Glucoside hydrolase derived from intestinal tracts of horses and capable of degrading sodium hyaluronate

Abstract

The invention discloses glucoside hydrolase which is derived from intestinal tracts of horses and can degrade sodium hyaluronate. The glucoside hydrolase is characterized in that the amino acid sequence of the glucoside hydrolase is SEQ ID NO.1. A target protein is expressed through a prokaryotic expression system, protein purification is carried out through an affinity chromatography method, the concentration of the target protein is measured to be 0.478 mg / mL through a Bradford method, the enzyme activity of the target protein is measured through a salicylic acid method (DNS), it is found that a degradation substrate of the target protein is sodium hyaluronate, N-acetyl glucosamine is generated, the enzyme activity is 62.8 U, and the specific activity is 2.09 U / mg.

Description

Technical Field

[0001] The invention belongs to the field of microbial molecular biology and relates to a glycoside hydrolase separated from horse intestines and capable of degrading sodium hyaluronate. Background Art

[0002] Carbohydrates exist in nature in many forms, from protein-linked glycans to cell walls of plants, fungi, and bacteria, as well as storage polymers such as starch and glycogen. Carbohydrate degradation in animals is closely related to their intestinal microorganisms, and most of the enzymes that they lack that can degrade carbohydrates are present in intestinal microorganisms [Wardman, JF, Bains, RK, Rahfeld, P. et al. Carbohydrate-active enzymes (CAZymes) in the gut microbiome. Nat Rev Microbiol 20, 542–556 (2022)]. Although most known industrial enzymes are obtained from microorganisms through pure culture isolation and screening, recent estimates indicate that more than 99% of microorganisms in the environment cannot be cultured by conventional microbiological methods. Therefore, the emergence of metagenomics in recent years provides a powerful tool to obtain the genetic and metabolic diversity of microorganisms in any environment, bypassing the limitations of current culture-dependent methods [Pabbathi NPP, Velidandi A, Tavarna T, et al. Role of metagenomicsin prospecting novel endoglucanases, accentuating functional metagenomics approach in second-generation biofuel production: a review. Biomass Convers Biorefin. 2023;13(2):1371-1398.], which is conducive to the discovery of potential carbohydrate enzymes in intestinal microorganisms. In a previous study, we used metagenomic sequencing to confirm that the horse cecal microbiota is an important source of unknown enzymes involved in complex carbohydrate degradation [Li C, Li X, Guo R, et al. Expanded catalogue of metagenome-assembled genomes reveals resistome characteristicsand athletic performance-associated microbes in horse. Microbiome. 2023;11(1):7. Published 2023 Jan 12.]. Therefore, it is crucial to discover and mine CAZymes encoded by the equine cecal microbiota using metagenomic data analysis.

[0003] There is a growing interest in CAZymes. So far, 189 glycoside hydrolases (GHs), 137 glycosyltransferases (GTs), 43 polysaccharide lyases (PLs), and 20 carbohydrate esterases (CEs), 17 auxiliary enzyme families (AAs), and 103 carbohydrate binding modules (CBMs) have been found in the Carbohydrate Active Enzyme Database (CAZy, www.cazy.org). They are widely used in food processing, industrial production, biofuels, environmental management and other fields [Wardman, JF, Bains, RK, Rahfeld, P. et al. Carbohydrate-active enzymes(CAZymes) in the gut microbiome. Nat Rev Microbiol 20, 542–556 (2022)].

[0004] The present application separates a glycoside hydrolase from the horse intestine that can efficiently degrade hyaluronic acid. Summary of the invention

[0005] The purpose of the invention is to explore a novel and efficient carbohydrate enzyme.

[0006] In order to achieve the above technical effects, the present invention adopts the following technical solutions: A glycoside hydrolase derived from horse intestines and capable of degrading sodium hyaluronate, characterized in that its amino acid sequence is SEQ ID NO.1.

[0007] The amino acid sequence of the glycoside hydrolase SEQ ID NO.1 is as follows: MIQSYNENRGQQFPFDVVIPTDEKNSKFVGTVVTHVRRRMPEAGTIYIVTKRILFDKIKRYVHDDDWQLIDENTMVAGLSEVFGYLKKKGCDSVNKVGWYFQQFLKLAFALTSYCKGYYLTWDSDTLPISELHFFQDGQPLFTMKKEYHRPYFNTL QRLIGLDKTSSKSFIAEHMIFKPEFVCEMIEEISQNTLPGKNWVEKIIQACDFDYEEHCFSEFETYGTFCTVRYPGYYGEQTLNTFRAGSLIRGRYVNDFIIERLSSDVDIASFEIYDAMFPYDIEKRIYIWKSRWKRLTNLSPSQALGLIWRRIFKK.

[0008] A recombinant expression vector of a glycoside hydrolase capable of degrading sodium hyaluronate derived from the intestinal tract of a horse, characterized in that the nucleotide sequence of the above-mentioned glycoside hydrolase capable of degrading sodium hyaluronate is inserted into an Escherichia coli expression vector to obtain a recombinant expression vector expressing the glycoside hydrolase capable of degrading sodium hyaluronate.

[0009] The E. coli expression vector is 119-PET-32a.

[0010] A recombinant strain, characterized in that the recombinant expression vector obtained according to claim 2 or 3 is introduced into Escherichia coli and screened to obtain it.

[0011] The Escherichia coli is DE3.

[0012] A method for preparing the glycoside hydrolase derived from horse intestine and capable of degrading sodium hyaluronate as claimed in claim 1, characterized in that it comprises the following steps: a. Collection of horse cecal contents, sequencing and assembly of horse cecal contents, bioinformatics screening of novel carbohydrate enzyme candidate genes, bioinformatics analysis of proteins encoded by candidate target genes and screening of candidate target gene bfc119; b. Construction of glycoside hydrolase recombinant expression vector; c. Prokaryotic expression of glycoside hydrolase protein; d. Purification of glycoside hydrolase protein to obtain glycoside hydrolase.

[0013] The present invention utilizes bioinformatics technology to screen and obtain a candidate protein bfc119 gene, and obtains the candidate protein bfc119 through prokaryotic expression and purification. Experimental verification shows that bfc119 has the ability to degrade sodium hyaluronate. In the intestine, its decomposition product N-acetylglucosamine can increase the abundance of Akkermansia, promote weight loss and enhance immunity.

[0014] The discovery of a novel carbohydrate enzyme includes the following steps: collection of horse cecal contents, sequencing and assembly of horse cecal contents, bioinformatics screening of novel carbohydrate enzyme candidate genes, bioinformatics analysis of proteins encoded by candidate target genes and screening of candidate target genes, prokaryotic expression and purification of carbohydrate enzyme gene bfc119, enzyme activity determination of carbohydrate enzyme gene bfc119, and prediction of carbohydrate enzyme gene bfc119 family members. The specific steps are as follows: Collection of horse cecal contents: Fresh horse cecal contents were collected at the slaughterhouse, placed in 50 ml centrifuge tubes, and stored in liquid nitrogen.

[0015] Sequencing and assembly of horse cecal contents: The horse cecal contents samples were sent to the company for sequencing and assembled using bioinformatics technology to obtain the metagenome assembled genome (MAG).

[0016] Bioinformatics screening of novel carbohydrate enzyme candidate genes: Use open source bioinformatics analysis tools such as cazy and PUL to annotate and predict carbohydrate enzymes in the metagenome assembled genome (MAG) and screen novel carbohydrate enzymes.

[0017] Bioinformatics analysis of proteins encoded by candidate target genes and screening of candidate target genes: Various physical and chemical property analyses and structural predictions of the candidate gene bfc119 were performed using various online websites such as Swiss-model.

[0018] Prokaryotic expression and purification of carbohydrate enzyme gene bfc119: The candidate protein bfc119 was expressed using a prokaryotic expression system, and the pure protein of bfc119 was obtained by affinity chromatography purification.

[0019] Determination of the enzymatic activity of the carbohydrate enzyme gene bfc119: The carbohydrate enzyme activity of the candidate protein 119 was verified using the DNS method.

[0020] Prediction of members of the bfc119 family of carbohydrate enzyme genes: comparison of the horse cecal protein library and the NR database using blastp.

[0021] According to a further technical solution of the present invention, the novel carbohydrate enzyme bfc119 can efficiently degrade sodium hyaluronate and produce N-acetylglucosamine.

[0022] The present invention uses bioinformatics to mine a new carbohydrate enzyme family from the assembled genome of the horse cecum metagenomic genome, screens a candidate new carbohydrate enzyme gene bfc119, then expresses the target protein through a prokaryotic expression system, and purifies the protein by affinity chromatography. The concentration of the target protein is measured to be 0.478 mg / mL by the Bradford method, and the enzymatic activity of the target protein 119 is determined by the salicylic acid method (DNS). It is found that the degradation substrate of the target protein 119 is sodium hyaluronate, and N-acetylglucosamine is produced, the enzyme activity is 62.8 U, and the specific activity is 2.09 U / mg.

[0023] The present invention aims to enrich the existing carbohydrate enzyme gene library, improve the utilization efficiency of lignocellulose, and develop new and efficient enzyme preparations. The present invention discovers a new and efficient carbohydrate enzyme, which has a high efficiency in degrading sodium hyaluronate, and lays a research foundation for improving the utilization efficiency of carbohydrates and developing new and efficient enzyme preparations. At the same time, the N-acetylglucosamine produced has the effect of increasing the abundance of probiotics, and has broad application prospects in the fields of medicine, food, and agriculture.

[0024] Compared with the existing inventions, the present invention has the following beneficial effects: 1. The carbohydrate enzyme of the present invention is derived from horse cecum, and compared with chemically synthesized products, it is natural and non-toxic; 2. The present invention uses bioinformatics technology to discover a new type of carbohydrate enzyme, and experiments have proved that it can efficiently degrade sodium hyaluronate, and its product is N-acetylglucosamine, which can promote the growth of intestinal probiotics Akkermansia, and has the effects of weight loss and improving immunity; BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Distribution of carbohydrate enzymes in the cecum of horses.

[0026] Figure 2 MAG 117.bin 13 Carbohydrate enzyme distribution.

[0027] Figure 3-1 Secondary structure of the carbohydrase BFC119.

[0028] Figure 3-2 3D modeling of the carbohydrase BFC119.

[0029] Figure 4-1 PET-32a+-119 plasmid extraction and enzyme digestion; Among them: AM: marker, 1 and 2 are PET-32a+-119 plasmid extraction; BM: marker, 1: PET-32a+-119 plasmid extraction, 2: PET-32a+-119 enzyme digestion.

[0030] Figure 4-2 Carbohydrate enzyme bfc119 induced expression; Wherein: M: protein marker, 1: uninduced bacterial solution, 2: supernatant of induced bacterial solution, 3: induced bacteria.

[0031] Figure 5 Prediction of members of the BFC119 family of carbohydrate enzymes.

[0032] Figure 6 The hydrolysis activity of the target protein BFC119 towards different substrates. DETAILED DESCRIPTION

[0033] The above contents of the present invention are further described in detail below through embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example 1: Collection and processing of horse cecal content samples 1. Collection of equine cecal content samples A total of 242 cecal intestinal samples from indigenous Ili horses, thoroughbred horses, Tibetan horses, Shetland ponies, and Ili racehorses were collected for this study. The horses were raised in different counties in China. Indigenous Ili horses (n = 110, Ili horses) and Tibetan horses (n = 10, Tibetan horses) grazed on natural pastures and fed a diet of grass. Thoroughbred horses (n = 58, Thoroughbred horses) were stabled and fed a mixed hay and concentrate feed.

[0035] 2. Processing of horse cecal content samples All samples were frozen in liquid nitrogen, transported to the laboratory, and stored in a −80°C freezer until DNA extraction. To obtain high-quality microbial DNA from intestinal contents, DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) method.

[0036] Example 2: Bioinformatics analysis 1. Sequencing and assembly of horse cecum samples We used the metaWRAP-Binning module to generate bins, and after deduplication (average nucleotide identification (ANI) ≤ 99%) and quality assessment, we obtained a subset of MAGs that met or exceeded the previously established medium quality standards (completeness ≥ 50%, contamination ≤ 10%). Another subset of genomes was generated from two sequenced samples (HGM35 and HGM77) by nanopore sequencing, so we assembled MAGs using Illumina and Nanopore sequencing technologies.

[0037] 2. Bioinformatics screening of novel carbohydrate enzyme candidate genes The metagenome assembly genome (MAG) constructed from the metagenomic data of horse cecum was used as the research object. PULpy was used to predict the PUL of MAG, and the MAG with the most polysaccharide utilization sites (PULs) was screened. The MAG was annotated with CAZY using run_dbcan2, and proteins with a similarity of 10%-30% with CAZY were selected through blast comparison. They were further analyzed with the NR database to obtain hypothetical proteins, and hypothetical genes were selected as candidate target genes according to the distribution of carbohydrate enzymes on the gene cluster.

[0038] 3. Bioinformatics analysis of proteins encoded by candidate target genes and screening of candidate target genes Swiss-model was used to perform homology modeling on the screened hypothetical proteins, and SOPMA and SWISS-MODEL were used to predict the secondary and tertiary structures of the proteins, respectively. Protparam was used to predict the theoretical molecular weight (Mw) and isoelectric point (PI) of the target gene, and protScale online tool was used to predict the hydrophilicity of the amino acid sequence. SignalP 3.0 was used to predict the N-terminal signal peptide of the amino acid sequence, TMHMM 2.0 was used to predict the transmembrane domain, and Glycosylation Predictor was used to predict the glycosylation site. The candidate target gene bfc119 was screened using the above results.

[0039] Example 3: Prokaryotic expression and purification of carbohydrase bfc119 1. Prokaryotic Expression of Carbohydrate Enzyme BFC119 1. Construction of engineered bacteria 500 μl of E. coli DH5α containing the recombinant plasmid was taken into 20 ml LB liquid medium containing ampicillin and cultured at 37°C and 180 r / min. After the plasmid was extracted with a plasmid extraction kit. The plasmid was double-digested with restriction endonucleases BamH Ⅰ and EcoR Ⅰ and incubated in a 37°C water bath for 90 min. Detection was performed using 1% agarose gel electrophoresis with a voltage of 120 V and an electrophoresis time of 25 min. The plasmid was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing and identification. After successful sequencing, the plasmid with the correct sequencing result was transformed into the E. coli DE3 strain to induce expression.

[0040] 2. Protein Expression of Carbohydrate Enzyme BFC119 The strain was inoculated into LB liquid medium containing ampicillin and cultured at 37°C overnight. The bacterial solution was inoculated into 800 mL LB liquid medium and cultured at 180 rpm and 37°C until OD 600 When the p-value reaches 0.6, IPTG (isopropyl-β- d -thiogalactoside) is added to induce the expression of the target protein for 16 h. The bacterial solution is centrifuged at 8000 r / min for 10 min to collect the cells, and 30 ml of lysis buffer is added to mix and lyse overnight. The overnight lysate is frozen in liquid nitrogen for 5 min, and completely thawed in a 37°C water bath. This is repeated three times, and finally ultrasonically broken in an ice bath for 1 h, and centrifuged at 8000 r / min for 15 min to remove the supernatant. The precipitate is mixed with 8 mol / L urea and dissolved at 4°C overnight.

[0041] Example 4: Purification and refolding of carbohydrase bfc119 1. Purification of Carbohydrase BFC119 The overnight lysate was centrifuged at low speed to obtain the supernatant, filtered with a 0.22 μm filter membrane, and 10 volumes of ddH2O were added to wash the nickel column, and the 20% ethanol used to store the nickel column was washed away. 10 volumes of elution solution and washing solution were added in sequence, 5 mL of filtered supernatant was added, and 10 volumes of washing solution were added to remove impurities. The effluent was collected as a control, and 10 volumes of elution solution were added to elute the target protein, and the effluent was collected. 12% SDS-PAGE electrophoresis was used for detection. The bacterial sample was 12,000 r / min for 1 min to remove the supernatant, 40 μl ddH2O and 10 μL 5× protein loading buffer were added, and the sample was placed in a boiling water bath for 10 min. When spotting, protein markers, uninduced bacterial solution, induced unpurified protein and purified protein were added in sequence, and electrophoresis was performed at room temperature at 120 V for 100 min, and visualized with Coomassie Brilliant Blue staining.

[0042] 2. Renaturation of Carbohydrate Enzyme BFC119 Inclusion Bodies The purified protein solution was placed in a dialysis bag and soaked with 6 mol / L urea, 4 mol / L urea, 2 mol / L urea, 1 mol / L urea, 0.5 mol / L urea and 1 mol / L PBS, respectively. The refolding solution was replaced every 12 hours, and then the solution was concentrated by centrifugation at 5,000 r / min using an ultrafiltration tube.

[0043] Example 5: Determination of the enzyme activity and specific activity of the carbohydrate enzyme gene bfc119 1. Determination of the enzymatic activity of the carbohydrate enzyme gene bfc119 Enzyme activity is defined as the amount of enzyme required to convert 1 micromole of substrate in 1 minute at the optimal temperature, which is one unit of enzyme activity (U). Enzyme activity determination formula: Enzyme activity (U) = (V × △OD 535 ) / (ε× V×t). Where V is the volume of the reaction solution (mL), OD 535 is the absorbance difference of the reaction solution at a specific wavelength, ε is the molar extinction coefficient of the enzymatic reaction (L / mol·cm), V is the volume of the enzyme solution to be tested (mL), and t is the reaction time (min).

[0044] Determine the activity of the target protease according to the reaction system: 100 μl of target protein, 200 μl of sodium hyaluronate, 37 ℃, 30 min. After the reaction is completed, add 400 μl of DNS in a boiling water bath for 5 min, wait for it to cool and measure its OD 535 The amount of reducing sugar produced by the degradation of carbohydrates by the target protein can be obtained according to the standard curve of reducing sugar. In the polysaccharide hydrolysis experiment, the selected polysaccharide substrates include carboxymethyl cellulose (CMC-Na), xylan, raffinose, soluble starch, sucrose, L-arabinose, sodium hyaluronate, α-lactose, D-(+)-maltose and p-nitrophenyl-β-D-galactoside.

[0045] 2. Determination of the specific activity of the carbohydrate enzyme gene bfc119 The formula of enzyme specific activity: specific activity (U / mg) = (c×K×V) / (m×t×m×1000) where c: concentration of enzyme product (mg / mL), K: dilution factor of substrate solution, V: volume of substrate solution (mL), m: mass of enzyme (mg), t: duration of enzyme reaction (min), M: molar mass of product, 1000: conversion of millimole (mmol) to micromole (umol). The experimental results of enzyme activity determination were averaged for three measurements, and all data were expressed as mean ± standard deviation of three independent experiments (n=3).

[0046] Example 6: Prediction of carbohydrase gene bfc119 family members In order to further understand the members with similarity to the target protein bfc119 in horse cecum, blastp was used (E value was 10 -5 ) The proteins encoded by the horse cecum metagenome were compared with the NR database.

Claims

1. A glycoside hydrolase derived from horse intestine that is capable of degrading sodium hyaluronate, characterized in that: Its amino acid sequence is SEQ ID NO.

1.

2. A recombinant expression vector of glycoside hydrolase from horse intestine capable of degrading sodium hyaluronate, characterized in that: The nucleotide sequence of the glycoside hydrolase capable of degrading sodium hyaluronate as claimed in claim 1 is inserted into an Escherichia coli expression vector to obtain a recombinant expression vector expressing the glycoside hydrolase capable of degrading sodium hyaluronate.

3. The recombinant expression vector of glycoside hydrolase capable of degrading sodium hyaluronate derived from horse intestine as claimed in claim 2, characterized in that: The E. coli expression vector is 119-PET-32a.

4. A recombinant strain, characterized in that: The recombinant expression vector obtained in claim 2 or 3 is introduced into Escherichia coli and screened to obtain the product.

5. The recombinant strain according to claim 4, characterized in that: E. coli is DE3.

6. A method for preparing the glycoside hydrolase derived from horse intestine and capable of degrading sodium hyaluronate as claimed in claim 1, characterized in that: The steps include: a. Collection of horse cecal contents, sequencing and assembly of horse cecal contents, bioinformatics screening of novel carbohydrate enzyme candidate genes, bioinformatics analysis of proteins encoded by candidate target genes and screening of candidate target gene bfc119; b. Construction of glycoside hydrolase recombinant expression vector; c. Prokaryotic expression of glycoside hydrolase protein; d. Purification of glycoside hydrolase protein to obtain glycoside hydrolase.

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