CN115960869B - Construction and application of xylanase and secretory expression strain thereof - Google Patents
Construction and application of xylanase and secretory expression strain thereof Download PDFInfo
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
The invention discloses xylanase and construction and application of a secretory expression strain thereof, and relates to the fields of molecular biology and biotechnology. The amino acid sequence of the xylanase is shown as SEQ ID NO. 1. The invention also provides application of the xylanase in preparing baked food. According to the codon preference of Trichoderma reesei, the coding gene of xylanase is subjected to codon optimization, and the optimized gene is named xynA1. The invention constructs plasmids CBH1T1.0 and CBH2T1.0, converts xynA1 into Trichoderma reesei genome by using the plasmids, replaces two cellulase genes cbh1 and cbh2 with highest secretion expression quantity in Trichoderma reesei in sequence, and successfully constructs engineering strains for secretion expression of xylanase. The engineering strain can be used for producing xylanase which can obviously increase specific volume of baked foods (such as bread).
Description
Technical Field
The invention relates to the fields of molecular biology and biotechnology, in particular to construction and application of xylanase and secretory expression strain thereof.
Background
Trichoderma reesei or Hypocreateocorina is a Wen Fu-philic filamentous fungus. The isolated strain was identified as Trichoderma reesei QM6a. Starting from the original strain QM6a, a series of mutation breeding is carried out, and a plurality of cellulase high-yield strains such as Rut-C30 are screened.
Trichoderma reesei is widely used because of its ability to naturally secrete large amounts of endogenous proteins to the outside of the cell, whereas endogenous cellulases are fermented to yields up to 100g/L. Trichoderma reesei has also been a common host for expression of both homologous and heterologous proteins due to various post-translational modifications. Cellulases produced by trichoderma reesei are widely used in various industries. For example, two cellulase cbh1 and cbh2 promoters with the highest expression levels have been successfully used to express heterologous proteins in Trichoderma reesei.
Xylan (xylan) is a heterogeneous polysaccharide present in plant cell walls, accounting for about 15% -35% of the dry weight of plant cells, and is the main component of plant hemicellulose. Xylanase (xylanases) refers to an enzyme that can degrade xylan into oligosaccharides and xylose, and is widely distributed in nature. At present, xylanase is mainly produced by fermentation by using fungi, bacteria and other microorganisms. Extracellular enzymes secreted by filamentous fungi are highest among fungi. The xylanase can be applied to the industries of food, brewing and feed. The xylanase can decompose cell walls and beta-glucan of raw materials in food, brewing or feed industry, reduce the viscosity of the materials, increase the specific volume of the materials, promote the release of effective substances, reduce non-starch polysaccharide in feed grains and promote the absorption and utilization of nutrient substances.
In the prior art, xylanase has been applied to the preparation of baked foods, and the xylanase with better effect and capability of remarkably increasing specific volume of the baked foods is provided, so that the xylanase is more suitable for actual baking requirements.
Disclosure of Invention
The invention aims to provide a xylanase and construction and application of a secretory expression strain thereof, so as to solve the problems in the prior art.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides xylanase, the amino acid sequence of which is shown as SEQ ID NO. 1.
The invention also provides a coding gene of the xylanase.
Further, the nucleotide sequence of the coding gene is shown as SEQ ID NO. 2.
The invention also provides an expression vector comprising the coding gene.
Further, the expression vector also comprises a promoter and terminator sequence of a Trichoderma reesei (Trichoderma reesei) cellulase coding gene.
The invention also provides a microbial strain, which comprises the expression vector.
Further, the microbial strain is trichoderma reesei.
The invention also provides application of the coding gene, the expression vector or the microbial strain in preparation of xylanase.
The invention also provides application of the xylanase in preparing baked food.
The invention also provides a construction method of the trichoderma reesei engineering strain for secreting and expressing xylanase, which comprises the following steps:
(1) Constructing and obtaining the expression vector;
(2) And (3) converting the expression vector into Trichoderma reesei to obtain the Trichoderma reesei engineering strain.
The invention discloses the following technical effects:
the invention discloses xylanase and discloses a coding gene which is optimized according to the codon preference of trichoderma reesei and is named xynA1. The invention constructs plasmids CBH1T1.0 and CBH2T1.0, converts xynA1 into Trichoderma reesei genome by using the plasmids, replaces two cellulase genes cbh1 and cbh2 with highest secretion expression quantity in Trichoderma reesei in sequence, and successfully constructs engineering strains for secretion expression of xylanase. The engineering strain can be used for producing xylanase which can obviously increase specific volume of baked foods (such as bread).
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart showing construction of expression plasmids in the present invention; a: CBH1T1.0 and CBH2T1.0 plasmids; b: expression vectors CBH1T1.0-xynA1 and CBH2T1.0-xynA1;
FIG. 2 is a flow chart of the construction of engineering strain TrxynA1 according to the invention;
FIG. 3 is the activity level of xylanase produced by fermentation from the engineering strain of example 4;
FIG. 4 is a graph showing the effect of different xylanases on bread specific volume in example 5.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
In an embodiment of the method of the invention, the medium and reagents are formulated as follows:
(1) LuriaBertani (LB) culture medium formula: 5g of yeast powder, 10g of peptone, 10g of sodium chloride and tap water to 1L, and naturally pH.
(2) PDA culture medium formula: 200g of potato, 20g of glucose (or xylose), 20g of agar and tap water to 1L, and natural pH.
(3) The formula of the fermentation medium comprises: 40g of lactose (or sophorose mixture), 10g of cellulose powder, 5g of bran, 5g of corn steep liquor, 3g of peptone, 2g of yeast powder, (NH) 4 ) 2 SO 4 3g，KH 2 PO 4 2.0g，CaCl 2 0.34g，MgSO 4 ·7H 2 O0.3g, mandels microelement solution 1mL, and Tween 80 1mL.
(4) Formula of Mandels microelement liquid (1000X): feSO 4 ·7H 2 O5g，CoCl·6H 2 O2g，ZnSO 4 ·7H 2 O1.4g，MnSO 4 ·H 2 O1.6g, purified water to 1L.
(5) Culture medium for joint transfer:
a.5M KOH:7.013gKOH powder was sized to 25mL.
b.1MMES:21.32gMES, water was added to a volume of 100mL, pH5.3 (5 MKOH-adjusted) and sterilized by passing through the membrane.
c. Acetosyringone solution: 0.01962g was weighed, dissolved in DMSO and fixed to a volume of 0.5mL, and sterilized by filtration.
d.MMsalts(1L)：NaCl0.375g、(NH 4 ) 2 SO 4 1.250g、K 2 HPO 4 5.125g、KH 2 PO 4 3.625g、CaCl 2 ·2H 2 O0.165g、MgSO 4 ·7H 2 O1.250g and FeSO 4 ·7H 2 O0.0062g。
e.M-100Trace(0.5L)：H 3 BO 3 30mg、MnCl 2 ·4H 2 O70mg、ZnCl 2 200mg、Na 2 MoO 4 ·2H 2 O20mg、FeCl 3 ·6H 2 O50mg and CuSO 4 ·5H 2 O200mg。
f.M-100Salt(1L)：KCl8g、KH 2 PO 4 16g、Na 2 SO 4 4g、CaCl 2 1g、MgSO 4 ·7H 2 O2g and M-100Trace8mL.
Im liquid medium: 80 mM, 0.36g glucose, 1.26g glycerol, adding water to a constant volume of 192mL, packaging into 8 bottles, sterilizing, and 24mL each bottle; 0.025mL acetosyringone solution and 1mM MES (1M, sterilization by passing through the membrane) were added per bottle at the time of inoculation, and the main function was to adjust the pH value.
Im solid medium: 160 mM glucose, 0.36g glucose, 2.52g glycerol, adding water to a volume of 384mL, subpackaging 4 bottles, 96mL each bottle, adding 1.5g agar, 115 ℃ and sterilizing for 20 min. Before the plate was poured, 0.1mL of acetosyringone solution and 4 mM MES (1M, membrane sterilization) were added per bottle.
i.M-100 liquid medium: 62.5. 62.5mLM-100salt,10g xylose, 3g KNO 3 Adding water to a constant volume of 1L, and sub-packaging into 20 bottles of 50mL each bottle. Sterilizing at 115 deg.C for 20 min.
The Trichoderma reesei starting strain used in the method examples of the present invention was Trichoderma reesei Rut-C30 (ATCC 56765). This protocol is also applicable to other Trichoderma reesei strains.
The plasmid extraction kit is purchased from AXYGEN company, the gel recovery kit is purchased from MAGEN company, the seamless cloning kit is selected from full-scale gold company, and the DNA restriction endonuclease and the ligase are required to be NEB company. And similar products of other companies can be used for replacing the products.
The experimental procedure, which does not specify specific conditions in the following examples, is generally followed by conventional conditions, such as "molecular cloning: the conditions described in the laboratory Manual (NewYork: cold spring harbor laboratory Press, 1989).
The amino acid sequence of the xynA1 protein in the following examples is shown in SEQ ID No. 1.
SEQ ID NO.1：
MVSFSALFLALSAAVGVLSAPVEQAEYANDSFNETLHELVERSTPSSTGWSNGYYYSFWTDGGGDVTYTNGAGGSYTVQWSNVGNFVGGKGWNPGSTRTINYGGSFNPSGNGYLAIYGWTQNPLIEYYIVESYGNYNPGSGGTYKGTVTSDGGTYDIYTSVRYNAPSIEGTKTFTQFWSVRQSKRVGGSVTTANHFAAWSRLGMSLGNHNYQIVATEGYQSSGSSSITVY。
The nucleotide sequence of the xynA1 gene is shown as SEQ ID NO.2, and the sequence is optimized according to the codon preference of Trichoderma reesei.
SEQ ID NO.2：
ATGGTCTCCTTCAGCGCCCTGTTCCTCGCCCTCTCTGCTGCTGTCGGCGTCCTCAGCGCTCCTGTCGAGCAGGCTGAGTACGCTAACGACAGCTTCAACGAGACCCTGCACGAGCTGGTCGAGCGATCTACCCCTAGCAGCACCGGCTGGTCTAACGGCTACTACTACTCCTTCTGGACCGACGGCGGCGGCGACGTCACCTACACCAACGGCGCTGGCGGCAGCTACACTGTCCAGTGGTCTAACGTCGGCAACTTCGTCGGCGGCAAGGGCTGGAACCCTGGCAGCACCCGAACTATCAACTACGGCGGCAGCTTCAACCCCAGCGGCAACGGCTACCTGGCTATCTACGGCTGGACCCAGAACCCCCTCATCGAGTACTACATCGTCGAGAGCTACGGCAACTACAACCCCGGCTCCGGCGGCACCTACAAGGGCACCGTCACCAGCGATGGCGGCACCTACGATATCTACACCAGCGTCCGCTACAACGCCCCCTCTATCGAGGGCACTAAGACCTTCACCCAGTTCTGGTCCGTCCGCCAGAGCAAGCGAGTCGGCGGCAGCGTCACCACCGCTAACCACTTTGCCGCCTGGAGCCGACTCGGCATGTCTCTCGGCAACCACAACTACCAGATCGTCGCCACCGAGGGCTACCAGTCTTCCGGCAGCTCTAGCATCACCGTCTACTAA。
Example 1 construction of plasmids CBH1T1.0 and CBH2T1.0
The skeleton of the constructed vector is LmL2.0a (disclosed in the document "Zhangetal. Light-induciblegenetic engineeringandcontrolofnon-homologo use-joininginindustrialeukaryotic microorganisms: lmL3.0and OFN1.0.scientific reports.2016, 6:20761"), and contains Cre-loxP elements, so that the repeated use of the screening mark can be realized.
1. Construction of expression plasmid CBH1T1.0
(1) The primers Pcbh1-F and Pcbh1-R were used to amplify the promoter sequence Pcbh1, 1392bp of cbh1 using Trichoderma reesei genome as template.
Pcbh1-F(SEQ ID NO.3)：
5’-GATTACGAATTCTTAATTAATTCTGGAGACGGCTTGTTGAATCAT-3’；
Pcbh1-R(SEQ ID NO.4)：
5’-ATTATACGAAGTTATCTAGATGCGCAGTCCGCGGTTGA-3’。
Amplification reaction system: 10 XPCRBuforforKOD-Plus-Neo 5. Mu.L; 2mM dNTPs 5. Mu.L; 25mM MgSO 4 3 μL; 1.5. Mu.L of primer (10. Mu.Meach); trichoderma reesei genome modelPlate (20 ng) 1. Mu.L; KOD-Plus-Neo (1U/. Mu.L) 1. Mu.L.
The reaction procedure: 94 ℃ for 2min; 30 cycles were run at 98℃for 10sec,58℃for 30sec,68℃for 45 sec; and at 68℃for 5min.
(2) The terminator sequence Tcbh1, 1120bp of cbh1 was amplified using primers Tcbh1-1 and Tcbh1-2, with Trichoderma reesei genome as template.
Tcbh1-1(SEQ ID NO.5)：
5’-ACTAGTGAGCTCATTTAGCTCCGTGGCGAAAGCCT-3’；
Tcbh1-2(SEQ ID NO.6)：
5’-AGTGCCAAGCTTATTTCATCGTAACCGAGAATCCAGAGCTG-3’。
Amplification reaction system: 10 XPCRBuforforKOD-Plus-Neo 5. Mu.L; 2mM dNTPs 5. Mu.L; 25mM MgSO 4 3 μL; 1.5. Mu.L of primer (10. Mu.Meach); trichoderma reesei genome template (20 ng) 1. Mu.L; KOD-Plus-Neo (1U/. Mu.L) 1. Mu.L.
The reaction procedure: 94 ℃ for 2min; 30 cycles were run at 98℃for 10sec,58℃for 30sec,68℃for 35 sec; and at 68℃for 5min.
(3) Constructing an expression vector by taking LmL2.0a as a framework, firstly carrying out double enzyme digestion by using restriction enzyme PacI/XbaI, carrying out homologous recombination by using VazymeOneStepClonekit, and connecting into a promoter sequence Pcbh1 of cbh1; then restriction enzyme SwaI is used for single enzyme digestion, vazymoOneSteplonekit is used for homologous recombination, and a terminator sequence Tcbh1 of cbh1 is connected, namely the expression plasmid CBH1T1.0 (A in figure 1).
2. Construction of expression plasmid CBH2T1.0
(1) The primers Pcbh2-F and Pcbh2-R were used to amplify the promoter sequence Pcbh2, 1266bp of cbh2 using the Trichoderma reesei genome as template.
Pcbh2-F(SEQ ID NO.7)：
5’-GATTACGAATTCTTAATTAAGCAATAAGACGCATCTCAGGTCACA-3’；
Pcbh2-R(SEQ ID NO.8)：
5’-TACATTATACGAAGTTATTTAAATGGTGCAATACACAGAGGGTGATCTTAC-3’。
Amplification reaction system: 10 XPCRBuforforKOD-Plus-Neo 5. Mu.L; 2mM dNTPs 5. Mu.L; 25mM MgSO 4 3 μL; primer (10 mu Mea)ch) 1.5. Mu.L; trichoderma reesei genome template (20 ng) 1. Mu.L; KOD-Plus-Neo (1U/. Mu.L) 1. Mu.L.
The reaction procedure: 94 ℃ for 2min; 30 cycles were run at 98℃for 10sec,58℃for 30sec,68℃for 45 sec; and at 68℃for 5min.
(2) The terminator sequence Tcbh2, 885bp of cbh2 was amplified using primers Tcbh2-1 and Tcbh2-2, with Trichoderma reesei genome as template.
Tcbh2-1(SEQ ID NO.9)：
5’-ACTAGTGAGCTCATTTGCTTTCGTGACCGGGCTTCAAA-3’；
Tcbh2-2(SEQ ID NO.10)：
5’-AGTGCCAAGCTTATTTATGCGATGCGGCTCAAGACTTC-3’。
Amplification reaction system: 10 XPCRBuforforKOD-Plus-Neo 5. Mu.L; 2mM dNTPs 5. Mu.L; 25mM MgSO 4 3 μL; 1.5. Mu.L of primer (10. Mu.Meach); trichoderma reesei genome template (20 ng) 1. Mu.L; KOD-Plus-Neo (1U/. Mu.L) 1. Mu.L.
The reaction procedure: 94 ℃ for 2min; 30 cycles were run at 98℃for 10sec,58℃for 30sec,68℃for 30 sec; and at 68℃for 5min.
(3) Constructing an expression vector by taking LmL2.0a as a framework, firstly, carrying out single enzyme digestion by utilizing restriction enzyme SwaI, carrying out homologous recombination by utilizing Vazyme OneStepClonekit, and connecting into a terminator sequence Tcbh2 of cbh2; double digestion with restriction enzyme PacI/XbaI and homologous recombination with VazymeOneStepClonekit are performed, and the promoter sequence Pcbh2, which is the expression plasmid CBH2T1.0 (FIG. 1A), is ligated into cbh 2.
The expression vector used herein mainly provides the function of the Agrobacterium tumefaciens conjugative transfer site, so, in addition to LmL2.0a, any of the following Agrobacterium binary vectors may be used: PZP100, pPZP201B, pPZP BK, pBI121, pCAMBIA, pPK2, lml2.1, but not just these vectors.
EXAMPLE 2 construction of expression vectors for xylanase genes
(1) The primers xynA1-1-1 and xynA1-2 were used to amplify the C1-xynA1 sequence using an artificially synthesized DNA (designated Shanghai JieR Biotechnology Co., ltd.) containing the xynA1 gene sequence shown in SEQ ID NO.2 as a template.
xynA1-1-1(SEQ ID NO.11)：
5’-ACCGCGGACTGCGCATCATGGTCTCCTTCAGCGCCCTGTT-3’；
xynA1-2(SEQ ID NO.12)：
5’-ATTATACGAAGTTATCTATTAGTAGACGGTGATGCTA-3’。
Amplification reaction system: 10 XPCRBuforforKOD-Plus-Neo 5. Mu.L; 2mM dNTPs 5. Mu.L; 25mM MgSO 4 3 μL; 1.5. Mu.L of primer (10. Mu.Meach); 1. Mu.L of artificially synthesized DNA template (5 ng); KOD-Plus-Neo (1U/. Mu.L) 1. Mu.L.
The reaction procedure: 94 ℃ for 2min; 30 cycles were run at 98℃for 10sec,58℃for 30sec,68℃for 30 sec; and at 68℃for 5min.
C1-xynA1 sequence (SEQ ID NO. 13):
ACCGCGGACTGCGCATCATGGTCTCCTTCAGCGCCCTGTTCCTCGCCCTCTCTGCTGCTGTCGGCGTCCTCAGCGCTCCTGTCGAGCAGGCTGAGTACGCTAACGACAGCTTCAACGAGACCCTGCACGAGCTGGTCGAGCGATCTACCCCTAGCAGCACCGGCTGGTCTAACGGCTACTACTACTCCTTCTGGACCGACGGCGGCGGCGACGTCACCTACACCAACGGCGCTGGCGGCAGCTACACTGTCCAGTGGTCTAACGTCGGCAACTTCGTCGGCGGCAAGGGCTGGAACCCTGGCAGCACCCGAACTATCAACTACGGCGGCAGCTTCAACCCCAGCGGCAACGGCTACCTGGCTATCTACGGCTGGACCCAGAACCCCCTCATCGAGTACTACATCGTCGAGAGCTACGGCAACTACAACCCCGGCTCCGGCGGCACCTACAAGGGCACCGTCACCAGCGATGGCGGCACCTACGATATCTACACCAGCGTCCGCTACAACGCCCCCTCTATCGAGGGCACTAAGACCTTCACCCAGTTCTGGTCCGTCCGCCAGAGCAAGCGAGTCGGCGGCAGCGTCACCACCGCTAACCACTTTGCCGCCTGGAGCCGACTCGGCATGTCTCTCGGCAACCACAACTACCAGATCGTCGCCACCGAGGGCTACCAGTCTTCCGGCAGCTCTAGCATCACCGTCTACTAATAGATAACTTCGTATAAT。
(2) The primers xynA1-2-1 and xynA1-2 are used for amplifying the C2-xynA1 sequence by taking the artificially synthesized DNA containing the xynA1 gene sequence shown in SEQ ID NO.2 as a template.
xynA1-2-1(SEQ ID NO.14)：
5’-CCCTCTGTGTATTGCACCATGGTCTCCTTCAGCGCCCTGTT-3’；
The primer xynA1-2 sequence is as indicated previously.
Amplification reaction system: 10 XPCRBuforforKOD-Plus-Neo 5. Mu.L; 2mM dNTPs 5. Mu.L; 25mM MgSO 4 3 μL; 1.5. Mu.L of primer (10. Mu.Meach); 1. Mu.L of artificially synthesized DNA template (5 ng); KOD-Plus-Neo (1U/. Mu.L) 1. Mu.L.
The reaction procedure: 94 ℃ for 2min; 30 cycles were run at 98℃for 10sec,58℃for 30sec,68℃for 30 sec; and at 68℃for 5min.
C2-xynA1 sequence (SEQ ID NO. 15):
CCCTCTGTGTATTGCACCATGGTCTCCTTCAGCGCCCTGTTCCTCGCCCTCTCTGCTGCTGTCGGCGTCCTCAGCGCTCCTGTCGAGCAGGCTGAGTACGCTAACGACAGCTTCAACGAGACCCTGCACGAGCTGGTCGAGCGATCTACCCCTAGCAGCACCGGCTGGTCTAACGGCTACTACTACTCCTTCTGGACCGACGGCGGCGGCGACGTCACCTACACCAACGGCGCTGGCGGCAGCTACACTGTCCAGTGGTCTAACGTCGGCAACTTCGTCGGCGGCAAGGGCTGGAACCCTGGCAGCACCCGAACTATCAACTACGGCGGCAGCTTCAACCCCAGCGGCAACGGCTACCTGGCTATCTACGGCTGGACCCAGAACCCCCTCATCGAGTACTACATCGTCGAGAGCTACGGCAACTACAACCCCGGCTCCGGCGGCACCTACAAGGGCACCGTCACCAGCGATGGCGGCACCTACGATATCTACACCAGCGTCCGCTACAACGCCCCCTCTATCGAGGGCACTAAGACCTTCACCCAGTTCTGGTCCGTCCGCCAGAGCAAGCGAGTCGGCGGCAGCGTCACCACCGCTAACCACTTTGCCGCCTGGAGCCGACTCGGCATGTCTCTCGGCAACCACAACTACCAGATCGTCGCCACCGAGGGCTACCAGTCTTCCGGCAGCTCTAGCATCACCGTCTACTAATAGATAACTTCGTATAAT。
(3) Carrying out restriction enzyme XbaI single enzyme digestion on the existing plasmid CBH1T1.0, carrying out homologous recombination by using VazymeOne StepCloneKit, and connecting the restriction enzyme with a C1-xynA1 sequence obtained by PCR amplification to obtain an expression vector CBH1T1.0-xynA1 (FIG. 1B); the restriction enzyme SwaI on the existing plasmid CBH2T1.0 was subjected to single cleavage, homologous recombination was performed using the VazymeOneStepClonekit, and the resulting C2-xynA1 sequence was ligated into PCR amplification to obtain an expression vector CBH2T1.0-xynA1 (FIG. 1B).
EXAMPLE 3 construction of Trichoderma reesei engineering Strain secreting xylanase
The embodiment provides a Trichoderma reesei genetic engineering bacterium for expressing recombinant proteins, which is an engineering bacterium obtained by transforming Trichoderma reesei with agrobacterium tumefaciens carrying recombinant protein expression vectors to obtain transformants and screening and culturing, wherein the specific preparation steps are as follows:
firstly, the xylanase expression vector CBH1T1.0-xynA1 is electrically transformed into agrobacterium tumefaciens AGL-1 (purchased from China center for type culture collection), and kanamycin (50 mu g/mL) is used for screening positive agrobacterium tumefaciens; positive root cancer agro-pole of LB liquid cultureTransferring the fungus to liquid IM, and diluting Trichoderma reesei spore to 10 5 ～10 8 A gradient concentration spore suspension in the range of individual/mL. The suspension of Agrobacterium tumefaciens and Trichoderma reesei spores in IM was mixed in equal volumes and spotted onto nitrocellulose membranes of solid IndustionM media plates. After 48h co-cultivation at 28℃the membranes were transferred to PDA medium supplemented with 20g/L glucose, hygromycin (75. Mu.g/mL) and cephalosporin (300. Mu.g/mL) by a super clean bench sterile procedure, the plates were incubated at 28℃for 7 days, spores were collected, diluted to a concentration that grew on the plates in monoclonal form, PDA plates (containing 20g/L glucose, 75. Mu.g/mL hygromycin and 150. Mu.g/mL cephalosporin) were smeared, after 48h incubation at 28℃the monoclonals were picked up in PDA well plates (containing 20g/L glucose, 75. Mu.g/mL hygromycin and 150. Mu.g/mL cephalosporin), spores were collected, the mycelia were scraped off for 7 days, and the sequence was verified for replacement of the cbh1 gene by PCR (see FIG. 2).
Inoculating the obtained positive recombinant Trichoderma reesei strain into M100 liquid (containing 20g/L xylose) culture medium to induce resistance gene deletion, shake culturing at 28deg.C at 200rpm for 48 hr, selecting small amount of mycelium points on PDA (containing 20g/L xylose) plate, culturing at 28deg.C for 7 days, collecting spores, gradient diluting, coating on PDA plate (containing 20g/L glucose or xylose), and culturing at 28deg.C for 48 hr. The monoclonal was picked up in PDA well plates (containing 20g/L glucose or xylose) and incubated at 28℃for 24h. The hyphal agar pellet from the well plate was picked up to PDA well plate (containing 20g/L glucose, 75. Mu.g/mL hygromycin) to verify the success of the resistance deletion. The strain with successful resistance loss is the first generation engineered strain (figure 2). After 7 days of culture at 28 ℃, spores of the first-generation engineered strain were collected.
Next, xylanase expression vector CBH2T1.0-xynA1 was introduced into the first-generation engineered strain as described above, and finally a second-generation engineered strain, namely, an engineered strain TrxynA1 (FIG. 2) for secretion expression of xylanase was obtained.
EXAMPLE 4 production of xylanase by engineering Strain
Inoculating the obtained engineering strain TrxynA1 into a fermentation medium, and fermenting in a tank. Fermenter culture methods are described in reference "Chenetal. Engineering of Trichoderma reesei for enhancement of ddugreevesed conjugation of lignocerulosidobiomassia bytoplasmic of the celllulasaseactionacitor ACE3. Biotechnology Biofuel.2020, 13:62". And after fermentation, taking fermentation liquor, centrifuging at 12500rpm for 10min, and taking supernatant to obtain xylanase crude enzyme liquor. The enzyme activity of the xylanase crude enzyme solution was measured (FIG. 3). Enzyme activity unit: the amount of enzyme required to degrade and release 1. Mu. Mol of reducing sugar per minute from a solution of oat xylan at a concentration of 10mg/mL at 50℃and pH 5.0 is one vitality unit (U), the reducing sugar being in xylose equivalent. The results show (FIG. 3) that the engineered strain secretes a greater amount of xylanase than the unmodified Trichoderma reesei Rut-C30 control group of the invention.
And (3) drying the xylanase crude enzyme liquid produced by the engineering strain, and adding a general protective agent to prepare solid xylanase powder, wherein the enzyme powder activity reaches 160000U/g.
EXAMPLE 5 use of xylanase
Bread baking experiments were performed with the xylanase powder prepared in example 4 and two commercial xylanase powders SBE-01X (enzyme activity 180000U/g) and SBE-02X (enzyme activity 180000U/g), respectively, with no enzyme addition as blank.
The dough formulation for making bread in this example is: 1000g of wheat flour (flour), 10g of yeast powder, 16g of salt, 580g of water, 60g of sugar and 30g of vegetable oil, wherein the addition amount of xylanase is 20ppm. .
The bread preparation steps are as follows: the ingredients of the dough with the formula are added into a dough kneading bowl and stirred for 18min to form uniform dough. Placing the dough on a balance, dividing into 50 g/piece, kneading into round shape, fermenting at 38deg.C for 60min, and baking in a oven at 180deg.C for 10min to obtain bread product.
The resulting breads were cooled at room temperature for 2 hours, and the mass and volume of each experimental group of breads were measured, respectively. Specific volume is the volume of bread per mass of bread (mL/g). The results show (fig. 4) that the three groups of xylanase additions all significantly increased the specific volume of bread; under the same adding amount and similar enzyme activity conditions, the xylanase xynA1 disclosed by the invention has better performance in the aspect of increasing the specific volume of bread compared with two commercial xylanases.
From the results of the above examples, it can be seen that: the xylanase xynA1 produced by the engineering strain for secreting and expressing xylanase disclosed by the invention has excellent acting force with flour substrates, can be applied to the dough treatment process in the food industry, and improves the quality of bread.
In conclusion, after the trichoderma reesei is genetically modified, the engineering strain for secreting and expressing xylanase is successfully constructed, and xylanase produced by the engineering strain can be applied to the food industry and improves the quality of baked foods.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (1)
1. The application of xylanase in preparing baked food is characterized in that the amino acid sequence of xylanase is shown as SEQ ID NO. 1;
the xylanase is used to increase the specific volume of the baked good.
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