molecular template and theoretical reference for the discovery of such multi-functional tool enzymes.

Description

Technical Field

[0001] This invention relates to a disaccharide exoglycosidase with galactosidase activity and its applications, belonging to the field of biotechnology. Background Technology

[0002] Agar is a natural polysaccharide extracted from red algae such as Gracilaria and Porphyra, with agarose as its main component. Agarose is a linear, straight-chain molecule composed of disaccharide units linked by α-1,3 glycosidic bonds between β-D-galactopyranose residues (G) and their derivatives, such as 3,6-endo-L-galactopyranose residues (A). Repeated disaccharide units are then linked by β-1,4 glycosidic bonds to form polysaccharides. Due to its large production volume, agar, along with alginate and carrageenan, is considered one of the three major marine algae polysaccharides. Agarose has a significant water-absorbing and gelling property, and is widely used in the food industry (as a thickener, coagulant, and stabilizer) and in biotechnology fields such as microbial culture (a core component of solid culture media). However, high molecular weight agarose also has significant drawbacks, such as high viscosity, low solubility, and poor absorption, which greatly limits its wider application in the biological field. It is worth noting that agar polysaccharides can be hydrolyzed into low molecular weight oligosaccharides by chemical, physical, or enzymatic methods, and have better water solubility and higher bioavailability, showing greater application value and potential. For example, in the food industry, they can be used as natural preservatives and sweeteners; in the pharmaceutical industry, they can be used as raw materials for anti-tumor, anti-allergy, or anti-inflammatory purposes; and in the medical aesthetics and chemical industry, they can be used as the main ingredient in whitening, moisturizing, or anti-aging products.

[0003] Currently, among the methods for preparing oligosaccharides, chemical methods (such as acid, alkali, or oxidation methods) or physical methods (such as ultrasonic or infrared methods) are relatively mature in the degradation of agar polysaccharides, but they also have certain problems, such as: harsh and difficult-to-control conditions, uneven molecular weight distribution of products, and serious environmental pollution. In contrast, although enzymatic hydrolysis has the disadvantage of higher cost, it has advantages such as high yield, mild reaction conditions, and well-defined product structure. As a green and environmentally friendly modern bioproduction technology, enzymatic hydrolysis has significant potential for application in large-scale industrial production.

[0004] Agarases can specifically catalyze the hydrolysis of glycosidic bonds within agar polysaccharide molecules to generate low-molecular-weight oligosaccharides. Based on the difference in the type of glycosidic bonds catalyzed, agarases can be divided into two categories: (1) α-agarase (EC3.2.1.158): selectively catalyzes the hydrolysis of α-1,3 glycosidic bonds in agar polysaccharide chains, and the resulting oligosaccharide product has a reducing end of 3,6-intraether-L-galactopyranose (A) in agarooligosaccharides (AOS); (2) β-agarase (EC3.2.1.81): specifically catalyzes the hydrolysis of β-1,4 glycosidic bonds in agar polysaccharide chains, and the resulting oligosaccharide product has a reducing end of β-D-galactopyranose (G) in neoagaro-oligosaccharides (NAOS). After degrading agar polysaccharides, the new agar oligosaccharides generated by β-agarase exhibit stronger stability and other characteristics. Therefore, current enzymological research mainly focuses on β-agarase, with a relatively clear enzymatic mechanism, and the classification based on sequence characteristics in the CAZy database is also quite clear. This enzyme can be further classified into exonucleases, such as members of the GH50 and GH118 families; and endonucleases, such as members of the GH16 and GH96 families. Members within the same family exhibit a regularity of conserved amino acid sequences and identical or similar catalytic site residues. For example, the GH16 and GH50 families each have their own conserved catalytic modules and Glu(E) catalytic site residues. In addition, the new agar oligosaccharides, products of β-agarase hydrolysis of agar polysaccharides, show significant application potential in the food industry (such as the development of oligosaccharide functional additives), drug carrier synthesis (such as the construction of targeted delivery systems), and cosmetic raw material preparation (such as the extraction of natural moisturizing factors). Therefore, with the advancement of molecular enzymology engineering technology, the modified β-agarase has further improved catalytic efficiency, continuously optimized stability, and continuously reduced application costs, which is expected to promote the industrialization of related products.

[0005] Among glycosyl hydrolases, galactosidases are functionally complementary to disaccharide exoglycosidases and can completely degrade neoagar oligosaccharides into monosaccharides. α-Galactosidases, in particular, can further catalyze the hydrolysis of α-1,3 glycosidic bonds within the neoagar disaccharide (NA2) unit, yielding two monosaccharides, A and G, for further utilization by the body, thus forming the basic metabolic enzyme system of agarose. Typically, the genes for these enzymes are also linked on the chromosomes of prokaryotes. Because the glycosidic bonds catalyzed by these two types of enzymes alternate within the agar polysaccharide molecule and are of different types, their industrial applications differ.

[0006] In summary, existing research on the development of β-agarase and galactosidase focuses on the following core directions: (1) the discovery of enzyme-producing strains and enzyme resources; (2) the optimization of heterologous expression conditions to increase enzyme yield and meet production needs; and (3) improving enzyme catalytic efficiency, substrate specificity, thermal stability, and acid and alkali resistance through molecular modification methods such as gene truncation and site-directed mutagenesis. However, a slight shortcoming of existing research is that it has not yet been clearly revealed whether a single enzyme possesses both of the above-mentioned different activities.

[0007] The present invention provides a disaccharide exoglycosidase β-agarase with galactosidase activity, which not only has broad application prospects in the pharmaceutical and food industries, but also provides a molecular template and theoretical reference for the discovery of such multi-functional tool enzymes. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a disaccharide exoglycosidase with galactosidase activity and its applications.

[0009] The technical solution of the present invention is as follows: A disaccharide exonuclease ORF0276 with galactosidase activity has the amino acid sequence shown in SEQ ID NO.2.

[0010] The disaccharide exoglycosidase ORF0276, which also has galactosidase activity, contains 737 amino acids and has a molecular weight of 83.7 kDa.

[0011] The disaccharide exoglycosidase ORF0276, which also possesses galactosidase activity, produces oligosaccharide end products, primarily neo-agarobiose, during the hydrolysis of agar polysaccharides, exhibiting mainly exoglycosidase activity focused on disaccharide production. It also produces a small amount of monosaccharides, suggesting α-galactosidase activity. Overall, this enzyme is predominantly β-galactosidase with secondary galactosidase activity. Furthermore, this enzyme can catalyze the degradation of β-1,4 glycosidic bonds in lactose, producing both galactose and glucose monosaccharides, thus demonstrating β-galactosidase activity.

[0012] The optimal temperature for degradation of agar polysaccharides by the disaccharide exokinase ORF0276, which also has galactosidase activity, is 50℃, and the optimal pH is 7.0. It has certain stability below 40℃ and can tolerate a pH range of 5.0 to 10.0, demonstrating strong pH tolerance.

[0013] A coding gene that encodes the aforementioned disaccharide exonuclease β-agarase ORF0276, which also possesses galactosidase activity.

[0014] According to a preferred embodiment of the present invention, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0015] A recombinant expression vector containing the above-mentioned encoding gene.

[0016] According to a preferred embodiment of the present invention, the plasmid vector of the recombinant expression vector is pET-30a(+).

[0017] A recombinant bacterial strain comprising the above-described coding gene or the above-described recombinant expression vector.

[0018] According to a preferred embodiment of the present invention, the host bacterium of the recombinant strain is Escherichia coli DH5α or Escherichia coli. E. coli BL21(DE3).

[0019] The application of the above-mentioned encoding genes, recombinant expression vectors, or recombinant strains in the preparation of disaccharide exonuclease β-agarase ORF0276 with galactosidase activity.

[0020] The above-mentioned disaccharide exokinase ORF0276, which also has galactosidase activity, is used in the degradation of agar polysaccharides.

[0021] The above-mentioned disaccharide exokinase ORF0276, which also has galactosidase activity, is used in the preparation of new agarobirosaccharides.

[0022] Beneficial effects: (1) This invention discloses for the first time the method of obtaining Pseudoalteromonas ( Pseudoalteromonas An enzyme ORF0276 and its encoding gene, which has both galactosidase and disaccharide exonuclease activities, was obtained from the genome of strain Q02 (sp.).

[0023] (2) The disaccharide exoglycosidase ORF0276 with galactosidase activity has the characteristics of strong thermal stability, strong pH tolerance and stable physicochemical properties, and therefore has the potential for industrial application.

[0024] (3) The disaccharide exoglycoside β-agarase ORF0276 provided by this invention, which also has galactosidase activity, is classified into the GH50 family of β-agarases based on the characteristics of its amino acid sequence. When degrading agar polysaccharide substrates, it can act on the β-1,4 glycosidic bonds of the sugar chain and produce products mainly composed of neo-agarbiose, which can be applied to the industrial preparation of neo-agarbiose (NA2).

[0025] (4) The research of this invention provides a molecular template and theoretical reference for the discovery of such tool enzymes with multiple activities. Attached Figure Description

[0026] Figure 1 Evolutionary position analysis diagram for enzyme ORF0276; Figure 2Electrophoresis diagram of recombinant expression and purification of recombinase rORF0276; In the figure: M: protein molecular weight standard, the band sizes from top to bottom are 120 kDa, 100 kDa, 80 kDa, 60 kDa, 50 kDa, 40 kDa, 30 kDa, 20 kDa, 12 kDa; Lane 1: control E. coli induced by IPTG. E. coli BL21(DE3) strain (containing only pET-30a(+) plasmid) cells before cell disruption, loading volume 5 µL; Lane 2: IPTG-induced Escherichia coli E. coli BL21(DE3) strain (containing pET30a-ORF0276 plasmid) before cell disruption, loading volume 5 µL; Lane 3: IPTG-induced Escherichia coli E. coli After cell wall disruption, the supernatant of BL21(DE3) strain (containing pET30a-ORF0276 plasmid) was loaded in 5 µL; Lane 4: Recombinase rORF0276 purified by nickel column was loaded in 5 µL. Figure 3 The graph shows the effect of temperature on the activity of recombinant enzyme rORF0276 in degrading agar polysaccharides. Figure 4 The graph shows the effect of pH on the activity of recombinant enzyme rORF0276 in degrading agar polysaccharides. Figure 5 This is a graph showing the effect of temperature on the stability of recombinant enzyme rORF0276. Figure 6 This is a graph showing the effect of pH on the stability of the recombinant enzyme rORF0276. Figure 7 Bar graph showing the effect of metal ions and chemical reagents on the activity of recombinant enzyme rORF0276 in degrading agar polysaccharides; Figure 8 The graph shows the effect of NaCl on the activity of recombinant enzyme rORF0276 in degrading agar polysaccharides. Figure 9 TLC analysis of oligosaccharide products during the degradation of agar polysaccharides by recombinant enzyme rORF0276; In the figure: M on the horizontal axis: new agar oligosaccharide standard; Lane 1: product of negative control reaction at 0 min; Lane 2: product of negative control reaction at 72 h; Lanes 3-12: represent products of recombinant enzyme rORF0276 reacting with agar polysaccharides at 0 min, 15 min, 30 min, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h, respectively. Figure 10Fluorescence-high performance liquid chromatography (HPLC) chromatograms of the characteristics of agar polysaccharide degradation products by recombinant enzyme rORF0276, where A is the chromatogram of agar polysaccharide degradation products at different times, and B is the chromatogram of agar polysaccharide degradation products at 0 and 72 h of reaction. Figure 11 Mass spectrometry analysis of the oligosaccharide final products of agar polysaccharide degradation by recombinant enzyme rORF0276, where A is the primary mass spectrum of the 2-AB labeled oligosaccharide product; B is the structural formula of the 2-AB labeled neo-agglobiose and 2-AB labeled agarbiose; C is the secondary mass spectrum of the target ion; and D is the structural formula of the 2-AB labeled β-D-galactose. Figure 12 TLC analysis of lactose degradation mode of recombinant enzyme rORF0276; In the figure: M (Marker): Neo-Agar oligosaccharide (NAOS) standard; 1: 0.10% glucose standard solution; 2: 0.10% galactose standard solution; 3: 0.10% lactose standard solution; 4: reaction product of recombinant enzyme rORF0276 with lactose. Detailed Implementation

[0027] The following examples are provided to fully disclose some common techniques for implementing the present invention, and not to limit the scope of the invention. Unless otherwise stated, molecular weight in the present invention refers to average molecular weight, and temperature is in degrees Celsius.

[0028] Source of experimental materials: Pseudomonas aeruginosa ( Pseudoalteromonas strain Q02 (sp.) was derived from a highly efficient polysaccharide-degrading bacterium screened from coastal sediments near Qingdao Zhanqiao Pier by our research group. Molecular identification confirmed it to be... Pseudoalteromonas This bacterium, named strain Q02, is referred to as *Pseudomonas aeruginosa* Q02 in the following examples. This strain is currently deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, China, with depositary number CGMCC No. 16720. This strain has been disclosed in patent CN109593672A (application number 201811528550.5), and is not related to microbial preservation in this invention.

[0029] Unless otherwise specified, the experimental materials used in the examples are all common commercially available products.

[0030] Example 1 Extraction of genomic DNA from Pseudomonas aeruginosa strain Q02 The *Pseudomonas aeruginosa* strain Q02 was streaked onto artificial seawater LB solid medium plates and incubated upside down at 30°C for 16–24 h until single colonies grew. Single colonies of *Pseudomonas aeruginosa* strain Q02 were picked and inoculated onto artificial seawater LB liquid medium and incubated at 30°C and 200 rpm for 24 h. Genomic DNA was then obtained using the Novizan gene extraction kit according to the manufacturer's instructions.

[0031] The above-mentioned artificial seawater LB solid culture medium has the following components per liter: 10g tryptone, 5g yeast extract, 3g KH2PO4, 7g K2HPO4, 2g (NH4)2SO4, 30g NaCl, 0.01g FeSO4·7H2O, 0.01g MgSO4·7H2O, and 15g agar powder (agar gel); dissolve in ddH2O and bring the volume to 1L, adjust the pH to 7.2, and then autoclave at 121℃ for 20 min.

[0032] The aforementioned artificial seawater LB liquid culture medium is based on the components of a solid culture medium without agar powder.

[0033] Example 2 Genome scanning and sequence analysis of Pseudomonas alterniflora strain Q02 The genomic DNA obtained in Example 1 was sequenced using pyrosequencing technology by Shanghai Meiji Biotechnology Co., Ltd. The DNA sequencing results were analyzed using online software from the NCBI (National Center for Biotechnology Information, http: / / www.ncbi.nlm.nih.gov / ) website. The NCBI analysis software used was Open Reading Frame Finder (ORF Finder, http: / / www.ncbi.nlm.nih.gov / gorf / gorf.html) and Basic Local Alignment Search Tool (BLAST, http: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The results of the analysis using the above biological software showed that the genomic DNA of *Pseudomonas aeruginosa* strain Q02 carries multiple agarase encoding genes. Among them, there is a candidate agarase gene, ORF0276: the coding region of this gene is 2214 bp long, and its nucleotide sequence is shown in SEQ ID NO.1; the protein encoded by this gene contains 737 amino acids, and its amino acid sequence is shown in SEQ ID NO.2. The identified glycosyl hydrolases (GH) in the CAzY database were analyzed and downloaded online using BLASTp. Multiple sequence alignment was then performed using BioEdit 7.01 software, and a phylogenetic tree was constructed using the Neigh-hour-Joining method in MEGA 7.0 software to classify the enzyme ORF0276 into a family and analyze its evolutionary position. The results are as follows: Figure 1 As shown: Enzyme ORF0276 can be classified into the β-agarase GH50 family, and among the enzyme members identified in the GH50 family, enzyme ORF0276 is similar to those from... Paraglaciecola hydrolytica The sequence identity of S66T (GenBank: QEP52089.1) with exonuclease β-agarase activity was 61%, and the enzyme ORF0276 was similar to that from […]. Colwellia echini The sequence identity of A3 (GenBank: WP_148747661.1) with β-galactosidase activity was 62%. Therefore, sequence analysis suggests that enzyme ORF0276 may have both β-agarase and galactosidase activities.

[0034] Further analysis using the biology website ExPASY showed that the theoretical molecular weight of enzyme ORF0276 is approximately 83.7 kDa; online analysis using the signal peptide prediction software SignalP 5.0 (http: / / www.cbs.dtu.dk / services / SignalP / ) revealed that this protein has no signal peptide.

[0035] Example 3 Enzyme ORF0276 in Escherichia coli E. coli Recombinant expression in BL21(DE3) strain Using the genomic DNA obtained in Example 1 as a template, the gene was amplified by PCR, and a recombinant expression vector was constructed using the commercial plasmid pET-30a(+).

[0036] The primer sequences are as follows: Forward primers for amplifying the ORF0276 encoding gene ORF0276-F:5'-g catATG CACTTATTAGATGATGC-3'; Reverse primers for amplifying the ORF0276 encoding gene ORF0276-R: 5'-g ctcgag TTTTTTAAATCGTCGC-3'; The underlined part of the forward primer indicates the restriction endonuclease. Nde The specific site for I is indicated by the underlined restriction endonuclease on the reverse primer. Xho I specific site.

[0037] The high-fidelity DNA polymerase Prime STAR HS DNA Polymerase used was purchased from Takara Bio Inc. (Dalian) Co., Ltd., and the PCR reaction reagents used were operated in accordance with the product instructions provided by the company.

[0038] PCR reaction system: 2×Primer star GC buffer 5 µL, forward primer 0.35 µL, reverse primer 0.35 µL, template (1 ng / µL) 1 µL, ddH2O 3.3 µL, polymerase 0.1 µL, dNTP 0.8 µL.

[0039] PCR reaction conditions: 95℃ pre-denaturation for 4 min; 94℃ denaturation for 40 s, 60℃ annealing for 30 s, 72℃ extension for 150 s, 35 cycles; 72℃ extension for 10 min; 4℃ stability for 10 min.

[0040] The PCR products were treated with restriction endonucleases. Nde I and Xho I was subjected to double enzyme digestion, and the digested products were recovered by agarose gel electrophoresis. pET-30a(+) plasmid DNA (purchased from Invitrogen, USA) was also digested using the same method. Nde I and Xho I. The enzymes were double-digested, verified by agarose gel electrophoresis, and the digested product fragments were recovered.

[0041] Restriction endonucleases Nde I and Xho All enzymes were purchased from Takara Bio Engineering (Dalian) Co., Ltd., and the reaction system, temperature, and time of the enzymes and substrates used in the enzyme digestion were all operated in accordance with the product instructions provided by the company.

[0042] Will pass Nde I and XhoThe PCR product, after double digestion with enzyme I, was ligated with the pET-30a(+) plasmid vector, which had also undergone double digestion, using T4-DNA ligase. The ligation product was transformed into *E. coli* DH5α strain and plated on Luria-Bertani (LB) solid medium containing 50 μg / mL kanamycin. After incubation at 37°C for 16 h, single colonies were picked. These single colonies were then inoculated into Luria-Bertani (LB) liquid medium containing 50 μg / mL kanamycin and incubated at 37°C and 200 rpm for 16 h. The plasmid was then extracted. The plasmid was verified by PCR using the forward primer ORF0276-F and the reverse primer ORF0276-R. The result was an amplification product of approximately 2.2 kb, preliminarily confirming the correctness of the constructed recombinant plasmid. Sequencing of the recombinant plasmid showed that the pET-30a(+) vector... Nde I and Xho The gene sequence shown in SEQ ID NO.1 was inserted between the I restriction sites, and the insertion direction was correct. This proves that the constructed recombinant plasmid is correct. The recombinant plasmid is named pET30a-ORF0276 and will be used to generate the recombinase rORF0276.

[0043] The recombinant plasmid pET30a-ORF0276 was transformed into E. coli. E. coli BL21(DE3) strain (purchased from Invitrogen, USA) was evenly spread on LB solid medium plates (containing 50 μg / mL kanamycin) and incubated upside down at 37°C for 14 h. Single colonies were picked and placed in LB liquid medium containing 50 μg / mL kanamycin and incubated at 37°C and 200 rpm for 14 h with shaking. At an inoculum volume of 1.0%, the bacterial culture was inoculated into 100 mL of LB liquid medium containing 50 μg / mL kanamycin and incubated at 37°C and 200 rpm with shaking. When the bacterial culture reached OD... 600When the concentration was 0.6–0.8, IPTG (isopropyl galactothioglycoside) was added to a final concentration of 0.05 mM, and the cells were cultured at 16 °C and 220 rpm for 24 h to induce the expression of the target protein. Then, the cells were centrifuged at 8,000 × g and 4 °C for 10 min. After centrifugation, the bacterial strains were resuspended in an appropriate amount of Buffer A (50 mM Tris, 150 mM NaCl, pH 8.0) and mixed well. The resuspended bacterial solution was placed in a pre-chilled 50 mL beaker, and the cells were disrupted using an ultrasonic cell disruptor in an ice-water bath until the cells were clear. The disrupted bacterial solution was transferred to a round-bottom centrifuge tube and centrifuged at 15,000 × g and 4 °C for 30 min. The supernatant and precipitate were separated, the water-soluble fraction was collected, and the recombinant enzyme rORF0276 was adsorbed onto a Ni-agarose gel. Gradient elution was performed using Buffer A containing imidazole at concentrations of 10, 20, 50, 100, 250, and 500 mM, respectively, following the purification conditions specified in the gel's product manual. The purification status of the recombinase rORF0276 was assessed by polyacrylamide gel denaturing electrophoresis. Results are as follows: Figure 2 As shown: Recombinant plasmid pET30a-ORF0276 in E. coli After IPTG induction, the expression product of BL21(DE3) strain was water-soluble. The recombinase rORF0276 purified by nickel column affinity chromatography showed a single band on the electrophoresis gel, and the size was consistent with the theoretical value, indicating that the purity and size met the requirements of subsequent experiments.

[0044] The purified recombinant enzyme rORF0276 sample was placed in a dialysis bag with a minimum molecular weight cutoff of 10 kDa and dialyzed at 4°C with 2 L of Buffer A. The buffer was changed for the first 2 h, and then every 4 h thereafter, for a total of 5 dialysis cycles. The purified recombinant enzyme rORF0276 solution was obtained with a concentration of approximately 35 mg / L, which was used for subsequent experiments.

[0045] Example 4 Determination of the optimal temperature for recombinase rORF0276 A 0.20% (w / v, g / mL) agar solution was prepared using deionized water and dissolved by heating. The 0.20% agar substrate was then mixed with 150 mM pH 7.0 Tris-HCl buffer at a 1:1 volume ratio and incubated for 1 h in water baths at 0 ℃, 10 ℃, 20 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 60 ℃, and 70 ℃, respectively. For each temperature condition, 200 μL of the substrate and buffer mixture was added to 100 μL of the recombinant enzyme rORF0276 prepared in Example 3, and the mixture was mixed and incubated for another 4 h at the corresponding temperature. Three parallel samples were prepared for each temperature condition, with a control group containing a mixture of recombinant enzyme rORF0276 inactivated by boiling water bath.

[0046] DNS (3,5-dinitrosalicylic acid)-reducing sugar method for enzyme activity determination: Enzyme activity is determined by measuring the absorbance change of the colored product formed by the reaction of the aldehyde group produced by the reducing sugar with DNS. The absorbance (OD) of the colored product formed by the reaction of the aldehyde group with DNS is... 540 The activity of the enzyme is directly proportional to the concentration of reducing sugars. The concentration of reducing sugars can be indirectly determined by measuring changes in absorbance, thus allowing the enzyme activity to be inferred. The reaction temperature corresponding to the maximum absorbance is the optimum temperature for the recombinant enzyme rORF0276. Relative enzyme activity (RA) is defined as the percentage of each absorbance value relative to the maximum absorbance value.

[0047] The results are as follows Figure 3 As shown above, when enzyme activity was measured using agar as a substrate with equal mass concentrations, the recombinant enzyme rORF0276 showed increased activity in the range of 0℃ to 30℃, but only 50% of its maximum activity was observed at 30℃. In the range of 30℃ to 50℃, enzyme activity continued to increase, reaching its maximum at 50℃. In the range of 50℃ to 60℃, enzyme activity decreased, dropping to 20% of its maximum value at 60℃. Above 70℃, enzyme activity was completely lost. This indicates that the optimal temperature for the enzymatic reaction of enzyme ORF0276 is 50℃.

[0048] Example 5 Determination of the optimal pH of recombinase rORF0276 Agar was prepared with deionized water to a concentration of 0.20% (w / v, g / mL). After dissolving by heating, the 0.20% agar substrate was mixed with different buffer solutions at a 1:1 ratio and incubated at 50°C for 1 h. The buffer solutions included 150 mM NaAc-HAc buffer (pH values ​​5.0 and 6.0), 150 mM NaH2PO4-Na2HPO4 buffer (pH values ​​6.0, 7.0, and 8.0), and 150 mM Tris-HCl buffer (pH values ​​7.0, 8.0, 9.0, and 10.0). 200 μL of the above substrate and buffer solution was added to 100 μL of the recombinant enzyme rORF0276 prepared in Example 3, mixed well, and incubated at 50°C for another 4 h. Three parallel samples were prepared for each condition, with a control group containing a mixture of recombinant enzyme rORF0276 inactivated by boiling water bath. Enzyme activity was determined using the DNS-reducing sugar method. Relative enzyme activity (RA) was defined as the percentage of the average absorbance to the maximum absorbance in each group, with the pH value corresponding to the maximum absorbance being the optimal pH for the recombinant enzyme.

[0049] The results are as follows Figure 4 As shown, the optimal buffer for the recombinant enzyme rORF0276 in degrading agar polysaccharides is a 150 mM Tris-HCl buffer with a pH of 7.0. Within the pH range of 5–10, the relative enzyme activity of rORF0276 remains above 50%, indicating that the enzyme has broad pH reactivity. Under acidic conditions of 150 mM NaAc-HAc buffer at pH 5–6, the enzyme activity is approximately 70% of its maximum value. Under alkaline conditions of 150 mM Tris-HCl buffer at pH 8–10, the activity gradually decreases with increasing pH. This indicates that the optimal pH for the enzymatic reaction of enzyme ORF0276 is 7.0. While the enzyme activity decreases at pH 5–6 or pH 8–10, it retains about 50% of its activity, demonstrating strong pH tolerance within a range of 5.0–10.0.

[0050] Example 6 Temperature stability analysis of recombinase rORF0276 The recombinant enzyme rORF0276 solution prepared in Example 3 was pretreated by heating at 0 ℃, 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, and 70 ℃ for 0 min, 30 min, 1 h, 2 h, 4 h, 6 h, and 24 h, respectively. Then, 100 μL of each of the following solutions (0.20% (w / v, g / mL) agar substrate solution, 150 mM Tris-HCl (pH=7.0) buffer, and the pretreated enzyme solution were thoroughly mixed at a volume ratio of 1:1:1. The mixture was then incubated at 50 ℃ for 4 h, and enzyme activity was measured. Three parallel samples were prepared for each condition, with a control group consisting of a mixed solution containing recombinant enzyme rORF0276 inactivated by boiling water bath. The enzyme activity of the untreated enzyme solution was defined as 100% relative enzyme activity.

[0051] The results are as follows Figure 5 As shown, when recombinant enzyme rORF0276 was pretreated at 0℃~20℃ for 24 h, its enzyme activity remained above 50%; when pretreated at 30℃~40℃ for 2 h, and at above 50℃ for 0.5 h, the enzyme activity of recombinant enzyme rORF0276 remained at approximately 50%. This half-life determination indicates that enzyme ORF0276 has certain thermostability at low temperatures below 40℃, with a half-life of approximately 2 h at 40℃.

[0052] Example 7 pH stability analysis of recombinase rORF0276 The recombinant enzyme rORF0276 solution prepared in Example 3 was mixed with buffers of different pH values ​​at a volume ratio of 1:1 and pre-incubated at 0°C for 2 h. The buffers included 150 mM NaAc-HAc buffer (pH values ​​of 5.0 and 6.0), 150 mM NaH2PO4-Na2HPO4 buffer (pH values ​​of 6.0, 7.0 and 8.0), and 150 mM Tris-HCl buffer (pH values ​​of 7.0, 8.0, 9.0 and 10.0). Take 200 μL of the above enzyme solution and buffer solution, add 100 μL of agar substrate at a concentration of 0.20% (w / v, g / mL), mix thoroughly, and incubate at 50 ℃ for 4 h. Measure enzyme activity. Three parallel samples were prepared for each condition. The control group was a mixed solution containing recombinant enzyme rORF0276 inactivated by boiling water bath. The enzyme activity of the untreated enzyme solution was defined as 100% relative enzyme activity.

[0053] The results are as follows Figure 6As shown, the recombinant enzyme rORF0276 exhibited the highest relative activity after pretreatment with buffer solutions ranging from pH 5.0 to 10.0 for 2 h, followed by pretreatment with Tris-HCl buffer at pH 9, which was comparable to the activity of the untreated enzyme solution. Pretreatment with different buffer solutions ranging from pH 5.0 to 10.0 for 2 h maintained the activity of recombinant enzyme rORF0276 above 70%, indicating that enzyme ORF0276 has a wide pH tolerance range.

[0054] Example 8 Effects of metal ions and chemical reagents on the activity of recombinant enzyme rORF0276 A mixture of 0.20% (w / v, g / mL) agar substrate, the recombinant enzyme rORF0276 solution prepared in Example 3, and 150 mM pH 7.0 Tris-HCl buffer at a ratio of 1:1:1 (volume ratio) was prepared. Different metal ions were then added to the reaction system, with final concentrations of 1 mM or 10 mM. The mixture was then incubated at 50 °C for 4 h. Three parallel samples were prepared for each condition, with a control group consisting of a mixture containing recombinant enzyme rORF0276 inactivated by boiling water bath. Enzyme activity was measured using the DNS-reducing sugar method described above. The enzyme activity of recombinant enzyme rORF0276 without the addition of any metal ions was defined as 100% relative enzyme activity.

[0055] The results are as follows Figure 7 As shown, this enzyme, during the degradation of agar substrates, at K... + Na + Ca 2+ Mg 2+ Pb 2+ Fe 3+ Imidazole and SDS showed promoting effects at a concentration of 1 mM, and inhibiting effects at a concentration of 10 mM; Ag + Zn 2+ Cr 3 + Both EDTA additions of 1 mM and 10 mM showed significant inhibitory effects; Co 2+ Fe 2+ Mn 2+ Ni 3+ DTT, glycerol, and β-mercaptoethanol all promote enzyme activity at both 1 mM and 10 mM. Among them, Co... 2+ The highest relative enzyme activity reached over 400% when 10 mM was added, suggesting that the recombinant enzyme rORF0276 may contain enzymes similar to Co. 2+The bound residues. Therefore, based on the above results, appropriate concentrations of metal ions or chemical reagents can be selected as promoters for enzyme ORF0276.

[0056] Example 9 Effect of NaCl on the activity of recombinant enzyme rORF0276 A mixture of 0.20% (w / v, g / mL) agar substrate, the recombinant enzyme rORF0276 solution prepared in Example 3, and 150 mM pH 7.0 Tris-HCl buffer at a ratio of 1:1:1 (volume ratio) was prepared. NaCl was then added to the reaction system to final concentrations of 0 M, 0.1 M, 0.2 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, and 1.0 M, respectively. The reaction was carried out at 50 °C for 4 h. Three parallel groups were set up for each NaCl concentration. Enzyme activity was determined using the DNS-reducing sugar method described above. The mixed solution containing the recombinant enzyme rORF0276 inactivated by boiling water bath served as the control group. The enzyme activity at 0 M NaCl was defined as 100% relative enzyme activity.

[0057] The results are as follows Figure 8 As shown, NaCl exhibits extremely slight inhibitory or promoting effects on the recombinant enzyme rORF0276 within the 0–0.4 M range, while at higher concentrations (0.5–1.0 M), NaCl demonstrates a promoting effect on the enzyme. This seemingly contradictory characteristic suggests that the enzyme ORF0276 possesses a certain degree of salt tolerance and a wide salt tolerance range, possibly related to the fact that the enzyme's source strain, Q02, is a marine-derived polysaccharide-degrading bacterium.

[0058] Example 10 TLC analysis of oligosaccharide products from the degradation of agar polysaccharides by recombinase rORF0276 A 0.20% (w / v, g / mL) agar solution was prepared using deionized water and dissolved by heating. The 0.20% agar substrate was then mixed with 150 mM pH 7.0 Tris-HCl buffer at a 1:1 volume ratio and incubated at 50 °C for 1 h. 200 μL of the substrate and buffer mixture was then added to 100 μL of the recombinant enzyme rORF0276 prepared in Example 3. The mixture was thoroughly mixed and incubated at 50 °C for further reaction. Samples were taken at 0 min, 15 min, 30 min, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h after the reaction. After sampling, the samples were first boiled in water for 10 min, then transferred to an ice-water bath for 10 min, and centrifuged at 4 °C and 13,300 × g for 10 min. The supernatant was collected and stored at -20 °C for later use. Finally, the degradation products were analyzed by thin-layer chromatography (TLC). A mixed solution containing the recombinant enzyme rORF0276 inactivated by boiling water bath was used as a negative control.

[0059] The product was analyzed by thin-layer chromatography. 1 μL of supernatant was placed on a chromatography plate (TLC Silica 60 F254, MERK, Germany). The developing solvent was n-butanol:ethanol:water in a volume ratio of 2:1:1. The plate was stained with acetone solution of diphenylamine and then developed under high temperature conditions.

[0060] The results are as follows Figure 9 As shown in the figure, M on the horizontal axis represents the neo-agar oligosaccharide standard; lane 1 represents the product of the negative control reaction at 0 min; lane 2 represents the product of the negative control reaction at 72 h; lanes 3-12 represent the products of the reaction between recombinant enzyme rORF0276 and agar polysaccharide at 0 min, 15 min, 30 min, 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, and 72 h, respectively; in the figure, the Rf value of neo-agarobiose (NA2) in the neo-agar oligosaccharide standard is approximately 0.57. The chromatographic results show that with the extension of reaction time, the content of products with the same Rf as NA2 gradually increases; after 24 h or even longer reaction times, the content of the NA2 counterpart remains essentially unchanged, indicating that the recombinant enzyme rORF0276 degrades agar polysaccharide to ultimately generate oligosaccharide products mainly composed of NA2. However, around 12 hours into the reaction, diffusion was observed, with smaller oligosaccharide products being generated. This suggests that the neoagarbiose may have been further degraded into monosaccharides, leading to the hypothesis that the recombinant enzyme rORF0276 possesses α-galactosidase activity, which requires further verification. Therefore, the activity of enzyme ORF0276 is primarily characterized by disaccharide exoclease-type β-agarase activity, with glycoside hydrolase activity as a secondary component.

[0061] Example 11 Fluorescence-high performance liquid chromatography (HPLC) analysis of characteristic oligosaccharides generated by recombinase rORF0276 A 0.20% (w / v, g / mL) agar substrate was prepared using deionized water and dissolved by heating. The agar substrate was then mixed with 150 mM pH 7.0 Tris-HCl buffer at a 1:1 ratio and incubated at 50 °C for 1 hour. 200 μL of the substrate and buffer mixture was added to 100 μL of the recombinant enzyme rORF0276 prepared in Example 3. After mixing, the mixture was incubated at 50 °C for further reaction. Samples were taken at 0 min, 1 h, 6 h, 12 h, 24 h, 48 h, and 72 h after the reaction. For the control group, a heat-inactivated recombinant enzyme rORF0276 was added, and samples were taken at 72 h after the reaction. After sampling, the samples were first placed in a boiling water bath for 10 min, then transferred to an ice water bath for 10 min, and finally centrifuged at 4 °C and 13,300 × g for 10 min. The supernatant was then frozen at -80 °C for later use. The sample was added to a dimethyl sulfoxide (DMSO) solution containing excess o-aminobenzamide (2-AB) and sodium cyanoborohydride, mixed well, incubated at 60 °C for 3 h, and repeatedly extracted with chloroform to obtain oligosaccharide products with 2-AB fluorescently labeled reducing ends.

[0062] Equilibrate Superdex using a 0.20 mol / L NH4HCO3 solution. TM A 30 Increase10 / 300GL (GE, General Electric Company) molecular gel chromatography column was used at a flow rate of 0.40 mL / min. Oligosaccharide products labeled with fluorescence and digested at different times were loaded with 100 ng of sample (approximately 20 μL) using an autosampler (excitation light 330 nm, detection light 420 nm). The integrated area of ​​each oligosaccharide component was analyzed using HPLC software, and the relative molar concentration was calculated based on the theoretical molecular weight.

[0063] like Figure 10 As shown in Figure A, the time gradient of agar polysaccharide degradation by recombinant enzyme rORF0276 reveals that oligosaccharide products are produced quickly during degradation. These products gradually accumulate over time, consistently exhibiting an enzymatic hydrolysis signal with the same elution time as the 2-AB-NA2 peak in the marker (standard). This signal remains stable after 24 hours (up to 72 hours or longer), indicating that the enzymatic catalysis of agar polysaccharide degradation tends to stabilize. Figure 10 As shown in Figure B, under the above conditions, the oligosaccharide products of agar polysaccharide degradation by the 2-AB-labeled recombinant enzyme rORF0276 at 0 min and 72 h were finally detected, and a signal corresponding to 2-AB-NA2 was detected. These results suggest that the enzyme ORF0276 degrades agar polysaccharide and generates oligosaccharide products mainly composed of NA2.

[0064] Example 12 Mass spectrometry analysis of agar oligosaccharide products from recombinase rORF0276 First, the oligosaccharide product obtained from the degradation of recombinant enzyme rORF0276 for 72 h in Example 11 was 2-AB labeled, separated and purified by high-performance liquid chromatography-gel chromatography, and the target product was collected multiple times and further freeze-dried for concentration. Finally, the separated and purified sample was freeze-dried multiple times, dissolved in methanol, filtered through a 0.22 μm filter membrane, and injected into a Bruker Q-TOF mass spectrometer for mass spectrometry analysis.

[0065] Primary mass spectrometry analysis of the 2-AB labeled oligosaccharide products yielded the following results: Figure 11 As shown in Figure A, a distinct ionic signal was observed at m / z 467, suggesting it to be a 2-AB-labeled disaccharide derivative -[Na+]. + The signal indicates that the 2-AB-labeled oligosaccharide product may be either neo-agglobinose or agarobirobinose (structural formula as shown). Figure 11 As shown in B), but the structural information of its reduced end is unknown. Therefore, based on this, the target ion with m / z 467 in the first-stage mass spectrometry was further broken up and analyzed by second-stage mass spectrometry, and the results are as follows. Figure 11 As shown in Figure C, a significant ionic signal was observed at m / z 323, therefore it was determined to be the G sugar unit -[Na+] labeled with 2-AB. + The signal indicates that the reducing end of the disaccharide product labeled with 2-AB is β-D-galactopyranose (structural formula shown). Figure 11 (As shown in D); Meanwhile, the A sugar unit -[Na] labeled by 2-AB was not present in either the primary or secondary mass spectra of the labeled disaccharide product. + The signal (m / z of glycogen A is 162) was observed. These results indicate that the recombinant enzyme rORF0276 acts on agar polysaccharides and enzymatically hydrolyzes the resulting oligosaccharides. The reducing end linked to 2-AB is a G sugar (β-D-galactopyranose) rather than an A sugar (3,6-galactopyranose). Therefore, combined with mass spectrometry analysis, it is determined that the disaccharide product obtained from enzymatic hydrolysis is neo-agarbiose rather than agarbiose. This indicates that enzyme ORF0276 is a β-agarase, which catalyzes the β-1,4 glycosidic bonds in the agar polysaccharide chain, and the final product of agar polysaccharide degradation is neo-agarbiose.

[0066] Example 13 Analysis of lactose degradation patterns of recombinase rORF0276 First, a 0.20% (w / v, g / mL) lactose substrate solution was prepared using deionized water, sterilized, and cooled to room temperature. Markers were prepared using 0.10% glucose, 0.10% galactose, 0.10% lactose, and 0.10% neo-Agar oligosaccharide (NAOS) standards, respectively. Then, the substrate and 150 mM pH 7.0 Tris-HCl buffer were incubated at a 1:1 volume ratio at 50°C for 1 h. 200 μL of the substrate and buffer mixture was then added to 100 μL of the recombinant enzyme rORF0276 prepared in Example 3, and the reaction was incubated at 50°C for 24 h. After the reaction was complete, the enzyme was inactivated by boiling and centrifuged. The final product was analyzed by thin-layer chromatography (TLC). 2 μL of the reaction supernatant was collected on a chromatography plate (TLC Silica 60 F254, MERK, Germany). The developing solvent was n-butanol:ethanol:water in a volume ratio of 2:1:1, and the colorimetric reagent was a diphenylamine solution in acetone. The mixture was then developed at high temperature.

[0067] The results are as follows Figure 12 As shown, the final product Rf value of the reaction between recombinant enzyme rORF0276 and lactose substrate falls between galactose and lactose, indicating that the product is a mixture of galactose and lactose. This suggests that recombinant enzyme rORF0276 can partially degrade lactose substrate, generating equimolar amounts of galactose and glucose. This implies that enzyme ORF0276 is a disaccharide exokinase of the GH50 family that also possesses β-galactosidase activity.

[0068] The above experiments show that enzyme ORF0276 is a disaccharide exoglycosidase with both galactosidase and β-agarase activities.

Claims

1. A disaccharide exoglycosidase β-agarase ORF0276 with galactosidase activity, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

2. A gene encoding a gene, characterized in that, The encoding gene can encode the disaccharide exonuclease β-agarase ORF0276 with galactosidase activity as described in claim 1.

3. The encoding gene as described in claim 2, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

1.

4. A recombinant expression vector, characterized in that, It includes the coding gene as described in claim 2 or claim 3.

5. The recombinant expression vector as described in claim 4, characterized in that, The plasmid vector for the recombinant expression vector is pET-30a(+).

6. A recombinant bacterial strain, characterized in that, It includes the coding gene as described in claim 2 or claim 3, or the recombinant expression vector as described in claim 4.

7. The recombinant strain according to claim 6, characterized in that, The host bacteria of the recombinant strain is Escherichia coli DH5α or Escherichia coli. E. coli BL21(DE3).

8. The use of the encoding gene of claim 2 or claim 3, the recombinant expression vector of claim 4, or the recombinant strain of claim 6 in the preparation of the disaccharide exonuclease β-agarase ORF0276 with galactosidase activity as described in claim 1.

9. The application of the disaccharide exokinase ORF0276 with galactosidase activity as described in claim 1 in the degradation of agar polysaccharides.

10. The application of the disaccharide exokinase ORF0276 with galactosidase activity as described in claim 1 in the preparation of new agarobirosaccharides.

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