CN110684677B

CN110684677B - Trichoderma reesei engineering bacterium and preparation method and application thereof

Info

Publication number: CN110684677B
Application number: CN201911038024.5A
Authority: CN
Inventors: 刘森林; 陈伟钊
Original assignee: Shenzhen University
Current assignee: Guangdong Cancheng Agricultural Products Group Co ltd
Priority date: 2019-10-29
Filing date: 2019-10-29
Publication date: 2021-08-06
Anticipated expiration: 2039-10-29
Also published as: CN110684677A

Abstract

The invention discloses a trichoderma reesei engineering bacterium and a preparation method and application thereof. The trichoderma reesei engineering bacteria can efficiently express endoglucanase, and under the optimized condition, the extracellular enzyme activity of the endoglucanase expressed by the trichoderma reesei engineering bacteria reaches 31.46U/mL, is 7.6 times of the enzyme activity of 4.12U/mL of the endoglucanase of wild bacteria, and is 2.3 times of the enzyme activity of 13.6U/mL of escherichia coli; the endoglucanase expressed by the trichoderma reesei has higher tolerance to SDS, particularly in 0.5% SDS, the residual enzyme activity of the tEgh31 enzyme is close to 55%, and the residual enzyme activity of rEgh31 expressed by pronucleus is about 30%; and the specific activity (161.2U/mg) of the tEgh31 enzyme expressed by the trichoderma reesei is improved by about 19 percent compared with the specific activity (135.7U/mg) of the rEgh31 enzyme expressed by pronucleus. This shows that the endoglucanase expressed by the fungus has wider application prospect.

Description

Trichoderma reesei engineering bacterium and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a trichoderma reesei engineering bacterium as well as a preparation method and application thereof.

Background

Enzymes produced by eukaryotes and enzymes produced by prokaryotes differ in reaction conditions such as optimum pH and optimum temperature and in catalytic mechanism. Trichoderma reesei is a species of filamentous fungus, and is a multicellular eukaryotic microorganism. The Trichoderma reesei expression system has the advantages of high expression level, secretory expression, high-density fermentation and the like, and is widely applied to industrial production of enzymes at present.

According to the function of the promoter, the Trichoderma reesei expression system can be divided into a constitutive expression system and an inducible expression system (Gaoyun, 2002). Constitutive promoters often correspond to genes essential for the basic life activities in the cell, their synthesis rate being independent of metabolic, environmental, spatiotemporal effects; in contrast, inducible promoters are capable of promoting expression under specific conditions. Commonly used constitutive promoters include glyceraldehyde 3-phosphate dehydrogenase (GPDA) promoter and the like, and commonly used inducible promoters include exo-type β -glucoronidase (CBHI) promoter and the like. Miettinen-Oinonen et al (2002) successfully expressed endoglucanase EGI using CBHI strong promoter; wang et al (2004) integrated the endoglucanase eg3 gene into the Trichoderma reesei chromosome, and regulated the expression of the gene using CBHI strong promoter, to obtain a recombinase with high activity. The subject group von willebrand et al (2014) constructs an expression cassette formed by sequentially connecting a promoter of trichoderma reesei Pyruvate Decarboxylase (PDC), a signal peptide of cellobiohydrolase CBHI, an eg4 gene and a PDC terminator, and successfully realizes the high-efficiency expression of the 61 family glycoside hydrolase in trichoderma reesei.

The trichoderma reesei mainly produces acidic cellulase, and neutral and alkaline cellulase derived from bacteria is difficult to efficiently express in a trichoderma reesei expression system due to the pH adaptability problem, so that few reports about constitutive expression of endoglucanase in trichoderma reesei exist at present, and how to realize constitutive expression of bacterial endoglucanase Egh31 in trichoderma reesei by using a strong promoter Ppdc is a problem to be solved urgently.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention mainly aims to provide the trichoderma reesei engineering bacterium.

The invention also aims to provide a preparation method of the trichoderma reesei engineering bacteria.

The invention further aims to provide application of the trichoderma reesei engineering bacteria.

The invention successfully carries out high-efficiency constitutive expression on the endoglucanase gene derived from the prokaryotic bacterium streptomyces in a trichoderma reesei expression system, and the research on enzymology properties also shows that the fungus recombinase preparation possibly has some advantages in the industry.

The purpose of the invention is realized by the following technical scheme:

the invention provides a constitutive expression cassette of trichoderma reesei, which consists of a constitutive strong promoter (Ppdc) of a trichoderma reesei pyruvate decarboxylase gene, a signal peptide gene of a trichoderma reesei cellobiohydrolase I gene (cbh I), an exogenous target gene (endoglucanase gene egh31) and a trichoderma reesei pyruvate decarboxylase gene terminator (Tpdc), and is abbreviated as Ppdc-cbh I-egh 31-Tpdc;

the constitutive strong promoter (Ppdc) of the Trichoderma reesei pyruvate decarboxylase gene and the Trichoderma reesei pyruvate decarboxylase gene terminator (Tpdc) are disclosed in 201110196079.6, a Trichoderma reesei constitutive expression cassette, an expression vector, a recombinant strain and application thereof, and are sequentially shown as SEQ ID NO.1 and 2 in 201110196079.6.

The nucleotide sequence of the signal peptide gene of the trichoderma reesei cellobiohydrolase I gene (cbh I) is shown as SEQ ID No: 1 is shown.

The endoglucanase gene egh31 is an endoglucanase gene egh31 derived from Streptomyces sp.H31, the exogenous target gene endoglucanase gene egh31 is a neutral endoglucanase H31 gene disclosed in the patent '201510036517.0, a neutral endoglucanase and a coding gene and application thereof', and the nucleotide sequence and the coded amino acid sequence of the gene are sequentially shown as SEQ ID NO.1 and 2 in 201510036517.0;

the invention also provides an expression vector containing the trichoderma reesei constitutive expression cassette.

The invention also provides a recombinant strain containing the expression vector.

The recombinant strain is recombinant trichoderma reesei, namely trichoderma reesei engineering bacteria.

The starting strain of the recombinant strain is Trichoderma reesei QM 9414.

The invention also provides application of the recombinant strain in preparation of industrial enzyme preparations.

The industrial enzyme preparation is neutral endoglucanase tEgh 31.

A recombinant endoglucanase, named as tEgh31, is obtained by fermentation of the above Trichoderma reesei engineering bacteria.

The invention also provides the application of the recombinant endoglucanase in washing, papermaking and other industries.

The invention also provides a preparation method of the trichoderma reesei engineering bacteria, which comprises the following steps:

(1) constructing an endoglucanase gene expression cassette Ppdc-cbh I-egh 31-Tpdc, wherein the Ppdc-cbh I-egh 31-Tpdc represents an endoglucanase gene expression cassette constructed by using a constitutive strong promoter (Ppdc) and a terminator of a Trichoderma reesei pyruvate decarboxylase gene and a signal peptide gene of a cellobiohydrolase I gene (cbh I);

(2) constructing a recombinant plasmid pPIC-P-egh31-T by using the Ppdc-cbh I-egh 31-Tpdc expression cassette, wherein the pPIC-P-egh31-T represents a plasmid pPIC inserted with the endoglucanase gene expression cassette;

(3) and co-transforming the trichoderma reesei by using the plasmid pPIC-P-egh31-T and the plasmid pAN7-1 to obtain the recombinant trichoderma reesei containing the endoglucanase gene trichoderma reesei constitutive expression cassette, namely the trichoderma reesei engineering bacteria.

Preferably, the plasmid pPIC in step (2) is plasmid pPICZ alpha A.

Preferably, the Trichoderma reesei described in step (3) is Trichoderma reesei QM 9414.

Compared with the prior art, the invention has the following advantages and effects:

(1) the trichoderma reesei constitutive expression cassette disclosed by the invention can be used for expressing protein by utilizing the efficient synthesis and secretion capacity of trichoderma reesei, is high in protein expression quantity, and can avoid excessive hybrid protein generated in an expression product;

(2) the expression vector constructed by the Trichoderma reesei constitutive expression cassette can express the gene from bacteria without induction, and the expression product can be modified appropriately;

(3) the trichoderma reesei engineering bacteria can efficiently express endoglucanase; under the optimized condition, the extracellular enzyme activity of the endoglucanase expressed by the trichoderma reesei engineering bacteria reaches 31.46U/mL, is 7.6 times of the enzyme activity (4.12U/mL) of the endoglucanase of wild bacteria H31 and is 2.3 times of the enzyme activity (13.6U/mL) of the escherichia coli expression enzyme; compared with the endoglucanase expressed by escherichia coli, the endoglucanase expressed by the fungus trichoderma reesei has higher tolerance to SDS than the endoglucanase expressed by the escherichia coli, particularly in an SDS solution with the final concentration of 0.5%, the residual enzyme activity of the tEgh31 enzyme is close to 55%, and the residual enzyme activity of the rEgh31 expressed by pronucleus is about 30%. In addition, the specific activity (161.2U/mg) of the tEgh31 enzyme expressed by the fungus Trichoderma reesei is improved by about 19 percent compared with the specific activity (135.7U/mg) of the rEgh31 enzyme expressed by pronucleus. This shows that the endoglucanase expressed by the fungus has wider application prospect.

Drawings

FIG. 1 is a colony PCR validation of recombinant expression vector pPIC-P-egh 31-T; m: a DNA molecular weight marker; 1: and (3) PCR products.

FIG. 2 is a PCR validation of positive clones; m: a molecular weight marker; 1-10: a positive clone; 11: and (5) negative control.

FIG. 3 is an SDS-PAGE electrophoretic analysis of the supernatant of the transformants; 1: reesei QM9414 blank; 2-5: the fermentation supernatant of the positive transformants was concentrated 10-fold.

FIG. 4 is an electrophoretogram of affinity chromatography purification of fungal recombinase; m: labeling with molecular weight; 1. purified endoglucanase tpegh 31;

FIG. 5 is the effect of temperature on endoglucanases; a: the optimum reaction temperature of endoglucanase; b: thermostability of endoglucanases.

FIG. 6 is the effect of pH on endoglucanase activity; a: optimum reaction pH for endoglucanase; b: pH stability of endoglucanase.

FIG. 7 is the effect of metal ions on recombinant endoglucanases; the enzyme activity of the blank control (without addition of ions) was set to 100%, and the results were shown as the mean ± standard deviation (n-3).

FIG. 8 is the effect of SDS and EDTA concentration on recombinant endoglucanase; a: the effect of SDS on recombinant endoglucanases; b: effect of EDTA on recombinant endoglucanases.

FIG. 9 shows the effect of different concentrations of washing powder on recombinant endoglucanase.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The experimental procedures for specific experimental conditions not specified in the following examples are generally performed according to conventional experimental conditions or according to experimental conditions recommended by the manufacturers.

The egh31 gene in the embodiment refers to the neutral endoglucanase H31 gene disclosed in the patent 201510036517.0, a neutral endoglucanase and a coding gene and application thereof.

The cell lysate of the escherichia coli recombinant endoglucanase described in the examples is crude enzyme solution obtained in step (two) 10 of example 2 in patent 201510036517.0, a neutral endoglucanase and its coding gene and application.

Example 1

1 Material

1.1 strains and vectors

(1) Coli TOP10, E.coli BL21 Star (DE3) were purchased from Invitrogen.

(2) Trichoderma reesei QM9414 was purchased from American Type Culture Collection (ATCC).

(3) Streptomyces sp.H31 has been disclosed in patent 201510036517.0, a neutral endoglucanase and its coding gene and application.

(4) Coli expression vector pET-28a (+) protein expression vector was purchased from Invitrogen corporation.

(5) Coli ligation T vector was purchased from Takara.

(6) The Trichoderma reesei expression vector pPIC-PT is given a gift from professor Liu of the subject group, wherein the expression vector pPIC-PT is obtained by replacing the promoter and terminator of Trichoderma reesei cellobiohydrolase cbh I in the expression vector pPIC-P-T disclosed in the document "Kangkang. Penicillium Swolenin gene clone expression and cellulase activity promotion research [ D ], southern China agricultural university, 2010" with the constitutive strong promoter (Ppdc) and terminator Tpdc of Trichoderma reesei pyruvate decarboxylase gene (pdc).

(7) The Trichoderma reesei co-transfer vector pAN7-1 was offered by professor Wang Tianhong of Shandong university and was disclosed in patent "200910039532.5, a Trichoderma reesei expression cassette and recombinant strains and uses thereof". Plasmid pAN7-1 carries the fungal selection marker hygromycin resistance gene (hph) and the E.coli selection marker ampicillin resistance gene (bla).

1.2 reagents and formulations

(1) Mandel nutrient salt concentrate:

3.0g of urea, (NH)₄)₂SO₄ 14.0g、MgSO₄·7H₂O 3.0g、CaCl₂·7H₂O 4.0g、KH₂PO₄20.0g and deionized water to a constant volume of 1L.

(2) Mandels microelement concentrate:

FeSO₄·7H₂O 5.0g、ZnSO₄·7H₂O 1.6g、CoCl₂·6H₂O 3.7g、MnSO₄·H₂o1.6 g and deionized water are added to the solution to reach a constant volume of 1L.

(3)1M citrate buffer: 210.0g of citric acid, 78.0g of sodium hydroxide and deionized water are added to the solution until the volume is 1L, and the pH value of the solution is adjusted to 4.5.

(4) STC solution: sorbitol 108.4g, 1M Tris-HCl (pH 7.5-8.0)5mL, 1M CaCl₂50mL, adding deionized water to make the volume to 500 mL.

(5)60％PEG 4000：1M Tris-HCl(pH 7.5)200μL，1M CaCl₂1mL, 400012 g of PEG, and adding deionized water to make the volume to 20 mL.

(6)0.05mol/L pH 4.8 citric acid-sodium citrate buffer: the prepared 1M citric acid buffer was diluted 20-fold to 0.05M and the pH was adjusted to 4.8.

(7)1M Tris-HCI (pH 7.5-8.0): accurately weighing 12.11g of Tris, adding deionized water for dissolving, and adjusting the pH value to 7.5-8.0 by using HCl until the volume is 100 mL.

(8) 1% agarose gel: 1g agarose dissolved in 100mL 1 × TAE buffer, and then 1 u L Goldview.

(9) TE buffer solution: 10mmol/L Tris-HCl pH8.0, 1mmol/L EDTA pH8.0.

(10) Preparation of protein isolation buffer

1. And (3) an equilibrium buffer: 20mM Tris-HCl, 200mM NaCl, 20mM Imidazole, pH 8.0.

2. Elution buffer: 20mM Tris-HCl, 200mM NaCl, 500mM Imidazole, pH 8.0.

3. 0.05M NiSO₄、0.05M EDTA、0.5M NaOH、20％C₂H₅OH。

1.3 Medium

(1) LB culture medium: 0.5% of yeast powder, 1% of peptone, 1% of NaCl and additionally added with Agar 2% in a solid LB culture medium. When the transformant of the recombinant plasmid is screened, ampicillin (escherichia coli prokaryotic expression) with the final concentration of 50-100 mug/mL or Zeocin antibiotic (trichoderma reesei eukaryotic expression) with the final concentration of 25 mug/mL is added.

(2) PDA culture medium: used for solid culture of T.reesei QM 9414. 20% of potato leachate, Agar 2% and 2% of glucose (prepared separately, mixed after sterilization), boiling for 25-30 min, and filtering with 3-4 layers of gauze. When transformants of T.reesei QM9414 were selected, hygromycin B antibiotic was added to the mixture at a final concentration of 50-100. mu.g/mL.

(3) Mandels medium: is used for liquid culture of Trichoderma reesei QM 9414. 20mL/L of Mandels nutrient salt concentrated solution, 1mL/L of Mandels microelement concentrated solution, 1g/L of peptone, 50mL/L of 1M citric acid buffer solution (pH4.5), 801-2 g/L of tween and 10g/L of anhydrous glucose (prepared independently, sterilized and mixed). When the method is used for the rescreening culture of the trichoderma reesei transformant, hygromycin B antibiotics with corresponding concentration can be added.

(4) Protoplast regeneration medium: the regeneration method is used for regeneration of the trichoderma reesei protoplast. STC solution 50mL, 10 × glucose 10mL, Mandel culture medium 40mL, prepared separately, sterilized and mixed.

(5) Enzyme production medium (culture 7 d): the method is used for non-induction enzyme-producing culture of Trichoderma reesei QM9414 and transformants thereof. 20mL/L of Mandels nutrient salt concentrated solution, 1mL/L of Mandels microelement concentrated solution, 50mL/L of 1M citric acid buffer solution (pH4.5), 801-2 g/L of Tween, 10g/L of peptone, 4.5g/L of yeast extract and 70g/L of glucose (prepared separately, mixed after sterilization).

(6) Basic enzyme production culture medium: the method is used for non-induction enzyme-producing culture of Trichoderma reesei QM9414 and transformants thereof. 20mL/L of Mandels nutrient salt concentrated solution, 1mL/L of Mandels microelement concentrated solution, 50mL/L of 1M citric acid buffer solution (pH4.5), 10g/L of peptone, 801-2 g/L of tween, 4.5g/L of yeast extract and 10g/L of glucose (prepared separately, mixed after sterilization).

(7) Induction enzyme production culture medium: 10g/L of CMC-Na (sodium carboxymethylcellulose), 20mL/L of Mandels nutrient salt concentrated solution, 1mL/L of Mandels microelement concentrated solution, 50mL/L of 1M citric acid buffer solution (pH4.5), 1g/L of peptone, 4.5g/L of yeast extract, 801-2 g/L of tween and 3g/L of glucose (prepared separately, mixed after sterilization).

1.4 Main instrumentation

A PCR instrument: applied Biosystems Inc

A centrifuge: eppendorf Co Ltd

Electrophoresis apparatus: bio-rad Co

Vertical pressure steam sterilization pot: hirayama, Japan Ltd

Gel imaging system: Dolphin-DOC.

2 method

2.1 expression of endoglucanase Gene egh31 in Trichoderma reesei

2.1.1 Trichoderma reesei QM9414 culture method

Liquid culture: single colonies were inoculated into Mandels medium and shake-cultured at 28 ℃ and 250rpm until the hyphae turned yellow.

Solid culture: inoculating a proper amount of fermentation liquor to a PDA (PDA dextrose agar) plate, culturing at the constant temperature of 28 ℃ for 7-10 days, and washing or scraping spores by using sterile water for seed preservation.

2.1.2 construction of pPIC-P-egh31-T expression plasmid

In order to successfully realize the expression of the egh31 gene in trichoderma reesei and facilitate subsequent purification, a nucleic acid sequence corresponding to a 6 × His tag is introduced into the C-terminal of the egh31 gene, and primers are designed according to a vector enzyme cutting site, which is shown in table 1.

TABLE 1 primers for the Tegh31 gene

The amplified sequence was named Tegh31, digested with Sfi I enzyme and ligated to a plasmid of pPIC-PT digested simultaneously with Sfi I and Eam1105I to obtain pPIC-P-egh31-T expression plasmid.

2.1.3 preparation and transformation of Trichoderma reesei protoplasts

Preparation of trichoderma reesei protoplast:

(1) inoculating the Trichoderma reesei QM9414 spores to a PDA solid plate, and culturing for 3-5 days at the temperature of 28 ℃. Then inoculating the mature spores into sterile water at a ratio of 1.2X 10⁸The number of spores was inoculated into 50mL of Mandels medium and cultured at 28 ℃ at 250r/min for 16 hours.

(2) 5mL of spore germination liquid is taken and centrifuged at 10000r/min for 4 min. The obtained mycelium was extracted with 1mol/L MgSO₄And (6) washing.

(3) 50mg of lywallzyme was dissolved in 5mL of 1mol/L MgSO₄And filtering and sterilizing to prepare enzymatic hydrolysate for suspending the spores. Culturing at 28 deg.C and 100r/min for 3 hr, and observing enzymolysis of spore protoplast with microscope.

(4) Adding the STC solution with the same volume into the enzymolysis solution, evenly mixing, centrifuging to remove the supernatant, washing the precipitated protoplast by using the STC solution, and finally suspending the washed protoplast in 1mL of STC solution to obtain the protoplast.

Transformation of trichoderma reesei protoplasts:

(1) the protoplasts thus prepared were diluted appropriately to a final concentration of 10³And (4) sucking 50-150 mu L of diluent per mL, and coating the diluent in a protoplast regeneration culture medium plate for accurately calculating the regeneration rate.

(2) Finally, the protoplast concentration was adjusted to 10⁸And taking 100 mu L of the plasmid, adding 5 mu g of pPIC-P-egh31-T expression plasmid and 5 mu g of pAN7-1 plasmid respectively, and mixing uniformly for later use.

(3) The prepared sample was heat-shocked at 48 ℃ for 2min, and then 25. mu.L of 60% PEG 4000 was added and left at room temperature for 15 min.

(4) Finally, 10mL of STC solution is added, 5000r/min is carried out, and the supernatant is removed after centrifugation for 10 min. The protoplast pellet was resuspended in 1mL of STC solution, added to the protoplast regeneration medium, and cultured at 28 ℃ at 50r/min for 1 day.

(5) And (3) centrifuging the culture solution at 6000r/min for 20min, resuspending the protoplast precipitate by using 1mL of STC solution, coating the protoplast precipitate on a PDA solid plate containing 50-100 g/mL of hygromycin B, and culturing for 2-3 days at 28 ℃ to obtain the recombinant Trichoderma reesei transformant.

2.1.4 screening and Activity measurement of recombinant Trichoderma reesei transformants

Inoculating the recombinant Trichoderma reesei transformant obtained by transformation into a Mandelis culture medium (containing 50g/mL hygromycin B), culturing for 2 days at 28 ℃ at 250r/min, then transferring the recombinant Trichoderma reesei transformant to an induced enzyme production culture medium according to the inoculum size of 5%, continuing culturing for 5 days, centrifuging the fermentation liquor for 5min at 12000r/min, and taking the supernatant to perform enzyme activity determination of endoglucanase.

2.2 isolation and purification of the enzyme and study of the enzymatic Properties

2.2.1 separation and purification of endoglucanase protein

The recombinant endoglucanase expressed in Escherichia coli (rEgh 31) and the recombinant endoglucanase expressed in Trichoderma reesei (tEgh 31) used both contained 6 XHis-tag, and thus protein isolation and purification were possible by affinity chromatography.

The method comprises the following steps:

(1) the cell lysate (E.coli expression system) or the fermentation supernatant (Trichoderma reesei expression system) is dissolved in equilibration buffer. The cell lysate referred to herein is 201510036517.0, which is the crude enzyme solution obtained in step (II) 10 of example 2, and the fermentation supernatant referred to herein is 2.1.4.

(2) Subjecting the Ni affinity chromatography column HisTrap^TMEquilibrate to baseline with equilibration buffer.

(3) The sample was loaded onto a Ni affinity chromatography column at a flow rate of 1mL/min by a constant flow pump.

(3) Gradient elution is carried out on imidazole elution solution, elution peaks are collected to determine enzyme activity, and after overnight dialysis, the imidazole elution solution is concentrated by 10 times for standby.

2.2.2 enzyme activity determination method

Adding a proper amount of enzyme solution into 1% CMC-Na solution, reacting at 50 deg.C for 30min, adding DNS solution into boiling water bath for 10min, measuring absorbance at 540nm, and calculating reducing sugar by standard curve. The amount of enzyme required to produce 1.0mg of glucose per hour under the above conditions was defined as one unit of enzyme activity, expressed in U/mL.

Definition of specific activity: enzyme activity per mg of protein (U/mg).

2.2.3 electrophoretic analysis of proteins

(1) SDS-PAGE gel formulation

The specific method is shown in Table 2.

(2) Step of electrophoresis

1. Centrifuging the sample at 12000r/min to remove precipitate, adding 5 Xsample buffer solution, boiling in boiling water for 10min, centrifuging to remove precipitate, and collecting the supernatant.

2. Carefully loading the sample by using a sample loader, wherein the sample loading amount is selected according to actual needs, and the sample does not overflow the sample loading hole.

3. Electrophoresis was started immediately after the end of the loading. The sample is in the gel concentration stage, generally low voltage is selected, the voltage is increased after the sample enters the separation gel until the bromophenol blue indicator band is positioned at the bottom of the gel, and the electrophoresis is finished.

4. The gel is carefully removed and stained for a period of time depending on the stain concentration.

5. And after dyeing is finished, pouring out the dyeing solution, washing the dyeing solution for multiple times by using deionized water, and then adding the decoloring solution.

6. And (4) after the decoloration is finished, photographing and analyzing by using a gel imaging system.

The specific steps refer to patent 201510036517.0, neutral endoglucanase and its coding gene and application, step (II) 9 of example 2.

TABLE 2 SDS-PAGE gel formulation

Components	Concentrated gum (5%) (5mL)	Separation gel (12%) (10mL)
			H₂O	3.6	3.3
30% acrylamide	0.83	4.0
			1M Tris-HCL(pH 6.8)	0.42	Is free of
1.5M Tris-HCL(pH 8.8)	Is free of	2.5
			10％SDS	0.05	0.1
10％AP	0.05	0.1
			TEMED	0.005	0.004

2.2.4 method for measuring protein concentration

Protein concentration was determined using the BCA protein quantification assay kit.

(1) 5mL of Solution A and 0.1mL of Solution B were used for each reaction to prepare an A + B mixture, which was mixed well.

(2) 0.1mL of sample to be tested is added into 2mL of A + B mixed solution, and the reaction is carried out for 30 minutes at 37 ℃.

(3) After the reaction, the absorbance of light was measured at 562 nm.

(4) Protein concentration was calculated by standard curve.

2.2.5 optimum temperature and thermostability of endoglucanases

Each data in the course of the enzymatic property analyses herein was obtained by taking the average of three replicates.

(1) Optimum reaction temperature

The citric acid buffer solution with pH 7.0 is used as a buffer system, 1% CMC is used as a substrate, the reaction system is 1100 mu L, and the reaction time is 30 min. And (3) measuring the enzyme activity of the endoglucanase Egh31 within the temperature range of 30-80 ℃, calculating the highest enzyme activity to be 100%, further calculating the relative enzyme activity within other temperature ranges, and fitting to a curve for analysis.

(2) Thermal stability

Respectively taking 100 mu L of enzyme solution, preserving the heat for 60min at the temperature of 30-80 ℃, taking a citric acid buffer solution with the pH of 7.0 as a buffer system, taking 1% CMC as a substrate, taking a reaction system as 1100 mu L, measuring the enzyme activity of endoglucanase, calculating the highest enzyme activity as 100%, further calculating the relative enzyme activity in other temperature ranges, and fitting into a curve for analysis.

2.2.6 optimum pH and pH stability of endoglucanases

(1) Optimum reaction pH value

The reaction temperature is 60 ℃, 1% CMC is used as a substrate, the reaction system is 1100 mu L, and the reaction time is 30 min. And (3) measuring the enzyme activity of the endoglucanase Egh31 under the condition of pH 3-11, calculating the highest enzyme activity to be 100%, further calculating the relative enzyme activities of other reaction pH values, and fitting to a curve for analysis.

(2) Stability of pH value

100 μ L of the enzyme solution was added to 1mL of a buffer solution having a pH of 3 to 11, and the mixture was incubated at 37 ℃ for 60 min. Then adding a reaction substrate CMC-Na solution (the final concentration of the substrate is 1 percent) to react for 30min at 60 ℃, measuring the enzyme activity of the endoglucanase Egh31, calculating the highest enzyme activity to be 100 percent, further calculating the relative enzyme activities of other pH values, and fitting a curve to analyze.

2.2.7 Effect of different Metal ions on enzyme Activity

The citric acid buffer solution with pH 7.0 is used as a buffer system, 1% CMC is used as a substrate, the reaction system is 1100 mu L, and the reaction time is 30 min. Various metal ions (Co) are added into the system²⁺、Li⁺、Cu²⁺、Sn⁴⁺、Ag⁺、Mn²⁺、Ni²⁺、Zn²⁺、Ca²⁺、Al³⁺、Fe³⁺) And finally, the final concentration of metal ions in the reaction system is 10mM, then the enzyme activity of the endoglucanase Egh31 is measured, the enzyme activity of a control group without metal ions is calculated to be 100%, further the relative enzyme activity of other metal ions is calculated, and the influence of different metal ions on the enzyme activity is analyzed.

2.2.8 Effect of washing powder on enzyme Activity

Simulating the time and mode commonly used in washing clothes in China families, adding washing powder into a reaction system containing endoglucanase Egh31 to ensure that the final concentration of the washing powder is 1-5%, after preserving the washing powder in a warm bath at 37 ℃ for 60min, measuring the enzyme activity of endoglucanase Egh31 according to a conventional method, and calculating the highest enzyme activity to be 100%.

2.2.9 Effect of surfactant SDS and Metal chelator EDTA on enzyme Activity

SDS and EDTA with different concentrations are added into a reaction system containing endoglucanase Egh31, so that the final concentrations of the SDS and the EDTA are respectively 0, 0.5%, 1% and 2%, after the SDS and the EDTA are stored for 60min in a warm bath at 37 ℃, the enzyme activity of endoglucanase Egh31 is respectively measured according to a conventional method, and the highest enzyme activity is calculated as 100%.

3 results and analysis of the experiments

3.1 expression of endoglucanases in Trichoderma reesei

3.1.1 construction of pPIC-P-egh31-T expression vector

The egh31 gene full length is obtained by using full length primer amplification, and is cut by restriction endonuclease Sfi I, and then is connected to pPIC-PT vector which is cut by Sfi I and Eam 1105I. Coli TOP10 was transformed with the ligation products, and positive clones were picked for PCR validation. As shown in FIG. 1, PCR preliminary verification showed that the pPIC-P-egh31-T expression vector had been successfully constructed.

3.1.2 pPIC-P-egh31-T expression vector for Co-transformation of Trichoderma reesei QM9414 protoplast

Cotransforming the Trichoderma reesei QM9414 by the pPIC-P-egh31-T and the plasmid pAN7-1 according to the operation steps, coating transformants on a screening PDA plate containing 100 mu g/mL hygromycin B, culturing for 3-5 days at 28 ℃, and allowing Trichoderma reesei strains to appear on successfully cotransformed plates, wherein the strains are green and fully paved on the whole plates, and no colonies appear on the plates corresponding to a control group.

The egh31 gene full-length primer is used for carrying out PCR identification on the transformant, as shown in figure 2, most positive clones have a band at 770bp, and the size of the band is consistent with the egh31 gene full-length. Indicating that the co-transformation has been successful.

3.1.3 expression and detection of endoglucanase protein in recombinant Trichoderma reesei transformants

Randomly picking 4 successfully transformed transformants, inoculating the transformants to a Mandels culture medium (containing 50g/mL hygromycin B), culturing for 2 days at 28 ℃ at 250r/min, transferring the transformants to an enzyme production induction culture medium according to the inoculation amount of 5%, continuously culturing for 5 days, centrifuging the fermentation liquid for 5min at 12000r/min, concentrating the supernatant by 10 times, and performing SDS-PAGE protein electrophoresis, wherein the electrophoresis result is shown in figure 3.

From the electrophoresis results, a band is evident in lanes 2-5 at 29kDa, but is not shown in the blank control group, while the theoretical molecular weight of the recombinant endoxylanase tEgh31 is 28.2kDa, which indicates that the Egh31 gene is expressed in the T.reesei QM9414 strain.

10 positive transformants were selected for fermentation culture and assayed for endoglucanase activity. Wherein the highest enzyme activity of the transformant with the highest activity in the fermentation supernatant reaches 31.46U/mL.

3.2 isolation and purification of recombinant enzyme tEgh31 expressed by Trichoderma reesei

The protein separation and purification are carried out by the same method as the recombinant endoglucanase expressed by the Escherichia coli expression system. The electrophoresis result is shown in FIG. 4, a single band exists at about 29kDa, which is consistent with the predicted molecular weight, and it can be seen that the recombinant enzyme has been successfully separated and purified. The BCA method is utilized to measure the concentration of pure enzyme protein, and the specific activity of endoglucanase tEgh31 expressed in the Trichoderma reesei is calculated to be 161.2U/mg.

3.3 enzymological Properties and potential applications

The experiment was performed to analyze the optimum reaction temperature, thermal stability, optimum reaction pH, pH stability, metal ion suitability, surfactant and detergent tolerance, etc. of endoglucanase Egh 31.

Attempts to separate and purify the endoglucanase in the H31 fermentation broth by using different separation schemes such as anion exchange chromatography, cation exchange chromatography, hydrophobic chromatography and the like all fail, and the purification process is supposed to destroy the structure of the protein. Therefore, the contents of the experiments described in this section were analyzed enzymatically using the H31 fermentation broth and compared with the recombinant enzyme.

The H31 fermentation liquid is prepared by inoculating Streptomyces sp.H31 strain to liquid selection culture medium (10g/L CMC-Na (sodium carboxymethylcellulose), 5g/L yeast powder, 5g/L NaCl, 2g/L KH)₂PO₄，5g/L NH₄NO₃，0.3g/L MgSO₄，1g/L(NH₄)₂SO₄pH 9.0), culturing at 37 deg.C and 200rpm for 48h, freeze-centrifuging the 48h culture solution at 14000rpm for 20min, and collecting supernatant; and (3) carrying out ultrafiltration concentration on the supernatant by 10 times by using a 3000 dalton ultrafiltration concentration membrane to obtain a crude enzyme solution, namely H31 fermentation liquor.

3.3.1 optimum reaction temperature

The optimum reaction temperature analysis is carried out on the H31 fermentation liquor, the endoglucanase rEgh31 expressed in the purified escherichia coli and the endoglucanase tEgh31 expressed in the purified trichoderma reesei, and the result is shown in figure 5-A, wherein the optimum reaction temperature range of the endoglucanase is 55-60 ℃, the relative enzyme activity in the range is close to 100%, and the maximum reaction temperature is reached at 60 ℃. From the experimental results, the optimal reaction temperature of the original bacterial liquid and the recombinase is basically consistent.

3.3.2 thermal stability

The H31 fermentation broth, the endoglucanase rEgh31 expressed in purified Escherichia coli and the endoglucanase tEgh31 expressed in purified Trichoderma reesei were subjected to thermal stability analysis. As can be seen from FIG. 5-B, the enzyme activity was substantially unchanged when incubated at less than 50 ℃ for 60min, whereas the temperature stability of the enzyme decreased more rapidly when the temperature was higher than 60 ℃. The relative enzyme activity is over 60 percent at 55 ℃. After 60 ℃, the relative enzyme activity is reduced to a lower level. It can be seen from the results that the thermal stability of the H31 fermentation broth is slightly better than that of the two recombinant enzymes, presumably because the presence of certain factors in the fermentation broth is beneficial to maintain the enzyme structure, thereby improving the thermal stability.

3.3.3 optimum pH of endoglucanase

The optimum pH value analysis is carried out on the H31 fermentation liquor, the endoglucanase rEgh31 expressed in the purified escherichia coli and the endoglucanase tEgh31 expressed in the purified trichoderma reesei. And (3) respectively adjusting the pH of the reaction systems to 3-11, measuring the enzyme activity at 60 ℃, and analyzing the optimum pH of the enzyme reaction. As can be seen from FIG. 6-A, the enzyme activity of endoglucanase is greater than 40% at pH 5.0-9.5, wherein the enzyme activity reaches the maximum value at pH 7-7.5; under strong acid condition (pH 3), the enzyme still has 15% of residual enzyme activity, and under strong alkaline condition (pH 11), the relative enzyme activity of endoglucanase can still exceed 20%. And in the range of pH 4-10, the residual enzyme activity is higher, so that the pH adaptation range of the enzyme is wider. After the pH value is more than 7, the enzyme activity is more slowly reduced, and the catalytic effect of the enzyme is particularly good under neutral and weakly alkaline conditions.

3.3.4 pH stability of endoglucanases

The H31 fermentation broth, the endoglucanase rEgh31 expressed in purified Escherichia coli and the endoglucanase tEgh31 expressed in purified Trichoderma reesei were subjected to pH stability analysis. The endoglucanase was incubated at different pH for 60min and its enzyme activity was measured at 60 ℃. The results are shown in FIG. 6-B. Under the condition of pH 3-10, the relative enzyme activity of the enzyme is higher than 60%; 40% can still be achieved under the condition of pH 11. Therefore, the enzyme can keep relatively high relative enzyme activity under the condition of pH 3-11, and shows excellent pH stability. The enzyme can be inferred from the experimental result to meet the requirement of the enzyme for the washing industry, and has wide application prospect in the washing industry.

3.3.5 Effect of Metal ions on enzyme Activity

And analyzing the enzyme activities of the endoglucanase rEgh31 expressed in the purified escherichia coli and the endoglucanase tEgh31 expressed in the purified trichoderma reesei by using metal ions. The metal ions may influence the catalytic process of the enzyme, and the metal ions are added into the reaction system of the enzyme to analyze the influence of different kinds of metal ions on the enzyme. From the experimental results (FIG. 7), Ni²⁺The enzyme activity promotion effect on the endoglucanase is most obvious, and the enzyme activity can be improved by 1.5 times; ca²⁺、Zn²⁺The enzyme activity is greatly promoted; al (Al)³⁺、Mg²⁺Exhibit an inhibitory effect, and Fe³⁺The addition of (2) greatly reduces the enzyme activity and shows strong inhibition. Presumably due to Fe³⁺Occupying the binding site of the substrate and the enzyme, causing a change in the conformation of the enzyme, which in turn affects the catalytic function of the enzyme (Zhu Jiang et al, 2003).

3.3.6 Effect of surfactant SDS and Metal chelator EDTA on enzyme tolerance

The enzyme tolerance of the purified endoglucanase rEgh31 expressed in E.coli and the purified endoglucanase tEgh31 expressed in Trichoderma reesei was analyzed with SDS and EDTA. To further examine the resistance of endoglucanases to SDS and EDTA, SDS and EDTA were added to the enzymatic reaction system, and their effects on the enzymatic activity were measured. The experimental result shows that after the treatment for 60min by 0.5% SDS, the relative enzyme activity of the endoglucanase rEgh31 expressed by the escherichia coli is about 30%, while the relative enzyme activity of the endoglucanase tEgh31 expressed by the trichoderma reesei expression system is close to 55%, which indicates that the relative enzyme activity of the endoglucanase tEgh31 to SDS is higher than that of the endoglucanase rEgh 31. When the SDS concentration in the reaction system exceeded 1%, the enzyme activities of both recombinases rapidly declined (FIG. 8-A).

From FIG. 8-B, it can be seen that the metal chelator EDTA has a small effect on both recombinases. When the concentration of EDTA in the reaction system is up to 2%, the relative enzyme activity of the two recombinant enzymes is still up to more than 80%. According to the analysis of comprehensive experimental results, the recombinant endoglucanases of two different expression systems show good tolerance to SDS and EDTA, and have good industrial application prospects. Wherein the fungal expression of tEgh31 showed higher tolerance to SDS, it is hypothesized that there may be some post-expression modifications in the fungal expression system that increase the tolerance of the enzyme to SDS.

3.3.7 Effect of commercial detergent powder on enzyme Activity

The enzyme activities of the endoglucanase rEgh31 expressed in the purified Escherichia coli and the endoglucanase tEgh31 expressed in the purified Trichoderma reesei were analyzed by commercially available washing powder. To further test the potential of the enzyme as a detergent additive, different concentrations of detergent powder were added to the enzymatic reaction system, and the results are shown in FIG. 9. As can be seen from the figure, under the condition that the concentration of the washing powder is 2%, the relative enzyme activity is as high as about 80%, and when the concentration of the washing powder is as high as 5%, the relative enzyme activity of the two recombinant enzymes is still nearly 40%. It can be seen that the commercial detergent (Brand: tide, Baojie) has little inhibition of both recombinases. The results of the experiments are combined, and the enzyme meets the basic requirements of detergent additive enzyme.

4 small knot

1. The expression of the egh31 gene in Escherichia coli is realized. The gene is inserted into a prokaryotic expression vector pET-28a (+) and expressed in a strain of Escherichia coli E.coli BL21 Star (DE3), the induction temperature is preferably 23-26 ℃, and the enzyme activity of liquid fermentation (namely cell lysate) of recombinant bacteria is 13.6U/mL under the optimized culture condition. After protein separation and purification, the specific activity of the pure enzyme is 135.7U/mg.

2. Constructs pPIC-P-egh31-T expression vector, and transfers the expression vector into Trichoderma reesei QM9414 to successfully carry out high-efficiency constitutive expression. Under the optimized condition, the extracellular enzyme activity of the endoglucanase expressed by the trichoderma reesei after 5 days of fermentation reaches 31.46U/mL (the supernatant liquid of the liquid fermentation of the recombinant bacteria), and is about 7.4 times higher than the enzyme activity of the endoglucanase of the original strain streptomycete (namely the enzyme activity of H31 fermentation liquor, 4.12U/mL). After the protein is separated and purified, the specific activity is measured to be 161.2U/mg, and is improved by about 19 percent compared with the specific activity (135.7U/mg) of the enzyme protein expressed in the Escherichia coli.

3. The zymotic properties of the Streptomyces sp.H31 original strain zymotic fluid (namely H31 zymotic fluid) and the recombinant endoglucanase (after separation and purification) expressed in escherichia coli and trichoderma reesei are respectively measured, and the results show that the optimum pH value of the endoglucanase is 7.0 and the optimum temperature is 60 ℃. After incubation for 60min at 55 ℃, the enzyme activity can still be maintained by more than 80 percent; the pH stability is good, more than 70% of enzyme activity is still kept after incubation for 1 hour at 37 ℃ in a buffer solution with the pH of 3.0-10.0, and the enzyme is neutral endoglucanase with excellent property and wide pH tolerance. The enzyme shows excellent tolerance to surfactant SDS, metal chelator EDTA and commercial detergent bacteria.

In addition, compared with the endoglucanase expressed by escherichia coli, the endoglucanase expressed by the fungus trichoderma reesei has higher tolerance to SDS than the endoglucanase expressed by the escherichia coli, particularly in an SDS solution with the final concentration of 0.5%, the residual enzyme activity of the tEgh31 enzyme is close to 55%, and the residual enzyme activity of the rEgh31 expressed by pronucleus is about 30%. This shows that the endoglucanase expressed by the fungus has wider application prospect.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> Shenzhen university

<120> Trichoderma reesei engineering bacterium, and preparation method and application thereof

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<170> SIPOSequenceListing 1.0

<210> 1

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> nucleotide sequence of signal peptide gene of Trichoderma reesei cellobiohydrolase I gene (cbh I)

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atgtatcgga agttggccgt catcacggcc ttcttggcca cagctcgtgc t 51

<210> 2

<211> 53

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Tegh31 (Sfi I) upstream

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agcggccttc ttggccacag ctcgtgctga caccacgatc tgcgagccct tcg 53

<210> 3

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Tegh31 (6 × His) downstream

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Claims

1. A Trichoderma reesei engineering bacterium is characterized in that: the trichoderma reesei engineering bacteria contain an expression vector;

the expression vector contains a Trichoderma reesei constitutive expression cassette;

the starting strain of the trichoderma reesei engineering bacteria is trichoderma reeseiTrichoderma reesei QM9414；

The constitutive expression cassette of the trichoderma reesei is a constitutive strong promoter of a pyruvate decarboxylase gene of the trichoderma reeseiPpdcTrichoderma reesei cellobiohydrolase I genecbhⅠThe signal peptide gene and the endoglucanase gene of the exogenous target geneegh31And Trichoderma reesei pyruvate decarboxylase gene terminatorTpdcThe composition is abbreviated as Ppdc-cbh I-egh 31-Tpdc;

the endoglucanase geneegh31The encoded amino acid sequence is shown below:

MLLAAATPARADTTICEPFGSTVIQGRYVVQNNRWGTGAPQCVTATDTGFRVIQADGSVPTDGAPKSYPSVFNGCHYTNCSPGTRLPARISTISSAPSSISYGYVPGGVYNAAYDIWLDPTPRTDGVNRTEIMIWFNRVGPVQPIGSPVATATVGGRTWEVWTGSNGTNDVISFVAPSTITSWSFDVMDFVDQAVNRGLAQRDWYLTSVQAGFEPWRDGVGLAVHSFSSTVNVGGDPGGPGGPGAPAPACQVV。

2. the engineered trichoderma reesei strain of claim 1, wherein:

the Trichoderma reesei cellobiohydrolase I genecbhⅠThe nucleotide sequence of the signal peptide gene is shown as SEQ ID No: 1 is shown.

3. The engineered trichoderma reesei strain of claim 1, wherein: the starting vector of the expression vector is pPICZ alpha A.

4. The engineered Trichoderma reesei strain according to any one of claims 1 to 3, wherein: the endoglucanase geneegh31The nucleotide sequence of (a) is as follows:

ATGCTCCTCGCCGCCGCCACTCCCGCCCGGGCGGACACCACGATCTGCGAGCCCTTCGGGTCGACCGTGATCCAGGGCCGCTACGTCGTCCAGAACAACCGCTGGGGCACCGGCGCCCCCCAGTGCGTCACCGCGACGGACACCGGCTTCCGGGTCATCCAGGCCGACGGCTCGGTGCCCACCGACGGCGCTCCCAAGTCGTACCCGTCGGTCTTCAACGGCTGCCACTACACCAACTGTTCGCCCGGGACCAGGCTCCCCGCACGGATCAGCACCATCTCCAGCGCGCCCAGCAGCATCTCCTACGGCTACGTGCCGGGCGGTGTGTACAACGCCGCGTACGACATCTGGCTGGACCCGACGCCCCGCACCGACGGTGTCAACCGGACCGAGATCATGATCTGGTTCAACCGGGTCGGCCCGGTCCAGCCGATCGGCTCTCCGGTCGCCACCGCAACCGTCGGTGGGCGCACCTGGGAGGTGTGGACGGGCAGCAACGGCACCAACGACGTGATCTCCTTCGTCGCCCCGTCGACCATCACGAGCTGGAGCTTCGACGTCATGGACTTCGTCGACCAGGCCGTCAACCGGGGCCTGGCGCAGCGCGACTGGTACCTGACGAGCGTTCAGGCCGGCTTCGAACCGTGGCGGGACGGCGTCGGACTGGCGGTGCACTCCTTCTCCTCCACCGTGAACGTCGGCGGTGACCCCGGCGGGCCGGGCGGGCCGGGTGCCCCGGCGCCCGCCTGCCAGGTGGTGTAG。

5. use of the engineered Trichoderma reesei strain of any one of claims 1 to 3 in the preparation of an industrial enzyme preparation.

6. Use according to claim 5, characterized in that:

the industrial enzyme preparation is neutral endoglucanase tEgh 31.