CN114317499B - Acid beta-glucosidase, and coding gene and application thereof - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to acid beta-glucosidase, and a coding gene and application thereof. The research utilizes RNA-SEQ to successfully screen out GH3 family beta-glucosidase gene bgl3HB, the full length of the sequence is 2688bp, the nucleotide sequence is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2. The catalytic efficiency of the beta-glucosidase (Bgl 3HB for short) on the aryl glycoside substrate is far higher than that of the beta-glucosidase reported in the prior art, the beta-glucosidase keeps higher enzyme activity for a long time under a higher temperature condition, and the enzyme activity is kept unchanged after being placed in 50% ethanol for 6 hours, so that the beta-glucosidase has good industrial application prospect.

Description

Acid beta-glucosidase, and coding gene and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to acid beta-glucosidase, and a coding gene and application thereof.

Background

With the increasing demand and consumption of fossil energy, the reserve of fossil energy is increasingly reduced, the ecological environment is gradually destroyed, and the global energy crisis is also increasingly imminent. The second generation bioethanol is also one of the most representative alternative energy sources worldwide as a partial substitute for biofuel, and has attracted great attention. Plant cellulose capable of producing bioethanol from renewable biomass resources has become a recent research focus on new energy sources.

Plant biomass is the most widely distributed and abundant renewable biological resource in the world and will probably play the same role as 20 th century petroleum in the future. The most important part of the plant biomass is the plant cell wall, which accounts for more than 90% of all biochar reserves. The main components of the plant biomass are cellulose and hemicellulose, and if the cellulose is degraded into usable saccharide substances, the plant biomass has great practical significance for solving the problems of energy shortage, air pollution, feed resource shortage and the like facing the current world.

Mangrove (Mangrove) grows at a relatively narrow border between land and sea, a forest suitable for salt tolerant species and with soil being slightly acidic. The Hainan island mangrove ecological system has a large amount of plant biomass and rich litters, so that the soil environment with rich organic matters and the unique geographical environment have a large amount of microorganisms with acidophilic, salt-tolerant and high-yield cellulase. The mangrove environment is characterized by special circulation of nutrients and interaction of enzymes, because oxygen released by plant roots can be utilized at any time.

Liu Sixin et al (2011) found that Hainan mangrove soil pH is typically between 3.0 and 7.5. Some studies have shown that mangrove soil actinomycetes and fungi are more numerous, the most common filamentous fungi being trichoderma and aspergillus. Beta-glucosidase produced by filamentous fungi is widely used in industry due to its easy recovery and high activity potency. In recent years, since Trichoderma harzianum has excellent antibiotic and re-hosting effects, its research has been mainly conducted in that it can secrete volatile or nonvolatile toxic metabolites that inhibit the proliferation of pathogenic microorganisms. Few reports on the direction of Trichoderma harzianum beta-glucosidase are about, trichoderma harzianum strains are rarely used for producing cellulase, the potential of filamentous fungi for producing enzymes from cellulolytic complexes is studied, and the Trichoderma harzianum IOC-4038 can be observed to produce a large amount of endoglucanases while producing high levels of beta-glucosidase; elham finds that trichoderma harzianum beta-glucosidase can replace chemical bactericides to prevent and control soybean alternaria solani causing crop carbon rot; aboshosha et al treated Trichoderma harzianum with ultraviolet irradiation and Ethyl Methanesulfonate (EMS) to obtain a strain with high beta-glucosidase yield.

In practical production applications, such as in brewing processes, acid-resistant beta-glucosidase plays a key role in the enzymatic release of aroma compounds from glycoside precursors present in fruit juices, cocktails and wines. Acid beta-glucosidase is very important in the food and beverage industry and in the production of fuel ethanol from cellulosic materials. In addition, most commercial cellulases on the market do not have good thermostability, and beta-glucosidase is the rate-limiting enzyme in the cellulose hydrolysis process, which makes the demand and market for thermostable beta-glucosidase broader. Firstly, the enzymatic hydrolysis reaction temperature of the plant biomass is 30-50 ℃, and the thermally stable beta-glucosidase is favorable for the enzymatic reaction; and secondly, the enzyme hydrolysis reaction is carried out at a higher temperature, so that the pollution of mixed bacteria can be effectively avoided, and the beta-glucosidase with acid resistance and thermal stability has wide commercial value and application prospect.

Disclosure of Invention

In view of the above, the invention provides an acid beta-glucosidase, and a coding gene and application thereof. The catalytic efficiency of the acid beta-glucosidase (Bgl 3HB for short) on the aryl glycoside substrate is far higher than that of the beta-glucosidase reported in the prior art, and the acid beta-glucosidase can resist high temperature, acidophilic and high-concentration salt ions and has a promising industrial application prospect.

In order to achieve the above object, the present invention provides the following technical solutions:

an acidic beta-glucosidase has an amino acid sequence shown in SEQ ID NO. 2 or an amino acid sequence with the same or similar functions obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 2.

The invention utilizes RNA-seq sequencing to screen beta-glucosidase gene with high expression quantity and good diversity, and expresses the beta-glucosidase gene in wild pichia X33, and passes through anion exchange column UNOsphereTM Q Cartridges and nickel column Nuvia ^TM The IMAC Ni-Charged is separated and purified to obtain the beta-glucosidase with high activity, high temperature resistance, acidophilic and high salt ion tolerance, and the ammonia thereofThe base acid sequence is shown as SEQ ID NO. 2. The molecular size of the protein is about 90kD; the optimum reaction pH for Bgl3HB was 5.0 and the optimum reaction temperature was 40℃as determined by using pNPG as a substrate.

In some embodiments, the method for preparing the acid β -glucosidase comprises the steps of:

1) Cloning the coding gene of the beta-glucosidase shown in SEQ ID NO. 2 to an expression vector to obtain a recombinant vector;

2) And transforming the recombinant vector into host bacteria, inducing expression, and purifying by using an anion exchange chromatographic column and a nickel column to obtain the beta-glucosidase.

The invention also provides a gene encoding the beta-glucosidase, namely bgl3HB gene.

In some embodiments, the bgl3HB gene has any one of nucleotide sequences I) through III):

i) A nucleotide sequence shown as SEQ ID No. 1; or (b)

II) substitution, deletion and/or addition of one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.1 and expression of the protein shown in SEQ ID NO. 2; or (b)

III) a nucleotide sequence complementary to the sequence shown in SEQ ID No. 1.

The invention successfully screens out the GH3 family beta-glucosidase gene bgl3HB by using RNA-SEQ, the total length of the sequence is 2688bp, the nucleotide sequence is shown as SEQ ID NO.1, and the gene is derived from Trichoderma harzianum strain.

Meanwhile, the invention also provides a recombinant vector containing the bgl3HB gene and host bacteria transformed with the recombinant vector.

In some embodiments, the host bacteria include escherichia coli, pichia pastoris.

In addition, the invention also provides application of the beta-glucosidase, the gene, the recombinant vector or the host bacterium in preparing at least one substance of reducing sugar, glucose and bioethanol.

The invention also provides a method for preparing the reducing sugar and/or glucose, which takes cellulose raw material as a substrate, and uses cellulase and the beta-glucosidase to carry out enzymolysis to obtain the reducing sugar and/or glucose.

In some embodiments, the cellulosic feedstock comprises at least one of bagasse, corn stover, and grain residue, including but not limited to.

The invention provides beta-glucosidase, and a coding gene and application thereof. Compared with the prior art, the beta-glucosidase has far higher catalytic efficiency on aryl glycoside substrates than the existing beta-glucosidase, keeps higher enzyme activity for a long time under higher temperature conditions, can keep the enzyme activity unchanged after being placed in 50% ethanol for 6 hours, and has good application prospect.

Drawings

FIG. 1 shows the melting curve of RT-PCR amplification of reference gene (a) beta-tublin, (b) actin;

FIG. 2 shows the melting curve of QT-PCR amplification of the gene to be tested;

FIG. 3 shows a schematic representation of Bgl3HB protein domain analysis;

FIG. 4 shows Bgl3HB dbCAN database annotation results;

FIG. 5 shows Trichoderma harzianum total RNA agarose gel electrophoresis;

FIG. 6 shows agarose gel electrophoresis analysis of the expression sequence of the beta-glucosidase gene bgl3 HB; m: molecular weight standard, lane 1: bgl3HB gene;

FIG. 7 shows the results of plate culture of recombinant product pPICZαA/bgl3HB conversion competence;

FIG. 8 shows the results of colony PCR identification of pPICZ alpha A/bgl3HB recombinant plasmids; m: molecular weight standard, lane 1: pPICZ. Alpha.A/bgl 3HB recombinant plasmid;

FIG. 9 shows the growth of recombinant plasmid pPICZ alpha A/bgl3HB pichia pastoris transformants;

FIG. 10 shows the PCR identification of colonies of pPICZαA/bgl3HB/X33 Pichia pastoris transformants;

FIG. 11 shows the growth of recombinant pPICZαA/bgl2/X33 on YPD plates containing 400 μg/mL (a), 800 μg/mL (b), 1200 μg/mL (c), 2000 μg/mL (d) Zeocin;

FIG. 12 shows a Bgl3HB column chromatography purification Bgl3HB map;

FIG. 13 shows the result of SDS-PAGE analysis of Bgl3 HB; m170: 170Kda protein Marker;1 purified Bgl3HB

FIG. 14 shows pNP concentration standard curves;

FIG. 15 shows a DNS glucose standard curve;

FIG. 16 shows GOD-method glucose standard curves;

FIG. 17 shows a BCA method protein quantification standard curve;

FIG. 18 shows the effect of reaction pH on enzyme activity;

FIG. 19 shows the pH stability test results;

FIG. 20 shows the effect of reaction temperature on enzyme activity;

FIG. 21 shows the thermal stability test results;

FIG. 22 shows the effect of metal ions on enzyme activity;

FIG. 23 shows the effect of NaCl on Bgl3HB enzyme activity;

FIG. 24 shows Bgl3HB synergistic commercialized cellulase saccharification effect; (a) determining the reducing sugar content by DNS method; (b) glucose oxidase kit for determining glucose content.

Detailed Description

The invention provides a beta-glucosidase, and a coding gene and application thereof. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

The invention is further illustrated by the following examples:

example 1

Experimental methods

1. Trichoderma harzianum RNA-seq sequencing

1.1 preparation of samples and Unigene annotation

Culture media with Glucose (GLU) and microcrystalline cellulose (MCC) as the sole carbon sources induced expression of β -glucosidase, respectively, where GLU is the control group and MCC is the treatment group. Four basic databases were annotated for genes, GO was annotated, and CDS predictions were made using a Transdecoder.

1.2 quantitative and differential Gene expression analysis

The alignment software bowtie2 was used to compare the reads of each sample to the reference transcriptome. This step was used to compare expressed genes and their quantification for comprehensive evaluation and identification of transcriptomes.

1.3 screening and verification of the Gene of interest

Using FDR and log ₂ FC to screen for differential genes under the conditions of (1) FDR<0.05；(2)|log ₂ FC|>2; (3) the variability is significant.

And (3) carrying out sequence analysis on the screened genes, including signal peptide prediction and active domain analysis, designing specific primers according to the nucleic acid sequence of the screened genes, and verifying the gene expression condition through RT-PCR and QPCR.

2. Cloning and sequence analysis of bgl3HB Gene

2.1 extraction of total RNA of Trichoderma harzianum Strain and first strand cDNA Synthesis

E.Z.N.A was used. ^TM The total RNA of the Trichoderma harzianum strain obtained by the primary fermentation (OMEGA, model R6840-01) is extracted by using a Fungal RNA Kit, and the method comprises the following specific steps:

(1) After culturing for six days with a fermentation medium using MCC as a carbon source and urea and Yeast extract, (NH 4) 2SO4 as three nitrogen sources, the fungus balls are collected by a 1.5mL centrifuge tube; setting the rotation speed of a centrifuge to 12000xg, centrifuging for 10min to collect (less than or equal to 100 mg), pouring the culture medium obtained by centrifugation into a garbage cup rapidly, placing the centrifuge tube in liquid nitrogen to prevent RNase enzyme from cracking RNA, taking out the fungus balls in an enzyme-free environment cleaned in advance, grinding by using liquid nitrogen, adding 500 mu L (or 600 mu L according to the amount of the ground fungus body and the addition amount of the mixed liquid) of Buffer RB/beta-Mercap mixed liquid into a 1.5mL centrifuge tube in advance, adding the ground fungus body into the mixed liquid, and fully cracking the fungus body by using a vortex oscillator;

(2) To the mixture was added 500. Mu.L of Phenolwater and 100. Mu.L of 2M sodium acetate (pH 4.0), and the medium was homogenized using a high-speed vortex for 15s;

(3) Adding 200 mu L of chloroform into the mixed solution in the last step, uniformly mixing for 15s by using a vortex oscillator, and placing the mixed solution on ice for 10min;

(4) Taking out a 1.5mL centrifuge tube from the ice box, centrifuging for 15min at 12000xg and 4 ℃, taking care that the centrifuge tube is not rocked when moving, and at the moment, layering of the mixed solution can be seen;

(5) Taking out the 1.5mL centrifuge tube after centrifugation, taking care of not shaking, transferring 600 mu L of supernatant to a new RNase-free 1.5mL centrifuge tube, adding 600 mu L of 70% ethanol prepared in advance, and slowly blowing and uniformly mixing by using a pixel;

(6) Placing 2.0. 2.0mLCollecting tube on a 1.5mL centrifuge tube rack without enzyme treatment, sleeving an RNA binding column on 2.0mL Collecting tube, gently adding 1.2mL mixed solution obtained in the step (5) into the binding column, setting the rotating speed of a centrifuge to 10000g, adjusting the temperature to room temperature, centrifuging for 30s to bind DNA on the column, pouring filtrate in a Collecting tube into a garbage cup after centrifugation is completed, and repeatedly using the Collecting tube;

(7) Adding 500 mu L RNA Wash Buffer I into the binding column, setting the rotation speed of a centrifuge to 10000Xg, heating to room temperature, centrifuging for 30s, pouring filtrate in a Collecting tube into a garbage cup, and repeatedly using the Collecting tube;

(8) Adding 700 mu L of RNA Wash Buffer II diluted by absolute ethyl alcohol into a binding column to the center of the bottom of the binding column, setting the rotating speed of a centrifugal machine to 10000xg, heating to room temperature, centrifuging for 30s, pouring filtrate in a Collecting tube into a garbage cup, and repeatedly using the Collecting tube;

(9) Adding 500 mu L RNA Wash Buffer II again to the binding column and repeating the operation of the step (8);

(10) Changing a new Collecting tube, sleeving a binding column, setting the rotating speed of a centrifugal machine to 13000xg, setting the temperature to room temperature, and centrifuging the binding column for 1min by an empty column to spin-dry the matrix in the binding column;

(11) Finally, sleeving the binding column on a 1.5mL centrifuge tube treated by RNase-free, adding 50 mu L of DEPC-treated water to the center of a bottom membrane of the binding column, standing for 1-2min, setting the rotating speed of a centrifuge to 13000xg, setting the temperature to room temperature, and centrifuging for 1min;

(12) Taking a small amount of RNA sample, detecting the quality and concentration of RNA by using a NanoPhotometer N50 Touch, preparing 1% agarose gel, detecting the quality of the extracted RNA by electrophoresis under the condition of 100V voltage, determining that the quality of the sample is better, and subpackaging the sample in an RNase-free centrifuge tube at the temperature of-80 ℃ for storage when the concentration is higher.

Reverse transcription was performed using the R312-01/02 kit, the system was as follows:

TABLE 1

RNase-free ddH 2 O	to 8μL
		Total RNA	10pg-5μg

Adding the above reagent into RNase-free PCR tube, placing into preheated PCR instrument, heating at 65deg.C for 5min, standing on ice for 2min, and cooling.

TABLE 2

The mixed solution in the last step	8μL
		5×gDNA wiper Mix	2μL

The above reagent was added again to take out gDNA, and after mixing with a gentle balloon, the mixture was reacted in a PCR apparatus at 42℃for 2 minutes.

TABLE 3 Table 3

The mixed solution in the last step	10μL
		10×RT Mix	2μL
HiScript III Enzyme Mix	2μL
		Oligo(dT) 20 VN	1μL
RNase-free ddH 2 O	5μL

The procedure of Table 4 was performed by gently stirring with a blender.

TABLE 4 Table 4

37℃	45min
		85℃	5s

The product obtained after the PCR reaction is cDNA, and the cDNA is stored at-20 ℃ by using a RNase-free 1.5mL centrifugal tube after subpackaging, so that the follow-up test is convenient to carry out.

2.2 amplification of the Gene bgl3HB of interest

Screening the result of the transcriptome to obtain a gene bgl3HB with high expression level and up-regulated expression, respectively analyzing the nucleotide sequence of the gene bgl3HB by using Snapgene software, designing a Primer for PCR amplification of an Open Reading Frame (ORF) by using a Primer premier5.0, adding EcoRI-HF and XbaI endonuclease gene sequences at the 5 'end and the 3' end of the sequence, and ensuring that the bgl3HB is directionally connected with an expression vector pPICZ alpha A; the 3' end of the forward primer was introduced with a 6His sequence for protein purification, and the primer design sequence is shown in Table 5:

table 5 beta-glucosidase gene cloning primers

The PCR reaction system was as follows (all operations were performed on ice):

TABLE 6

The reaction procedure was as follows:

TABLE 7

The PCR products were run out and sequenced in Sangon. The remaining product was stored at-20 ℃.

2.3 transformation of E.coli competence

(1) Taking DH5 alpha purchased from Sangon out of an ultra-low temperature refrigerator, standing on ice for 30min, taking out gently, taking out after melting, carefully adding 1 mu L of plasmid with seed preserved in advance, gently sucking and blowing with a pixel, mixing well, and placing on ice for 30min;

(2) Taking out a 1.5mL centrifuge tube from ice, immediately putting the centrifuge tube into a preheated PCR instrument at 42 ℃ for heat shock 45s to form a gap for plasmid to enter, and then rapidly and gently transferring the 1.5mL centrifuge tube onto ice for 2min (taking care that the centrifuge tube is not required to shake);

(3) Adding 700 mu L of pre-prepared SOC liquid culture medium which is sterilized under high pressure and does not contain any antibiotics such as Zeocin and the like into a centrifuge tube, slightly mixing the liquid culture medium with a pipedor, and then placing the mixture into a shaking table at 37 ℃ and 180rpm for culturing for 1h to recover strains;

(4) 200. Mu.L of the product was pipetted with a syringe and dropped onto a syringe containing Zeocin (25. Mu.g. ML ^-1 ) Is spread on Luria-Bertani agar medium and uniformly coated.

(5) The dried plate was placed in an incubator at 37℃and after complete absorption of the bacterial liquid, the plate was inverted overnight.

2.4 Positive clone colony PCR verification

Single colonies on Luria-Bertani plates were picked for colony PCR, the reaction system was as follows:

TABLE 8

The PCR amplification reaction conditions were:

TABLE 9

The obtained product is subjected to 1% run nucleic acid gel electrophoresis detection, and clone without error is inoculated with 200 mug.mL ^-1 The Luria-Bertani broth of Zeocin was incubated at 37℃for 16h at 180rpm to allow the monoclonal growth to the logarithmic phase.

2.5 extraction of plasmid pPICZ alpha A

E.Z.N.A. ^TM Plasmid DNA mini Kit kit plasmid extraction:

(1) Centrifuging 10000xg of 5mL of bacterial liquid cultured in a test tube for 1min at room temperature, discarding supernatant, and centrifuging for 5 times;

(2) Adding 250 mu L of the RNase-added reagent I and then carrying out vortex oscillation;

(3) Continue to add 250 μl of reagent II, gently invert four and five times;

(4) Then 350 mu L of reagent III is added, and after the addition is finished, the reagent III is rapidly inverted for many times, so that white substances in the centrifuge tube are found to be formed;

(5) Centrifuging the centrifuge tube for 10min at 13000Xg at room temperature after the precipitate is formed;

(6) During centrifugation, the DNA binding column is arranged on a 2mL collecting pipe, a pipetting gun is carefully used for transferring the supernatant to a central membrane of the binding column, and then the binding column is centrifuged for 1min at 13,000Xg at room temperature, and the filtrate is poured into a garbage cup and then the binding column is reloaded into the collecting pipe;

(7) Adding 500 mu LHB buffer, centrifuging according to the conditions, and pouring the filtrate into a garbage cup;

(8) Sleeving the collecting pipe, continuously adding 700 mu L DNA Wash Buffer into the combined column, centrifuging according to the conditions, and pouring filtrate into a garbage cup;

(9) Repeating the previous step;

(10) Mounting the collecting pipe on a new collecting pipe, and centrifuging for 2min with 13000xg empty column;

(11) After the binding column was placed in a 1.5mL centrifuge tube, 50. Mu.L of the solution Buffer was added to the center of the bottom of the binding column membrane, and the mixture was allowed to stand at room temperature for 1min, centrifuged at 13000Xg for 1min, and plasmids were collected.

2.6 preparation and recovery of linearization vectors

The collected plasmid pPICZ. Alpha.A was subjected to double cleavage using EcoRI-HF and XbaI, the reaction system was as follows:

table 10

Carrying out a warm bath reaction at 37 ℃ for 6 hours, detecting an enzyme digestion product by running gel, and adopting a Gel Extraction Kit kit for gel recovery:

(1) Cutting off the target fragment quickly after running the gel;

(2) The gel block was transferred to a weighed 1.5mL centrifuge tube and the weight of gel block was obtained by continuing the weighing. Adding XP2 Binding Buffer with equal volume, placing on a floating plate, and water-bathing in a water bath kettle at 55deg.C for 7min, and mixing every 2min in the water bath process;

(3) Taking out the DNA Mini binding column in the water bath process and loading the DNA Mini binding column in a 2mL collecting pipe;

(4) The mixture obtained in the third step was added to the center of the DNA Mini binding column using a pixel. The centrifuge was centrifuged at 10,000Xg for 1min at room temperature. Discarding the filtrate in the collecting pipe, and then sleeving the column back into the 2mL collecting pipe for repeated use;

(5) Transferring 300 mu L of XP2 Binding Buffer into a column, centrifuging at 13000rpm for 1min at room temperature, and pouring the filtrate into a garbage can;

(6) The binding column was collected in a recovery header and 700. Mu.L of SPW Buffer diluted with absolute ethanol was transferred to the binding column. Centrifuging 10000xg of the filter liquor for 1min at room temperature by a centrifuge, and pouring the filter liquor into a garbage can;

(7) Repeating step (6);

(8) Sleeving the DNA Mini binding column on a new 2mL collecting tube, and spin-drying the binding column matrix by setting a centrifugal machine 13,000Xg for 2min at room temperature;

(9) The DNA Mini binding column was loaded onto a 1.5mL centrifuge tube without enzyme, 30. Mu.L of the Elution Buffer was added to the bottom of the DNA Mini binding column, and the column was allowed to stand for 1min and centrifuged at 13000Xg for 1min to completely elute the DNA.

(10) Recovery of plasmid run nucleic acid gel electrophoresis confirmed that the cleavage was successful.

2.7 construction of recombinant expression vectors

Recombinant vector kit constructed by C112 is adopted for recombination, and the dosage is as follows:

TABLE 11

The recombination reaction system is as follows:

table 12

X and Y are the vector amount and the insert amount, respectively. The product was reacted at 37℃for 30min, immediately cooled to 4℃on ice and stored at-20 ℃.

2.8 conversion and identification of recombinant products

(1) E.coli DH 5. Alpha. Component Cells were thawed on ice.

(2) The reagents in the recombination system were reacted in a PCR apparatus at 37℃for thirty minutes, taken out and cooled on ice.

(3) 10. Mu.L of the above product was gently mixed with 100. Mu.L of DH 5. Alpha. Thawed on ice to prevent cell death, and after 30min of standing on ice, gently removed.

(4) And (3) placing the product in a PCR instrument preheated in advance, and carrying out heat shock at 42 ℃ for 45s so that the recombinant product can pass through gaps of escherichia coli cells, and immediately placing the product on ice for 2-3min after heat shock so as to cool the product, wherein the situation that the cells are dead due to external force is avoided.

(5) After cooling, adding 700 mu L of SOC liquid culture medium into the centrifuge tube, and culturing at 180rpm for 1h to recover strains;

(6) The poured LLura-Bertani plate containing Zeocin was also placed in an incubator at 37 ℃.

(7) 200. Mu.L of the transformed competent cell droplets were pipetted with a pipeter to contain 25. Mu.g.mL ^-1 LLura-Bertani of ZeocinThe agar medium was dried with a spreading bar.

(8) Plates were placed in an incubator at 37℃until bacterial fluid was completely absorbed by the plates and the plates were turned over for overnight incubation.

(9) Several clones on the recombinant transformation plates were picked and transferred to Zeocin-resistant LLura-Bertani medium and incubated at 37℃for 12-16h at 180 rpm.

(10) Colony PCR was performed and the correct band of glycerol bacteria from the PCR product was sent for sequencing.

Efficient expression of bgl3HB Gene in Pichia pastoris X33

3.1 recombinant plasmid extraction

The plasmid was extracted as 2.5.

3.2 linearization of recombinant plasmids

In order to improve the connection efficiency of the recombinant plasmid on a Pichia pastoris X chromosome and facilitate the subsequent test screening of the high-copy bell clone, the recombinant plasmid is subjected to single enzyme digestion by using endonucleases PmeI and SacI, and a single enzyme digestion linearization system comprises:

TABLE 13

The PCR instrument is preheated to 37 ℃ and reacted for 30min, the mixture is reacted for 20min at 65 ℃ to thermally deactivate the endonuclease, and after the reaction is finished, the linearization result is detected by running gel and recovered.

3.3 preparation of Pichia pastoris competence

The specific preparation method of the pichia pastoris X33 competent cells comprises the following steps:

(1) Taking out the preserved strain X-33 from the ultralow temperature refrigerator, resuscitating, scribing the glycerinum on a YPD plate, sealing with a sealing film, and culturing in a incubator at 30 ℃;

(2) Inoculating the single colony of X-33 into YPD liquid culture medium, and culturing overnight at 30 ℃;

(3) Inoculating the bacterial liquid cultured in step (2) into 100mL of non-anti YPD liquid culture medium according to 0.1% inoculum size, setting the temperature of a shaking table to 30 ℃, setting the rotating speed to 180rpm, and shaking to an absorbance valueOD ₆₀₀ =about 1.5;

(4) Adding 25mL of bacterial liquid, centrifuging at 4 ℃ and 1500xg for 5min, pouring out the supernatant medium to collect cells, and flushing the bottom of a centrifuge tube with 25mL of precooled Sterile water to suspend the cells;

(5) Centrifuging under the same condition, adding 15mL of precooled Sterile water, and flushing suspended cells at the bottom of the centrifuge tube again;

(6) Centrifuging under the same condition, adding 10mL of precooled Sterile water, and repeating the above operation;

(7) Centrifugation was performed under the same conditions, and 10mL of cold 1M sorbitol was added to the mixture after pouring the water to dry, and the above procedure was repeated;

(8) The supernatant was decanted under the same centrifugation conditions, and the cells were gently mixed in suspension with 500. Mu.L of pre-chilled 1M sorbitol and dispensed using sterile 1.5mL centrifuge tubes, 80. Mu.L of each tube, and stored at-80 ℃.

3.4 transformation of Yeast competent cells by electric shock with recombinant plasmids

The linearized recombinant plasmid and the linearized empty shock were transferred into X33 competent cells.

The specific electric shock conversion process is as follows:

(1) In an ultra-clean workbench, gently mixing the prepared 80 mu L competent cells with 20 mu L linearization recombinant vector (5-20 mu g), and transferring into a precooled 0.2cm electric shock conversion cup;

(2) Placing on ice for 5min;

(3) During the period, 1M sterile sorbitol is placed on ice for precooling, an electric shock conversion cup is taken out, then is wiped by absorbent paper and is placed into an electric shock conversion instrument, and yeast parameters are recommended by a system for electric shock treatment;

(4) Immediately adding 1mL of precooled 1M sterile sorbitol after electric shock treatment, transferring to a sterile centrifuge tube after gentle mixing, sealing by a sealing film, and incubating in a 30 ℃ incubator for about 1 h;

(5) Prepared 100 mug.mL ^-1 The YPDS plate of bleomycin is placed in an incubator for preheating; 200. Mu.L of the post-transformation inoculum was spread and placed in a 30℃incubator until the plates developed transformants.

3.5 screening and verification of Yeast Positive transformants

The plate transformants were used as templates for PCR reactions with universal 3'AOX1 and 5' AOX1 primers and sent to Sangon for product sequencing verification. The AOX1 universal primer amplification system in pPICZ alpha A is as follows:

TABLE 14

The PCR amplification reaction conditions were:

TABLE 15

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After amplification, 10 mu LPCR products are taken for 1% gel running electrophoresis, whether the transformant is a positive clone or not is judged from the position of an electrophoresis band, and the rest products are sent to a biological company for sequencing verification.

3.6PTVA method for screening multiple copies

The positive clone pPICZalpha A-bgl3HB with correct sequencing verification is transferred to 200 mu g.mL ^-1 Positive clones with good growth were transferred to 400. Mu.g/mL cells in YPD agar medium of Zeocin using post-transformation vector amplification method (PTVA method) (Sunga et al 2010) at five-day intervals ^-1 、800μg·mL ^-1 、1200μg·mL ^-1 、2000μg·mL ^{- 1} The positive clones containing multiple copies were screened in YPD agar medium of Zeocin to adapt the cells to higher concentrations of antibiotics and the screened high copy clones were sent to sequencing company for validation. Correct clones were verified to be inoculated into 250ml lypd broth.

3.7 Induction secretion expression of Bgl3HB

The cells were resuspended in 50mL BMGY medium to 1000mL BMGY liquid medium, induced at 28℃and 180rpm for 6d, during which 2% methanol was added every 24h and the enzyme activity was detected. The culture broth was centrifuged at 12,000rpm for 30min at 4℃to collect the supernatant.

4. Purification of the recombinase Bgl3HB

4.1 sample treatment

The engineering strain X33-bgl3HB is transferred to BMMY culture medium to collect supernatant after 2d culture in BMGY culture medium, and placed on ice, filtered and sterilized by a 0.22 mu m filter membrane, and the obtained liquid is concentrated by a 50kDa ultrafiltration tube and replaced by buffer A, and stored at 4 ℃ for later use.

4.2Bio-ScaleTM Mini UNOsphere ^TM Q Cartridges column chromatography

By unolphere ^TM The crude enzyme was purified by Q media affinity chromatography (column model: UNOsphere Q column mL; buffer A: citrate buffer (pH 7.0); buffer B: high-salt citrate buffer (pH 7.0)), comprising the following steps:

(1) The pump flow rate was set to 6.0mL min ^-1 (288cm·h ^-1 )；

(2) Washing with buffer A for 2min;

(3) Washing with buffer B for 5min;

(4) Equilibrate with buffer B for 5min;

(5) Loading, namely, taking attention that bubbles are not generated in the loading period, and removing the bubbles by using a needle cylinder if the bubbles are generated;

(6) Washing the exchange column with buffer a and collecting the breakthrough peak;

(1) The flow rate is regulated to 1 mL/min ^-1 Stepwise eluting at 5%,10%,15%, and 20% in buffer B concentration of 1-20%, collecting eluate at different peak times, and determining OD ₄₀₀ nm absorbance value and SDS-PAGE denatured protein electrophoresis analysis purification effect;

(2) Thoroughly eluting with 1M buffer B to clean the hybrid protein;

(3) Sterilizing the exchange column with 1M NaOH, and maintaining the contact time for 40 min;

(4) Re-equilibrate the column with 1M buffer B;

(5) Washing the exchange column with 20% ethanol, removing the exchange column, and preserving at 4deg.C;

(6) The successfully purified proteins were transferred to a completely new 15kDa Amicon U1tra-15 ultrafiltration tube for replacement 2-3 times with HEPES (pH 7.0) solution for desalting and concentration.

4.3Bio-Scale ^TM Mini Nuvia ^TM IMAC Ni-Charged Cartridges column chromatography

By Nuvia ^TM IMAC Ni-Charged media affinity chromatography (column model: nuvia) ^TM IMAC column 5mL was subjected to secondary purification. The method comprises the following specific steps:

(1) With 5CV of equilibration buffer at 2mL min ^-1 Is a velocity balanced column;

(2) At 1 mL/min ^-1 Is loaded with sample lysate at a rate;

(3) 1mL min with 6CV of equilibration buffer ^-1 Is used for washing the exchange column at the speed of (1);

(4) The washing buffer was used at a rate of 2mL min with 6CV ^-1 Is used for washing the exchange column at the speed of (1);

(5) The solution was eluted with 10CV of elution buffer at 2mL min ^-1 Is used for eluting purified protein at a speed;

(6) With 5CV guanidine hydrochloride solution at 6 mL/min ^-1 Is used for cleaning the exchange column at the speed of (1);

(7) Sterilizing the exchange column with 1M NaOH, and maintaining the contact time for 40 min;

(8) 6mL min with 5CV of 0.1M EDTA ^-1 The exchange column is cleaned at a speed of (1) to strip metal ions;

(9) The solution was treated with 5CV of a 0.1M nickel sulfate solution at 6mL min ^-1 Regeneration of the column at a rate of (2);

(10) Washing the exchange column with 20% ethanol, removing the exchange column, and preserving at 4deg.C;

(11) The successfully purified proteins were transferred to a completely new 15kDa Amicon U1tra-15 ultrafiltration tube for replacement 2-3 times with HEPES (pH 7.0) solution for desalting and concentration.

4.4 SDS-PAGE analysis of purified proteins

50. Mu.L of purified Bgl3HB protein sample was taken, mixed with 10. Mu. L protein loading buffer, and boiled in boiling water for 8min to denature it. mu.L of the denatured mixed solution was spotted into a gel electrophoresis tank. Setting the constant voltage to 120V for 70min until the strip runs to the bottom of the gel tank, dyeing with Coomassie blue dye solution, and deducing the molecular size of the target protein according to protein marking standards.

4.5 study of the enzymatic Properties of Bgl3HB

4.5.1 enzyme Activity determination and protein quantification Standard Curve

Determination of Activity of different substrates of 4.5.1.1 beta-glucosidase

The degradation capacity of Bgl3HB was determined separately under the action of substrates of different types of aglycones, the substrates being as follows: fiber II, III, IV, pentasaccharide gentiobiose, sophorose, and laminarin, pNPG, daidzin, laminarin.

The method for measuring the activity of the beta-glucosidase comprises the following steps:

specific activity unit U.mg ^-1 Is defined by: enzyme activity units contained per mg of enzyme protein. The protein concentration of the enzyme is measured by the BCA method, and the enzyme activity of the beta-glucosidase is measured, so that the specific activity of the enzyme is calculated.

The enzyme activity of the pNP glycoside substrate is measured, and the reaction steps are as follows: mixing 100 μl of purified enzyme solution (diluted 10 times) with 100 μl of 5mM pNPG solution, reacting at optimum temperature for 10min, immediately adding 600 μl of 1M Na2CO3, stopping the reaction, boiling for 10min with enzyme solution as reference, and standing at OD ₄₀₀ Absorbance values were measured at nm wavelength.

The polysaccharide substrate was measured by DNS method and the reaction steps were as follows: 900. Mu.L of 0.5% soybean glycoside/laminarin substrate was reacted in a PCR instrument preheated to 55℃for 2min, then 100. Mu.L of 10-fold diluted enzyme solution was added, after 10min of reaction, 1.5mL of DNS was added, boiled for 5min and then OD was measured ₅₄₀ nm value.

The enzyme activity of the reducing oligosaccharide substrate is measured by adopting a GOD method, and the measuring reaction steps are as follows: 100. Mu.L of the properly diluted enzyme solution was reacted with 200. Mu.L of glucose oxidase/peroxidase assay reagent at 37℃for 30min, and 200. Mu.L of 6M H was added ₂ SO ₄ The reaction was stopped and then OD was measured ₅₄₀ nm value.

4.5.1.2 standard curve formulation

(1) The pNP standard curve was prepared with reference to the diversity and molecular engineering of the thermophilic fungus family 3. Beta. -glucosidase (Xia Wei, 2016).

Table 16 pNP calibration curve formulation

(2) DNS glucose standard curve production methods refer to diversity and molecular engineering of thermophilic fungi family 3 β -glucosidase (Xia Wei, 2016).

Table 17 formulation of glucose Standard Curve by DNS method

(3) The development of GOD-method glucose standard curves refers to the diversity and molecular engineering of the thermophilic fungi family 3 beta-glucosidase (Xia Wei, 2016).

Table 18 glucose standard curve preparation by GOD method

(4) Establishment of BCA method protein standard curve: see beyotide BCA kit.

4.5.2 software for enzyme Activity determination and data statistics

Enzyme activity was measured using a full wavelength microplate reader and data analysis was performed by GraphPad Prism 9.0.0.

4.5.3 protein concentration determination

Purified protein concentrations were determined using the Beyotime BCA kit.

4.5.4 reaction optimum pH and pH stability

The reaction was carried out at different pH (pH 2.2-10) to determine the specific enzyme activity of Bgl3 HB. mu.L of the buffer and 10. Mu.L of the purified enzyme solution were mixed and 100. Mu.L of 5 mM. Multidot.L was added thereto ^-1 pNPG was reacted at 50℃for 10min, 600. Mu.L of 1M Na was added ₂ CO ₃ Termination of the reaction and determination of OD ₄₀₀ Absorbance at nm, three replicates per reactionThe highest enzyme activity was defined as 100%. The different pH values required are provided by the following buffers: 10 mM.L ^-1 McIlvaine buffer (pH 2.2-8.0) and 50 mM.L ^-1 Gly-NaOH(pH9.0-10.0)。

The residual activity was determined at 50℃after incubating 10. Mu.L of enzyme solution with 90. Mu.L of 3 buffer substrates having the highest specific activity at 4℃for 10, 20, 30, 40, 50, 60 min.

4.5.5 reaction optimum temperature and temperature stability

Under the optimal pH condition, 90 mu L of buffer solution and 10 mu L of enzyme solution are evenly mixed and then 100 mu L of 5mM L of enzyme solution is added ^-1 pNPG, incubating the mixture at 20, 30, 40, 50, 60, 70, 80, 90℃for 10min, adding 600. Mu.L 1M Na ₂ CO ₃ Termination of the reaction and determination of OD ₄₀₀ Absorbance at nm. Three replicates per reaction, the highest enzyme activity was defined as 100%.

Under the condition of the optimal pH, 3 temperatures with higher relative enzyme activities are taken for incubation without substrates for 10, 20, 30, 40, 50 and 60 minutes, and the residual activity is measured at the optimal temperature.

4.5.6 tolerance to salt ions at different concentrations

Under the optimal reaction conditions, the salt ion concentration is measured to be 0, 200, 400, 600, 800, 1000, 2000, 3000, 4000, 5000 mM.L ^-1 Influence on enzyme activity under the conditions. Mixing 90. Mu.L of salt ion solution and 10. Mu.L of enzyme solution, adding 100. Mu.L of 5 mM. Multidot.L ^-1 pNPG, incubating the mixture at an optimal temperature for 10min, adding 600. Mu.L of 1M Na ₂ CO ₃ Termination of the reaction and determination of OD ₄₀₀ Absorbance at nm. Three replicates per reaction, the highest enzyme activity was defined as 100%.

4.5.7 resistance to different metal ions

Under the optimal reaction conditions, the concentration is 1 mM.L respectively ^-1 And 5 mM.L ^-1 Ca ²⁺ ，K ⁺ ，Al ³⁺ ，Ni ²⁺ ，Cu ²⁺ ，Mg ²⁺ ，Mn ²⁺ ，Co ²⁺ ，Zn ⁺ ，Fe ³⁺ Influence on enzyme activity. Mixing 90 μl of metal ion solution with different concentrations and 10 μl of enzyme solution, adding 100 μl of 5mM·l solution ^-1 pNPG, willIncubating the mixture at the optimal temperature for 10min, adding 600 μl 1M Na ₂ CO ₃ Termination of the reaction and determination of OD ₄₀₀ Absorbance at nm. Three replicates per reaction, the highest enzyme activity was defined as 100%.

4.5.8 substrate specificity analysis

The purified Bgl3HB was reacted with different biomass substrates. The hydrolysis activity was calculated as absolute value by measuring three times and averaging. The biomass substrates were 4 mM.L, respectively ^-1 Fiber two, three, four, five sugar, gentiobiose, alpha-sophorose laminarin, pNPG, daidzin, laminarin substrate, all substrates were purchased from Shanghai Seattle Biotechnology Co.

4.5.9 enzymatic reaction kinetic constant determination

At different concentrations (1 mM.L) ^-1 、2mM·L ^-1 、4mM·L ^-1 ) The sugar solution is used as a substrate to measure the enzyme activity. Results the kinetic constants K of the enzymatic reactions were calculated according to the double reciprocal plot method _m And V _max And enzyme-specific catalytic efficiency K _cat /K _m . The method comprises the following specific steps:

(1) Accurately diluting the purified enzyme solution to 1 mg.mL ^-1 And (5) standby application.

(2) And respectively adding substrates with different concentrations into the reaction system (2.3.1.1), and measuring the enzyme activity under standard conditions by taking the inactivated enzyme liquid as a blank.

(3) Mapping by using a double reciprocal method, wherein the formula isObtaining the Michaelis constant K _m 。

4.5.10Bgl3HB synergistic cellulase saccharification effect

The high-temperature alkali-treated bagasse is used as a raw material for an enzymatic saccharification test, and the saccharification effects of novel beta-glucosidase, general commercial cellulase Celluclast 1.5L and Novozyme 188 are compared, and the test design is as follows:

TABLE 19 Bgl3HB Single enzyme saccharification assay

The method comprises the following specific steps: bagasse was treated with 1% NaOH at 121℃for 30min, ddH ₂ O was washed to neutrality and 10mL 100mM Na was added ₂ HPO ₄ Citric acid buffer (pH 5.0 and pH 4.0, respectively) was mixed in 100mL shake flasks at a concentration of 2% (dry basis), and the enzyme components described above were added simultaneously to each flask and reacted at 40℃and 120 rpm. Each enzyme combination was removed at 4h, 12h, 16h, 24h, 48h, 72h and 96h, respectively, and the reaction was stopped with boiling water, and the hydrolysate was centrifuged at 12000rpm and its reducing sugar (DNS method) and glucose (GOD method) contents were determined.

(II) results of experiments

1RNA-seq sequencing results analysis and testing

1.1 raw sequencing data Mass analysis

The numbers of Bases sequenced by the two sample transcriptomes CK group and T1 group (where CK group is control group and T1 is test group) of trichoderma harzianum S7 were 7,0743,36,089 and 7,057,209,178, respectively, and the number of Bases was greater than 6G.

1.2 screening of the Gene bgl3HB of interest

Data obtained after sequencing RNA-Seq was subjected to FDR<0.05，|log ₂ FC|>2 and significantly different conditions were screened for 756 Unigenes, and since β -glucosidase secretion was induced efficiently in the medium with MCC as the sole carbon source, 5 genes predicted to be β -glucosidase were screened from the nucleic acid database NR by NCBI blast alignment of Unigenes sequences, and the selected genes were designated bgl3HB as Unigene0015203 with the highest fold difference as shown in Table 20 for the next study.

Table 20 differential Gene alignment

1.3bgl3HB expression verification case

According to the bgl3HB gene sequence predicted by RNA-Seq sequencing, quantitative Real-time PCR primers (Table 5) were designed, and RT-PCR and Q-PCR were performed using cDNA reverse transcribed from RNA extracted under conditions of 4 days of culture in an induction medium and a control medium as templates. The β -tubulin and actin genes were found to be expressed relatively constantly under different conditions from studies and thus served as reference genes (Sun Qing, 2015, hong Tao et al 2012). The primer specificity of the two reference genes is verified by RT-PCR, and the result is shown in figure 1, the melting curve is a single peak, which shows that the primer specificity is higher, no impurity interference exists, and the primer can be used as a stable reference for quantification.

RT-PCR showed melting curves of two differentially expressed genes as shown in FIG. 2, with melting curves as a single peak, indicating that the primer was not amplified non-specifically. All values were first delta Ct with internal control, then 2-delta Ct analysis was performed separately, with the glucose group as control. RT-PCR and RNA-Seq results showed that bgl3HB expression was up-regulated and fold differences were consistent. Thus, it was demonstrated that the gene expression data obtained based on RNA-Seq was reliable.

Cloning and sequence analysis of 2bgl3HB Gene

2.1bgl3HB nucleic acid sequence and protein Structure analysis

The nucleic acid molecule sizes of bgl3HB were determined to be 2388bp by RNA-Seq sequencing. Blastx alignment analysis of the gene bgl3HB shows that they are completely new β -glucosidase genes, as they have no high similarity to some of the sequences submitted in NCBI. The protein size and isoelectric point of the predicted bgl3HB via the website https:// web. Expasy. Org/computer_pi/are 86.67kDa,5.35, respectively. Sequence analysis of bgl3HB and analysis of SignalP 5.0 and TMHMM2.0 shows that 1-20 amino acid residues of bgl3HB sequence are signal peptide sequences.

Analysis of the protein domain in Bgl3HB coding region by SMART showed that Bgl3HB encodes a protein sequence containing Pfam Glyco-hydro-3 (120-375, E-value: 8.40E-27) domain, pfam Glyco-3-C (414-640, E-value: 3.70E-45) domain and Fn3_like (694-765, E-value: 3.46E-11) domain, as shown in fig. 3, glyco-hydro-3 and Glyco-3-C are domains belonging to GH3 family and involved in catalysis, possibly binding to β -glucan, indicating that Bgl3HB protein belongs to the third family of glycoside hydrolases, fn3-like domain appears at the C-terminus, whose specific function is not known, possibly related to thermal stability of the enzyme (Liu Chunyan, 2018), further explored.

Carbohydrate activity related enzymes of bgl3HB were also annotated by dbCAN, the dbCAN data source consisting essentially of CAZy database and CAT, and the annotated results are shown in fig. 4.

2.2 extraction of total RNA of Trichoderma harzianum Strain and first strand cDNA Synthesis

The preparation of complete, high purity RNA is a critical factor in the synthesis of high quality cDNA. Total RNA of good purity and quality was isolated from the cells cultured for 4d by induction using RNA extraction kit (OMEGA, model R6840-01) (shown in FIG. 5). After total RNA extraction, reverse transcription was immediately performed to obtain cDNA, which was stored at-20℃for use.

2.3 amplification of the Gene bgl3HB of interest

PCR was performed using the reverse transcribed cDNA as a template and bgl3HBEF and bgl3HBER as upstream and downstream primers, respectively, to obtain a gene expression sequence of 2388bp for the mature peptide of β -glucosidase, and the sequencing results were as follows. A small amount of the product was detected by electrophoresis on a 1% agar glycogel, and the results are shown in FIG. 6. And the plasmid pPICZ alpha A was transformed into E.coli DH5 alpha component Cells, positive clones were screened by colony PCR identification, and the collected plasmid pPICZ alpha A was double digested with EcoRI-HF and XbaI.

2.4 construction of recombinant expression vectors and transformation and identification of recombinant products

By usingII OneStep Cloning Kit (Nanjinouzan Co., ltd., model C112) and the recombinant product was designated pPICZαA/bgl3HB as shown in FIG. 7 and subjected to colony PCR assay (FIG. 8) and sent to Bio-company for sequencing.

Efficient expression and purification of 3bgl3HB gene in Pichia pastoris X33

3.1 screening and verification of Pichia and Positive transformants transformed with recombinant expression plasmids

Positive transformants were designated pPICZαA/bgl3HB/X33 as shown in FIG. 9. The transformants were confirmed by agarose gel electrophoresis and the results are shown in FIG. 10.

3.2PTVA method for screening multiple copies and verification

Recombinant bacteria pPICZalpha A/bgl3HB/X33 in 400 mu g.mL ^-1 、800μg·mL ^-1 、1200μg·mL ^-1 、2000μg·mL ^-1 The growth status in YPD plates of Zeocin is shown in FIG. 11. The result shows that the recombinant bacteria pPICZalpha A/bgl3HB/X33 is 800 mug.mL ^-1 The ZeocinYPD plate of (C) was partially dead at 1200. Mu.g.mL ^-1 Almost half of the bacteria had died on ZeocinYPD plates, but at 2000. Mu.g.mL ^-1 Can continue to grow normally on ZeocinyPD plates, and these normally grown monoclonal are put into the next test.

3.4 purification of the recombinase Bgl3HB in Pichia pastoris

The recombinant strain pPICZαA/bgl3HB/X33 was cultured under methanol induction conditions and found to have the highest enzyme activity after 6d of methanol induction. The protein amount of Bgl3HB was measured to be 0.22mg/mL, respectively; the highest enzyme specific activities are U.mg respectively ^-1 . After 6d of methanol-induced fermentation, collecting crude enzyme solution of Bgl3HB, concentrating by using 50kDa AmiconU1tra-15 ultrafiltration tube, replacing culture medium in crude enzyme solution with buffer A, and balancing UNOsphere with deaerated low-salt buffer A (pH 7.0) and deaerated high-salt buffer B (pH 7.0) ^TM The Q cartridge column was subjected to chromatography, and the results of the chromatography are shown in FIG. 12. Purified protein is subjected to Nuvia ^TM Performing secondary purification on IMAC Ni-charge column, and Nuvia ^TM The IMAC Ni-Charged column can specifically bind to the 6 His-tagged protein, and then elute the protein of interest through a high concentration of imidazole buffer (PBS C, pH 8.0). Bgl3HB Via Nuvia ^TM After secondary chromatography on IMAC Ni-Charged column, single collection peaks were obtained.

3.5 SDS-PAGE analysis and Mass Spectrometry identification of purified proteins

SDS-PAGE analysis was performed on the collected peak proteins by formulating a separation glue at a concentration of 6% and a concentration of 5% respectively, with a band size of 95.8kDa, indicating correct expression of the Bgl3HB protein and efficient collection and purification.

The purified two proteins were identified by mass spectrometry, and were found to be indeed the target proteins, and the sequencing results were as shown in fig. 13, to ensure the follow-up experiments.

3.6 study of the enzymatic Properties of Bgl3HB

3.6.1 enzyme Activity determination and protein quantitative Standard Curve

3.6.1.1 pNP standard curve

The standard curve is shown in fig. 14.

3.6.1.2 Glucose standard curve of DNS method

The standard curve is shown in fig. 15.

3.6.1.3 GOD glucose standard curve

The standard curve is shown in fig. 16.

3.6.1.4 BCA method protein quantitative standard curve

The standard curve is shown in fig. 17.

3.6.2 protein concentration determination

The protein concentration of Bgl3HB was determined to be 0.15 mg.multidot.mL, respectively ^-1 。

3.6.3 reaction optimum pH and pH stability

Buffer systems with different pH values are respectively prepared: 10 mM.L ^-1 Disodium hydrogen phosphate-citric acid buffer (pH 2-8), 50 mM.L ^-1 Glycine-sodium hydroxide buffer (pH 9-10). The enzyme activity of the purified Bgl3HB was measured in buffers with different pH values according to a 2.3.2 reaction system, and the test was repeated three times. As a result, as shown in FIG. 18, the reaction optimum pH for the purified enzyme was 4.0, and 38% of the highest enzyme activity was maintained at pH 2.0, indicating that the protein was resistant to acidic conditions, and was able to maintain most of the activity in the pH range of 2-6, while only 36% of the highest enzyme activity was left at pH 7.0, indicating that Bgl3HB was an acid enzyme.

The purified Bgl3HB was subjected to the acid treatment for 1h, 4h, 8h, 12h, 24h under the conditions of FIG. 19, and the remaining enzyme activities were measured in the above-mentioned manner. Bgl3HB still maintains 55% of relative enzyme activity after 24 hours of treatment at pH 3.0, and it is worth mentioning that the remaining enzyme activity after 24 hours of treatment at pH 4.0 remains above 85%, indicating that the protein can withstand extreme acidic environments to some extent.

3.6.4 reaction optimum temperature and temperature stability

The enzyme activity of the purified Bgl3HB was measured at various temperatures (4 ℃ C. -90 ℃ C.) according to a reaction system of 2.3.3, and the test was repeated three times. As shown in FIG. 20, the optimal reaction temperatures of Bgl3HB are respectively 50 ℃, the residual relative enzyme activities at 60 ℃ are respectively 64%, the residual relative enzyme activities at 70 ℃ are respectively 29%, the residual enzyme activities at 80 ℃ are respectively 21%, the residual enzyme activities at 90 ℃ are respectively 19%, and the beta-glucosidase produced by Trichoderma harzianum from Hainan mangrove generally has higher optimal reaction temperature and can withstand a certain high temperature environment to meet the natural survival conditions.

The purified Bgl3HB was heat treated under the conditions of fig. 21 for 1h, 4h, 8h, 12h, and 24h, respectively, and the residual enzyme activities were measured as described above. As shown in the test results, the enzyme activity of Bgl3HB is stable under the optimal temperature condition, the relative enzyme activities of two enzyme proteins are 72% after the treatment for 24 hours under the optimal temperature condition, and the results show that the Trichoderma harzianum Bgl3HB can maintain higher enzyme activity for a long time under the higher temperature condition.

3.6.5 resistance to different metal ions

Preparing 1mM L respectively ^-1 And 5 mMl.L ^-1 Cu ²⁺ ，Ca ²⁺ ，Ni ²⁺ ，Mg ²⁺ ，K ⁺ ，Al ³⁺ ，Mn ²⁺ ，Zn ⁺ ，Fe ³⁺ ，Co ²⁺ The enzyme activity was measured according to the reaction system of 2.3.7, and the enzyme activity of the blank group (without any metal ion added) was defined as 100%, and the test results are shown in FIG. 23, ca ²⁺ And Mn of ²⁺ Extremely obviously improves Bgl3Hb activity by 30.96 percent and 29.98 percent

3.6.6 tolerance to salt ions at different concentrations

The enzyme activity of Bgl3HB after purification was measured in accordance with the reaction system of 2.3.6 at different concentrations of salt ions (0-5000 mM/L), and the test was repeated three times, and the test results are shown in FIG. 23.Bgl3HB relative enzyme activity with increasing salt ion concentration tended to rise and then fall, at 400 mM.L ^-1 The salt ion concentration reached the highest, and 66% of enzyme activity remained at the highest concentration of salt ion. Bgl3HB relative enzyme activity increased with increasing salt ion concentration, indicating that salt ion pair Bgl3HB has different promotion effects. This is probably because salinity is the most important factor affecting the microbial community structure of mangrove wetland (Ceccon et al, 2019, tong et al, 2019a, fu et al, 2019), salinity is significantly inversely related to the activity and diversity of soil microorganisms, and as salinity and soil osmotic pressure increase, some microbial communities sensitive to salinity stress cannot survive, so that trichoderma harzianum isolated from mangrove forest soil can be tested to produce salt tolerance, even a certain concentration of salinity has a promoting effect on enzyme activity (Wang et al, 2010, tong et al, 2019 b).

3.6.7 substrate specificity assay

Bgl3HB was reacted with various substrates to measure the reactivity thereof with respect to the various substrates, and the inactivated enzyme after high temperature treatment was used as a control, and the activity unit was U.mg-1. As shown in the table, bgl3HB has the best effect on laminarin, and the enzyme activities are 129.79 U.mg-1 respectively; when disaccharides with different bonds are used as substrates, the selective size sequence of the enzymes is as follows: gentiobiose (beta-1, 6 bond) > sophorose (beta-1, 2 bond) > laminariabiose (beta-1, 3 bond) > cellobiose (beta-1, 4 bond) > cellopentose (beta-1, 4 bond) > cellotetraose (beta-1, 4 bond) > cellotriose (beta-1, 4 bond). The results demonstrate that Bgl3HB is a broad substrate specificity enzyme and is active on most of the cellulose substrates assayed, and Bgl3HB also has a strong ability to hydrolyze pNPG.

TABLE 21 Bgl3HB substrate specificity assay

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3.6.8 enzymatic reaction kinetic constant determination

The kinetic constants of the enzymatic reactions of Bgl3HB for pNPG, daidzin, laminarin are shown in Table 22. The substrate affinity and the catalytic efficiency of Bgl3HB to laminarin are far higher than those of other aryl glycosides, and the excellent degradation capability of Bgl3HB to laminarin, so that the Bgl3HB has great advantages in the aspect of producing biofuel by taking algae biomass as a raw material.

TABLE 22Bgl3HB different substrate enzyme reaction kinetic constants

3.7Bgl3HB synergistic cellulase saccharification effect

To evaluate the application value of Bgl3HB under practical conditions, the application potential of Bgl3HB and commercial Nov188 in saccharification of cellulosic feedstock was compared at ph4.0 and 50 ℃. As shown in FIG. 24, the control group, to which only the commercial enzyme Celluclast 1.5L was added, was incubated at 50℃for 96 hours at pH4.0, and 296.4. Mu. Mol of reducing sugar and 42.9. Mu. Mol of glucose were released (glucose conversion rate: 14.5%). Celluclast 1.5L, in synergy with Nov188, released 353.8. Mu. Mol of reducing sugar, 61.5. Mu. Mol of glucose (glucose conversion 17.4%) when 12BGU of beta-glucosidase was added. Bgl3HB combined with Celluclast 1.5L released only 344.0. Mu. Mol of reducing sugar and 58.1. Mu. Mol of glucose (glucose conversion 16.9%).

However, celluclast 1.5L and Bgl3HB improved the performance of synergistic enzymatic saccharification with the addition of 5mM NaCl. The yields of reducing sugar and fermentable glucose were 380.84. Mu. Mol and 67.6. Mu. Mol, respectively (glucose conversion 17.8%).

Notably, although the bagasse conversion of Bgl3HB was lower than commercial Novozyme 188, the glucose conversion of Bgl3HB was higher than commercial Novozyme 188. The bagasse conversion rate of Bgl3HB with 5mM NaCl was higher than that of commercial Novozyme 188, and the glucose conversion rate was also higher than that of commercial Novozyme 188. This is consistent with other results we studied, indicating that NaCl may increase the activity of Bgl3HB, possibly contributing to an increase in the hydrolytic capacity of Bgl3HB during saccharification. This effect may be due to the fact that the salt contributes to the stability of the enzyme. This important interaction will allow commercial cellulases to be hydrolysed with less enzyme, which will reduce the cost of industrial application.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

<110> university of Hainan

<120> an acid beta-glucosidase, and coding gene and application thereof

<130> MP21026382

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2388

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

atggtgaaca acgcagccct cgtcgccgct ctgtcggctc tgctgtctcc agctctggca 60

cagaacaatc agacatatgc caactactct gcccagggcc agcccgatct ctaccctcag 120

actcttgcca ctctcgaact ctcgttcccc gactgcgacc atggccccct gaagaacaac 180

ctcgtctgtg actcttcggc cggatacgtc gagcgagccc aggccctcat ctccctcttc 240

accctcgagg agctgattct caacacccag aactcgggcc ccggcgtgcc tcgcctgggt 300

cttccaaact accaagtctg gaacgaggct ctgcacggct tggaccgcgc caactttgcc 360

acaaagggcg gccaatacga atgggcaacc tccttcccca tgcccatcct gtcaatggca 420

gctctcaacc gcaccctgat ccaccagatt gcggacatca tctcgaccca ggctcgagca 480

ttcagcaaca ctggccgcta cggtctcgat gtctacgccc ccaacatcaa tggcttccgt 540

agccctctct ggggccgtgg acaggagact cccggtgaag atgccaacgt gctgacctct 600

gcctacacct acgagtacat caccggtatc cagggcggtg tagaccccga gaacctcaag 660

gttgccgcca cggccaagca ctttgccggc tacgatctcg agaactacaa caaccagtct 720

cgtctgggct tcgacgccat catcacccag caggacctcg ccgagtacta cactccccag 780

ttcctcgctg cgtcgcgcta cgcaaagtct cacagcttca tgtgcgccta caactccgtc 840

aacggcgtgc ccagctgcgc caacagcttc ttcctgcaga ccctgctgag agagagctgg 900

ggcttccccg aatatggcta cgtctcgtcc gattgcgatg ccgtctacaa cgtcttcaac 960

cctcacgact acgccagcaa catgtcttca gctgctgcct cctccctgag ggccggtacc 1020

gacattgact gcggtcagac atacccatgg cacttgaacg agtcctttgt ggctggcgag 1080

gtctcccgcg gcgagatcga gcgctccgtg actcgtctgt atgccaatct cgtccagctc 1140

ggatactttg acaagaagaa cgagtaccgc tcgctcggct ggaaggacgt cgtcaagacc 1200

gatgcttgga acatttcgta tgaggctgct gtcgagggca ttgtcctgct caagaacgac 1260

ggcactctcc ctctgtccaa gaaggtcaag agcatcgccc tgatcggacc ctgggccaat 1320

gccaccaccc agatgcaggg caactacttt ggcactcctc catacctcat cagccctctc 1380

gaggctgcca agaaggctgg ctacaaggtc aactttgcgc ttggaaccga catcgccagc 1440

accagcaccg ccggctttgc caaggctatt gccgccgctg agaagtctga tgccatcatc 1500

ttcgctggtg gtatcgacaa cacggttgaa caggagggcg ctgaccgcac ggacattgct 1560

tggcccggca accagctcga cctcatcaag tcgctcagca agctcaagaa gcctctcgtc 1620

gtcctgcaga tgggcggtgg ccaggttgac tcatcttctc tcaagagcaa caagaacgtc 1680

aactcccttg tctggggtgg atatcccggc cagtctggag gtgtcgctct ctttgacatc 1740

ttgtctggca agcgtgcccc cgctggacga ttggtctcaa cccagtaccc ggccagctac 1800

gttcacgagt tcccccagaa cgacatgaac ctccgccctg atggaaagaa gaaccccgga 1860

cagacttaca tctggtacac tggcaagcct gtctaccagt ttggtgacgg tatcttctac 1920

actactttca aggagagctt gtctggcaag tccaagagcc tcaagtacaa cgttgctgaa 1980

atcattgctg gtgcccaccc tgaatacacc tacagtgagc aggttccggt cttcaccttc 2040

actgccgaga ttaagaactc tggcaagact gagtccccat actcggccat gctcttcgtc 2100

cgcacttcca acgctggtcc tgccccctac cccaacaagt ggctggttgg attcgacaga 2160

cttgccgata tcaagcctgg tcactcctct acgctcagca tccctatccc catcagcgcc 2220

cttgcccgta ccgactctct tggaaacaag attgtctacc ctggcaagta tgagctggct 2280

ctcaacactg acgagtctgt caagctggag tttgagcttg tgggcgagga ggtgatcctc 2340

gagcactggc ctctggatca gcagcagatt caggatgcca ctccataa 2388

<210> 2

<211> 795

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Met Val Asn Asn Ala Ala Leu Val Ala Ala Leu Ser Ala Leu Leu Ser

1 5 10 15

Pro Ala Leu Ala Gln Asn Asn Gln Thr Tyr Ala Asn Tyr Ser Ala Gln

20 25 30

Gly Gln Pro Asp Leu Tyr Pro Gln Thr Leu Ala Thr Leu Glu Leu Ser

35 40 45

Phe Pro Asp Cys Asp His Gly Pro Leu Lys Asn Asn Leu Val Cys Asp

50 55 60

Ser Ser Ala Gly Tyr Val Glu Arg Ala Gln Ala Leu Ile Ser Leu Phe

65 70 75 80

Thr Leu Glu Glu Leu Ile Leu Asn Thr Gln Asn Ser Gly Pro Gly Val

85 90 95

Pro Arg Leu Gly Leu Pro Asn Tyr Gln Val Trp Asn Glu Ala Leu His

100 105 110

Gly Leu Asp Arg Ala Asn Phe Ala Thr Lys Gly Gly Gln Tyr Glu Trp

115 120 125

Ala Thr Ser Phe Pro Met Pro Ile Leu Ser Met Ala Ala Leu Asn Arg

130 135 140

Thr Leu Ile His Gln Ile Ala Asp Ile Ile Ser Thr Gln Ala Arg Ala

145 150 155 160

Phe Ser Asn Thr Gly Arg Tyr Gly Leu Asp Val Tyr Ala Pro Asn Ile

165 170 175

Asn Gly Phe Arg Ser Pro Leu Trp Gly Arg Gly Gln Glu Thr Pro Gly

180 185 190

Glu Asp Ala Asn Val Leu Thr Ser Ala Tyr Thr Tyr Glu Tyr Ile Thr

195 200 205

Gly Ile Gln Gly Gly Val Asp Pro Glu Asn Leu Lys Val Ala Ala Thr

210 215 220

Ala Lys His Phe Ala Gly Tyr Asp Leu Glu Asn Tyr Asn Asn Gln Ser

225 230 235 240

Arg Leu Gly Phe Asp Ala Ile Ile Thr Gln Gln Asp Leu Ala Glu Tyr

245 250 255

Tyr Thr Pro Gln Phe Leu Ala Ala Ser Arg Tyr Ala Lys Ser His Ser

260 265 270

Phe Met Cys Ala Tyr Asn Ser Val Asn Gly Val Pro Ser Cys Ala Asn

275 280 285

Ser Phe Phe Leu Gln Thr Leu Leu Arg Glu Ser Trp Gly Phe Pro Glu

290 295 300

Tyr Gly Tyr Val Ser Ser Asp Cys Asp Ala Val Tyr Asn Val Phe Asn

305 310 315 320

Pro His Asp Tyr Ala Ser Asn Met Ser Ser Ala Ala Ala Ser Ser Leu

325 330 335

Arg Ala Gly Thr Asp Ile Asp Cys Gly Gln Thr Tyr Pro Trp His Leu

340 345 350

Asn Glu Ser Phe Val Ala Gly Glu Val Ser Arg Gly Glu Ile Glu Arg

355 360 365

Ser Val Thr Arg Leu Tyr Ala Asn Leu Val Gln Leu Gly Tyr Phe Asp

370 375 380

Lys Lys Asn Glu Tyr Arg Ser Leu Gly Trp Lys Asp Val Val Lys Thr

385 390 395 400

Asp Ala Trp Asn Ile Ser Tyr Glu Ala Ala Val Glu Gly Ile Val Leu

405 410 415

Leu Lys Asn Asp Gly Thr Leu Pro Leu Ser Lys Lys Val Lys Ser Ile

420 425 430

Ala Leu Ile Gly Pro Trp Ala Asn Ala Thr Thr Gln Met Gln Gly Asn

435 440 445

Tyr Phe Gly Thr Pro Pro Tyr Leu Ile Ser Pro Leu Glu Ala Ala Lys

450 455 460

Lys Ala Gly Tyr Lys Val Asn Phe Ala Leu Gly Thr Asp Ile Ala Ser

465 470 475 480

Thr Ser Thr Ala Gly Phe Ala Lys Ala Ile Ala Ala Ala Glu Lys Ser

485 490 495

Asp Ala Ile Ile Phe Ala Gly Gly Ile Asp Asn Thr Val Glu Gln Glu

500 505 510

Gly Ala Asp Arg Thr Asp Ile Ala Trp Pro Gly Asn Gln Leu Asp Leu

515 520 525

Ile Lys Ser Leu Ser Lys Leu Lys Lys Pro Leu Val Val Leu Gln Met

530 535 540

Gly Gly Gly Gln Val Asp Ser Ser Ser Leu Lys Ser Asn Lys Asn Val

545 550 555 560

Asn Ser Leu Val Trp Gly Gly Tyr Pro Gly Gln Ser Gly Gly Val Ala

565 570 575

Leu Phe Asp Ile Leu Ser Gly Lys Arg Ala Pro Ala Gly Arg Leu Val

580 585 590

Ser Thr Gln Tyr Pro Ala Ser Tyr Val His Glu Phe Pro Gln Asn Asp

595 600 605

Met Asn Leu Arg Pro Asp Gly Lys Lys Asn Pro Gly Gln Thr Tyr Ile

610 615 620

Trp Tyr Thr Gly Lys Pro Val Tyr Gln Phe Gly Asp Gly Ile Phe Tyr

625 630 635 640

Thr Thr Phe Lys Glu Ser Leu Ser Gly Lys Ser Lys Ser Leu Lys Tyr

645 650 655

Asn Val Ala Glu Ile Ile Ala Gly Ala His Pro Glu Tyr Thr Tyr Ser

660 665 670

Glu Gln Val Pro Val Phe Thr Phe Thr Ala Glu Ile Lys Asn Ser Gly

675 680 685

Lys Thr Glu Ser Pro Tyr Ser Ala Met Leu Phe Val Arg Thr Ser Asn

690 695 700

Ala Gly Pro Ala Pro Tyr Pro Asn Lys Trp Leu Val Gly Phe Asp Arg

705 710 715 720

Leu Ala Asp Ile Lys Pro Gly His Ser Ser Thr Leu Ser Ile Pro Ile

725 730 735

Pro Ile Ser Ala Leu Ala Arg Thr Asp Ser Leu Gly Asn Lys Ile Val

740 745 750

Tyr Pro Gly Lys Tyr Glu Leu Ala Leu Asn Thr Asp Glu Ser Val Lys

755 760 765

Leu Glu Phe Glu Leu Val Gly Glu Glu Val Ile Leu Glu His Trp Pro

770 775 780

Leu Asp Gln Gln Gln Ile Gln Asp Ala Thr Pro

785 790 795

Claims (10)

1. An acid beta-glucosidase with the amino acid sequence shown in SEQ ID NO. 2.

2. The method for producing an acidic β -glucosidase according to claim 1, comprising:

1) Cloning the coding gene of the acid beta-glucosidase in claim 1 into an expression vector to obtain a recombinant vector;

2) And transforming the recombinant vector into host bacteria, inducing expression, and purifying by using an anion exchange chromatographic column and a nickel column to obtain the beta-glucosidase.

3. A gene encoding the acid β -glucosidase of claim 1.

4. A gene according to claim 3, characterized in that it has the nucleotide sequence of any one of I) to III):

I) A nucleotide sequence shown as SEQ ID No. 1; or (b)

II), substitution, deletion and/or addition of one or more nucleotides in the nucleotide sequence shown in SEQ ID No.1 and expression of the nucleotide sequence of the protein shown in SEQ ID No. 2.

5. A recombinant vector comprising the gene according to claim 3 or 4.

6. A host bacterium transformed with the recombinant vector of claim 5.

7. The host bacterium according to claim 6, wherein the host bacterium comprises Escherichia coli or Pichia pastoris.

8. Use of the acid β -glucosidase of claim 1, the gene of claim 3 or 4, the recombinant vector of claim 5, or the host bacterium of claim 6 or 7 for the preparation of at least one of reducing sugar, glucose, bioethanol.

9. A method for preparing reducing sugar and/or glucose, which is characterized in that cellulose raw material is taken as a substrate, and cellulase and the acid beta-glucosidase of claim 1 are utilized for enzymolysis to obtain the reducing sugar and/or glucose.

10. The method of claim 9, wherein the cellulosic feedstock comprises at least one of bagasse, corn stover, and grain residue.