CN107663520A - Lift the cellulase of activity - Google Patents
Lift the cellulase of activity Download PDFInfo
- Publication number
- CN107663520A CN107663520A CN201610614570.9A CN201610614570A CN107663520A CN 107663520 A CN107663520 A CN 107663520A CN 201610614570 A CN201610614570 A CN 201610614570A CN 107663520 A CN107663520 A CN 107663520A
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- CN
- China
- Prior art keywords
- cellulase
- activity
- sequence
- eg1t
- amino acid
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- 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
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- 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)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
Abstract
For the present invention on a kind of cellulase for lifting activity, its amino acid sequence is that the tyrosine of the position of sequence number 2 the 161st is changed to the sequence for substituting modification with histidine.Cellulase provided by the invention can effectively lift activity, therefore can further reduce production cost, and increase its industrial application value.
Description
Technical field
The present invention is on a kind of cellulase, espespecially a kind of cellulase for lifting activity.
Background technology
Cellulose is one of main composition of plant cell wall, and primary biological mass-energy on the earth
(biomass) source, therefore, the zymoproteins for being capable of effective decomposition of cellulose many now are in different industry
On application it is also quite varied.It by glucose is unit that cellulose, which is, with β-Isosorbide-5-Nitrae-glycosidic bond
The long chain polysaccharides that (β-Isosorbide-5-Nitrae-glycosidic bond) is bonded, these polysaccharide bodies, which are organized in concert, to be arranged in
Close crystal type cellulose, and then resist extraneous decomposition.It is however, many herbvore in living nature
Animal and microorganism etc. are needed by resolve into the polysaccharide fiber element in plant cell wall can be by body
Systemic grape monose, to be used as existence energy source.The catalyst mechanism of cellulase mainly by
Acid-base reaction, effect is hydrolyzed in the β-Isosorbide-5-Nitrae-glycosidic bond for connecting two monose, and then it is fine to decompose polysaccharide
Dimension element.And cellulase is substantially divided into three classes, respectively endoglucanase (endo-glucanase),
Exoglucanase (cellobiohydrolase) and glucuroide (β-glucosidase).Endo-glucanase
Long chain cellulose can randomly be cut into the oligosaccharides of many small fragments by enzyme；And exoglucanase then can be from
The reducing end or non-reducing end of long chain cellulose are decomposed, and its primary product is cellobiose；As for Portugal
Polyglycoside enzyme can then be decomposed into cellobiose the glucose of monose.
The application of cellulase industrially is quite varied, whether in food, feed or textile industry, very
Biomass energy of greatest concern till now can extremely be used.For different industrial applications, cellulase also needs
Have and meet its different applicable elements and scope.For example：It is adapted to slant acidity and resistance on feed industry
The zymoprotein of temperature, but be then the cellulase of preference meta-alkalescence in textile industry.Therefore, find out more
It is the target that academic or industrial circle is made great efforts to meet different industrial required zymoproteins.At present in many correlations
Research in, in order to obtain more preferably enzyme, in addition to being screened in nature, being exactly will be existing
Zymoprotein transformed.Mainly there are two big Reconstruc-tion policies now, one is random mutation or by enzyme base
Because of random alignment, under specific action condition, the zymoprotein for more meeting its action condition is filtered out.
This tactful benefit is need not to further investigate the structure or mechanism of action of enzyme, but directly in specific condition
Go down to find out more preferable zymoprotein at random；However, its shortcoming is to need substantial amounts of manpower and time to go
Largely screen or there are good a large amount of screening techniques to coordinate.Another Reconstruc-tion policy be then by
There is critical amino acid for enzymatic activity or characteristic to find out by research enzymatic structure and mechanism of action, and
It is mutated and is tested for these specific amino acids, and then obtains the stronger transformation zymoprotein of feature.
This advantage is to be not required to spend time and manpower mass mutation is with screening the step of, but needs first to understand this enzyme
Protein structure and its mechanism of action, can just find out tool transformation potentiality specific amino acids.
Therefore, the present invention is intended to have critical amino acid for enzymatic activity by research enzymatic structure to find out
And genetic modification is carried out, effectively to lift the activity of cellulase, and then the industry for increasing cellulase should
With value.
The content of the invention
It is an object of the invention to transform existing cellulase, using structural analysis and site-directed mutagenesis technique,
Effectively to lift the activity of cellulase, and then increase the industrial application value of cellulase.
For the above-mentioned purpose, a broader embodiment of the invention is to provide a kind of cellulase, its ammonia
Base acid sequence is that the tyrosine of the position of sequence number 2 the 161st is changed to the sequence for substituting modification with histidine.
In one embodiment, the gene for encoding the sequence number 2 is from straw mushroom (Volvariella volvacea)
It is separated out and optimized eg1t genes.
In one embodiment, the cellulase is an endoglucanase.
In one embodiment, the amino acid sequence of the cellulase is as shown in sequence number 6.
Another broader embodiment of the present invention is divided to provide a kind of nucleic acid for encoding foregoing cellulase
Son.
For the another broader embodiment of the present invention to provide a kind of restructuring plastid, it includes foregoing nucleic acid
Molecule.
Brief description of the drawings
Fig. 1 shows pristine fibre element enzyme eg1t nucleotide sequence and amino acid sequence.
Fig. 2 shows primer sequences used by point mutation technology.
Fig. 3 displays are mutated improved cellulase Y161H nucleotide sequence and amino acid sequence.
Fig. 4 shows shaking flask activity of the original protein eg1t and mutain Y161H in BMMY.
Fig. 5 shows the original protein eg1t and mutain Y161H of same protein amount expression activitiy.
Fig. 6 shows original protein eg1t most suitable action pH.
Fig. 7 shows mutain Y161H most suitable action pH.
Embodiment
Embodying some exemplary embodiments of feature of present invention and advantage will describe in detail in the explanation of back segment.
It should be understood that the present invention can have various changes in different aspects, it does not all depart from the present invention
Scope, and it is therein explanation and accompanying drawing be illustrated as being used in itself, and be not used to limitation the present invention.
The endo glucanase gene Cel5A of trichoderma reesei (Trichoderma reesei) is with high activity
Cellulase, therefore be often studied and restoration and reuse in one of cellulase of textile industry.The present invention
The endoglucanase gene eg 1t of straw mushroom (Volvariella volvacea) is subjected to protein structure emulation
(modeling) zymoprotein simulation architecture, is obtained and then by the Cel5A structures of trichoderma reesei and straw mushroom
Eg1t simulation architectures are compared, and find out similar sequences (consensus residue), and attached in active region
Near pick out may be transformed the influential amino acid of activity.
The method of present invention transformation cellulase and its resulting modified cellulose enzyme are will be described below.
First, by the endoglucanase eg1t amino acid sequences of straw mushroom (Volvariella volvacea) with
SWISS-MODEL softwares carry out structure simulation, after obtaining protein simulation architecture, then with PyMOL
Endoglucanase Cel5A (the PDB ID of software and trichoderma reesei (Trichoderma reesei):3QR3)
Structure alignment (alignment) is carried out, in trichoderma reesei Cel5A zymoprotein structure, near active region
Histidine (Histidine, H) on 150th position and the tryptophan (Tryptophan, W) on the 185th position
There is structure stack (stacking), may have an impact for activity, and in straw mushroom eg1t zymoprotein structures,
Corresponding structure is the tyrosine (Tyrosine, Y) and the tryptophan of the 252nd position of the 161st position
(Tryptophan, W), therefore the tyrosine (Tyrosine, Y) on the 161st position of straw mushroom eg1t is mutated
For histidine (Histidine, H), and mutain (Y161H) is expressed and active testing.
The present invention carries out mutation transformation with point mutation technology.Optimize first and synthesize one section of endo-glucanase
Enzyme eg1t genes, wherein (nucleotide sequence is compiled with sequence as shown in figure 1, eg1t genes include 1002 bases
Number 1 sign) and 333 amino acid (amino acid sequence is indicated with sequence number 2), this section of gene both ends are simultaneously
It is engaged on EcoRI and NotI restriction enzymes position on pPICZA carriers.Fig. 2 is used by point mutation technology
Primer sequences, the tyrosine (Tyrosine, Y) of the position of eg1t genes the 161st is sported into histidine
(Histidine, H), wherein F are forward introduction, and R is reverse introduction, and primer sequences are compiled with sequence respectively
Numbers 3 and 4 signs.Mutated improved cellulase Y161H nucleotide sequence and amino acid sequence is such as
Shown in Fig. 3, wherein, equally comprising 1002 bases, (nucleotide sequence is with sequence for cellulase Y161H genes
Column number 5 indicates) and 333 amino acid (amino acid sequence is indicated with sequence number 6), and the 161st ammonia
Base acid sports histidine (Histidine, H) by tyrosine (Tyrosine, Y).
After the DNA plastids for being mutated transformation are linearized using PmeI restriction enzymes, turned with electricity
(electroporation) mode is sent into yeast Pichia pastoris X33, is then coated onto the bacterium solution after transition
On YPD flat boards containing 100 μ g/ml zeocin antibiotic, it is positioned over 30 DEG C of incubator and carries out two days
Culture.Single bacterium colony is selected afterwards to 5ml YPD in 30 DEG C of cultures, is inoculated in 50ml BMGY
30 DEG C are cultivated one day.Next inducible protein that thalline is shifted to 20ml BMMY progress four days is expressed,
Sample within every 24 hours and add 0.5% methanol, bacterium solution is centrifuged with 3500rpm and collects supernatant, carries out albumen
Measurement and cellulase activity measure.
The active testing mode of cellulase is to take 1% carboxymethyl cellulose (Carboxymethyl
Cellulose, CMC, pH 6.0,0.05M kaliumphosphate buffers) 0.2ml and debita spissitudo cellulase egg
White liquor (dilution buffer be 0.05M kaliumphosphate buffers, pH 6.0) 0.2ml, after being well mixed at 50 DEG C
Effect 15 minutes, it is subsequently added into 1.2ml 1%DNS and 5 minutes is boiled in 100 DEG C of boiling water to stop instead
Should and colour generation, then cool 10 minutes in cold water, in the wavelength of OD 540 measure light absorption value, then is converted into
Unit of enzyme activity (unit).Wherein, the standard curve of enzymatic activity is by glucose standards solution 0-0.35
Formulated between (μ g/m), and 1unit definition is the zymoprotein amount needed for 1 μm of ole product of release per minute.
Fig. 4 shows shaking flask activity of the original protein eg1t and mutain Y161H in BMMY, wherein two
Strain expression yeast strain Y161H-7 and Y161H-20 zymoprotein supernatant are selected to carry out active survey
Examination, as a result finds under the methanol induction of four days, mutain Y161H-7 and Y161H-20 fiber
Plain enzymatic activity is higher than original protein eg1t.
Further adjust mutain Y161H-7 and Y161H-20 and original protein eg1t enzyme liquid concentration one
After cause, Activity determination is carried out at 0.05M kaliumphosphate buffers, pH 6.0 and 50 DEG C, and compare it
Relative activity value.Fig. 5 is the original protein eg1t and mutain Y161H that show same protein amount work
Property compares, and from graphical results, enzymatic activity is determined under identical zymoprotein amount, mutain
Y161H-7 and Y161H-20 cellulase activity is all apparently higher than original protein eg1t.
In addition, the present invention also further determines the Optimun pH of mutain and original protein, it is surveyed
Method for testing is the 0.05M lemons comprising 3.5~pH of pH 6.0 with different buffer solutions by appropriate zymoprotein liquid
0.05M kaliumphosphate buffers (the potassium of acid buffer (citrate buffer), 5.8~pH of pH 8.0
Phosphate buffer, KP buffer), 8.0~pH of pH 9.0 0.05M sodium tetraborates/hydrochloride buffer
(Na-tetraborate/HCl buffer) and 9.0~pH of pH 10 0.05M sodium carbonate buffers
After (Na-carbonate buffer) dilution, reacted 15 minutes at 50 DEG C, 4 DEG C of coolings 10 are placed on after taking-up
Minute places room temperature and risen again 10 minutes again, 50 DEG C of Enzyme assay is carried out again afterwards, with highest enzymatic activity
As 100% control, different pH value percent relative activities are calculated respectively.Fig. 6 shows original protein eg1t
Most suitable action pH, Fig. 7 shows mutain Y161H most suitable action pH, from graphical results,
The most suitable action pH scopes of mutain Y161H and original protein eg1t are constant, all in 5.5~pH of neutral pH
5.8 scope.
In summary, in order to increase the industrial application value of cellulase, the present invention compare straw mushroom eg1t and
Trichoderma reesei Cel5A protein structure, the catastrophe point near active region is filtered out, by straw mushroom eg1t albumen
Tyrosine (Tyrosine, Y) on 161st position sports histidine (Histidine, H), through further
Expression and active testing find, mutain Y161H cellulase activity is apparently higher than original protein
Eg1t, and most suitable action pH scope is constant.Therefore, cellulase mutain Y161H provided by the invention
The activity of cellulase can be effectively lifted, therefore can further reduce production cost, and increases its commercial Application
Value.
Even if the present invention is described and can be appointed by those of ordinary skill in the art and applied in detail by above-described embodiment
Craftsman think and be it is all as modification, it is so neither de- as attached claims are intended to Protector.
Claims (6)
1. a kind of cellulase, its amino acid sequence is to change the tyrosine of the position of sequence number 2 the 161st
With the sequence of histidine substitution modification.
2. cellulase as claimed in claim 1, wherein the gene for encoding the sequence number 2 is from grass
Mushroom (Volvariella volvacea) it is separated out and optimized eg1t genes.
3. cellulase as claimed in claim 1, the wherein cellulase are an endoglucanase.
4. the amino acid sequence of cellulase as claimed in claim 1, the wherein cellulase such as sequence
Shown in numbering 6.
A kind of 5. nucleic acid molecules for encoding cellulase as claimed in claim 1.
6. one kind restructuring plastid, it includes nucleic acid molecules as claimed in claim 5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610614570.9A CN107663520A (en) | 2016-07-29 | 2016-07-29 | Lift the cellulase of activity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610614570.9A CN107663520A (en) | 2016-07-29 | 2016-07-29 | Lift the cellulase of activity |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107663520A true CN107663520A (en) | 2018-02-06 |
Family
ID=61115618
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610614570.9A Pending CN107663520A (en) | 2016-07-29 | 2016-07-29 | Lift the cellulase of activity |
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| CN (1) | CN107663520A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111826367A (en) * | 2019-04-16 | 2020-10-27 | 湖北大学 | Enzyme activity enhanced cellulase |
| CN116970591A (en) * | 2023-09-19 | 2023-10-31 | 中国科学院天津工业生物技术研究所 | Thermophilic endo-cellulase mutant and preparation method thereof |
Citations (3)
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|---|---|---|---|---|
| WO1999010481A2 (en) * | 1997-08-26 | 1999-03-04 | Genencor International, Inc. | Mutant thermomonospora spp. cellulase |
| CN102732540A (en) * | 2011-04-08 | 2012-10-17 | 林忠平 | Positioning mutant gene of Trichoderma viride endoglucanase and application thereof |
| CN105802854A (en) * | 2014-12-30 | 2016-07-27 | 中国科学院上海生命科学研究院 | Cellulase high-yielding bacterial strain and application thereof |
-
2016
- 2016-07-29 CN CN201610614570.9A patent/CN107663520A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999010481A2 (en) * | 1997-08-26 | 1999-03-04 | Genencor International, Inc. | Mutant thermomonospora spp. cellulase |
| CN102732540A (en) * | 2011-04-08 | 2012-10-17 | 林忠平 | Positioning mutant gene of Trichoderma viride endoglucanase and application thereof |
| CN105802854A (en) * | 2014-12-30 | 2016-07-27 | 中国科学院上海生命科学研究院 | Cellulase high-yielding bacterial strain and application thereof |
Non-Patent Citations (4)
| Title |
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| DING,S等: ""endoglucanase[Volvariella volvace]"", 《GENBANK DATABASE》 * |
| JUNJIE YAN等: ""Functional and structural analysis of Pichia pastoris-expressed Aspergillus niger 1,4-β-endoglucanase"", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》 * |
| 张达等: ""草菇糖苷水解酶家族5纤维素酶的定向改造"", 《中国优秀硕士学位论文全文数据库（电子期刊）》 * |
| 王超凯等: ""编码草菇中性内切葡聚糖酶I的基因克隆与表达研究"", 《食品与发酵工业》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111826367A (en) * | 2019-04-16 | 2020-10-27 | 湖北大学 | Enzyme activity enhanced cellulase |
| CN111826367B (en) * | 2019-04-16 | 2022-01-14 | 湖北大学 | Enzyme activity enhanced cellulase |
| CN116970591A (en) * | 2023-09-19 | 2023-10-31 | 中国科学院天津工业生物技术研究所 | Thermophilic endo-cellulase mutant and preparation method thereof |
| CN116970591B (en) * | 2023-09-19 | 2023-12-05 | 中国科学院天津工业生物技术研究所 | Thermophilic endo-cellulase mutant and preparation method thereof |
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