CN101372693A - Heat resisting cellulase gene, recombinant engineering bacterium, heat resisting cellulase and use - Google Patents

Heat resisting cellulase gene, recombinant engineering bacterium, heat resisting cellulase and use Download PDF

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CN101372693A
CN101372693A CNA2008100509035A CN200810050903A CN101372693A CN 101372693 A CN101372693 A CN 101372693A CN A2008100509035 A CNA2008100509035 A CN A2008100509035A CN 200810050903 A CN200810050903 A CN 200810050903A CN 101372693 A CN101372693 A CN 101372693A
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cellulase
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冯雁
王校楠
王玉国
张作明
郑柏松
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Jilin University
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Jilin University
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Abstract

The invention belongs to the bioengineering field, which particularly relates to a heat-resistant cellulase gene, a recombinant vector, a cellulase gene engineering strain, a heat-resistance cellulase derived from Fervidobacterium and the application thereof. The heat-resistant cellulase (FnCel5A and FnNHCel5A) carries out high-efficiency catalytic reaction under the high temperature condition (70-80 DEG C), the heat-resistant cellulase has very good stability and has the advantages of low storage and transport cost, accelerating kinetic reaction and low requirement standard for the cooling system of a reactor in production. Meanwhile, the FnCel5A and the FnNHCel5A are the heat-resistant cellulase with single endonuclease activity and can be widely applied to the field of cellulose processing. The heat-resistant recombinant cellulase can also be applied to the aspects of detergents, waster water treatment, animal feeds, and the like, or applied to the textile industry, the recycled paper production industry, the industry of extracting active components from plants, the juice processing industry, etc.

Description

Resisting cellulase gene, recombinant bacterial strain, resisting cellulase and application
Technical field
The invention belongs to bioengineering field, be specifically related to derive from resisting cellulase gene, resisting cellulase, the recombinant vectors of flicker Bacillaceae (Fervidobacterium) bacterium, the genetic engineering bacterium of production resisting cellulase, and this resisting cellulase is in the application in fields such as fiber process.
Background technology
Mierocrystalline cellulose is a kind of fibrous, tough and tensile, water-insoluble material, extensively is present in the cell walls of plant.Mierocrystalline cellulose is undoubtedly organic compound the abundantest on the earth.Nature Mierocrystalline cellulose main source has cotton, fiber crops, trees, wild plant or the like, also has the cane that derives from each kind of plant greatly in addition, as straw, straw, sorghum stalk, bagasse or the like.Mierocrystalline cellulose is the main upholder of plant.The same with starch, Mierocrystalline cellulose also is a kind of polysaccharide, and its molecular weight is between 5.0 * 10 4To 4.0 * 10 9Between, roughly be equivalent to 8.0 * 10 3-1.0 * 10 4Individual glucosyl residue.The straight chain that cellulosic molecule is formed by connecting with β (1 → 4) glycosidic link by many β-D-glucose molecule.Parallel to each other between straight chain, very easily form hydrogen bond between the interchain hydroxy group of glucose, make complete Mierocrystalline cellulose have highly water-fast character.
Mierocrystalline cellulose can not be by most of animal digestions, and this is that glucose unit passes through β (1 → 4) glycosidic link and connects and composes because of Mierocrystalline cellulose, and has only the lytic enzyme of hydrolyzing alpha glycosidic link in most of animal body.Some animals can come digest cellulose by the symbiotic microorganism in the digestive tube, and for example ruminating animal (ox, sheep, camel or the like) has a cud structure, and can the eccrine fiber plain enzyme of the microorganism in the cud helps the food of ruminating animal digest cellulose class.
A large amount of cellulases derive from microorganism.Cellulase can be divided into endo cellulase, circumscribed cellulase and beta-glucosidase, and the plain enzyme of three fibrids acts synergistically each other, effectively the hydrolysis natural cellulose.The exploitation and use efficient, cheap cellulase preparation, be the key that Mierocrystalline cellulose is utilized.The cellulase of present industrial use is the product that derives from fungi fermentation mostly, is to play a role under a kind of medium temperature condition, contains the mixing enzyme preparation of the plain enzyme of three fibrids simultaneously, and cellulolytic efficient is good inadequately; Simultaneously, in existing cellulase Application Areas (for example fabric treating, washing, softening, artificial silk processing, paper process etc.), use the plain zymin of traditional mixed fiber, may cause unnecessary damage to the fibrous body structure, be unfavorable for the cellulosic technological process of control hydrolysis, so, one-component or use the cellulase of multiple one-component in proportion, especially use single endo cellulase component, for effective control with improve production technique and have unusual meaning.
Derive from the resisting cellulase of thermophile bacteria, perhaps be called thermophilic cellulase,, paid close attention to by people day by day, become the focus of cellulase fundamental research and development of new cellulase preparation owing to have higher enzyme activity and thermostability.
About Zimadzhunt L 340, be after the Taq archaeal dna polymerase is successfully used to polymerase chain reaction (PCR) from first kind of Zimadzhunt L 340 in 1985, be grown in the positive paid more and more attention of microorganism under the special high thermal environment.Many Zimadzhunt L 340s (thermophilic enzyme, 55-80 ℃) with hydrolytic enzyme activities have obtained exploitation in succession, and have brought into play vital role in a lot of fields.In recent years, people separate from the thermophilic archeobacteria of oceanic heat flow and obtain super Zimadzhunt L 340 (hyperthermophilic enzyme, 80-113 ℃), for modern enzyme engineering technology has represented new application prospect.Zimadzhunt L 340 not only has the incomparable advantage of chemical catalyst, and is strong as catalytic efficiency height and Substratspezifitaet, and the excellent stability of enzyme.Thereby it can overcome in warm enzyme (mesophilic enzyme, 20-55 ℃) and cold-adapted enzyme (psychrophilic enzyme,-2-20 ℃) the unsettled phenomenon of biological property that in application process, usually occurs, thereby a lot of high-temperature chemical reaction processes are achieved, this will greatly promote the development of biotechnology industry, thus the raising of the level of motivating technical transformation of the factory with financial strength and quality of life.
Utilize Zimadzhunt L 340 as biological catalyst following advantage to be arranged: the preparation cost of (1) zymin reduces.Because the stability of Zimadzhunt L 340 is high, thereby can at room temperature separate and purify and packed and transported, and can keep active muchly.(2) accelerated kinetic reaction.Along with the raising of temperature of reaction, molecular motion speeds up, and the enzyme catalysis ability is strengthened.(3) standard that requires to the reactor cooling system reduces, thereby has reduced energy consumption.(4) improved degree of purity of production.Under the Zimadzhunt L 340 catalytic reaction condition (above 70 ℃), assorted bacterium existence is seldom arranged, thereby reduced the pollution of bacterium metabolite product.Because the pyroreaction activity of Zimadzhunt L 340, and to the strong resistance of organic solvent, stain remover and denaturing agent, make its potentiality that all are widely used at aspects such as food, medicine, process hides, oil production and waste treatment.
Separate flicker Bacillaceae (Fervidobacterium) bacterium of natural extreme thermal environments such as self-heating fountain, belong to thermophilic eubacterium, from the flicker Bacillus bacteria, separate the Zimadzhunt L 340 that obtains and have good zymologic property and application potential more.To the genomic analysis of more piece flicker bacillus (Fervidobacterium nodosum), we find may comprise several thermophilic cellulose enzyme genes in the genome of this bacterium.The applied molecular biology technology, we clone and express one of them cellulose enzyme gene, expressing protein has the vigor of very high hydrolyzed carboxymethylcellulo, e sodium, and further enzymology confirms, this albumen is the good heat-resisting endo cellulase of a kind of character.
Because thermophile bacteria artificial culture difficulty, growth cycle is longer, and the content of cellulase is very low, therefore is unfavorable for directly utilizing thermophile bacteria to produce resisting cellulase.The resisting cellulase gene changed over to can breed fast, in the simple normal temperature host of culture condition, can address these problems effectively.
Summary of the invention
One of purpose of the present invention provides a kind of resisting cellulase gene;
Two of purpose of the present invention provides recombinant plasmid and the recombinant bacterial strain that is made up by above-mentioned heat resistance fiber plain gene;
Three of purpose of the present invention provides a kind of strong heat-resisting endo cellulase (FnCel5A) of good stability, thermotolerance that utilizes above-mentioned engineering bacteria preparation;
The 4th purpose of the present invention provides above-mentioned resisting cellulase at the Mierocrystalline cellulose manufacture field and such as the application in industrial or agricultural, medicine and the scientific research.
Main contents of the present invention comprise: the cultivation of thermophilic bacterium (Fervidobacterium nodosum strain Rt17-B1), genomic extraction, angle with PCR method and to get the expression in the host bacterium of goal gene, construction of recombinant plasmid, recombinant plasmid, the fermentation culture of engineering bacteria, results, extraction, purifying, vitality test and the property research of resisting cellulase.
The resisting cellulase that we studied belongs to Zimadzhunt L 340, and its gene comes from thermophilic eubacterium (Fervidobacteriumnodosum strain Rt17-B1).
We will (nucleotide sequence of gene be shown in SEQ ID NO 1 from the resisting cellulase precursor protein gene of thermophilic eubacterium (Fervidobacterium nodosum strain Rt17-B1), by the aminoacid sequence of the resisting cellulase precursor protein of its coding shown in SEQ ID NO 2), (its nucleotide sequence is shown in SEQ ID NO 3 for resisting cellulase mature protein gene behind the removal signal peptide sequence, by the aminoacid sequence of the resisting cellulase maturation protein of its coding shown in SEQ IDNO4) be loaded in pET-11a (Novagen company product, plasmid map is referring to Fig. 2 A, the nucleotide sequence in clone/expression zone is referring to Fig. 2 C) or pET-15b (Novagen company product, plasmid map is referring to Fig. 2 B, the nucleotide sequence in clone/expression zone is referring to Fig. 2 D) on the carrier, obtain two recombinant plasmid pCel5A and pNHCel5A, be transferred to then among intestinal bacteria E.coli strain BL 21-CodonPlus (DE3)-RIL (Stratagene company product) host, obtain two strain engineering bacteria BLFnCel5A and BLFnNHCel5A.These two kinds of engineering bacterias have obtained the resisting cellulase of great expression through fermentation culture, purifying, be respectively resisting cellulase maturation protein FnCel5A (its aminoacid sequence is shown in SEQ ID NO 4) and albumen N-end and be added with the resisting cellulase recombinant protein FnNHCel5A (its aminoacid sequence shown in SEQ IDNO6, the nucleotide sequence of corresponding resisting cellulase recombinant protein gene is shown in SEQ ID NO 5) of Histag.
The feature and intestinal bacteria (Escherichia coli) the BL21 bacterial strain of engineering bacteria involved in the present invention (BLFnCel5A, BLFnNHCel5A bacterial strain) are basic identical, specifically describe as follows:
(1) morphological features: direct rod shape, the thalline size is (1.1～1.5) μ m * 2.0～6.0 μ m, and is single or paired.Gram-negative.Move or do not move with peritrichous, can be referring to figure seven;
(2) cultural characteristic: amphimicrobian has and breathes and the two kinds of metabolic types that ferment.Bacterium colony on nutrient agar medium is smooth, low protruding, moistening, grey, glossy, the full edge in surface, disperses easily in physiological saline;
(3) physiological and biochemical property: chemoorganotrophy; Oxidase negative, acetate can be used as the sole carbon source utilization, and Citrate trianion can not utilize; Glucose and other carbohydrate fermentation produce pyruvic acid, further are being converted into lactic acid, acetate and formic acid, and the formic acid part can be decomposed into the CO of equivalent by hydrogenlyase 2And H 2
Further, the redundant organism, the varient that obtain through gene recombination, sudden change, sequence assembly, means such as chimeric, contain in its gene with the nucleotides sequence shown in SEQ ID NO 1 or SEQ ID NO 3 or the SEQ ID NO 5 and show homology more than 60%, further preferably be at least 80% homology, more preferably be at least 90% homology, even more preferably be at least 95% homology.
Further, derived protein, varient albumen through the acquisition of means such as molecular biology, genetically engineered, aminoacid sequence shown in its aminoacid sequence and SEQ ID NO 2 or SEQ ID NO 4 or the SEQ ID NO 6 has the homology more than 60%, further preferably be at least 80% homology, more preferably be at least 90% homology, even more preferably be at least 95% homology.
Has 93% homology as the resisting cellulase recombinant protein and the resisting cellulase maturation protein of aminoacid sequence shown in SEQ ID NO4 of aminoacid sequence shown in SEQ ID NO 6.
(FnNHCel5A possesses the character identical with FnCel5A for resisting cellulase FnCel5A, FnNHCel5A that we obtained, just vigor is low slightly), can be in hot conditions (70～80 ℃) efficient catalytic reaction down, and enzyme is stable very good, apply in the production, have carrying cost low, accelerate kinetic reaction, to advantages such as the reactor cooling systematic requirement criteria are low.Simultaneously, FnCel5A, FnNHCel5A are the resisting cellulases with single endonuclease activity, have widely at the Mierocrystalline cellulose manufacture field and use.
In addition, this recombinant heat-proof cellulase also all has considerable application potential in a lot of fields.For example: can be applied to the Mierocrystalline cellulose in hydrolyzing plant source, cellulose conversion is become and can perhaps change into valuable chemical products such as alcohol fuel for the carbohydrate of fermentation.Also this recombinant heat-proof cellulase can be applied to aspects such as washing composition, wastewater treatment, animal-feed, perhaps be applied in the industry such as textile industry, recycled writing paper production, effective ingredients in plant extraction, fruit juice processing.
Description of drawings
Fig. 1: PCR product electrophorogram;
Fig. 2: pET-11a and pET-15b plasmid structural representation;
Fig. 3: recombinant plasmid pCel5A building process;
Fig. 4: recombinant plasmid pNHCel5A building process;
Fig. 5: DNA electrophorogram;
Fig. 6: the gene DNA sequencing result of coding FnCel5A;
Fig. 7: engineering bacteria BLFnCel5A thalli morphology;
Fig. 8: resisting cellulase SDS-polyacrylamide gel electrophoresis band;
Fig. 9: the suitableeest catalyzed reaction temperature curve of resisting cellulase FnCel5A;
Figure 10: the suitableeest catalyzed reaction pH value of resisting cellulase FnCel5A curve;
Figure 11: resisting cellulase FnCel5A is at 70 ℃ of following thermostability curves;
Figure 12: resisting cellulase FnCel5A is at 75 ℃ of following thermostability curves;
Figure 13: resisting cellulase FnCel5A is at 80 ℃ of following thermostability curves.
As shown in Figure 1, swimming lane 1 is DL2000 DNA Marker (available from a TaKaRa biotech firm) electrophoresis result, and swimming lane 2 is a FnCel5A maturation protein sequence gene amplified production electrophoresis result;
As shown in Figure 2, wherein A figure is the pET-11a plasmid map; B figure is the pET-15b plasmid map; C figure is the regional nucleotide sequence synoptic diagram of clone/expression in the pET-11a plasmid; D figure is the regional nucleotide sequence synoptic diagram of clone/expression in the pET-15b plasmid.
As shown in Figure 5, swimming lane 1 is represented the electrophoresis result of the gene DNA segment (about 1kb) of linear dna form of plasmid vector pET-11a and coding FnCel5A; Swimming lane 2 is represented the electrophoresis result of the gene DNA segment (about 1kb) of linear dna form of plasmid vector pET-15b and coding FnCel5A; Swimming lane 3 is represented the electrophoresis result of DL2000 DNA Marker (Takara company product), contrasts the together size of electrophoretic DNA of measurement as the spectrum of a standard.
As shown in Figure 6, wherein A is the gene 5 '-rectify to sequencing result of FnCel5A, and B is gene 3 '-end backward sequencing result of FnCel5A;
As shown in Figure 8, swimming lane 1 is represented the supernatant liquor behind the engineering bacteria BLFnCel5A bacterial cell disruption, swimming lane 2 is represented the precipitation after 60 ℃ of 10min of supernatant liquor handle behind the BLFnCel5A bacterial cell disruption, swimming lane 3 is represented the supernatant liquor after 60 ℃ of 10min of swimming lane 1 sample handle, protein band after swimming lane 4 represents FnNHCel5A through the Ni-NTA affinity chromatography, swimming lane 5 represents FnCel5A through the protein band behind the ion exchange chromatography.
Embodiment
Embodiment 1:
1, the cultivation of thermophilic bacterium (Fervidobacterium nodosum strain Rt17-B1)
The source of thermophilic bacterium (more piece flicker bacillus, Fervidobacterium nodosum Rt17-B1): DSM 5306＜ATCC 35602＜B.K.C.Patel, Rt17-B1.Hot spring; New Zealand
Culture condition is DSMZ Medium 144 substratum, and 70 ℃, anaerobism is cultivated, and concrete operations are:
According to the culture medium prescription that DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbHGerman Collection of Microorganisms and Cell Cultures) provides, the preparation bacteria culture medium:
No. 144 substratum of DSMZ: high temperature anaerobic bacteria culture medium
NH 4Cl 0.900g
NaCl 0.900g
MgCl 2·6H 2O 0.400g
KH 2PO 4 0.750g
K 2HPO 4 1.500g
Trace element solution (seeing for details hereinafter) 9.000ml
FeSO 4·7H 2O 3.000mg
Resazurin (Resazurin) 1.000mg
Vitamin solution (seeing for details hereinafter) 5.000ml
Na 2S·9H 2O 1.000g
Yeast extract 3.000g
Protolysate peptone 10.000g
Glucose 5.000g
Add distilled water to 1000.000ml
Substratum disposes in 100% nitrogen, and glucose is sterilized separately, final pH value 7.2～7.4.
Trace element solution:
Nitrilotriacetic acid(NTA) 12.800g
FeCl 2·4H 2O 0.2009
MnCl 2·4H 2O 0.100g
CoCl 2·6H 2O 0.170g
CaCl 2·2H 2O 0.100g
ZnCl 2 0.100g
CuCl 2 0.020g
H 3BO 3 0.010g
Na 2MoO 4·2H 2O 0.010g
NiCl 2·6H 2O 0.026g
NaCl 1.000g
Na 2SeO 3·5H 2O 0.020g
Add distilled water to 1000.0ml
During initial the preparation, dissolve nitrilotriacetic acid(NTA), and be adjusted to pH6.5 with KOH.
Vitamin solution:
Vitamin H (vitamin H) 2.000mg
Folic acid (vitamins B) 2.000mg
Vitamins B 610.000mg
Thiamines (VITMAIN B1) 2H 2O 5.000mg
Riboflavin 5.000mg
Nicotinic acid (nicotinic acid) 5.000mg
D-calcium pantothenate 5.000mg
Vitamins B 120.100mg
P-aminobenzoic acid 5.000mg
Thioctic Acid 5.000mg
Add distilled water to 1000.0ml
Will be from the more piece flicker bacillus (Fervidobacteriumnodosum Rt17-B1 DSMZ numbers DSM 5306) of DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbHGerman Collection of Microorganisms and Cell Cultures), reference literature (Patel BK, et al.Fervidobacteriumnodosum new-genus new-species, a new chemoorganotrophic caldoactive anaerobicbacterium.Arch.Microbiol.141:63-69,1985.) cultivate.Culturing process may be summarized to be:
(1) prescription that provides by DSMZ, preparation DSMZ144 substratum (high temperature anaerobic bacteria culture medium), substratum carries out the high-temperature steam sterilising treatment under 115 ℃, the condition of 15min;
(2) in substratum, add 10%Na by VN=200:1 2S solution;
(3) in substratum, feed N 2(more than the 3min);
(4) measure by 1% the bacterium that connects, insert Fervidobacterium nodosum Rt17-B1 kind daughter bacteria liquid, airtight then;
(5) under 60 ℃ of conditions, cultivate, until reaching the ideal cell density.
2, genomic extraction
With the bacterial cultures of the more piece that obtains above flicker bacillus (Fervidobacterium nodosum Rt17-B1 DSMZ numbers DSM5306) in 10, centrifugal 1 minute of 000rpm, use up supernatant liquor, collect bacterial sediment, (what use in this example is the bacterial genomes DNA extraction test kit that TIANGEN company produces with bacterial genomes DNA extraction test kit, thebacteria genomic DNA purification kit), extract genomic dna according to product description, standby.
3, angling of goal gene got
(1) design of primers
The full gene sequencing engineering of thermophilic bacterium Fervidobacterium nodosum strain Rt17-B1 is finished (Project ID:16719 at US DOE Joint Genome Institute[Complete]), is announcing (Genbank numbers CP00071) on the Internet.By the internet database analysis, in its genome, contain the gene of several sections cellulases of may encoding.We have selected one of them possible cellulose enzyme gene (sequence area is 1698601～1699632, and concrete sequence is referring to sequence table SEQ ID NO 1, and in this patent, we are referred to as resisting cellulase precursor protein gene) as research object.
SEQ ID NO 1 gene order is estimated 343 amino acid of coding, the aminoacid sequence of the resisting cellulase precursor protein of its coding shown in SEQ ID NO 2, by the online Analysis Service of SignaIP 3.0 softwares ( Http:// www.cbs.dtu.dk/services/SignaIP/), 24 amino acid of predicted protein N-end are signal peptide sequences, obtain the resisting cellulase maturation protein after removing this signal peptide sequence, its aminoacid sequence is shown in sequence table SEQ ID NO 4, and the gene order of corresponding this resisting cellulase maturation protein of coding is shown in sequence table SEQ ID NO 3.
By Pfam website data Analysis Service (http://pfam.janelia.org), predict that this fragment gene coding is a cellulase that belongs to glycosyl hydrolase family 5 (glycosyl hydrolase family 5) member.Glycosyl hydrolase enzyme nomenclature according to the Henrissat proposition, we are the maturation protein called after FnCel5A of this enzyme (aminoacid sequence is shown in sequence table SEQ ID NO 4), and the recombinant protein that maturation protein N-end is added with HisTag is named into NHFnCel5A (aminoacid sequence is shown in sequence table SEQ ID NO 6); Cel shows that the suitableeest substrate of the lytic enzyme of this genes encoding is a Mierocrystalline cellulose, " 5 " represent that the lytic enzyme of this genes encoding is to belong to glycosyl hydrolase the 5th family, " A " represent this gene be this experiment from Fervidobacterium nodosum, obtain first belong to the cellulase of glycosyl hydrolase the 5th family, Fn is writing a Chinese character in simplified form of Fervidobacterium nodosum, and NH shows at zymoprotein N-end and is added with the little peptide of HisTag.
According to the sequence characteristic of this gene, and the signal peptide sequence of the expressing protein of sequence correspondence and maturation protein sequence characteristic, we have designed two sections primer (upstream primer CAG CGC that have restriction enzyme site CAT ATGGAC CAA AGTGTG AGT AA, the line part is a Nde I restriction enzyme site; Downstream primer GAC GTC GGA TCCTTA TTT TCCAAG TGC Ag, the line part is a BamH I restriction enzyme site), entrust synthetic this a pair of primer of Dalian TaKaRa biotech firm to be used to angle and get resisting cellulase mature protein gene (aminoacid sequence of the resisting cellulase maturation protein of this genes encoding is referring to SEQ ID NO 4), angle the method that goal gene is taked pcr amplification of getting.
(2) pcr amplification and the purifying of goal gene (SEQ ID NO 3)
In 50 μ l Pfu or TaKaRa Ex Taq reaction system (compound method is referring to the reagent specification sheets), be template with described F.nodosum Rt17-B1 genomic dna above, upstream primer CAG CGC CAT ATGGAC CAAAGT GTGAGT AA and downstream primer GAC GTC GGA TCCTwo primers of TTAT TT TCC AAG TGC Ag are as amplimer, and the method by PCR increases.
PCR reaction conditions: 95 ℃ of pre-sex change 10 minutes; Thermal cycle conditions be 95 ℃ 1 minute, 60 ℃ 1 minute, 72 ℃ 1 minute, 30 circulations are carried out in reaction; Extended 10 minutes 72 ℃ of insulations at last.According to such experimental design, this full length gene by pcr amplification is 960bp, detects the PCR product with 1.0% agarose gel electrophoresis, and the result is shown in swimming lane among Fig. 12.After confirmation obtains the DNA cloning product of goal gene, use PCR product purification test kit (available from the PCR cleaning agents box of Hangzhou Wei Tejie company) that amplified production is carried out the DNA purifying ,-20 ℃ of preservations, standby.
4, construction of recombinant plasmid
(1) expresses the proteic pCel5A construction of recombinant plasmid of ripe resisting cellulase (Fig. 3)
With the resisting cellulase gene product DNA purifying behind the pcr amplification, the DNA behind the purifying carries out endonuclease reaction with restriction enzyme BamH I and Nde I successively, and experimental implementation is with reference to the TaKaRa product description.Enzyme cuts complete, add an amount of load sample liquid (6X sample-loading buffer, 30mM EDTA, 36% glycerine, 0.05% dimethylbenzene green grass or young crops, 0.05% tetrabromophenol sulfonphthalein, mix by 1:5 with nucleic acid solution), electrophoresis on 1.0% sepharose, using dna gel and reclaim the dna fragmentation of test kit (available from Hangzhou Wei Tejie company dna gel recovery test kit) after the recovery enzyme is cut, is the dna segment of coding resisting cellulase gene.
The enzyme that should use the same method is cut the pET-11a plasmid, use alkaline phosphatase (CAP) to handle plasmid then, in 0.8% sepharose, detect linear plasmid, and (use dna gel and reclaim the dna fragmentation of test kit (available from Hangzhou Wei Tejie company dna gel recovery test kit) after the recovery enzyme is cut) plasmid after enzyme is cut of purifying.
PET-11a plasmid after the dna segment of the coding resisting cellulase gene of above-mentioned recovery and enzyme cut mixes, and uses the T4 dna ligases at 16 ℃ and carries out ligation.Plasmid after connecting is changed in the intestinal bacteria XL bacterial strain, on the solid agar plate that contains 50-100mg/L microbiotic penbritin (Amp), be coated with, 37 ℃ of static cultivations are about 12 hours, until the single bacterium colony that positive colony occurs, choose the monoclonal cell bacterium colony, shaking culture is spent the night in being added with the antibiotic liquid nutrient medium of penbritin.With plasmid extraction kit plasmid is proposed, use restriction enzyme Nde I and BamH I hydrolysis plasmid again, verify through agarose electrophoresis then, the result is referring to shown in Figure 5, from figure swimming lane 1 we can see as a result, through behind the double digestion, the gene DNA of former pET-11a plasmid of the digested again one-tenth of recombinant plasmid and coding FnCel5A, coming to the same thing of its result and Fig. 1 swimming lane 2 proves that the plasmid that extracts is our plasmid required, that be connected with resisting cellulase gene fragment and expression vector.The resisting cellulase gene is checked order, and the result proves the recombinant plasmid pCel5A that has obtained containing the resisting cellulase goal gene with consistent according to the result of SEQ ID NO 3 designs.
(2) it is basic identical with top pCel5A building process that expression N-holds pNHCel5A construction of recombinant plasmid (Fig. 4) operation of the resisting cellulase recombinant protein that is added with HisTag peptide section, and the carrier that only is to use has changed pET-15b into by pET-11a.
(3) order-checking
Recombinant plasmid pCel5A and pNHCel5A behind the structure entrust Shanghai to give birth to worker biotech firm and carry out sequencing.When corresponding gene of the present invention was checked order, this used order-checking instrument was ABI PRISM 3730, and sequencing reagent is BigDye terminator v3.1.ABI company to the performance specification of instrument is: under the normal circumstances, except beginning about 10 to 30 bases, at 650bp with the interior accuracy rate that can reach more than 98.5%, generally only carry out positive anti-chain (gene forward and reverse, perhaps claim 5 ' of gene-end and 3 '-end) order-checking simultaneously, under the situation that two sequences fit like a glove, can guarantee that just institute's calling sequence 100% is reliable.
As Fig. 2 C and Fig. 2 D, signal to the regional nucleotide sequence of clone/expression in pET-11a and the pET-15b plasmid, resisting cellulase gene forward is inserted between Nde I and the BamH I restriction enzyme site, 5 ' of gene-end increases, sequencing reaction to inserting can to use primer T7 promoter primer#69348-3,5 '-end to the resisting cellulase gene that inserts carries out the forward order-checking, obtains the forward sequence sequencing result of gene; 3 ' of gene-end increases, sequencing reaction to inserting to use primer T7 terminator primer #69337-3, and 3 '-end of the resisting cellulase gene that inserts is carried out backward sequencing, obtains the reverse sequence sequencing result of gene.Sequencing result as shown in Figure 6, wherein Fig. 6 (A) is from gene 5 '-the rectify result who obtains to order-checking, applied analysis software Chromas 1.62 (Technelysium Pty Ltd, Http:// www.technelysium.com.au/chromas.html) check sequencing result, order-checking spectrogram shown in Fig. 6 (A) is that the startup codon ATG from gene begins, back 3 nucleotide sequences of the sequence C ATATG that the restriction enzyme site Nde I of gene insertion is just discerned begin, and the gene 5 ' that obtains-terminal nucleotide forward sequence as shown in Figure 6A.Fig. 6 (B) is the result from gene 3 '-the end backward sequencing obtains, shown order-checking spectrogram is that the complementary sequence TTA from the terminator codon TAA of gene begins, the nucleotide sequence that obtains is shown in Fig. 6 B, this is the reverse sequence of gene, by sequence analysis software, for example FastPCR Professional (Primer Digital Ltd) according to the nucleotide base principle of complementarity, obtains the forward reading result of gene 3 '-terminal sequence.The result of two-way order-checking is through splicing, and it is consistent with SEQ ID NO 3 sequences to obtain gene order, and SEQID NO 3 sequences are the sequences that are derived from SEQ ID NO 1 sequence and coding FnCel5A maturation protein.Such result has just confirmed two gene orders that recombinant plasmid is contained, can express the maturation protein FnCel5A of SEQ ID NO 4 described aminoacid sequences respectively, and this maturation protein N-end is added with the recombinant protein FnNHCel5A (its aminoacid sequence table is shown in SEQ ID NO 6) of HisTag peptide section.
(4) the proteic similarities and differences of resisting cellulase of recombinant plasmid pCel5A and the coded expression of pNHCel5A.
With resisting cellulase mature protein gene sequence SEQ ID NO 3, be inserted into respectively between the Nde I and two restriction enzyme sites of BamH I in the clone of pET-11a and pET-15b/expression zone (referring to Fig. 2 C, D), constituted recombinant plasmid pCel5A and pNHCel5A.
With reference to clone/expression regional sequence constructional feature of pET-11a and pET-15b, last terminator codon (taa) that the sequence of expressing protein will be first methionine(Met) of rbs back (Met, corresponding codon are atg) in the SEQ ID NO 3.Like this, the pCel5A expressed proteins of can encoding is the FnCel5A maturation protein, and aminoacid sequence is shown in SEQ ID NO 4; And the recombinant protein that pNHCel5A can encode and express is that the N-of the maturation protein FnCel5A of SEQ ID NO 4 codings holds the recombinant protein FnNHCel5A that is added with HisTag peptide section, and its aminoacid sequence is shown in SEQ ID NO 6.
The gene order of FnCel5A and two kinds of resisting cellulases of FnNHCel5A is basic identical, different is for convenience application of nickel affinity chromatography (Ni-NTA Agarose) to recombinant protein separate, purifying, the N-end of FnNHCel5A is added with the HisTag peptide section that affinity interaction is arranged with the nickel affinity chromatography material.The protein sequence of two kinds of resisting cellulases has 93% homology, all is resisting cellulase, and both character is basic identical.Further, according to theoretical prediction, have more than 95%, more than 90%, more than 80% even the albumen of 60% above homology, the character of the cellulase identical or close with FnCel5A arranged also with FnCel5A sequence (SEQ IDNO4).
5, recombinant plasmid is in the host bacteria (expression among the E.colistrain BL 21-CodonPlus (DE3)-RIL)
The recombinant plasmid that will contain goal gene changes in the host bacterium, the first-selected intestinal bacteria (E.coli) of host bacterium, and best selection is E.coli strain BL 21-CodonPlus (DE3)-RIL.Conversion has E.coli strain BL21-CodonPlus (the DE3)-RIL competent cell of goal gene, be coated with screening containing on the solid plate of penbritin, the positive transformed bacteria of picking, place 5ml to contain the liquid nutrient medium of penbritin, the selection of liquid nutrient medium can be 2YT, the LB substratum that often use in the laboratory, and 37 ℃ of shaking culture are spent the night; Next day, measure the saturated bacterium liquid of 0.1ml by 1% inoculation and join in the 10ml fresh liquid substratum, 37 ℃ are continued shaking culture up to A 600It is 0.6～1 o'clock, add isopropylthio β-D-galactoside (IPTG), final concentration is 1mmol/L, 28 ℃ shaking culture 12-16 hour, centrifugal (10,000rpm, 1min) collect thalline, carry out the 15%SDS-polyacrylamide gel electrophoresis, the electrophorogram that obtains has protein band near being presented at the molecules of interest amount, and protein content is very big; Simultaneously, bacterial sediment is resuspended with the distilled water of 5-20 times of volume, behind the ultrasonic disruption, and the bacterial cell disruption liquid (including expressed proteins) that obtains, as substrate, 995 μ l1% (w/v) CMC are placed water-bath preheating 15min with sodium carboxymethyl-cellulose (CMC).Add 5 μ l enzyme liquid (being bacterial cell disruption liquid), with the abundant mixing of substrate, insulation 5min; Immediately sample is put into ice-water bath; 4 ℃ of centrifugal 1min of 12000rpm get 1ml DNS reagent and add 500 μ l reaction solutions; Boiling water bath 5min, cold water is cooled to room temperature.Every pipe adding distil water is diluted to 10ml, reads A 520Absorbance, calculate the glucose concn that produces in the reaction system according to the glucose typical curve.Analyze by the amount of the reducing sugar that generates in the certain hour in the detection reaction system and to express proteic cellulase activity.The growing amount of reducing sugar adopts the DNS method to measure experimental implementation reference literature (Miller GL (1959) Use of dinitrosalicyclic acid reagent for determination ofreducing sugar.Biotechnol Bioeng Symp 5:193-219).The mensuration of zymoprotein concentration adopts the Bradford detection method.Through measuring, two kinds of recombinant bacterial strains can great expression have the albumen of cellulase activity, illustrate and obtained the two kinds of recombined engineering bacteria strains that can express cellulase protein, be respectively the engineering bacteria BLFnCel5A that is loaded with the pCel5A recombinant plasmid in the host bacterium, can express resisting cellulase maturation protein (name and be FnCel5A); Also have the engineering bacteria BLFnNHCel5A that is loaded with the pNHCel5A recombinant plasmid in the host bacterium, can express the recombinant heat-proof cellulase protein (FnNHCel5A) that the N-end is added with HisTag peptide section.
Picking engineering bacteria involved in the present invention a little, place 5ml to contain the liquid nutrient medium of penbritin, the selection of liquid nutrient medium can be 2YT, the LB substratum that often use in the laboratory, also can be industrial fermentation substratum (as malt extract medium, corn steep liquor substratum etc.), 37 ℃ of shaking culture be spent the night; Next day, the inoculum size by 1% joins saturated bacterium liquid in the 100-200ml fresh liquid substratum, and 37 ℃ are continued shaking culture up to A 600Be 0.6～1 o'clock, add isopropylthio β-D-galactoside (IPTG), final concentration is 1mmol/L, 28 ℃ shaking culture 12-16 hour, it is centrifugal that (6,000rpm, 15min) collects thalline by 4 ℃.The resisting cellulase albumen of expressing is present in the somatic cells with the form of soluble protein, and smudge cells can obtain the thick enzyme product of resisting cellulase.
6, the abduction delivering of engineering bacterium fermentation and target protein
The picking engineering bacteria a little, place 50ml to contain the liquid nutrient medium of penbritin, the selection of liquid nutrient medium can be that 2 * YT that often use in the laboratory (prepares every liter of substratum, should add 16g tryptone, 10g yeast extract, 5gNaCl in the 900ml deionized water, use 5molL -1NaOH (about 0.2ml) adjust pH to 7.0 is settled to 1L with deionized water, at 1.05kg/cm 2Steam sterilizing 20min under the high pressure), LB (prepares every liter of substratum, should add 10g tryptone, 5g yeast extract, 10gNaCl in the 950ml deionized water, use 5molL -1NaOH (about 0.2ml) adjust pH to 7.0 is settled to 1L with deionized water, at 1.05kg/cm 2Steam sterilizing 20min under the high pressure) substratum also can be an industrial fermentation substratum (as malt extract medium, corn steep liquor substratum etc.), and 37 ℃ of shaking culture are spent the night, and make seed liquor; The seed liquor of tentatively amplifying is inserted in the liquid nutrient medium of 2L with 1% ratio, and the air shaking table is cultivated under 37 ℃ of 180rpm/min conditions, (A when treating that thalli growth arrives logarithmic phase 600Be 0.6～1) add IPTG and reach 1mM to final concentration, and the temperature that reduces shaking table is 28 ℃, thalline induced make it produce a large amount of target proteins, avoids producing the inclusion body of zymoprotein simultaneously, cultivates and can gather in the crops thalline after 12-16 hour.
7, the expression method of other resisting cellulases
Except expressing the resisting cellulase, also can take the other biological technique means to express with above-mentioned method.
Can it is generally acknowledged that integrating the back dna sequence dna can more stably keep in cell by encoding the proteic gene integration of resisting cellulase to host chromosome.
Host cell is not confined to Bacillus coli cells, the gene of resisting cellulase can be loaded in the host cell of other types yet, expresses cellulose enzyme gene.Host cell can be a higher organism, the cell of animal, plant for example, but be preferably microorganism cells, for example bacterium or fungi (comprising yeast) cell.
Suitably the example of bacterium is: gram positive bacterium, and as Bacillus subtillis, lactobacillus, bacillus licheniformis, bacillus brevis, bacillus stearothermophilus, bacillus alcalophilus, bacillus amyloliquefaciens, bacillus coagulans, bacillus circulans, Bacillus lautus, Bacillus megatherium, Tribactur, shallow Streptomyces glaucoviolaceus or mouse ash streptomycete etc.; Or gram negative bacterium, as colon bacillus, pseudomonas etc.By for example protoplast transformation, or can realize the conversion of bacterium by use experience attitude cell in a manner known way.
Yeast cell can advantageously be selected from the kind of saccharomyces or Schizosaccharomyces, for example yeast saccharomyces cerevisiae; Also can select yeast cell such as pichia spp, candiyeast.
Fungi can be the kind that belongs to Aspergillus, for example aspergillus oryzae or aspergillus niger; Also can be the kind of Trichoderma, for example Trichodermareesei.
Above-mentioned organism or cell, be loaded with the resisting cellulase gene that the present invention relates to after, equally also can express, produce resisting cellulase albumen involved in the present invention; Such organism just becomes the genetic engineering bacterium that can express, produce resisting cellulase, genetically engineered eukaryotic cell, genetically engineered prokaryotic cell prokaryocyte, genetically engineered animal, genetically engineered plants, genetically engineered microorganism etc.
Embodiment 2: the purifying schema of resisting cellulase
(1) acquisition of crude enzyme liquid
With the above-described IPTG abduction delivering of using, cultivate the engineering bacteria nutrient solution 6 after 12-16 hour, centrifugal 15 minutes of 000rpm, bacterial sediment is resuspended with the distilled water of 5-20 times of volume, ultrasonic disruption; In 60 ℃ of heat treated 10-30 minutes, (12,000rpm 5min), collected supernatant and gets crude enzyme liquid for the centrifugal intestinal bacteria foreign protein of removing sex change and cell debris with broken liquid.Counter sample is used SDS-PAGE (15%) protein electrophoresis and is detected, structure is shown in swimming lane 1～3 among accompanying drawing Fig. 8: swimming lane 1 is the supernatant liquor behind the engineering bacteria BLFnCel5A bacterial cell disruption, under the background of e. coli bl21 tropina, with the position of FnCel5A protein with the equimolecular quantity size of Theoretical Calculation on, a very large protein electrophoresis band of protein content is arranged, the target protein FnCel5A of great expression that Here it is; Swimming lane 2 is the precipitations after the supernatant liquor behind the BLFnCel5A bacterial cell disruption is handled through 60 ℃, 10min, can see proteic electrophoresis band in other the most e. coli bl21 thalline except resisting cellulase FnCel5A, this explanation is by after 60 ℃, the thermal treatment of 10min, except resisting cellulase FnCel5A, other most foreign proteins have been removed by thermoprecipitation in the bacterial cell disruption liquid; Swimming lane 3 is after the supernatant liquor behind the engineering bacteria BLFnCel5A bacterial cell disruption is handled through 60 ℃, 10min, remove the supernatant liquor after precipitating partly, can see by thermal treatment, after having removed a large amount of foreign proteins with sedimentary form, target protein is that the content of resisting cellulase FnCel5A is significantly improved, and the proteic purpose of preliminary purification has been played in thermal treatment.
(2) ion exchange chromatography
Be loaded with the engineering bacteria BLFnCel5A of pCel5A plasmid, express the maturation protein (FnCel5A) of resisting cellulase, use ion exchange chromatography (Q Sepharose Fast Flow) to resisting cellulase separate, purifying.Use the proteic purity of SDS-PAGE (15%) electrophoresis detection, the result is shown in swimming lane among Fig. 85, can't see the electrophoresis band of other foreign proteins basically this moment, the electrophoresis band that has only a large amount of resisting cellulase FnCel5A in the electrophorogram, illustrate through the resisting cellulase FnCel5A albumen behind separation, the purifying, very pure, reached electrophoretically pure purity grade.
(2) affinity column chromatography
Be loaded with the engineering bacteria BLFnNHCel5A of pNHCel5A plasmid, can express the recombinant protein (FnNHCel5A) that the N-end contains HisTag, application of nickel affinity chromatography (Ni-NTA Agarose) to recombinant protein separate, purifying.Use the purity of SDS-PAGE (15%) electrophoresis detection recombinant protein, the result is shown in swimming lane among Fig. 84.Can't see the electrophoresis band of other foreign proteins basically this moment, the electrophoresis band that has only a large amount of resisting cellulase FnNHCel5A in the electrophorogram, illustrate through the resisting cellulase FnCel5A albumen behind separation, the purifying, very pure, reached electrophoretically pure purity grade.
Embodiment 3: the character of recombinant heat-proof cellulase
Resisting cellulase FnCel5A and the FnNHCel5A that the present invention relates to have essentially identical character, but also have fine distinction, below mainly the character of resisting cellulase is explained with FnCel5A:
(1) cellulase activity of hydrolyzed carboxymethylcellulo, e (CMC)
With sodium carboxymethyl-cellulose (CMC) as substrate, use resisting cellulase of the present invention to the certain density CMC aqueous solution, under certain physico chemical factor, react, analyze the vigor of cellulase behind the certain hour by the amount of the reducing sugar that generates in the detection reaction system.The growing amount of reducing sugar adopts the DNS method to measure experimental implementation reference literature (Miller GL (1959) Use of dinitrosalicyclic acid reagent for determination of reducing sugar.BiotechnolBioeng Symp 5:193-219).The mensuration of zymoprotein concentration adopts the Bradford detection method.
The definition of enzyme activity unit (U): it is 1U that per minute catalytic decomposition substrate produces the required enzyme amount of 1 μ mol reducing sugar.
The definition of specific activity of enzyme: the enzyme activity (U/mg or U/g) that every mg or every g protein are contained.
The measuring cellulase that arrives involved in the present invention, at high temperature show the vigor (than vigor more than 800U/mg) of very high hydrolysis CMC, the vigor that it has been generally acknowledged that hydrolysis CMC is usually used in characterizing inscribe β-1,4-dextranase (Endo-β-1,4-glucanase, EC 3.2.1.4) vigor is so determine that tentatively enzyme involved in the present invention is heat-resisting endo cellulase.
(2) resisting cellulase optimal reactive temperature
Temperature of reaction is that the important factor that influences the enzyme catalysis vigor, especially thermostable enzyme reaction vigor at high temperature is higher than low temperature far away.With CMC as the enzymic catalytic reaction substrate, we investigated resisting cellulase in the phosphoric acid buffer of pH7.0 differing temps to the influence of enzyme activity.In 37 ℃-95 ℃ temperature range, measure the ratio of resisting cellulase and live, the result as shown in Figure 9, when temperature reached 80 ℃, this enzyme showed maximum catalysis activity.
(3) optimal pH of resisting cellulase
Environment pH value influences the conformation of charged amino acid whose dissociated state and enzyme in the enzyme molecule, and then influences the catalysis activity of enzyme., measuring the ratio of resisting cellulase in the pH4-8 scope and live as substrate with CMC, is X-coordinate with pH, and enzyme is lived and is the ordinate zou mapping relatively, and the result as shown in figure 10.The result shows that the optimal pH of this enzyme is 5.8.
(4) thermostability of resisting cellulase
The enzyme of purifying is dissolved in Citrate trianion-phosphate buffered saline buffer of pH5.8, places 70 ℃, 75 ℃ and 80 ℃ to be incubated certain hour down, measure its residual enzyme activity then, the result is respectively as Figure 11, Figure 12 and shown in Figure 13.The result shows that in the stage that begins to be incubated, enzyme activity all increases, this may because Zimadzhunt L 340 be to use engineering bacteria in, express under the cold condition, improve temperature and help the folding again of Zimadzhunt L 340 after insulation for some time and activate; Resisting cellulase at the vigor of 70 ℃, 75 ℃ insulation losses after 7 days seldom, what vigor reduced in 80 ℃ of insulations 6 hours is few, this thermostability that this enzyme is described is higher.
(5) metal ion is to the influence of enzyme activity
9 kinds of mineral ion (Zn have been tested 2+, Mg 2+, Co 2+, Ni 2+, Ca 2+, Ba 2+, Na +, NH 4 +, Mn 2+) when concentration is respectively 5mmol/L to the influence of enzyme activity, surveying the temperature of living is 80 ℃, the damping fluid of use is pH5.8 Citrate trianion-phosphate buffered saline buffer.As 100%, the vigor of comparing that adds behind the metal ion is listed in the following table 1 with the ratio vigor of the control enzyme liquid that do not add these metal ions, and the result shows Ca 2+, NH 4 +, Mn 2+Resisting cellulase is had slight activation, and other ions enzyme vigor have restraining effect, Zn 2+Enzyme activity there is very significant inhibitory effect.
Table 1. metal ion is to the influence of enzyme activity
Figure A200810050903D00161
Attached: resisting cellulase gene nucleotide series table involved in the present invention and by the resisting cellulase aminoacid sequence table of this genetic expression
(1) SEQ ID NO.1 resisting cellulase precursor protein gene nucleotide series table
＜110〉Jilin University
＜120〉resisting cellulase gene, recombinant bacterial strain, resisting cellulase and application
<160>6
<210>1
<211>1032
<212>DNA
＜213〉eubacterium (Fervidobacterium nodosum)
<220>
<221>CDS
<222>(1)...(1032)
<400>1
ATG?AAG?AAA?AAA?ATA?TTG?AAT?GTT?TTG?CTT?GGT?TTT?GCT?TTA?ATT?TTT
Met?Lys?Lys?Lys?Ile?Leu?Asn?Val?Leu?Leu?Gly?Phe?Ala?Leu?Ile?Phe
1 5 10 15
CTT?ATG?AAT?GGT?AAT?TGC?AAA?GCT?GAC?CAA?AGT?GTG?AGT?AAT?GTT?GAT
Leu?Met?Asn?Gly?Asn?Cys?Lys?Ala?Asp?Gln?Ser?Val?Ser?Asn?Val?Asp
20 25 30
AAA?AGT?AGT?GCT?TTT?GAA?TAT?AAT?AAA?ATG?ATT?GGA?CAT?GGG?ATA?AAC
Lys?Ser?Ser?Ala?Phe?Glu?Tyr?Asn?Lys?Met?Ile?Gly?His?Gly?Ile?Asn
35 40 45
ATG?GGG?AAT?GCT?TTA?GAA?GCC?CCT?GTA?GAA?GGT?TCT?TGG?GGA?GTT?TAT
Met?Gly?Asn?Ala?Leu?Glu?Ala?Pro?Val?Glu?Gly?Ser?Trp?Gly?Val?Tyr
50 55 60
ATT?GAG?GAT?GAA?TAT?TTT?AAA?ATA?ATA?AAA?GAA?AGA?GGC?TTT?GAT?TCT
Ile?Glu?Asp?Glu?Tyr?Phe?Lys?Ile?Ile?Lys?Glu?Arg?Gly?Phe?Asp?Ser
65 70 75 80
GTA?AGG?ATT?CCT?ATC?AGA?TGG?TCT?GCA?CAC?ATT?TCT?GAG?AAG?TAT?CCT
Val?Arg?Ile?Pro?Ile?Arg?Trp?Ser?Ala?His?Ile?Ser?Glu?Lys?Tyr?Pro
85 90 95
TAC?GAA?ATT?GAT?AAA?TTC?TTT?TTA?GAC?AGA?GTG?AAA?CAC?GTT?GTC?GAT
Tyr?Glu?Ile?Asp?Lys?Phe?Phe?Leu?Asp?Arg?Val?Lys?His?Val?Val?Asp
100 105 110
GTC?GCG?TTG?AAG?AAT?GAT?TTG?GTT?GTA?ATA?ATC?AAT?TGT?CAT?CAT?TTC
Val?Ala?Leu?Lys?Asn?Asp?Leu?Val?Val?Ile?Ile?Asn?Cys?His?His?Phe
105 120 125
GAG?GAA?TTA?TAT?CAA?GCC?CCT?GAT?AAA?TAT?GGT?CCT?GTA?TTA?GTT?GAA
Glu?Glu?Leu?Tyr?Gln?Ala?Pro?Asp?Lys?Tyr?Gly?Pro?Val?Leu?Val?Glu
130 135 140
ATT?TGG?AAA?CAA?GTT?GCC?CAA?GCT?TTT?AAA?GAT?TAT?CCA?GAC?AAA?TTG
Ile?Trp?Lys?Gln?Val?Ala?Gln?Ala?Phe?Lys?Asp?Tyr?Pro?Asp?Lys?Leu
145 150 155 160
TTC?TTT?GAA?ATA?TTT?AAC?GAA?CCA?GCT?CAA?AAT?TTG?ACT?CCG?ACT?AAG
Phe?Phe?Glu?Ile?Phe?Asn?Glu?Pro?Ala?Gln?Asn?Leu?Thr?Pro?Thr?Lys
165 170 175
TGG?AAT?GAG?CTT?TAT?CCA?AAA?GTT?TTA?GGT?GAA?ATT?CGA?AAA?ACG?AAT
Trp?Asn?Glu?Leu?Tyr?Pro?Lys?Val?Leu?Gly?Glu?Ile?Arg?Lys?Thr?Asn
180 185 190
CCA?TCA?AGA?ATT?GTA?ATT?ATA?GAC?GTT?CCA?AAT?TGG?TCG?AAC?TAC?AGC
Pro?Ser?Arg?Ile?Val?Ile?Ile?Asp?Val?Pro?Asn?Trp?Ser?Asn?Tyr?Ser
195 200 205
TAC?GTA?AGA?GAG?TTA?AAG?CTT?GTT?GAT?GAT?AAA?AAT?ATA?ATT?GTT?TCA
Tyr?Val?Arg?Glu?Leu?Lys?Leu?Val?Asp?Asp?Lys?Asn?Ile?Ile?Val?Ser
210 215 220
TTC?CAT?TAT?TAC?GAA?CCT?TTT?AAT?TTT?ACT?CAC?CAA?GGT?GCT?GAA?TGG
Phe?His?Tyr?Tyr?Glu?Pro?Phe?Asn?Phe?Thr?His?Gln?Gly?Ala?Glu?Trp
225 230 235 240
GTT?AGT?CCA?ACG?CTT?CCA?ATT?GGC?GTT?AAA?TGG?GAA?GGA?AAA?GAT?TGG
Val?Ser?Pro?Thr?Leu?Pro?Ile?Gly?Val?Lys?Trp?Glu?Gly?Lys?Asp?Trp
245 250 255
GAA?GTG?GAA?CAG?ATT?AGA?AAT?CAT?TTC?AAA?TAT?GTT?AGT?GAG?TGG?GCA
Glu?Val?Glu?Gln?Ile?Arg?Asn?His?Phe?Lys?Tyr?Val?Ser?Glu?Trp?Ala
260 265 270
AAG?AAA?AAC?AAT?GTT?CCG?ATA?TTT?TTA?GGT?GAG?TTT?GGC?GCA?TAT?TCA
Lys?Lys?Asn?Asn?Val?Pro?Ile?Phe?Leu?Gly?Glu?Phe?Gly?Ala?Tyr?Ser
275 280 285
AAA?GCA?GAT?ATG?GAA?TCA?CGG?GTG?AAA?TGG?ACC?AAA?ACT?GTT?AGG?AGA
Lys?Ala?Asp?Met?Glu?Ser?Arg?Val?Lys?Trp?Thr?Lys?Thr?Val?Arg?Arg
290 295 300
ATC?GCT?GAA?GAA?TTC?GGA?TTT?TCG?CTT?GCA?TAT?TGG?GAA?TTT?TGT?GCG
Ile?Ala?Glu?Glu?Phe?Gly?Phe?Ser?Leu?Ala?Tyr?Trp?Glu?Phe?Cys?Ala
305 310 315 320
GGG?TTC?GGG?TTG?TAC?GAT?AGA?TGG?ACA?AAA?ACA?TGG?ATA?GAA?CCT?CTT
Gly?Phe?Gly?Leu?Tyr?Asp?Arg?Trp?Thr?Lys?Thr?Trp?Ile?Glu?Pro?Leu
325 330 335
ACT?ACC?TCT?GCA?CTT?GGA?AAA?TAA(1032)
Thr?Thr?Ser?Ala?Leu?Gly?Lys *
340
(2) SEQ ID NO.2 resisting cellulase precursor protein aminoacid sequence table
<210>2
<211>343
<212>PRT
＜213〉eubacterium (Fervidobacterium nodosum)
<220>
<222>(1)...(343)
<400>2
Met?Lys?Lys?Lys?Ile?Leu?Asn?Val?Leu?Leu?Gly?Phe?Ala?Leu?Ile?Phe
1 5 10 15
Leu?Met?Asn?Gly?Asn?Cys?Lys?Ala?Asp?Gln?Ser?Val?Ser?Asn?Val?Asp
20 25 30
Lys?Ser?Ser?Ala?Phe?Glu?Tyr?Asn?Lys?Met?Ile?Gly?His?Gly?Ile?Asn
35 40 45
Met?Gly?Asn?Ala?Leu?Glu?Ala?Pro?Val?Glu?Gly?Ser?Trp?Gly?Val?Tyr
50 55 60
Ile?Glu?Asp?Glu?Tyr?Phe?Lys?Ile?Ile?Lys?Glu?Arg?Gly?Phe?Asp?Ser
65 70 75 80
Val?Arg?Ile?Pro?Ile?Arg?Trp?Ser?Ala?His?Ile?Ser?Glu?Lys?Tyr?Pro
85 90 95
Tyr?Glu?Ile?Asp?Lys?Phe?Phe?Leu?Asp?Arg?Val?Lys?His?Val?Val?Asp
100 105 110
Val?Ala?Leu?Lys?Asn?Asp?Leu?Val?Val?Ile?Ile?Asn?Cys?His?His?Phe
105 120 125
Glu?Glu?Leu?Tyr?Gln?Ala?Pro?Asp?Lys?Tyr?Gly?Pro?Val?Leu?Val?Glu
130 135 140
Ile?Trp?Lys?Gln?Val?Ala?Gln?Ala?Phe?Lys?Asp?Tyr?Pro?Asp?Lys?Leu
145 50 155 160
Phe?Phe?Glu?Ile?Phe?Asn?Glu?Pro?Ala?Gln?Asn?Leu?Thr?Pro?Thr?Lys
165 170 175
Trp?Asn?Glu?Leu?Tyr?Pro?Lys?Val?Leu?Gly?Glu?Ile?Arg?Lys?Thr?Asn
180 185 190
Pro?Ser?Arg?Ile?Val?Ile?Ile?Asp?Val?Pro?Asn?Trp?Ser?Asn?Tyr?Ser
195 200 205
Tyr?Val?Arg?Glu?Leu?Lys?Leu?Val?Asp?Asp?Lys?Asn?Ile?Ile?Val?Ser
210 215 220
Phe?His?Tyr?Tyr?Glu?Pro?Phe?Asn?Phe?Thr?His?Gln?Gly?Ala?Glu?Trp
225 230 235 240
Val?Ser?Pro?Thr?Leu?Pro?Ile?Gly?Val?Lys?Trp?Glu?Gly?Lys?Asp?Trp
245 250 255
Glu?Val?Glu?Gln?Ile?Arg?Asn?His?Phe?Lys?Tyr?Val?Ser?Glu?Trp?Ala
260 265 270
Lys?Lys?Asn?Asn?Val?Pro?Ile?Phe?Leu?Gly?Glu?Phe?Gly?Ala?Tyr?Ser
275 280 285
Lys?Ala?Asp?Met?Glu?Ser?Arg?Val?Lys?Trp?Thr?Lys?Thr?Val?Arg?Arg
290 295 300
Ile?Ala?Glu?Glu?Phe?Gly?Phe?Ser?Lys?Ala?Tyr?Trp?Glu?Phe?Cys?Ala
305 310 315 320
Gly?Phe?Gly?Leu?Tyr?Asp?Arg?Trp?Thr?Lys?Thr?Trp?Ile?Glu?Pro?Leu
325 330 335
Thr?Thr?Ser?Ala?Leu?Gly?Lys *
340
(3) SEQ ID NO.3 resisting cellulase mature protein gene nucleotides sequence tabulation
<210>3
<211>963
<212>DNA
＜213〉eubacterium (Fervidobacterium nodosum)
<220>
<221>CDS
<222>(1)...(963)
<400>3
ATG?GAC?CAA?AGT?GTG?AGT?AAT?GTT?GAT?AAA?AGT?AGT?GCT?TTT?GAA?TAT
Met?Asp?Gln?Ser?Val?Ser?Asn?Val?Asp?Lys?Ser?Ser?Ala?Phe?Glu?Tyr
1 5 10 15
AAT?AAA?ATG?ATT?GGA?CAT?GGG?ATA?AAC?ATG?GGG?AAT?GCT?TTA?GAA?GCC
Asn?Lys?Met?Ile?Gly?His?Gly?Ile?Asn?Met?Gly?Asn?Ala?Leu?Glu?Ala
20 25 30
CCT?GTA?GAA?GGT?TCT?TGG?GGA?GTT?TAT?ATT?GAG?GAT?GAA?TAT?TTT?AAA
Pro?Val?Glu?Gly?Ser?Trp?Gly?Val?Tyr?Ile?Glu?Asp?Glu?Tyr?Phe?Lys
35 40 45
ATA?ATA?AAA?GAA?AGA?GGC?TTT?GAT?TCT?GTA?AGG?ATT?CCT?ATC?AGA?TGG
Ile?Ile?Lys?Glu?Arg?Gly?Phe?Asp?Ser?Val?Arg?Ile?Pro?Ile?Arg?Trp
50 55 60
TCT?GCA?CAC?ATT?TCT?GAG?AAG?TAT?CCT?TAC?GAA?ATT?GAT?AAA?TTC?TTT
Ser?Ala?His?Ile?Ser?Glu?Lys?Tyr?Pro?Tyr?Glu?Ile?Asp?Lys?Phe?Phe
65 70 75 80
TTA?GAC?AGA?GTG?AAA?CAC?GTT?GTC?GAT?GTC?GCG?TTG?AAG?AAT?GAT?TTG
Leu?Asp?Arg?Val?Lys?His?Val?Val?Asp?Val?Ala?Leu?Lys?Asn?Asp?Leu
85 90 95
GTT?GTA?ATA?ATC?AAT?TGT?CAT?CAT?TTC?GAG?GAA?TTA?TAT?CAA?GCC?CCT
Val?Val?Ile?Ile?Asn?Cys?His?His?Phe?Glu?Glu?Leu?Tyr?Gln?Ala?Pro
100 105 110
GAT?AAA?TAT?GGT?CCT?GTA?TTA?GTT?GAA?ATT?TGG?AAA?CAA?GTT?GCC?CAA
Asp?Lys?Tyr?Gly?Pro?Val?Leu?Val?Glu?Ile?Trp?Lys?Gln?Val?Ala?Gln
105 120 125
GCT?TTT?AAA?GAT?TAT?CCA?GAC?AAA?TTG?TTC?TTT?GAA?ATA?TTT?AAC?GAA
Ala?Phe?Lys?Asp?Tyr?Pro?Asp?Lys?Leu?Phe?Phe?Glu?Ile?Phe?Asn?Glu
130 135 140
CCA?GCT?CAA?AAT?TTG?ACT?CCG?ACT?AAG?TGG?AAT?GAG?CTT?TAT?CCA?AAA
Pro?Ala?Gln?Asn?Leu?Thr?Pro?Thr?Lys?Trp?Asn?Glu?Leu?Tyr?Pro?Lys
145 150 155 160
GTT?TTA?GGT?GAA?ATT?CGA?AAA?ACG?AAT?CCA?TCA?AGA?ATT?GTA?ATT?ATA
Val?Leu?Gly?Glu?Ile?Arg?Lys?Thr?Asn?Pro?Ser?Arg?Ile?Val?Ile?Ile
165 170 175
GAC?GTT?CCA?AAT?TGG?TCG?AAC?TAC?AGC?TAC?GTA?AGA?GAG?TTA?AAG?CTT
Asp?Val?Pro?Asn?Trp?Ser?Asn?Tyr?Ser?Tyr?Val?Arg?Glu?Leu?Lys?Leu
180 185 190
GTT?GAT?GAT?AAA?AAT?ATA?ATT?GTT?TCA?TTC?CAT?TAT?TAC?GAA?CCT?TTT
Val?Asp?Asp?Lys?Asn?Ile?Ile?Val?Ser?Phe?His?Tyr?Tyr?Glu?Pro?Phe
195 200 205
AAT?TTT?ACT?CAC?CAA?GGT?GCT?GAA?TGG?GTT?AGT?CCA?ACG?CTT?CCA?ATT
Asn?Phe?Thr?His?Gln?Gly?Ala?Glu?Trp?Val?Ser?Pro?Thr?Leu?Pro?Ile
210 215 220
GGC?GTT?AAA?TGG?GAA?GGA?AAA?GAT?TGG?GAA?GTG?GAA?CAG?ATT?AGA?AAT
Gly?Val?Lys?Trp?Glu?Gly?Lys?Asp?Trp?Glu?Val?Glu?Gln?Ile?Arg?Asn
225 230 235 240
CAT?TTC?AAA?TAT?GTT?AGT?GAG?TGG?GCA?AAG?AAA?AAC?AAT?GTT?CCG?ATA
His?Phe?Lys?Tyr?Val?Ser?Glu?Trp?Ala?Lys?Lys?Asn?Asn?Val?Pro?Ile
245 250 255
TTT?TTA?GGT?GAG?TTT?GGC?GCA?TAT?TCA?AAA?GCA?GAT?ATG?GAA?TCA?CGG
Phe?Leu?Gly?Glu?Phe?Gly?Ala?Tyr?Ser?Lys?Ala?Asp?Met?Glu?Ser?Arg
260 265 270
GTG?AAA?TGG?ACC?AAA?ACT?GTT?AGG?AGA?ATC?GCT?GAA?GAA?TTC?GGA?TTT
Val?Lys?Trp?Thr?Lys?Thr?Val?Arg?Arg?Ile?Ala?Glu?Glu?Phe?Gly?Phe
275 280 285
TCG?CTT?GCA?TAT?TGG?GAA?TTT?TGT?GCG?GGG?TTC?GGG?TTG?TAC?GAT?AGA
Ser?Leu?Ala?Tyr?Trp?Glu?Phe?Cys?Ala?Gly?Phe?Gly?Leu?Tyr?Asp?Arg
290 295 300
TGG?ACA?AAA?ACA?TGG?ATA?GAA?CCT?CTT?ACT?ACC?TCT?GCA?CTT?GGA?AAA
Trp?Thr?Lys?Thr?Trp?Ile?Glu?Pro?Leu?Thr?Thr?Ser?Ala?Leu?Gly?Lys
305 310 315 320
TAA(963)
*
(4) SEQ ID NO.4 resisting cellulase maturation protein aminoacid sequence table
<210>4
<211>320
<212>PRT
＜213〉eubacterium (Fervidobacterium nodosum)
<220>
<222>(1)...(320)
<400>4
Met?Asp?Gln?Ser?Val?Ser?Asn?Val?Asp?Lys?Ser?Ser?Ala?Phe?Glu?Tyr
1 5 10 15
Asn?Lys?Met?Ile?Gly?His?Gly?Ile?Asn?Met?Gly?Asn?Ala?Leu?Glu?Ala
20 25 30
Pro?Val?Glu?Gly?Ser?Trp?Gly?Val?Tyr?Ile?Glu?Asp?Glu?Tyr?Phe?Lys
35 40 45
Ile?Ile?Lys?Glu?Arg?Gly?Phe?Asp?Ser?Val?Arg?Ile?Pro?Ile?Arg?Trp
50 55 60
Ser?Ala?His?Ile?Ser?Glu?Lys?Tyr?Pro?Tyr?Glu?Ile?Asp?Lys?Phe?Phe
65 70 75 80
Leu?Asp?Arg?Val?Lys?His?Val?Val?Asp?Val?Ala?Leu?Lys?Asn?Asp?Leu
85 90 95
Val?Val?Ile?Ile?Asn?Cys?His?His?Phe?Glu?Glu?Leu?Tyr?Gln?Ala?Pro
100 105 110
Asp?Lys?Tyr?Gly?Pro?Val?Leu?Val?Glu?Ile?Trp?Lys?Gln?Val?Ala?Gln
105 120 125
Ala?Phe?Lys?Asp?Tyr?Pro?Asp?Lys?Leu?Phe?Phe?Glu?Ile?Phe?Asn?Glu
130 135 140
Pro?Ala?Gln?Asn?Leu?Thr?Pro?Thr?Lys?Trp?Asn?Glu?Leu?Tyr?Pro?Lys
145 150 155 160
Val?Leu?Gly?Glu?Ile?Arg?Lys?Thr?Asn?Pro?Ser?Arg?Ile?Val?Ile?Ile
165 170 175
Asp?Val?Pro?Asn?Trp?Ser?Asn?Tyr?Ser?Tyr?Val?Arg?Glu?Leu?Lys?Leu
180 185 190
Val?Asp?Asp?Lys?Asn?Ile?Ile?Val?Ser?Phe?His?Tyr?Tyr?Glu?Pro?Phe
195 200 205
Asn?Phe?Thr?His?Gln?Gly?Ala?Glu?Trp?Val?Ser?Pro?Thr?Leu?Pro?Ile
210 215 220
Gly?Val?Lys?Trp?Glu?Gly?Lys?Asp?Trp?Glu?Val?Glu?Gln?Ile?Arg?Asn
225 230 235 240
His?Phe?Lys?Tyr?Val?Ser?Glu?Trp?Ala?Lys?Lys?Asn?Asn?Val?Pro?Ile
245 250 255
Phe?Leu?Gly?Glu?Phe?Gly?Ala?Tyr?Ser?Lys?Ala?Asp?Met?Glu?Ser?Arg
260 265 270
Val?Lys?Trp?Thr?Lys?Thr?Val?Arg?Arg?Ile?Ala?Glu?Glu?Phe?Gly?Phe
275 280 285
Ser?Lys?Ala?Tyr?Trp?Glu?Phe?Cys?Ala?Gly?Phe?Gly?Leu?Tyr?Asp?Arg
290 295 300
Trp?Thr?Lys?Thr?Trp?Ile?Glu?Pro?Leu?Thr?Thr?Ser?Ala?Leu?Gly?Lys
305 310 315 320
*
(5) SEQ ID NO.5 resisting cellulase recombinant protein gene nucleotide series table
<210>5
<211>1023
<212>DNA
＜213〉eubacterium (Fervidobacterium nodosum)
<220>
<221>CDS
<222>(1)...(1023)
<400>5
ATG?GGC?AGC?AGC?CAT?CAT?CAT?CAT?CAT?CAC?AGC?AGC?GGC?CTG?GTG?CCG
Met?Gly?Ser?Ser?His?His?His?His?His?His?Ser?Ser?Gly?Leu?Val?Pro
1 5 10 15
CGC?GGC?AGC?CAT?ATG?GAC?CAA?AGT?GTG?AGT?AAT?GTT?GAT?AAA?AGT?AGT
Arg?Gly?Ser?His?Met?Asp?Gln?Ser?Val?Ser?Asn?Val?Asp?Lys?Ser?Ser
20 25 30
GCT?TTT?GAA?TAT?AAT?AAA?ATG?ATT?GGA?CAT?GGG?ATA?AAC?ATG?GGG?AAT
Ala?Phe?Glu?Tyr?Asn?Lys?Met?Ile?Gly?His?Gly?Ile?Asn?Met?Gly?Asn
35 40 45
GCT?TTA?GAA?GCC?CCT?GTA?GAA?GGT?TCT?TGG?GGA?GTT?TAT?ATT?GAG?GAT
Ala?Leu?Glu?Ala?Pro?Val?Glu?Gly?Ser?Trp?Gly?Val?Tyr?Ile?Glu?Asp
50 55 60
GAA?TAT?TTT?AAA?ATA?ATA?AAA?GAA?AGA?GGC?TTT?GAT?TCT?GTA?AGG?ATT
Glu?Tyr?Phe?Lys?Ile?Ile?Lys?Glu?Arg?Gly?Phe?Asp?Ser?Val?Arg?Ile
65 70 75 80
CCT?ATC?AGA?TGG?TCT?GCA?CAC?ATT?TCT?GAG?AAG?TAT?CCT?TAC?GAA?ATT
Pro?Ile?Arg?Trp?Ser?Ala?His?Ile?Ser?Glu?Lys?Tyr?Pro?Tyr?Glu?Ile
85 90 95
GAT?AAA?TTC?TTT?TTA?GAC?AGA?GTG?AAA?CAC?GTT?GTC?GAT?GTC?GCG?TTG
Asp?Lys?Phe?Phe?Leu?Asp?Arg?Val?Lys?His?Val?Val?Asp?Val?Ala?Leu
100 105 110
AAG?AAT?GAT?TTG?TT?GTA?ATA?ATC?AAT?TGT?CAT?CAT?TTC?GAG?GAA?TTA
Lys?Asn?Asp?Leu?Val?Val?Ile?Ile?Asn?Cys?His?His?Phe?Glu?Glu?Leu
105 120 125
TAT?CAA?GCC?CCT?GAT?AAA?TAT?GGT?CCT?GTA?TTA?GTT?GAA?ATT?TGG?AAA
Tyr?Gln?Ala?Pro?Asp?Lys?Tyr?Gly?Pro?Val?Leu?Val?Glu?Ile?Trp?Lys
130 135 140
CAA?GTT?GCC?CAA?GCT?TTT?AAA?GAT?TAT?CCA?GAC?AAA?TTG?TTC?TTT?GAA
Gln?Val?Ala?Gln?Ala?Phe?Lys?Asp?Tyr?Pro?Asp?Lys?Leu?Phe?Phe?Glu
145 150 155 160
ATA?TTT?AAC?GAA?CCA?GCT?CAA?AAT?TTG?ACT?CCG?ACT?AAG?TGG?AAT?GAG
Ile?Phe?Asn?Glu?Pro?Ala?Gln?Asn?Leu?Thr?Pro?Thr?Lys?Trp?Asn?Glu
165 170 175
CTT?TAT?CCA?AAA?GTT?TTA?GGT?GAA?ATT?CGA?AAA?ACG?AAT?CCA?TCA?AGA
Leu?Tyr?Pro?Lys?Val?Leu?Gly?Glu?Ile?Arg?Lys?Thr?Asn?Pro?Ser?Arg
180 185 190
ATT?GTA?ATT?ATA?GAC?GTT?CCA?AAT?TGG?TCG?AAC?TAC?AGC?TAC?GTA?AGA
Ile?Val?Ile?Ile?Asp?Val?Pro?Asn?Trp?Ser?Asn?Tyr?Ser?Tyr?Val?Arg
195 200 205
GAG?TTA?AAG?CTT?GTT?GAT?GAT?AAA?AAT?ATA?ATT?GTT?TCA?TTC?CAT?TAT
Glu?Leu?Lys?Leu?Val?Asp?Asp?Lys?Asn?Ile?Ile?Val?Ser?Phe?His?Tyr
210 215 220
TAC?GAA?CCT?TTT?AAT?TTT?ACT?CAC?CAA?GGT?GCT?GAA?TGG?GTT?AGT?CCA
Tyr?Glu?Pro?Phe?Asn?Phe?Thr?His?Gln?Gly?Ala?Glu?Trp?Val?Ser?Pro
225 230 235 240
ACG?CTT?CCA?ATT?GGC?GTT?AAA?TGG?GAA?GGA?AAA?GAT?TGG?GAA?GTG?GAA
Thr?Leu?Pro?Ile?Gly?Val?Lys?Trp?Glu?Gly?Lys?Asp?Trp?Glu?Val?Glu
245 250 255
CAG?ATT?AGA?AAT?CAT?TTC?AAA?TAT?GTT?AGT?GAG?TGG?GCA?AAG?AAA?AAC
Gln?Ile?Arg?Asn?His?Phe?Lys?Tyr?Val?Ser?Glu?Trp?Ala?Lys?Lys?Asn
260 265 270
AAT?GTT?CCG?ATA?TTT?TTA?GGT?GAG?TTT?GGC?GCA?TAT?TCA?AAA?GCA?GAT
Asn?Val?Pro?Ile?Phe?Leu?Gly?Glu?Phe?Gly?Ala?Tyr?Ser?Lys?Ala?Asp
275 280 285
ATG?GAA?TCA?CGG?GTG?AAA?TGG?ACC?AAA?ACT?GTT?AGG?AGA?ATC?GCT?GAA
Met?Glu?Ser?Arg?Val?Lys?Trp?Thr?Lys?Thr?Val?Arg?Arg?Ile?Ala?Glu
290 295 300
GAA?TTC?GGA?TTT?TCG?CTT?GCA?TAT?TGG?GAA?TTT?TGT?GCG?GGG?TTC?GGG
Glu?Phe?Gly?Phe?Ser?Leu?Ala?Tyr?Trp?Glu?Phe?Cys?Ala?Gly?Phe?Gly
305 310 315 320
TTG?TAC?GAT?AGA?TGG?ACA?AAA?ACA?TGG?ATA?GAA?CCT?CTT?ACT?ACC?TCT
Leu?Tyr?Asp?Arg?Trp?Thr?Lys?Thr?Trp?Ile?Glu?Pro?Leu?Thr?Thr?Ser
325 330 335
GCA?CTT?GGA?AAA?TAA(1023)
Ala?Leu?Gly?Lys *
340
(6) SEQ ID NO.6 resisting cellulase recombinant protein aminoacid sequence table
<210>6
<211>340
<212>PRT
＜213〉eubacterium (Fervidobacterium nodosum)
<220>
<221>(1)...(340)
<400>6
Met?Gly?Ser?Ser?His?His?His?His?His?His?Ser?Ser?Gly?Leu?Val?Pro
1 5 10 15
Arg?Gly?Ser?His?Met?Asp?Gln?Ser?Val?Ser?Asn?Val?Asp?Lys?Ser?Ser
20 25 30
Ala?Phe?Glu?Tyr?Asn?Lys?Met?Ile?Gly?His?Gly?Ile?Asn?Met?Gly?Asn
35 40 45
Ala?Leu?Glu?Ala?Pro?Val?Glu?Gly?Ser?Trp?Gly?Val?Tyr?Ile?Glu?Asp
50 55 60
Glu?Tyr?Phe?Lys?Ile?Ile?Lys?Glu?Arg?Gly?Phe?Asp?Ser?Val?Arg?Ile
65 70 75 80
Pro?Ile?Arg?Trp?Ser?Ala?His?Ile?Ser?Glu?Lys?Tyr?Pro?Tyr?Glu?Ile
85 90 95
Asp?Lys?Phe?Phe?Leu?Asp?Arg?Val?Lys?His?Val?Val?Asp?Val?Ala?Leu
100 105 110
Lys?Asn?Asp?Leu?Val?Val?Ile?Ile?Asn?Cys?His?His?Phe?Glu?Glu?Leu
105 120 125
Tyr?Gln?Ala?Pro?Asp?Lys?Tyr?Gly?Pro?Val?Leu?Val?Glu?Ile?Trp?Lys
130 135 140
Gln?Val?Ala?Gln?Ala?Phe?Lys?Asp?Tyr?Pro?Asp?Lys?Leu?Phe?Phe?Glu
145 150 155 160
Ile?Phe?Asn?Glu?Pro?Ala?Gln?Asn?Leu?Thr?Pro?Thr?Lys?Trp?Asn?Glu
165 170 175
Leu?Tyr?Pro?Lys?Val?Leu?Gly?Glu?Ile?Arg?Lys?Thr?Asn?Pro?Ser?Arg
180 185 190
Ile?Val?Ile?Ile?Asp?Val?Pro?Asn?Trp?Ser?Asn?Tyr?Ser?Tyr?Val?Arg
195 200 205
Glu?Leu?Lys?Leu?Val?Asp?Asp?Lys?Asn?Ile?Ile?Val?Ser?Phe?His?Tyr
210 215 220
Tyr?Glu?Pro?Phe?Asn?Phe?Thr?His?Gln?Gly?Ala?Glu?Trp?Val?Ser?Pro
225 230 235 240
Thr?Leu?Pro?Ile?Gly?Val?Lys?Trp?Glu?Gly?Lys?Asp?Trp?Glu?Val?Glu
245 250 255
Gln?Ile?Arg?Asn?His?Phe?Lys?Tyr?Val?Ser?Glu?Trp?Ala?Lys?Lys?Asn
260 265 270
Asn?Val?Pro?Ile?Phe?Leu?Gly?Glu?Phe?Gly?Ala?Tyr?Ser?Lys?Ala?Asp
275 280 285
Met?Glu?Ser?Arg?Val?Lys?Trp?Thr?Lys?Thr?Val?Arg?Arg?Ile?Ala?Glu
290 295 300
Glu?Phe?Gly?Phe?Ser?Lys?Ala?Tyr?Trp?Glu?Phe?Cys?Ala?Gly?Phe?Gly
305 310 315 320
Leu?Tyr?Asp?Arg?Trp?Thr?Lys?Thr?Trp?Ile?Glu?Pro?Leu?Thr?Thr?Ser
325 330 335
Ala?Leu?Gly?Lys?*
340

Claims (12)

1. the resisting cellulase precursor protein gene of a nucleotide sequence shown in SEQ ID NO 1.
2. the resisting cellulase precursor protein of an aminoacid sequence shown in SEQ ID NO 2.
3. the resisting cellulase mature protein gene of a nucleotide sequence shown in SEQ ID NO 3.
4. contain the recombinant plasmid of the resisting cellulase mature protein gene of nucleotide sequence shown in the SEQ ID NO 3, it is characterized in that: be recombinant plasmid pCel5A.
5. contain the resisting cellulase engineering bacteria of the described recombinant plasmid of claim 4, it is characterized in that: be the engineering bacteria of in intestinal bacteria Ecoli, expressing.
6. the resisting cellulase maturation protein of an aminoacid sequence shown in SEQ ID NO 4.
7. contain the recombinant plasmid of the resisting cellulase recombinant protein gene of nucleotide sequence shown in the SEQ ID NO 3, it is characterized in that: be recombinant plasmid pNHCel5A.
8. contain the resisting cellulase engineering bacteria of the described recombinant plasmid of claim 7, it is characterized in that: be the engineering bacteria of in intestinal bacteria E.coli, expressing.
9. resisting cellulase recombinant protein that has 90% above homology with aminoacid sequence shown in the SEQ ID NO 4.
10. the resisting cellulase recombinant protein that has 90% above homology with aminoacid sequence shown in the SEQ ID NO 4 as claimed in claim 9 is characterized in that: be the resisting cellulase recombinant protein for aminoacid sequence shown in SEQ ID NO 6.
11. the described resisting cellulase albumen of claim 6 is in the application of Mierocrystalline cellulose manufacture field.
12. claim 9 or 10 described resisting cellulase recombinant proteins are in the application of Mierocrystalline cellulose manufacture field.
CNA2008100509035A 2008-07-01 2008-07-01 Heat resisting cellulase gene, recombinant engineering bacterium, heat resisting cellulase and use Pending CN101372693A (en)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102168097A (en) * 2010-01-28 2011-08-31 四川贝安迪生物基因工程有限公司 Gene for encoding protein capable of improving heat resistances of plants and microorganisms and application thereof
CN102399767A (en) * 2011-11-04 2012-04-04 中国农业科学院饲料研究所 Cellulose Cel5A with wide pH range as well as gene and application
CN102771630A (en) * 2012-06-07 2012-11-14 宁夏伊品生物科技股份有限公司 Fermentation and coating of lysine and cellulose
CN111826368A (en) * 2020-07-23 2020-10-27 中国农业科学院农产品加工研究所 Mutant enzyme of type I pullulanase and preparation method and application thereof
CN112501148A (en) * 2020-12-14 2021-03-16 黑龙江中医药大学 Cellulase and application thereof
CN116254210A (en) * 2022-10-27 2023-06-13 天津科技大学 Method for producing lactic acid monomer by mixing and fermenting bagasse as raw material

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102168097A (en) * 2010-01-28 2011-08-31 四川贝安迪生物基因工程有限公司 Gene for encoding protein capable of improving heat resistances of plants and microorganisms and application thereof
CN102168097B (en) * 2010-01-28 2014-02-12 四川贝安迪生物基因工程有限公司 Gene for encoding protein capable of improving heat resistances of plants and microorganisms and application thereof
CN102399767A (en) * 2011-11-04 2012-04-04 中国农业科学院饲料研究所 Cellulose Cel5A with wide pH range as well as gene and application
CN102399767B (en) * 2011-11-04 2013-05-01 中国农业科学院饲料研究所 Cellulose Cel5A with wide pH range as well as gene and application
CN102771630A (en) * 2012-06-07 2012-11-14 宁夏伊品生物科技股份有限公司 Fermentation and coating of lysine and cellulose
CN102771630B (en) * 2012-06-07 2013-10-16 宁夏伊品生物科技股份有限公司 Fermentation and coating of lysine and cellulose
CN111826368A (en) * 2020-07-23 2020-10-27 中国农业科学院农产品加工研究所 Mutant enzyme of type I pullulanase and preparation method and application thereof
CN111826368B (en) * 2020-07-23 2021-11-23 中国农业科学院农产品加工研究所 Mutant enzyme of type I pullulanase and preparation method and application thereof
CN112501148A (en) * 2020-12-14 2021-03-16 黑龙江中医药大学 Cellulase and application thereof
CN112501148B (en) * 2020-12-14 2021-09-14 黑龙江中医药大学 Cellulase and application thereof
CN116254210A (en) * 2022-10-27 2023-06-13 天津科技大学 Method for producing lactic acid monomer by mixing and fermenting bagasse as raw material

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