Glucoside hydrolase capable of degrading cellobiose and maltotriose

Abstract

The invention discloses a glucoside hydrolase capable of degrading cellobiose and maltotriose. The glucoside hydrolase is characterized in that the amino acid sequence of the glucoside hydrolase is SEQ ID NO. 1. The strain has relatively high activity on cellobiose and maltotriose, the enzyme activities are respectively 0.1 U and 0.102 U, and the specific activities are respectively 80.4 U / mg and 82.0 U / mg. The invention aims to enrich the existing carbohydrate enzyme gene bank and develop a novel efficient enzyme preparation. According to the present invention, the novel efficient carbohydrate enzyme is explored, and has efficient degradation ability on cellobiose and maltotriose, such that the reference is provided for the exploration of the novel enteric microorganism encoded CAZYmes. CAZymes generated by animal intestinal microorganisms are beneficial to decomposition of complex carbohydrates, the carbohydrates are difficult to decompose in a digestive system of a host, and the carbohydrate active enzyme obtained through excavation is expected to improve the digestion efficiency. By studying CAZymes in intestinal microorganisms, a novel enzyme preparation is expected to be developed and used in food industry, feed industry or as a therapeutic agent.

Description

Technical Field

[0001] This invention belongs to the field of microbial molecular biology and relates to a glycoside hydrolase isolated from the intestines of horses that can efficiently degrade cellobiose and maltotriose. Background Technology

[0002] Carbohydrate active enzymes act on glycoconjugates, oligosaccharides, and polysaccharides, breaking down large carbohydrate molecules into smaller products, accompanied by the release of ATP. The function and colonization of CAZymes are adapted to the local carbohydrate composition of various organs and tissues. Therefore, microbial CAZymes may play an important role in normal human physiological activities. [ZhouZY, Xu X, Zhou Y. [Research progress on carbohydrate active enzymes of humanmicrobiome]. Hua Xi Kou Qiang Yi Xue Za Zhi. 2019 Dec 1;37(6):666-670.] Bacteria in the digestive tract of equines have a great influence on their digestive system, especially the bacteria in the cecum. The massive number of microorganisms symbiotic in the enlarged cecum is an important source for developing highly efficient cellulase preparations. The quantity and types of these microorganisms affect the human body's digestion, absorption, and utilization of nutrients. The cecum has a capacity that is more than ten times that of a normal gastrointestinal tract. It is also the largest fermentation zone in equines, where an extremely rich and highly complex anaerobic microbial community is also found. This microbiome plays a crucial role in the animal digestive system. On one hand, the quantity and variety of bacteria directly affect the body's digestion, absorption, and utilization of nutrients; on the other hand, the large number of enzymes produced by this microbiome play an important role in the metabolism of dietary fiber. Li et al. used metagenomics to study the equine gut microbiome, confirming that the cecal microbiome is an important source of unknown enzymes involved in the degradation of complex carbohydrates [Li C, Li X, Guo R, et al. Expanded catalogue of metagenome-assembled genomes reveals resistome characteristics and 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 gut microbiota using metagenomic data analysis.

[0003] In recent years, with increasing attention to the functions of microorganisms, research on gut microbiota has also grown. A large number of microorganisms exist in the intestines of ruminants, playing a vital role in the host's nutritional metabolism and immune function, and are a key factor influencing health. To date, cellulose-decomposing bacteria have been reported isolated from the large intestine or rumen of animals such as cattle, horses, rabbits, tapirs, capybaras, rhinoceroses, monkeys, elephants, rats, and pigs [Neumann AP, McCormick CA, Suen G. Fibrobacter communities in the gastrointestinal tracts of diverse hindgut-fermenting herbivores are distinct from those of therumen.] Environ Microbiol . 2017;19(9):3768-3783. ]. Although dozens of cellulose-decomposing bacteria have been isolated and identified from animals and humans, research on these strains is not yet complete [Froidurot A, Julliand V. Cellulolytic bacteria in the largeintestine of mammals. ]. Gut Microbes . 2022;14(1):2031694.]. Therefore, in-depth research on the genome of animal gut microbiota and the discovery of novel carbohydrate-active enzymes that can efficiently degrade cellulose have important scientific research value and practical production significance. Summary of the Invention

[0004] This invention discloses a glycoside hydrolase capable of degrading cellobiose and maltotriose, the amino acid sequence of which is SEQ ID NO.1.

[0005] The amino acid sequences of the glycoside hydrolases that degrade cellobiose and maltotriose are as follows: MDRRTFLKSASMKGTAIVTASAVGTEMLHAAESIGASDVASGKRNAPKAKRLPEDLQELVKDSSLLRKPDNLTVACYTFPNYHASALHDKIYGPGWTEYNLVRSARPWFQGHAQPRGPLLGEMDESKPGTWEKYNELCKQSGIDVLIWDWYWYNNEPCLHEALENGFLRASNRNDVKFACMWTNHPWYVLYPTLLPNGYKAYPPSFAPADGS LKECWQSLSYIISRYCHLENYWRIDDKPVVCIWDPNRLEKNIGVDGVKQLFAELTEFARKLGHKGLHFHSSGFYSPNSKEVGYNTAGSYNPFTWVADNYQPKNIELPDYGVAAADVAFKL WPKHHDDFAIPYLPSLSPGWDSTPRYIPPVSRPDQPNRDAWPNCVILDNENPASFKALVQSAFAYLNKHKDVPPILTIACFNEWTEGHYLLPDNRFGYGMLDALAEAVGKSDNHQIHGFF.

[0006] A recombinant expression vector for a glycoside hydrolase capable of degrading cellobiose and maltotriose, characterized in that the nucleotide sequence of the glycoside hydrolase capable of degrading cellobiose and maltotriose described in claim 1 is inserted into the pET-28a vector to obtain a recombinant expression vector for the glycoside hydrolase capable of degrading cellobiose and maltotriose, wherein the obtained recombinant expression vector is pET-28a(+)-H113.

[0007] A recombinant bacterial strain is characterized by: cloning and expressing the recombinant expression vector pET-28a(+)-H113, transforming it into Escherichia coli, and then screening to obtain the strain.

[0008] The Escherichia coli strain is DH5α.

[0009] The strain was Escherichia coli BL21(DE3).

[0010] Building upon existing research, this invention screened for a functional hypothetical protein, H113, using bioinformatics and other methods. Physicochemical properties, signal peptide prediction, and secondary and tertiary structure prediction were then performed on H113. The protein was subsequently expressed in prokaryotes. Enzyme activity assays revealed that H113 can efficiently hydrolyze cellobiose and maltotriose, laying the foundation for the development of novel industrial enzymes.

[0011] A novel carbohydrate-active enzyme comprises the following steps: carbohydrate-active enzyme annotation, screening of hypothetical proteins, bioinformatics analysis of target protein H113, prokaryotic expression of target protein H113, protein purification of target protein H113, enzyme activity assay of target protein H113, and prediction of members of the carbohydrate enzyme H113 family.

[0012] The specific steps are as follows: Carbohydrate-active enzyme annotation: The acquired data was classified using GTDB to screen for Bacteroides. The CAZymes family was predicted using the default settings of the CAZymes analysis toolbox.

[0013] Screening of hypothetical proteins: The online software PULpy and SWISS-MODEL was used to annotate, screen, and determine the structural similarity of hypothetical proteins to carbohydrate-active enzymes.

[0014] Bioinformatics analysis of the target protein H113: Physicochemical properties and structure prediction of the protein were performed using online software such as SOPMA and SWISS-MODEL.

[0015] Prokaryotic expression of target protein H113: The target protein H113 was induced to be expressed using a prokaryotic expression system.

[0016] Protein purification of target protein H113: Pure H113 protein was obtained by affinity chromatography.

[0017] Enzyme activity assay of target protein H113: The activity of carbohydrate-active enzyme H113 was determined using the DNS method.

[0018] Prediction of members of the H113 family of carbohydrate enzymes: comparison of equine cecal protein library and NR database using blastp.

[0019] A further technical solution of the present invention is that the carbohydrate-active enzyme can efficiently degrade cellobiose and maltotriose.

[0020] A method for preparing the glycoside hydrolase of claim 1, capable of degrading cellobiose and maltotriose, is characterized by comprising the following steps: a. Data screening and bioinformatics analysis of carbohydrate-active enzymes; b. Construction of recombinant expression vector for glycoside hydrolases; c. Prokaryotic expression of glycoside hydrolase protein; d. The glycoside hydrolase protein is purified to obtain the glycoside hydrolase.

[0021] Compared with existing inventions, the present invention has the following beneficial effects: 1. The carbohydrate enzyme of this invention is derived from horse cecum, and compared with chemically synthesized products, it has the characteristics of being natural and non-toxic. 2. The novel carbohydrate enzyme discovered in this invention can efficiently degrade cellobiose and maltotriose, and is expected to be developed into a novel enzyme preparation for use in the food industry, feed industry or as a therapeutic agent. Attached Figure Description

[0022] Figure 1-1 The secondary structure of carbohydrate enzyme H113.

[0023] Figure 1-2 3D modeling of carbohydrate enzyme H113.

[0024] Figure 1-3 Prediction of the hydrophilicity / hydrophobicity of carbohydrate enzyme H113.

[0025] Figure 1-4 Prediction of the signal peptide of carbohydrate enzyme H113.

[0026] Figure 1-5 Prediction of transmembrane domains of carbohydrate enzyme H113.

[0027] Figure 1-6 Prediction of glycosylation sites in carbohydrate enzyme H113.

[0028] Figure 1-7 Prediction of phosphorylation sites of carbohydrate enzyme H113.

[0029] Figure 2-1 pET-28a(+)H113 plasmid extraction and enzyme digestion; Where: AM: marker, 1 and 2 are extracted from PET-28a+-H113 plasmid; BM: marker, 1: PET-28a+-H113 plasmid extraction, 2: PET-28a+-H113 enzyme digestion.

[0030] Figure 2-2 Carbohydrate enzyme H113 induced expression; Where: M: protein marker, 1: uninduced bacterial culture, 2: supernatant of induced bacterial culture, 3: bacterial cells after induction.

[0031] Figure 3 Predicted members of the H113 family of carbohydrate enzymes.

[0032] Figure 4 Hydrolytic activity of target protein H113 on different substrates. Detailed Implementation

[0033] The following embodiments further illustrate the above-described content of the present invention in detail. Obviously, the described embodiments are merely a part of the embodiments of the present invention, and not all of them. 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.

[0034] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0035] Example 1: Data screening of carbohydrate-active enzymes 1. Annotation of carbohydrate-active enzymes First, we performed GTDB classification on the acquired data to filter out Bacteroides. We then used the default settings of the CAZymes analysis toolbox to predict the CAZymes family. The annotated carbohydrate-active enzymes were sorted and filtered according to sequence similarity. Sequences with ≤30% sequence similarity and annotated as hypothetical proteins in the NCBI database were selected for further screening.

[0036] 2. Hypothesis protein screening We used PULpy annotation to screen and sort the polysaccharide utilization sites of the obtained protein sequences, and then marked the hypothetical protein sequences identified in the previous step at these polysaccharide utilization sites for further screening. We further determined the structural similarity between the hypothetical proteins and carbohydrate-active enzymes using the SWISS-MODEL online software. Finally, hypothetical proteins were selected based on the following criteria: not included in the CAZy database, sequence similarity ≤30%, annotated as hypothetical proteins in the NCBI database, and located in gene clusters at polysaccharide utilization sites with a certain degree of structural similarity to carbohydrate-active enzymes. These hypothetical proteins were then used for further analysis.

[0037] Example 2: Bioinformatics Analysis of the Target Protein 1. Bioinformatics analysis of the target protein The physicochemical properties of proteins were predicted using the ProtParam online tool; the hydrophilicity / hydrophobicity of proteins were predicted using the ProtScale online software; the signal peptide and transmembrane domain of proteins were predicted using the SignalP3.0 and TMHMM online tools, respectively; the glycosylation sites and phosphorylation sites of proteins were predicted using the NetNGlyc 1.0 and NetPhos 2.0 online tools, respectively; and the secondary and tertiary structures of proteins were predicted using the SOPMA and SWISS-MODEL online software, respectively.

[0038] Example 3: Prokaryotic expression of target protein H113 1. Ligation of the target gene and the vector The pET-28a(+)-H113 recombinant expression vector was constructed. Plasmids containing the target gene and the vector were extracted from the cloning strain DH5a using a plasmid miniprep kit. Both the target gene fragment and the vector DNA fragment were digested with restriction endonucleases EcoRI and XhoI. After digestion, the digested DNA products were recovered (using a Sangon Biotech (Shanghai) DNA recovery kit), and the concentration of the recovered products was measured using a Nanodrop 2000. The ligation system was calculated based on the concentration of the digested products, with a vector:target gene ratio of 3:1. After mixing, ligation was performed at 16℃ for 2 hours.

[0039] 2. Transformed expression strain The ligated recombinant plasmid pET-28a(+)-H113 was transformed into *E. coli* strain DH5α. Prepared competent cells were immediately placed on ice. The ligation product (no more than 1 / 10 of the transformation volume) was added to the competent cells, gently mixed, and placed on ice for 45 min. The ligation system was then heat-shocked in a 42°C water bath for 90 sec, and quickly transferred to ice for 3-5 min. 1 ml of LB broth was added to the competent cells, and the cells were cultured at 37°C with shaking for 1 h. After 1 h, the cells were centrifuged at 8000 rpm for 15 min. The supernatant was discarded, and approximately 100 µl of culture medium was collected in a tube. The culture medium and precipitate were mixed thoroughly, and an appropriate amount of culture was spread onto LB broth containing Kansas protein. + In solid culture medium, place the culture upright for 30 min, then incubate at 37°C for no more than 17 h. Pick colonies and place them in 1 ml of LB (containing Kansas) solution. + In EP tubes of culture medium, incubate on a shaker for 4 h, then inoculate into 20 ml LB (containing Kans) culture medium. + Incubate overnight in liquid culture medium; for bacterial preservation: add 500 µl of 50% glycerol to 500 µl of bacterial culture, mix well, and store at -80℃. Use a plasmid miniprep kit to extract recombinant plasmids from the cloning strain DH5a and transform them into Escherichia coli strain DE3 for expression.

[0040] 3. Inducing protein expression The expressed DE3 strain was inoculated into 20 ml LB (containing Kans) + Incubate overnight in liquid culture medium, then take 4 ml of the overnight culture and add it to 400 ml of LB (containing Kans). +In liquid culture medium, the cells were cultured in a shaker at 37°C until the logarithmic growth phase (OD600 = 0.6~0.8). IPTG was added, and expression was induced at 37°C for 18 h. The induced cells were centrifuged at 8000 rpm for 10 min at 4°C, and the precipitate was collected. The cells were washed with PBS, resuspended in 20 mL of non-denaturing lysis buffer (50 mM Tris, 500 mM NaCl, adjusted to 7.5 with hydrochloric acid), and incubated overnight at 4°C. The cells were then disrupted using an ultrasonic cell disruptor at 40% power for 30 min (3 s on, 5 s off).

[0041] 3. Protein purification Centrifuge the dissolved material at low speed to collect the supernatant. Filter the supernatant through a 0.22 μm filter membrane. Add 10 volumes of ddH2O to wash the nickel column, washing away the 20% ethanol used to store the nickel column. Add 10 volumes of elution buffer and washing buffer sequentially. Add 5 mL of the filtered supernatant, and continue adding 10 volumes of washing buffer to remove impurities. Collect the eluent as a control. Add 10 volumes of elution buffer to elute the target protein, and collect the eluent. Detect using 12% SDS-PAGE electrophoresis. For bacterial samples, perform electrophoresis at 12,000 r / min for 1 min. Remove the supernatant, add 40 μL of ddH2O and 10 μL of 5× protein loading buffer, and incubate in a boiling water bath for 10 min. During loading, add protein markers, uninduced bacterial culture, induced unpurified protein, and purified protein in sequence. Electrophore at 80 V for 30 min at room temperature, then at 120 V for 100 min. Visualize using Coomassie Brilliant Blue staining.

[0042] Example 4: Determination of enzyme activity and specific activity of carbohydrate enzyme H113 1. Determination of the enzyme activity of carbohydrate enzyme H113 Enzyme activity is defined as the amount of enzyme required to convert 1 micromolar of substrate in 1 minute at the optimal temperature; one unit of enzyme activity (U) is defined as this amount of enzyme. The formula for enzyme activity determination is: Enzyme activity (U) = (V × ΔOD) 540 ) / (ε×V×t). Where V is the volume of the reaction liquid (mL), OD 540 ε is the absorbance difference of the reaction solution at a specific wavelength, ε is the molar extinction coefficient of the enzyme-catalyzed reaction (L / mol·cm), V is the volume of the enzyme solution to be tested (mL), and t is the reaction time (min).

[0043] The target protease activity was determined using the following reaction system: 200 μl target protein, 300 μl cellobiose / maltotriose, incubated at 37 ℃ for 30 min. After the reaction was complete, 500 μl DNS was added and the mixture was boiled in a water bath for 10 min. The OD value was then measured after cooling. 540The amount of reducing sugar produced by the degradation of carbohydrates by the target protein can be obtained from the standard curve of reducing sugars. In the polysaccharide hydrolysis experiment, the selected polysaccharide substrates include carboxymethyl cellulose (CMC-Na), xylan, raffinose, soluble starch, sucrose, L-arabinose, α-lactose, D-(+)-maltose, amylopectin, amylopectin, sucrose, miciside, salicin, trehalose, mannose, arbutin, and methyl-α-D-galactopyranoside.

[0044] 1. Determination of the specific activity of carbohydrate enzyme H113 The formula for specific enzyme activity is: 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, and 1000: conversion of millimoles (mmol) to micromoles (µmol). The experimental results for enzyme activity assays are calculated by averaging three measurements. All data are expressed as mean ± standard deviation of three independent experiments (n=3).

[0045] Example 5: Prediction of members of the H113 family of carbohydrate enzymes To further understand the members in the equine cecum that are similar to the target protein H113, blastp (E value 10) was used. -5 The distribution of proteins encoded by the metagenomics of the equine cecum was compared with that in the NR database.

Claims

1. A glycoside hydrolase capable of degrading cellobiose and maltotriose, characterized in that: The amino acid sequence is SEQ ID NO.

1.

2. A recombinant expression vector for a glycoside hydrolase capable of degrading cellobiose and maltotriose, characterized in that, The amino acid sequence of the glycoside hydrolase capable of degrading cellobiose and maltotriose in claim 1 is inserted into a pET-28a vector to obtain a recombinant expression vector of the glycoside hydrolase capable of degrading cellobiose and maltotriose, and the obtained recombinant expression vector is pET-28a(+)-H113.

3. A method for producing the glycoside hydrolase capable of degrading cellobiose and maltotriose according to claim 1, characterized by: The method comprises the following steps: a. Data screening and bioinformatics analysis of carbohydrate active enzymes; b. Construction of a glycoside hydrolase recombinant expression vector; c. Prokaryotic expression of glycoside hydrolase protein; d. Purification of glycoside hydrolase protein, i.e. obtaining glycoside hydrolase.

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