CN115851677A - Pichia pastoris engineering bacterium for high yield of beta-glucosidase and application thereof - Google Patents
Pichia pastoris engineering bacterium for high yield of beta-glucosidase and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a pichia pastoris engineering bacterium for high yield of beta-glucosidase. The amino acid sequence of the beta-glucosidase mutant is shown as SEQ ID No.4. Introducing the vector containing the mutant gene into a host by means of genetic engineering to obtain a genetically engineered bacterium, fermentation performance proves that the activity of the beta-glucosidase fermentation enzyme can reach more than 51000U/mL. The optimum pH of beta-glucosidase expressed by the mutant is 4.0, is reduced by 0.5 compared with that before mutation, the optimum temperature is 45 ℃, the beta-glucosidase is treated for 24 hours under the condition of pH 3.5, the relative activity still remains about 80 percent, the acid resistance is improved compared with that before mutation, and the beta-glucosidase is more beneficial to being applied in the cellulose bioconversion industry.
Description
The technical field is as follows:
the invention relates to the technical field of biology, in particular to a pichia pastoris engineering bacterium for high yield of beta-glucosidase.
Background art:
beta-glucosidase (beta-D-glucoside hydrolase, EC 3.2.1.21), a type of cellulase, can catalyze and hydrolyze terminal non-reducing beta-D-glycosidic bond from sugar-containing compounds to release beta-D-glucose and corresponding monosaccharide, oligosaccharide or complex sugar, and is a protein with biocatalyst function. The beta-glucosidase gene is widely existed in various microorganisms in nature, including bacteria, actinomycetes, yeast, filamentous fungi and the like, and also exists in some animals. Beta-glucosidase has an important role in degrading cellulose, and is widely used in other fields such as food, wine making, medicine and chemical industries.
Beta-glucosidase plays a key role in cellulose saccharification and hydrolysis as an important enzyme for cellulose biotransformation, but the proportion of the beta-glucosidase in a cellulase system produced by microorganisms is less than 1 percent, so that the beta-glucosidase becomes a key factor for degrading cellulose into monosaccharide, and the hydrolysis efficiency of cellulose is improved by adding the beta-glucosidase into a cellulase hydrolysis system industrially.
The current beta-glucosidase product is mainly derived from natural mould. Because the natural bacterial strain has poor enzyme production performance, the beta-glucosidase product cannot meet the market demand. The pichia pastoris is a eukaryotic expression host which is rapidly developed in recent years, has low nutritional requirement, is suitable for a large-scale production mode of high-density fermentation, particularly secretes little protein per se, and is favorable for purifying an expression product. Therefore, the invention obtains the beta-glucosidase mutant with improved acid resistance by modifying the Aspergillus niger for producing the beta-glucosidase of the company by a genetic engineering means, and has great significance for meeting the requirement of cellulose bioconversion industrial production and reducing the production cost by constructing corresponding Pichia pastoris engineering bacteria to express the Pichia pastoris mutant.
The invention content is as follows:
the invention aims to provide a beta-glucosidase mutant with improved acid resistance and a pichia pastoris engineering strain thereof. Aims to solve the problems of low enzyme activity and unsatisfactory cellulose biotransformation application of beta-glucosidase in industrial production.
The invention is realized by the following technical scheme:
a beta-glucosidase mutant with improved acid resistance is obtained by screening Aspergillus niger PT003 preserved in the laboratory of the applicant through a nitrosoguanidine mutagenesis method, cloning a beta-glucosidase mutant coding gene from a mutagenized strain, constructing a recombinant plasmid, and expressing in Pichia pastoris GS115 to obtain a Pichia pastoris engineering strain.
One of the technical schemes provided by the invention is as follows: is a beta-glucosidase mutant, the beta-glucosidase mutant is obtained by mutating serine at the 60 th position into leucine and valine at the 303 th position into alanine on the basis of PT003 wild type beta-glucosidase shown in SEQ ID No.2, and the amino acid sequence of the beta-glucosidase mutant is shown in SEQ ID No. 4;
the invention also provides a coding gene of the glucosidase mutant;
furthermore, the coding gene of the beta-glucosidase mutant has a nucleotide sequence shown in SEQ ID No. 3.
The enzymatic properties of the beta-glucosidase mutant are as follows:
(1) Optimum pH: the enzyme activity is stable at pH 3.5-5.5, and the optimum action pH is 4.0;
(2) Optimum temperature: the enzyme activity is stable at 35-55 ℃, and the optimal action temperature is 45 ℃;
(3) Acid resistance: the enzyme activity is still more than 80% after 24 hours under the condition that the pH value is 3.5.
The third technical scheme provided by the invention is as follows: the beta-glucosidase mutant coding gene is reconstructed into a recombinant vector or a recombinant strain containing the beta-glucosidase mutant coding gene, the recombinant vector is efficiently expressed in pichia pastoris to obtain the recombinant strain producing the high-activity beta-glucosidase, and the high-activity beta-glucosidase is obtained through technologies such as fermentation and extraction;
further, the host cell for expressing the beta-glucosidase mutant is pichia pastoris GS115;
further, an expression vector for expressing the beta-glucosidase mutant is a pPIC9K plasmid;
preferably, the recombinant strain is obtained by connecting a beta-glucosidase mutant coding gene shown in SEQ ID No.3 with an expression vector pPIC9K and expressing the gene in Pichia pastoris GS115.
The fourth technical scheme provided by the invention is as follows: the application of the recombinant vector or the recombinant strain is the third technical scheme, in particular to the application in fermentation production of the beta-glucosidase mutant shown in SEQ ID No.4.
The fifth technical scheme provided by the invention is as follows: is the application of the beta-glucosidase mutant shown in SEQ ID No.4, in particular to the application in catalyzing the hydrolysis of glycosidic bonds, and more particularly to the application in the cellulose biotransformation industry.
The following definitions are used in the present invention:
1. nomenclature for amino acid and DNA nucleic acid sequences
The accepted IUPAC nomenclature for amino acid residues is used, in either the three letter or single letter code format. DNA nucleic acid sequences employ the accepted IUPAC nomenclature.
2. Identification of beta-glucosidase mutants
"amino acid substituted at the original amino acid position" is used to indicate the mutated amino acid in the mutant. As S60L, it is indicated that the amino acid at position 60 is replaced by Ser in the wild type to Leu, the numbering of the positions corresponding to the numbering of the amino acid sequence of the wild type β -glucosidase in SEQ ID No. 2. In the present invention, G-1 represents a wild-type β -glucosidase, and G-2 represents a β -glucosidase mutant, and the information is as shown in the following table.
| Beta-glucosidase | Amino acid mutation site | Amino acid SEQ ID No. | Nucleotide SEQ ID No. |
| Wild type G-1 | Ser60、Val303 | 2 | 1 |
| Mutant G-2 | Ser60Leu、Val303Ala | 4 | 3 |
Has the advantages that:
the invention discloses a novel beta-glucosidase mutant, which has the characteristics of high enzyme activity and high acid resistance, and the enzyme activity of a mutant fermentation liquid can reach more than 51000U/mL.
The optimum reaction pH of the beta-glucosidase obtained by the invention is 4.0, the enzyme activity is highest, and compared with the beta-glucosidase of wild type PT003, the optimum reaction pH is reduced by 0.5, which indicates that the acid resistance is improved.
The beta-glucosidase obtained by the invention still has the enzyme activity of more than 80 percent after 24 hours under the condition of pH 3.5, and compared with the beta-glucosidase of wild PT003, the acid resistance is obviously improved.
Description of the drawings:
FIG. 1 pH optimum curve;
FIG. 2 is a temperature optimum curve;
fig. 3 acid resistance curve.
The specific implementation mode is as follows:
the present invention is illustrated in greater detail by the specific examples, which are given by way of illustration only and are not intended to limit the scope of the invention. Modifications may be made by those skilled in the art, which would be within the principles of the invention, and such modifications are to be considered within the scope of the invention. The molecular biological experiment method not specifically described in this example can be referred to "molecular cloning Experimental Manual".
The mutant is obtained by screening a Aspergillus niger PT003 strain preserved in the laboratory of the applicant after nitrosoguanidine mutagenesis, and the site of the mutation obtained by gene sequencing is that serine at the 60 th position is mutated into leucine, and valine at the 303 th position is mutated into alanine. The invention firstly obtains the coding gene of the beta-glucosidase mutant, connects the coding gene of the mutant with a pPIC9K carrier to construct recombinant plasmid, transfers the recombinant plasmid into corresponding host bacteria GS115 for heterologous expression, and ferments to obtain the corresponding beta-glucosidase of the mutant. The beta-glucosidase has high enzyme activity and is suitable for industrial production and cellulose bioconversion industries.
1. Experimental materials and reagents:
experimental strains and vectors: the gene source strain: aspergillus niger PT003 available from the same company; expression host bacteria and vectors: GS115 and pPIC9K were purchased from Invitrogen, USA; host bacteria: DH 5. Alpha. In the formula.
The main reagents are as follows: DNA polymerase, T4 DNA ligase, a DNA gel recovery kit, a plasmid extraction kit, an RNA extraction kit, pNPG, DNAmarker, agarose, ampicillin, IPTG, X-gal: all purchased from Shanghai Producers; various restriction enzymes: purchased from NEB corporation.
An experimental instrument: gel imager (Bio-Rad), protein electrophoresis apparatus (Bio-Rad), nucleic acid electrophoresis apparatus (Bio-Rad), PCR amplification apparatus (Bio-Rad), and high speed centrifuge (Eppendorf).
2. The beta-grape adopted by the invention the glycosidase activity determination method comprises the following steps:
(1) Taking 200 μ L of 0.2M acetic acid-sodium acetate buffer (pH 5.0), adding 50 μ L of diluted fermentation supernatant (enzyme solution) into the sample tube, adding 50 μ L of buffer into blank control, and preheating and balancing in 50 deg.C water bath for 5min; (2) At the same time, the pNPG solution (5.0 mM) is preheated and balanced at 50 ℃;
(3) Adding 250 mu L of preheated balanced pNPG substrate solution into the pre-balanced solution of the enzyme solution sample to form 500 mu L of enzyme reaction system, and reacting for 10min at 50 ℃ in a constant-temperature water bath kettle;
(4) 500 μ L of 1.0M Na was added immediately 2 CO 3 Enhancing color development of the solution, stopping reaction, uniformly mixing, and standing for 5min at room temperature;
(5) The absorbance A405 of p-Nitrophenol (p-Nitrophenol) is measured by a microplate reader at 405 nm. Three replicates were made for each sample.
Definition of enzyme activity unit: the amount of enzyme required to hydrolyze a substrate to give 1. Mu. MoL of p-Nitrophenol (p-Nitrophenol) per minute was one enzyme activity unit (U/mL) under the measurement conditions (pH 5.0, 50 ℃ C., unless otherwise specified) using pNPG as a substrate. Namely:
the unit of enzyme activity (U/mL) = (CxN)/T
In the formula, N: dilution times of raw enzyme liquid; t: reaction time (min); c: the different absorbance values A405 correspond to the concentration of p-nitrophenol (. Mu. Mol/mL) on the standard curve.
The nucleotide sequence of the wild beta-glucosidase G-1 encoding gene disclosed by the invention and the embodiment is shown in SEQ ID No. 1:
atgggttcagcaacagcttcaaccttgcctccggactttctctggggtttcgcgactgccagctaccagattgaaggcgcagt
aaccgaagatggccgtggtccttctatctgggacactttctgcaaaatccctggcaagatagccgggggagccaacggag
acgtagcatgcgactcgtaccaccgtacagctgaagacatcgcactgttaaaggaatgcggtgctcaggcttaccgcttctc
aatctcatggtcgcgcatcattcccctcggaggccgcaacgaccccatcaacgataagggtgtccaacattatgtcaagttc
gtcgacgatcttctagcagcggggatcacaccccttgtgacactgttccactgggatctccccgatgcattagacaagcgct
acggtgggctcctcaacaaggaggagttcgttgcagatttcgccaactacgcgcgagtcatgttccgggccctgggctcaa
aagtcaagcattggatcaccttcaatgagccgtggtgttccagtgtccttgggtataacgtcggtcagtttgctcccggtcgg
acgagtgatcggagcaagagcgcagagggagatagctcgagggagtgctggatcgtggggcacaatatcctcgtggct
catggagctgcggtgaagatctaccgagaggagttcaagagtagagatggtggggagatcgggatcacacttaacggag
actgggctgagccctgggatcccgagaatccggccgacatcgaagcctgcgaccgcaagatcgaattcgccatttcctgg
tttgcagaccccatctatcatggaaggtatccagacagcatgataaagcagcttggcgaccgactccccagctggaccgca
gaagacatcgctctcgtccacggcagcaatgacttctatggcatgaaccactactgcgccaactacatcaaagccaaaacc
ggcgaagcggatcccaacgatactgccggaaatctggagatcctgctcaagaacaagaagggcgagttcatcggcccag
agacacagtctgcatggctgagaccgtatgcacttgggttccggaagctgttgaaatggctaagcgatagatacggtcagc
ctaagatctacgttactgagaatgggacgagcttgaagggtgagaatgacttgccggtggaggagctgctgaaggatgaat
tccggacgcagtacttccgtgattatattgctgctatggctgatgcgtacacgctggatggggtgaatgtgagggcttatatg
gcttggagtttgatggataacttcgaatgggctgaaggttacgaaacccggtttgggtcgacctacgtcgactacgaacatg
gccagaagaggatcccgaaggacagtgcgaaacagatcggccagattttcagccagtatatcgagaagaaataa
the amino acid sequence of the wild type beta-glucosidase G-1 is shown in a sequence table SEQ ID No. 2:
MGSATASTLPPDFLWGFATASYQIEGAVTEDGRGPSIWDTFCKIPGKIAGGANG
DVACDSYHRTAEDIALLKECGAQAYRFSISWSRIIPLGGRNDPINDKGVQHYV
KFVDDLLAAGITPLVTLFHWDLPDALDKRYGGLLNKEEFVADFANYARVMFR
ALGSKVKHWITFNEPWCSSVLGYNVGQFAPGRTSDRSKSAEGDSSRECWIVG
HNILVAHGAAVKIYREEFKSRDGGEIGITLNGDWAEPWDPENPADIEACDRKIE
FAISWFADPIYHGRYPDSMIKQLGDRLPSWTAEDIALVHGSNDFYGMNHYCA
NYIKAKTGEADPNDTAGNLEILLKNKKGEFIGPETQSAWLRPYALGFRKLLK
WLSDRYGQPKIYVTENGTSLKGENDLPVEELLKDEFRTQYFRDYIAAMADA
YTLDGVNVRAYMAWSLMDNFEWAEGYETRFGSTYVDYEHGQKRIPKDSAK
QIGQIFSQYIEKK
the nucleotide sequence of the coding gene of the beta-glucosidase mutant G-2 is shown as SEQ ID No. 3:
atgggttcagcaacagcttcaaccttgcctccggactttctctggggtttcgcgactgccagctaccagattgaaggcgcagt
aaccgaagatggccgtggtccttctatctgggacactttctgcaaaatccctggcaagatagccgggggagccaacggag
acgtagcatgcgacttgtaccaccgtacagctgaagacatcgcactgttaaaggaatgcggtgctcaggcttaccgcttctc
aatctcatggtcgcgcatcattcccctcggaggccgcaacgaccccatcaacgataagggtgtccaacattatgtcaagttc
gtcgacgatcttctagcagcggggatcacaccccttgtgacactgttccactgggatctccccgatgcattagacaagcgct
acggtgggctcctcaacaaggaggagttcgttgcagatttcgccaactacgcgcgagtcatgttccgggccctgggctcaa
aagtcaagcattggatcaccttcaatgagccgtggtgttccagtgtccttgggtataacgtcggtcagtttgctcccggtcgg
acgagtgatcggagcaagagcgcagagggagatagctcgagggagtgctggatcgtggggcacaatatcctcgtggct
catggagctgcggtgaagatctaccgagaggagttcaagagtagagatggtggggagatcgggatcacacttaacggag
actgggctgagccctgggatcccgagaatccggccgacatcgaagcctgcgaccgcaagatcgaattcgccatttcctgg
tttgcagaccccatctatcatggaaggtatccagacagcatgataaagcagcttggcgaccgactccccagctggaccgca
gaagacatcgctctcgcccacggcagcaatgacttctatggcatgaaccactactgcgccaactacatcaaagccaaaacc
ggcgaagcggatcccaacgatactgccggaaatctggagatcctgctcaagaacaagaagggcgagttcatcggcccag
agacacagtctgcatggctgagaccgtatgcacttgggttccggaagctgttgaaatggctaagcgatagatacggtcagc
ctaagatctacgttactgagaatgggacgagcttgaagggtgagaatgacttgccggtggaggagctgctgaaggatgaat
tccggacgcagtacttccgtgattatattgctgctatggctgatgcgtacacgctggatggggtgaatgtgagggcttatatg
gcttggagtttgatggataacttcgaatgggctgaaggttacgaaacccggtttgggtcgacctacgtcgactacgaacatg
gccagaagaggatcccgaaggacagtgcgaaacagatcggccagattttcagccagtatatcgagaagaaataa
the amino acid sequence of the beta-glucosidase mutant G-2 is shown in a sequence table SEQ ID No. 4:
MGSATASTLPPDFLWGFATASYQIEGAVTEDGRGPSIWDTFCKIPGKIAGGANG
DVACDLYHRTAEDIALLKECGAQAYRFSISWSRIIPLGGRNDPINDKGVQHYV
KFVDDLLAAGITPLVTLFHWDLPDALDKRYGGLLNKEEFVADFANYARVMFR
ALGSKVKHWITFNEPWCSSVLGYNVGQFAPGRTSDRSKSAEGDSSRECWIVG
HNILVAHGAAVKIYREEFKSRDGGEIGITLNGDWAEPWDPENPADIEACDRKIE
FAISWFADPIYHGRYPDSMIKQLGDRLPSWTAEDIALAHGSNDFYGMNHYCA
NYIKAKTGEADPNDTAGNLEILLKNKKGEFIGPETQSAWLRPYALGFRKLLK
WLSDRYGQPKIYVTENGTSLKGENDLPVEELLKDEFRTQYFRDYIAAMADA
YTLDGVNVRAYMAWSLMDNFEWAEGYETRFGSTYVDYEHGQKRIPKDSAK
QIGQIFSQYIEKK
the present invention is further explained below by means of specific embodiments.
Example 1 acquisition of Gene encoding beta-glucosidase mutant G-2
Aspergillus niger PT003 of a strain of Aspergillus niger that produces beta-glucosidase that this company preserves, through the mutagenic screening of nitrosoguanidine, get a strain with acid-fast beta-glucosidase activity, design PCR primer according to the nucleotide sequence of the wild type beta-glucosidase G-1 encoding gene, 5 'end add restriction enzyme site Xho I,3' end add restriction enzyme site Not I, get the encoding gene of the mutant G-2 through PCR, get its nucleotide sequence as SEQ ID No.3 through sequencing, the corresponding amino acid sequence is SEQ ID No.4.
In the present invention, the mutation site information is obtained by comparing the nucleotide sequences of the wild type and the mutant as shown in the following table.
EXAMPLE 2 construction of recombinant vector pPIC9K-G-2
Respectively carrying out Xho I and Not I enzyme digestion on a mutant G-2 encoding gene (SEQ ID No. 3) and a plasmid pPIC9K, recovering a product, mixing the recovered G-2 encoding gene and the pPIC9K in proportion, connecting the mixture by using T4 ligase at 16 ℃ overnight, transforming Escherichia coli DH5 alpha competent cells by using the connecting product, coating the transformed product on an LB (ampicillin-containing) solid plate, carrying out inverted culture at 33 ℃ overnight, selecting a single colony to an LB liquid culture medium, carrying out culture at 33 ℃, obtaining a target sequence by colony PCR, displaying the target sequence as a nucleotide sequence shown in SEQ ID No.3 by sequencing comparison, namely, the obtained recombinant vector contains a correct mutant gene, and naming the obtained recombinant vector as pPIC9K-G-2.
EXAMPLE 3 transformation of Pichia pastoris with recombinant plasmids
1. Preparation of Pichia pastoris GS115 competent cell
(1) Selecting a single colony of a pichia pastoris plate, inoculating the single colony in 5mL of YPD culture medium, and oscillating overnight at 30 ℃ at 220 r/min;
(2) Inoculating 0.5mL of overnight cultured bacterial liquid into 50mL of freshly prepared YPD medium, shaking-culturing at 30 deg.C and 220r/min to OD 600 The value reaches 1.3 to 1.5;
(3) Centrifuging the culture solution at 4 deg.C and 3000r/min for 5min;
(4) Discarding the supernatant, adding 50mL of ice-precooled sterile water, and oscillating to resuspend the thalli;
(5) Centrifuging at 4 ℃ and 3000r/min for 5min, removing supernatant, sucking residual liquid on the tube wall, adding 25mL of ice-precooled sterile water, and oscillating to resuspend thalli;
(6) Centrifuging at 4 ℃ and 3000r/min for 5min, removing supernatant, sucking residual liquid on the tube wall, adding 10mL of 1mol/L sterile sorbitol solution precooled on ice, and resuspending thalli;
(3) Centrifuging at 4 deg.C and 3000r/min for 5min, discarding supernatant, sucking off residual liquid on tube wall, adding 1mL of 1mol/L sterile sorbitol solution precooled on ice (adding glycerol to final concentration of 15%), shaking and mixing;
(8) 100 μ L/tube into sterile EP jars and stored frozen at-30 deg.C (freshly prepared competent cells are more effective).
2. Transformation of linearized plasmids
The positive clone obtained in example 2 was extracted to obtain recombinant plasmid pPIC9K-G-2, which was digested with Sal I to obtain linearized plasmid. Freshly prepared (or-30 ℃ frozen) GS115 competent cells were placed in an ice bath and allowed to thaw completely.
(1) Removing 100 μ L of competent cells into a new sterile EP tube, adding 10 μ L of linearized plasmid, mixing by gentle blowing, sucking out and transferring into a 0.2 cm-type electroporation transfer cup;
(2) Placing the transformation cup in ice bath for 5-10min, and keeping the temperature at low temperature;
(3) Electroporation transformation shock conditions: 1500V,200 omega, 25 muF, discharge time of about 5ms, once electric shock;
(4) After electric shock, 1mol/L sorbitol solution precooled at 4 ℃ in 1mL is added into an electric shock conversion cup immediately, and is blown and beaten uniformly by a liquid transfer gun and placed in an ice bath;
(5) MD Medium (1.34% YNB; 4X 10) was spread aseptically on a clean bench -5 % biotin; 2% glucose plate), 150. Mu.L/plate, coated platePlate inverted culture at 30 deg.C for 3-4 days;
(6) Two recombinant strains are obtained by screening on an MD plate, a target sequence is obtained by colony PCR, the sequence comparison shows that the target sequence is a nucleotide sequence shown as SEQ ID NO.3, namely the obtained recombinant strains contain correct mutant genes, and the two recombinant strains are respectively named as N-01 and N-02.
Example 4 inducible expression of Yeast containing recombinant plasmid pPIC9K-G-2
BMGY medium formulation: 1.5% yeast extract, 2.5% peptone, 0.1mol/L phosphate buffer pH 6.0, 1.34% YNB, 4X 10 -5 % biotin, 1% glycerol, and the balance water.
BMMY culture medium formula: 1.5% yeast extract, 2.5% peptone, 0.1mol/L phosphate buffer pH 6.0, 1.34% YNB, 4X 10 -5 % biotin, 0.6% methanol, balance water.
The recombinant strain N-01, N-02 and the recombinant strain ND-01 constructed from the wild-type beta-glucosidase encoding gene (nucleotide sequence shown in SEQ ID NO. 1) by the same method as in example 3 were inoculated into a flask containing 30mL of BMGY medium, and cultured at 30 ℃ and 220r/min to OD 600 And (3) centrifuging to collect thalli, re-suspending the thalli by using 35mL of BMMY induction culture medium, continuously culturing for 50 hours at the temperature of 30 ℃ and at the speed of 220r/min, centrifuging fermentation liquor, and measuring the enzyme activity of beta-glucosidase in supernatant, wherein the result is shown in the table below.
| Bacterial strains | Beta-glucosidase enzyme activity (U/mL) |
| ND-01 | 8123 |
| N-01 | 13519 |
| N-02 | 13334 |
Example 5 fermentation Performance verification of recombinant bacterium N-02
The recombinant strain N-02 obtained in example 3 was used as a production strain.
The culture medium formula of the seeding tank comprises: 3.8% glycerol, 1.4% ammonium dihydrogen phosphate, 0.3% potassium dihydrogen phosphate, 0.5% magnesium sulfate, 0.6% potassium sulfate, 0.08% calcium sulfate, 0.4% potassium hydroxide, and the balance water, pH 4.5;
the fermentation tank culture medium formula is as follows: 3.8% glycerol, 1.4% ammonium dihydrogen phosphate, 0.3% potassium dihydrogen phosphate, 0.5% magnesium sulfate, 0.6% potassium sulfate, 0.08% calcium sulfate, 0.4% potassium hydroxide, and the balance water, pH 4.5;
carbon source: 50% glycerol;
methanol: pure methanol;
seed tank culture: the culture temperature is 30 ℃, the initial rotating speed is 200r/min, and the initial air volume is 2m 3 Ventilating, stirring and culturing, wherein the pH value is 4.5, the dissolved oxygen is kept at 30-40%, when the dissolved oxygen is lower than 30%, the rotating speed and the air volume are increased for control, and when the wet weight is increased to 85g/L, transplanting seeds;
culturing in a fermentation tank: the culture temperature is 30 ℃, the initial rotating speed is 200r/min, and the initial air volume is 2m 3 Ventilating and stirring for culture, wherein the inoculation amount is 10%, the pH value is 4.5, in the period of 0-22h, 50% of glycerol serving as a carbon source is added according to the flow rate of 600g/h, the dissolved oxygen is kept at 30-40%, and when the dissolved oxygen is lower than 30%, the rotating speed and the air volume are increased for control, so that the thallus is cultured until the wet weight is 230g/L; after the 22h period, stopping supplementing the carbon source, rebounding the dissolved oxygen to more than 80%, and keeping for 0.5h; then, methanol is added according to the flow rate of 220g/h, dissolved oxygen is kept at 20-30%, and the culture is carried out until the total fermentation period is 160h and the fermentation is finished.
A50L fermentation tank amplification verification experiment is carried out by adopting the fermentation method, the fermentation period is 160h, the fermentation enzyme production conditions of 3 batches are shown in the table below, and the average enzyme production level is 52106U/mL, so that the strain not only can highly produce the acid beta-glucosidase, but also has certain stability in fermentation performance and enzyme activity of the produced beta-glucosidase.
Fermentation enzyme production condition of 3 batches of genetically engineered bacteria
| Batches of | Fermentation period (h) | Fermentation vigor (U/mL) |
| 1 | 160 | 51892 |
| 2 | 160 | 52443 |
| 3 | 160 | 51985 |
Example 6 enzymatic Properties of beta-glucosidase
(1) Optimum pH for action
Taking the supernatant of the N-02 fermentation liquor obtained in the embodiment 4 as a mutated beta-glucosidase sample, taking the supernatant of the ND-01 fermentation liquor as a wild type control sample, adopting the beta-glucosidase activity determination method adopted by the invention, determining the relative enzyme activities under the conditions of different pH values of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0 at the temperature of 50 ℃, and taking the highest enzyme activity of the beta-glucosidase measured by the two enzymes as a reference. As can be seen from FIG. 1, the beta-glucosidase mutant of the invention has stable enzyme activity within the pH range of 3.5-5.0, the optimum pH value is 4.0, and the optimum pH value is reduced by 0.5 compared with the contrast. The results show that compared with the beta-glucosidase before mutation, the beta-glucosidase obtained after mutation has higher enzyme activity under higher acidic condition, has wider pH range of action, and is more suitable for cellulose bioconversion industry.
(2) Optimum temperature of action
Taking the supernatant of the N-02 fermentation broth obtained in the example 4 as a mutated beta-glucosidase sample, taking the supernatant of the ND-01 fermentation broth as a wild type control sample, adopting the beta-glucosidase activity determination method adopted by the invention, determining the enzyme activities at different temperatures of 30 ℃, 35 ℃, 40 ℃, 45 ℃,50 ℃, 55 ℃ and 60 ℃ under the condition of pH 4.0, and calculating the relative enzyme activities by taking the highest enzyme activities of the beta-glucosidase measured by the two enzymes as the reference, wherein the results are shown in figure 2.
(3) Acid resistance
Taking the supernatant of the N-02 fermentation liquor obtained in the example 4 as a mutated beta-glucosidase sample, taking the supernatant of the ND-01 fermentation liquor as a wild type control sample, taking the enzyme activity of the beta-glucosidase which is not treated as a reference of 100%, respectively carrying out heat preservation treatment at 45 ℃ under the condition of pH 3.5 on the two samples, measuring the enzyme activity at the temperature of 50 ℃ and the pH of a buffer solution of 5.0 every 2 hours, and calculating the residual enzyme activity, wherein as can be seen from a graph 3, after 24 hours, the relative activity of the beta-glucosidase mutant still remains more than 80%, and compared with the wild type beta-glucosidase, the acid resistance of the mutant beta-glucosidase is greatly improved, so that the beta-glucosidase mutant has good acid resistance. The results show that compared with the method before mutation, the improvement of acid resistance enables the mutated beta-glucosidase to be more suitable for being applied in the cellulose bioconversion industry.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, various changes, combinations and improvements can be made in the above embodiments without departing from the patent concept, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent shall be subject to the claims.
Claims (10)
1. A beta-glucosidase mutant is characterized in that the beta-glucosidase mutant has an amino acid sequence shown as SEQ ID NO.4.
2. The gene encoding the β -glucosidase mutant of claim 1.
3. The encoding gene of claim 2, wherein the encoding gene has a nucleotide sequence shown in SEQ id No. 3.
4. A recombinant vector or a recombinant strain comprising the coding gene of claim 2.
5. The recombinant vector of claim 4, wherein the expression vector is a pPIC9K plasmid.
6. The recombinant strain of claim 4, wherein the host cell is Pichia pastoris GS115.
7. The recombinant strain of claim 4, wherein the recombinant strain is obtained by connecting a beta-glucosidase mutant encoding gene shown in SEQ ID No.3 with an expression vector pPIC9K and expressing the gene in Pichia pastoris GS115.
8. Use of the recombinant vector or the recombinant strain of claim 4 for producing the β -glucosidase mutant of claim 1.
9. Use of the β -glucosidase mutant of claim 1.
10. The use of a mutant β -glucosidase as described in claim 9, wherein the mutant β -glucosidase is used in catalyzing hydrolysis of glycosidic bonds or in cellulose bioconversion industry.
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