CN111172136A - β -glucosidase with high specific enzyme activity and high thermal stability and preparation method and application thereof - Google Patents

β -glucosidase with high specific enzyme activity and high thermal stability and preparation method and application thereof Download PDF

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CN111172136A
CN111172136A CN201910910909.3A CN201910910909A CN111172136A CN 111172136 A CN111172136 A CN 111172136A CN 201910910909 A CN201910910909 A CN 201910910909A CN 111172136 A CN111172136 A CN 111172136A
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glucosidase
amino acid
acid sequence
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gly
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方诩
牛康乐
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Yantai Huakang Rongzan Biotechnology Co ltd
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    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2445—Beta-glucosidase (3.2.1.21)
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01021—Beta-glucosidase (3.2.1.21)

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Abstract

the invention improves the C terminal of beta-glucosidase polypeptide to make polypeptide self-assemble to form protein polymer, which improves the specific enzyme activity and thermal stability of beta-glucosidase obviously, the half-life time t of beta-glucosidase at 65 deg. C is verified by experiment1/2is 101-125h, is 306.1-378.8 times of β -glucosidase Cel1b before modification, improves the specific enzyme activity at 30 ℃ to 4.33U/mg from 0.05U/mg before modification, and improves the specific enzyme activity at 65 ℃ to 64.46U/mg from 0.02U/mg before modification, thereby being more suitable for industrial production and having good practical application value.

Description

β -glucosidase with high specific enzyme activity and high thermal stability and preparation method and application thereof
Technical Field
the invention belongs to the technical field of bioengineering, and particularly relates to β -glucosidase with high specific enzyme activity and high thermal stability, and a preparation method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
The enzyme is a high-efficiency biocatalyst, is composed of polypeptide, has the advantages of high efficiency, specificity, mild catalysis condition and the like, and therefore has important value in industrial application, such as wide application of glycosidase, lipase and pectinase in the aspects of food, medicine, leather, daily chemical industry and the like, but the thermal stability of the enzyme becomes a key factor limiting the industrial application of the enzyme.
In order to improve the thermal stability of enzyme, researchers have proposed many very important strategies, such as site-directed mutagenesis, random mutagenesis, DNA family recombination, direct evolution, chimera construction, chemical modification and other methods, but they generally have the problems of complicated construction, low success rate, large workload, high cost, long period and the like. In addition, the prior method improves the thermal stability of the enzyme and reduces the specific enzyme activity of the enzyme.
the β -glucosidase is an enzyme which can hydrolyze glycosidic bonds combined with terminal non-reducing residues in β -D-glucoside and oligosaccharide and release β -D-glucose and corresponding ligands at the same time.
Disclosure of Invention
the invention improves the C terminal of beta-glucosidase polypeptide to make polypeptide self-assemble to form protein polymer, which improves the specific enzyme activity and thermal stability of beta-glucosidase obviously, to make it have good practical application value.
in a first aspect of the present invention, there is provided a β -glucosidase having high specific enzyme activity and high thermal stability, which is obtained by modifying β -glucosidase Cel1b (GenBank: AAP57758.1) produced by trichoderma reesei (trichoderma reesei) strain, specifically, by replacing amino acids at positions 479-484 and replacing amino acids at positions 479-484 with IPEELAHLADLKF in the amino acid sequence of β -glucosidase Cel1 b.
preferably, the amino acid sequence of the β -glucosidase is shown as SEQ ID NO. 1. the β -glucosidase of the invention enables polypeptides to self-assemble to form a protein polymer by modifying the C terminal of the original β -glucosidase Cel1b, thereby effectively improving the specific enzyme activity and the thermal stability of the polypeptides.
in a second aspect of the present invention, there is provided a β -glucosidase analog having the same biological activity as the β -glucosidase, wherein the β -glucosidase analog is a polypeptide sequence or protein having biological activity formed by fusing the β -glucosidase with another compound or fusing another polypeptide or protein with an amino acid sequence of the β -glucosidase.
in a third aspect of the present invention, there is provided a β -glucosidase derivative, wherein the amino acid sequence of the derivative has an identity of 70% or more and a similarity of 90% or more with the main amino acid sequence of the β -glucosidase, and the derivative is a β -glucosidase having the same biological activity as the β -glucosidase after a group of one or more amino acids in the amino acid sequence of the β -glucosidase is replaced with another group.
in a fourth aspect of the present invention, there is provided a variant of β -glucosidase having an amino acid sequence with 70% or more identity and 90% or more similarity to the main amino acid sequence of β -glucosidase, wherein the variant is an amino acid sequence having one or more amino acid or nucleotide changes, or a nucleotide sequence encoding the same, wherein the changes comprise deletion, insertion or substitution of amino acids or nucleotides at any position in the middle of the amino acid sequence or nucleotide sequence, or addition of amino acids or nucleotides at both ends of the amino acid sequence or nucleotide sequence, and wherein the variant of β -glucosidase has the same biological activity as the above-mentioned β -glucosidase.
in a fifth aspect of the invention there is provided a nucleotide encoding said β -glucosidase, β -glucosidase analogue, β -glucosidase derivative or β -glucosidase variant, comprising any one of the following group:
a) a nucleotide encoding a polypeptide having the amino acid sequence or an analogue, derivative or variant thereof;
b) a nucleotide complementary to the nucleotide of a);
c) a nucleotide having 75% or more sequence identity to the nucleotide in a) or b).
The nucleotide is prepared by adopting an artificial synthesis method.
in a sixth aspect of the present invention, there is provided a method for producing the above-mentioned β -glucosidase, β -glucosidase analog, β -glucosidase derivative or β -glucosidase variant, which comprises producing the same from a genetically engineered bacterium by genetic recombination.
Further, the preparation method comprises the following steps:
synthesizing a nucleotide encoding the β -glucosidase, β -glucosidase analog, β -glucosidase derivative, or β -glucosidase variant;
introducing the nucleotide into an expression vector to construct a recombinant expression vector, then introducing the recombinant expression vector into a host for culturing and collecting.
in a seventh aspect of the present invention, there is provided a use of the above-mentioned β -glucosidase, β -glucosidase analog, β -glucosidase derivative, or β -glucosidase variant in the fields of food, feed, energy, and bioconversion, particularly in a high temperature environment.
the invention has the beneficial technical effects that the polypeptide is self-assembled to form a protein polymer by modifying the C terminal of β -glucosidase polypeptide, so that the specific activity and the thermal stability of β -glucosidase are obviously improved, and the half-life period t of the modified beta-glucosidase at 65 ℃ is verified by experiments1/2is 101-125h which is 306.1-378.8 times of β -glucosidase Cel1b before modification, the specific enzyme activity at 30 ℃ is improved to 4.33U/mg from 0.05U/mg before modification, and the specific enzyme activity at 65 ℃ is improved to 64.46U/mg from 0.02U/mg before modificationthe beta-glucosidase is more suitable for the industrial production, particularly the requirement of the industrial production in a high-temperature environment, is beneficial to improving the reaction catalysis efficiency and shortening the production time cost, and therefore has good practical application value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a model diagram of the three-dimensional structure of a modified beta-glucosidase in example 1 of the present invention.
FIG. 2 is an SDS-PAGE pattern of β -glucosidase after affinity purification according to example 2 of the present invention.
FIG. 3 is a graph showing the comparison of the optimum catalytic temperatures of β -glucosidase of example 2 of the present invention and wild-type β -glucosidase.
FIG. 4 is a graph comparing the thermostability of the modified β -glucosidase of example 3 of the present invention and wild-type β -glucosidase at 65 degrees.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, 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 application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise. It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. The experimental procedures, if specific conditions are not indicated in the following detailed description, are generally in accordance with conventional procedures and conditions of molecular biology within the skill of the art, which are fully explained in the literature. See, e.g., Sambrook et al, "molecular cloning: the techniques and conditions described in the laboratory Manual, or according to the manufacturer's recommendations.
as described above, specific enzyme activity and thermostability of the existing β -glucosidase still remain to be improved.
in view of the above, in one embodiment of the present invention, there is provided a β -glucosidase having high specific enzyme activity and high thermal stability, which is obtained by performing amino acid substitutions at positions 479 to 484, specifically, at positions 479 to 484 in the amino acid sequence of β -glucosidase Cel1b, to IPEELAHLADLKF.
in another embodiment of the present invention, the amino acid sequence of the β -glucosidase is shown in SEQ ID No. 1. the β -glucosidase self-assembles polypeptides to form a protein multimer by modifying the C-terminus of the original β -glucosidase, thereby effectively improving the specific enzyme activity and thermal stability of the polypeptides.
in yet another embodiment of the present invention, there is provided a β -glucosidase analog having the same biological activity as the β -glucosidase, wherein the β -glucosidase analog is a polypeptide sequence or protein having biological activity formed upon fusion of the β -glucosidase with another compound or fusion of another polypeptide or protein with an amino acid sequence of the β -glucosidase.
in still another embodiment of the present invention, there is provided a β -glucosidase derivative having an amino acid sequence with identity of 70% or more (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (complete) sequence) to the main amino acid sequence of β -glucosidase and similarity of 90% or more (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more), wherein the derivative is a β -glucosidase having the same biological activity as the β -glucosidase after a group of one or more amino acids in the amino acid sequence is substituted with another group.
in yet another embodiment of the present invention, there is provided a variant of β -glucosidase having an amino acid sequence with identity of > 70% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (complete) sequence) to the main amino acid sequence of β -glucosidase, similarity of > 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence or nucleotide sequence encoding it having one or more amino acid or nucleotide changes comprising deletion, insertion or substitution of an amino acid or nucleotide at any position in the middle of the sequence, or addition of an amino acid or nucleotide at both ends of the sequence, said variant having the same biological activity as the β -glucosidase described above.
in yet another embodiment of the present invention, there is provided a nucleotide encoding said β -glucosidase, β -glucosidase analog, β -glucosidase derivative, or β -glucosidase variant, comprising any one of the following group:
(a) a nucleotide encoding a polypeptide having the amino acid sequence or an analogue, derivative or variant thereof;
(b) a nucleotide complementary to the nucleotide of (a);
(c) a nucleotide that is more than or equal to 75% (e.g., 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (complete) sequence) identical to the nucleotide of (a) or (b).
The nucleotide is prepared by adopting an artificial synthesis method.
in still another embodiment of the present invention, there is provided a method for producing the above-mentioned β -glucosidase, β -glucosidase analog, β -glucosidase derivative, or β -glucosidase variant, the method comprising at least:
synthesizing a nucleotide encoding the β -glucosidase, β -glucosidase analog, β -glucosidase derivative, or β -glucosidase variant;
introducing the nucleotide into an expression vector to construct a recombinant expression vector, then introducing the recombinant expression vector into a host for culturing and collecting.
In yet another embodiment of the present invention, the expression vector is any one or more of a viral vector, a plasmid, a phage, a phagemid, a cosmid, an F-cosmid, a phage, or an artificial chromosome; the viral vector may comprise an adenoviral vector, a retroviral vector, or an adeno-associated viral vector, the artificial chromosomes comprising a Bacterial Artificial Chromosome (BAC), a bacteriophage P1 derived vector (PAC), a Yeast Artificial Chromosome (YAC), or a Mammalian Artificial Chromosome (MAC); further preferably a plasmid; even more preferably pET-28a plasmid;
in yet another embodiment of the present invention, the host includes, but is not limited to, bacteria, fungi and eukaryotic cells, further selected from the group consisting of Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Trichoderma reesei, and Penicillium oxalicum; further preferred is Escherichia coli BL21(DE 3).
in yet another embodiment of the present invention, there is provided the use of the above-mentioned β -glucosidase, β -glucosidase analog, β -glucosidase derivative, or β -glucosidase variant in the fields of food, feed, energy, and bioconversion, particularly where the above-mentioned application is a high temperature environment of not less than 30 ℃ (e.g., 35 ℃ -45 ℃, 45 ℃ -55 ℃, or 55 ℃ -65 ℃), and further not less than 65 ℃ (e.g., 65 ℃ -70 ℃, 70 ℃ -75 ℃, 75 ℃ -80 ℃, 80 ℃ -85 ℃, 85 ℃ -90 ℃ or higher), or 65 ℃, 66 ℃, 67 ℃, 68 ℃, 69 ℃, 70 ℃, 71 ℃, 72 ℃, 73 ℃, 74 ℃, 75 ℃, 76 ℃, 77 ℃, 78 ℃, 79 ℃, 80 ℃, 81 ℃, 82 ℃, 83 ℃, 84 ℃, 85 ℃, 86 ℃, 87 ℃, 88 ℃, 89 ℃, 90 ℃).
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The test methods in the following examples, which are not specified under specific conditions, are generally carried out under conventional conditions. pET-28A plasmid vectors were purchased from Novagen.
example 1 thermostability engineering of 1 β -glucosidase Cel1b
the sequence of the Trichoderma reesei β -glucosidase Cel1b is shown in SEQ ID NO. 2.
a) the C-terminal 6 amino acid sequence of β -glucosidase Cel1b was deleted, and exemplary sequences are as follows:
……YFAWALLDNLEWSDGYGPRFGVTFTDYTTLKRTPKKSALVLKDMFAA RQRVKVAA
b) the truncated β -glucosidase Cel1b was ligated at its C-terminus with a sequence of 13 amino acids, designated Cel1b-H13, exemplified by the following:
……YFAWALLDNLEWSDGYGPRFGVTFTDYTTLKRTPKKSALVLKDMFAA RQ IPEELAHLADLKF
c) the three-dimensional structure of the chimeric protein was predicted by the software SWISS-MODEL, as shown in fig. 1.
Example 2
The nucleotide sequences corresponding to the amino acid sequences of Cel1b and Cel1b-H13 were synthesized and ligated to pET-28a plasmid by the TEDA method (Xiaoet al, Nucleic Acids Research,47: e15,2019) to construct recombinant plasmids pET-28a-TrCel1b and pET-28a-TrCel1 b-H13. Introducing the recombinant plasmid into Escherichia coli BL21(DE3), culturing the obtained Escherichia coli containing the recombinant plasmid to OD600When the concentration is 0.6, an IPTG solution with the concentration of 100mM is added, the addition amount of the IPTG solution is one thousandth of the mass of the culture medium, and the induction is carried out for 16 hours at the temperature of 16 ℃. Then, the cells were centrifuged at 7000rpm for 10min, and the cells were collected and washed with 100mM disodium hydrogenphosphate/sodium dihydrogenphosphate (PBS) buffer at pH 7.4washing the thallus twice, purifying β -glucosidase of the chimera with improved heat stability by an ion exchange chromatography mode after the ultrasonication (the result is shown in figure 2), wherein the amino acid sequence is shown in SEQ ID NO.1, adding the purified protein into 100mM PBS buffer solution of 5mM p-nitrophenyl glucoside for reaction, the pH is 7.4, the result of measuring the catalytic activity at 0-90 ℃ is shown in figure 3, and the optimal catalytic temperature of β -glucosidase of the chimera after the modification reaches 75 ℃, which is far higher than that of the wild beta-glucosidase Cel1 b.
Example 3
100 mu l of the target protein (modified β -glucosidase) purified in example 2 and 100mM of wild-type (WT) β -glucosidase were added to 100mM PBS buffer solution containing 5mM of p-nitrophenyl glucoside, the pH was 7.4, the temperature was 65 ℃, after different times of reaction, 150 mu l of 10 WT% sodium bicarbonate solution was used to terminate the reaction, an appropriate volume was taken to measure the OD value at a light wavelength of 420nm, and the hydrolase activity was measured, and the polypeptide concentration was measured using Bradford kit (Shanghai Biotechnology SK 3041-1). the enzyme activity was defined as 1mM pNP produced by conversion per minute as one unit of enzyme activity (U). The results are shown in FIG. 4, the activity of wild-type β -glucosidase was completely lost after incubation at 65 ℃ for 20min, while the activity of β -glucosidase of the modified chimera was completely lost after incubation at 65 ℃ for 150h, and t thereof was completely lost1/2is 101-125h, which is 306.1-378.8 times of wild β -glucosidase Cel1 b.
TABLE 1 comparison of specific enzyme activities of β -glucosidase before and after modification
Specific activity (U/mg) 30℃ 65℃
Cel1b 0.05 0.02
Cel1b-H13 4.33 64.46
It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.
SEQUENCE LISTING
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Lys Val Ala Ala

Claims (10)

1. A β -glucosidase with high specific enzyme activity and high thermal stability, which is characterized in that the β -glucosidase is obtained by replacing 479-484 th amino acids in β -glucosidase Cel1b amino acid sequence, in particular, replacing IPEELAHLADLKF the 479-484 th amino acids.
2. the β -glucosidase with high specific enzyme activity and high thermal stability as claimed in claim 1, wherein the amino acid sequence of the β -glucosidase is shown in SEQ ID NO. 1.
3. a β -glucosidase analog having the same biological activity as the β -glucosidase of claim 1 or 2, wherein the β -glucosidase analog is a polypeptide sequence or protein having biological activity formed upon fusion of the β -glucosidase with another compound or fusion of another polypeptide or protein with an amino acid sequence of the β -glucosidase.
4. A beta-glucosidase derivative, characterized in that its amino acid sequence has 70% or more identity and 90% or more similarity to the main beta-glucosidase amino acid sequence of claim 1 or 2, which is β -glucosidase having the same biological activity as the beta-glucosidase after replacing a group of one or several amino acids in the amino acid sequence with another group.
5. a variant β -glucosidase having an amino acid sequence with 70% identity or more and 90% similarity or more to the main amino acid sequence of the β -glucosidase according to claim 1 or 2, which variant is an amino acid sequence with one or several amino acid or nucleotide changes comprising deletion, insertion or substitution of amino acids or nucleotides at any position in the middle of the sequence or addition of amino acids or nucleotides at both ends of the sequence in the amino acid sequence or nucleotide sequence encoding it, which variant β -glucosidase has the same biological activity as the above-mentioned β -glucosidase.
6. nucleotides encoding the β -glucosidase of claim 1 or 2, the β -glucosidase analogue of claim 3, the β -glucosidase derivative of claim 4 or the β -glucosidase variant of claim 5, preferably comprising any one of the following group:
(a) a nucleotide encoding a polypeptide having the amino acid sequence or an analogue, derivative or variant thereof;
(b) a nucleotide complementary to the nucleotide of (a);
(c) a nucleotide having 75% or more identity to the nucleotide in (a) or (b).
7. A process for producing the β -glucosidase of claim 1 or 2, the β -glucosidase analog of claim 3, the β -glucosidase derivative of claim 4, or the β -glucosidase variant of claim 5, which comprises producing the β -glucosidase from a genetically engineered bacterium by genetic recombination.
8. The method of claim 7, wherein the method comprises at least:
synthesizing a nucleotide encoding the β -glucosidase, β -glucosidase analog, β -glucosidase derivative, or β -glucosidase variant;
introducing the nucleotide into an expression vector to construct a recombinant expression vector, then introducing the recombinant expression vector into a host for culturing and collecting.
9. The method according to claim 7, wherein the expression vector is any one or more of a viral vector, a plasmid, a phage, a phagemid, a cosmid, an F cosmid, a phage, or an artificial chromosome; the expression vector is further preferably a plasmid; even more preferably pET-28a plasmid;
said host comprises bacteria, fungi and eukaryotic cells, further selected from Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Trichoderma reesei and Penicillium oxalicum; further preferred is Escherichia coli BL21(DE 3).
10. use of the β -glucosidase of claim 1 or 2, the β -glucosidase analog of claim 3, the β -glucosidase derivative of claim 4, or the β -glucosidase variant of claim 5 in the field of food, feed, energy, bioconversion;
preferably, the application field is a high-temperature environment; further preferably, the high-temperature environment is not lower than 30 ℃; even more preferably, the high temperature environment is no less than 65 ℃ (most preferably 75 ℃).
CN201910910909.3A 2019-09-25 2019-09-25 β -glucosidase with high specific enzyme activity and high thermal stability and preparation method and application thereof Withdrawn CN111172136A (en)

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Application publication date: 20200519