CN1485426A - Novel externally tangent-beta-1,4-glucanase/internally tangent-beta-1,4-xylanase and application thereof - Google Patents
Novel externally tangent-beta-1,4-glucanase/internally tangent-beta-1,4-xylanase and application thereof Download PDFInfo
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- CN1485426A CN1485426A CNA021372837A CN02137283A CN1485426A CN 1485426 A CN1485426 A CN 1485426A CN A021372837 A CNA021372837 A CN A021372837A CN 02137283 A CN02137283 A CN 02137283A CN 1485426 A CN1485426 A CN 1485426A
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Abstract
A new exo-beta-1, 4-glucanase/endo-beta-1, 4-xylanase, polynucleotide encoding the enzyme and a method of preparing the enzyme with DNA recombination technology. The invention also discloses carrier and host cell of the exo-beta-1, 4-glucanase/endo-beta-1, 4-xylanase, polynucleotide, and applications in producing glucose and monose.
Description
Technical field
The present invention relates to biological field.More specifically, the present invention relates to a kind of new circumscribed-β-1,4-dextranase/inscribe-β-1, the polynucleotide of 4-zytase, this enzyme of encoding and produce the method for this kind of enzyme through the DNA recombinant technology.The invention still further relates to and contain this circumscribed-β-1,4-dextranase/inscribe-β-1, the carrier of 4-zytase and host cell and the purposes aspect production simple sugars and glucose thereof.
Background technology
Mierocrystalline cellulose is the abundantest reproducible energy of nature.To change into simple sugars or glucose without any chemically treated Mierocrystalline cellulose biology, produce alcohol, and be the most desirable and effectively utilize one of method of natural cellulose resource as fermenting carbon source.
It has been generally acknowledged that the bio-transformation Mierocrystalline cellulose generates the synergy that glucose needs three kinds of different enzymes at least: (1) endoglucanase (E.C.3.2.1.4 also is referred to as the Mierocrystalline cellulose restriction endonuclease for Endo-1,4-β-D-glucanase).It acts on cellulosic noncrystalline domain, random hydrolysis β-1, the 4-glycosidic link generates the short oligosaccharide of band non-reducing end, (2) dextran excision enzyme (Exo-1,4-β-D-glucanase, E.C.3.2.1.91), it acts on the non-reduced terminal hydrolysis β-1 of cellulosic molecule, and the 4-glycosidic link produces cellobiose, (3) beta-glucosidase (β-D-glucosidase, E.C.3.2.1.21), it is hydrolyzed to glucose with cellobiose.
What at present, research was comparatively concentrated is the cellulase system of fungi and bacterium.The cellulase system of Li Shi wood in the filamentous fungus mould (T.reesei) and viride (T.viride) and species such as Clostridium thermocellum (C.thermocellum) and cellulomonas fimi is complicated, the restriction endonuclease that multiple subclass is arranged, excision enzyme and glucuroide [ThomasM.Wood, Biochemical.SocietyTransactions.1992,20,46-53], for example viride has the restriction endonuclease [Beldman of 6 kinds of hypotypes, G.et al Eur.J.Biochem.1985,146:301-308].The cellulase of Clostridium thermocellum need form the huge corpus fibrosum (Cellulosome) of molecular weight with a plurality of protein could play catalyzed reaction [Felix C.R and L.G.Ljiungdahl, Annu.Rev.Microbiol.1993,47:791-819], obviously complicated like this enzyme system brings great difficulty will inevitably for research and application.On the other hand, the restriction endonuclease of the one-component of purifying and excision enzyme or can not hydrolysis generate simple sugars without chemically treated natural cellulose, the hydrolysis vigor is extremely low, will be without chemically treated vegetable fibre as the synergy ability hydrolysis that needs the plain enzyme of 14 fibrids in the cellulase system of Li Shi wood mould (T.reesei) at least.
Traditional viewpoint thinks that animal does not have the cellulase system of oneself, and the cellulase that needs to rely on fungal component becomes monose with cellulose hydrolysis, needs for vital movement.But termite and small lobsters intrinsic Mierocrystalline cellulose restriction endonuclease [Hirofumi, W.Gaku, T, nathan, L Nature, 1998,394:330-331 have been found the end of the nineties; Keren A.B.et al., Gene, 1999,239,317-324].In addition, 12 kinds of incision enzyme genes [del Compillo, Curr.Top.Dev.Boil.1999,46:39-61] in Arabidopis thaliana, have also been found.The tunicin enzyme may become the focus of new applied research.
Annual on earth by fixation of C O
2Photosynthesis just can form 10,000,000,000 tons with the dried plant material on being, wherein the composition of these materials is over half is by Mierocrystalline cellulose, secondly is hemicellulose (mainly being made of xylan).If add the depleted Mierocrystalline cellulose that mankind's activity causes, as straw, wheat straw etc., its amount then will calculate with astronomical figure.How to adopt on biotechnology comprehensive utilization plant dry matter or the cellulosic problem of depleted, cellulase and zytase (hemicellulase) play keying action.Effectively that these are natural cellulose conversion is that simple sugars is the key of Mierocrystalline cellulose as the renewable energy resources.Present cellulase also far can not adapt to plant cellulose is converted into the demand of simple sugars as the renewable energy resources.Therefore, this area press for having of exploitation different sources can high-level efficiency cellulolytic new cellulase, and the technology of new production glucose.
Summary of the invention
Purpose of the present invention just provides a kind of new circumscribed-β-1,4-dextranase/inscribe-β-1, the 4-zytase with and fragment, analogue and derivative, and encoding sequence.
Another object of the present invention provides produces circumscribed-β-1,4-dextranase/inscribe-β-1, the method for 4-zytase with and uses thereof.
Another object of the present invention provides the new production simple sugars and the technology of glucose.
In a first aspect of the present invention, novel isolated circumscribed-β-1 is provided, 4-dextranase/inscribe-β-1, the 4-zytase, it comprises: polypeptide or its conservative property variation polypeptide or its active fragments or its reactive derivative with SEQ ID NO:2 aminoacid sequence.Preferably, this enzyme is selected from down group: the polypeptide that (a) has SEQ ID NO:2 aminoacid sequence; (b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have the circumscribed and/or xylan inscribe function of dextran by (a) polypeptides derived.
In a second aspect of the present invention, provide the coding isolating circumscribed-β-1,4-dextranase/inscribe-β-1, the polynucleotide of 4-zytase, these polynucleotide comprise a nucleotide sequence, this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group: (a) encode above-mentioned circumscribed-β-1,4-dextranase/inscribe-β-1, the polynucleotide of 4-zytase; (b) with polynucleotide (a) complementary polynucleotide.Preferably, this polynucleotide encoding has the polypeptide of aminoacid sequence shown in the SEQ ID NO:2.More preferably, the sequence of these polynucleotide is be selected from down group a kind of: the sequence with 77-1261 position among the SEQ ID NO:3; Sequence with 1-1293 position among the SEQ IDNO:3.
In a third aspect of the present invention, the carrier that contains above-mentioned polynucleotide is provided, and has been transformed or host cell of transduceing or the host cell that is directly transformed or transduce by above-mentioned polynucleotide by this carrier.
In a fourth aspect of the present invention, circumscribed-β-1 is provided, 4-dextranase/inscribe-β-1, the preparation method of 4-zytase, this method comprises: (a) be fit to express this circumscribed-β-1,4-dextranase/inscribe-β-1 under the condition of 4-zytase, is cultivated the above-mentioned host cell that is transformed or transduce; (b) from culture, isolate this circumscribed-β-1,4-dextranase/inscribe-β-1,4-zytase.
In a fifth aspect of the present invention, provide the present invention circumscribed-β-1,4-dextranase/inscribe-β-1, the purposes of 4-zytase, its encoding sequence, carrier or host cell, they can be used for production simple sugars and glucose, especially utilize to be raw material as plant dry matters such as straw.
In a sixth aspect of the present invention, a kind of technology of new production glucose is provided, has comprised step: (a) with the above-mentioned circumscribed-β-1 of the present invention, 4-dextranase/inscribe-β-1, the host cell of 4-zytase or conversion or transduction is handled cellulose materials, thereby produces simple sugars; (b) isolate described simple sugars.Preferably, described cellulose materials is the cellulose materials without any Chemical Pretreatment.
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 be inscribe-Glu C proteolysis enzymic hydrolysis Fushou spiral shell circumscribed-β-1,4-dextranase/inscribe-β-1, the segmental SDS-polyacrylamide gel electrophoresis of the range of hydrolysed peptides that the 4-zytase is generated (SDS-PAGE) collection of illustrative plates.
Fig. 2 shown change over to Fushou spiral shell circumscribed-β-1,4-dextranase/inscribe-β-1, the host cell of 4-zytase cDNA sequence has the activity of hydrolyzed xylan.
Fig. 3 shown chlorion to Fushou spiral shell circumscribed-β-1,4-dextranase/inscribe-β-1, the active influence of 4-zytase [circumscribed-β-1,4-dextranase].
Embodiment
The inventor is through extensive and deep research, from from Fushou spiral shell (AmpullariumCrossean), obtaining effectively hydrolysis does not produce simple sugars through any chemically treated straw cellulase system, Mierocrystalline cellulose excision enzyme and restriction endonuclease and beta-glucosidase have been isolated first.Wherein, a kind of isolating Fushou spiral shell Mierocrystalline cellulose excision enzyme also has polyxylose lytic enzyme (hemicellulase) vigor, promptly be the bifunctional enzyme that a kind of enzyme has two kinds of enzyme activities, according to the principle that enzyme classification is learned, this enzyme should belong to the 10th glycoside hydrolysis enzyme family (family 10).Therefore can claim that this enzyme is circumscribed-β-1,4-dextranase/inscribe-β-1,4-zytase (exo-β-1,4-glucanase/endo-β-1,4-xylanase), the vigor of the excision enzyme of this enzyme is not suppressed by the high density product, and this bifunctional enzyme of SDS-PAGE homogeneous can generate simple sugars by the hydrolysis straw, also having the activity of height ratio hemicellulase alive simultaneously, is to find one of hemicellulase that vigor is the highest at present.Finished the present invention on this basis.
In the present invention, term " Fushou spiral shell Mierocrystalline cellulose excision enzyme " " Fushou spiral shell dextran excision enzyme " " Fushou spiral shell circumscribed-1; 4-callose excision enzyme ", or " circumscribed-β-1; 4-dextranase/inscribe-β-1, the 4-zytase " be used interchangeably, and all refers to have circumscribed-β-1,4-dextranase/inscribe-β-1, the albumen or the polypeptide of 4-zytase aminoacid sequence (SEQ ID NO:2).They comprise the circumscribed-β-1 that contains or do not contain initial methionine, 4-dextranase/inscribe-β-1,4-zytase.Here inscribe-β-1, the 4-zytase also can be referred to as hemicellulase.
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
As used herein, " isolating circumscribed-β-1,4-dextranase/inscribe-β-1,4-zytase albumen or polypeptide " be meant circumscribed-β-1,4-dextranase/inscribe-β-1,4-zytase are substantially free of natural relative other albumen, lipid, carbohydrate or other material.Those skilled in the art can use this circumscribed-β-1 of purified technology of protein purifying of standard, 4-dextranase/inscribe-β-1,4-zytase.Basically pure polypeptide can produce single band on non-reduced polyacrylamide gel.
Of the present invention circumscribed-β-1,4-dextranase/inscribe-β-1, the 4-zytase can be the reorganization, natural, synthetic, preferably the reorganization.Of the present invention circumscribed-β-1,4-dextranase/inscribe-β-1,4-zytase can be the products of natural purifying, or the product of chemosynthesis, or use recombinant technology from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell), to produce.The host used according to the recombinant production scheme, polypeptide of the present invention can be glycosylated, maybe can be nonglycosylated.Polypeptide of the present invention also can comprise or not comprise initial methionine residues.
The present invention also comprises this circumscribed-β-1,4-dextranase/inscribe-β-1, the fragment of 4-zytase, derivative and analogue.As used herein, term " fragment ", " derivative " and " analogue " be meant keep basically of the present invention natural circumscribed-β-1,4-dextranase/inscribe-β-1, the biological function that the 4-zytase is identical or active polypeptide.Polypeptide fragment of the present invention, derivative or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue), and the amino-acid residue of such replacement can be also can not encoded by genetic code, or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical, or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period, polyoxyethylene glycol for example) merge formed polypeptide, or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (as leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying).According to the instruction of this paper, these fragments, derivative and analogue belong to the known scope of those skilled in the art.
In the present invention, and term " Fushou spiral shell is circumscribed-β-1, and 4-dextranase/inscribe-β-1,4-zytase " refer to have circumscribed-β-1,4-dextranase/inscribe-β-1, the SEQ ID NO:2 polypeptide of sequence of 4-xylanase activity.This term also comprises having circumscribed-β-1,4-dextranase/inscribe-β-1, the variant form 4-zytase, SEQ IDNO:2 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprise Fushou spiral shell circumscribed-β-1,4-dextranase/inscribe-β-1, the active fragments of 4-zytase and reactive derivative.
The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, can-β circumscribed with Fushou spiral shell-1 under high or low tight degree condition, 4-dextranase/inscribe-β-1, the coded albumen of DNA of 4-xylan enzyme dna hybridization.The present invention also provides other polypeptide, as comprise Fushou spiral shell circumscribed-β-1,4-dextranase/inscribe-β-1,4-zytase or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also comprised Fushou spiral shell circumscribed-β-1,4-dextranase/inscribe-β-1, the soluble fragments of 4-zytase.Usually, this fragment has Fushou spiral shell circumscribed-β-1,4-dextranase/inscribe-β-1,4-zytase sequence at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.
The invention also provide Fushou spiral shell circumscribed-β-1,4-dextranase/inscribe-β-1, the analogue of 4-zytase or polypeptide.These analogues and natural Fushou spiral shell be circumscribed-β-1, and 4-dextranase/inscribe-β-1, the difference of 4-zytase can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps have both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.
(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
In the present invention, " Fushou spiral shell is circumscribed-β-1; and 4-dextranase/inscribe-β-1; the conservative property variation polypeptide of 4-zytase " refer to compare with the aminoacid sequence of SEQ ID NO:2, there are 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to Table A and produce.
Table A
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
The coding Fushou spiral shell circumscribed-β-1,4-dextranase/inscribe-β-1, the polynucleotide of 4-zytase mature polypeptide comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide+various additional code sequences; The encoding sequence of mature polypeptide (with optional additional code sequence)+non-coding sequence.
Term " coding Fushou spiral shell circumscribed-β-1; 4-dextranase/inscribe-β-1; polynucleotide of 4-zytase " can be comprise the Fushou spiral shell of only encoding circumscribed-β-1,4-dextranase/inscribe-β-1, the polynucleotide of 4-zytase also can be the polynucleotide that also comprise additional code and/or non-coding sequence.
The invention still further relates to the varient of above-mentioned polynucleotide, its coding has the polypeptide of identical aminoacid sequence or fragment, analogue and the derivative of polypeptide with the present invention.The varient of these polynucleotide can be the allelic variant of natural generation or the varient that non-natural takes place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of its encoded polypeptides in fact.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide of the present invention.In the present invention, " stringent condition " is meant: (1) than hybridization under low ionic strength and the comparatively high temps and wash-out, as 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, as 50% (v/v) methane amide, 0.1% calf serum/0.1%Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has circumscribed-β-1, and 4-dextranase activity and inscribe-β-1, the 4-xylanase activity.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably more than at least 100 Nucleotide.The amplification technique (as PCR) that nucleic acid fragment can be used for nucleic acid with determine and/or separate the coding Fushou spiral shell circumscribed-β-1,4-dextranase/inscribe-β-1, the polynucleotide of 4-zytase.
Fushou spiral shell of the present invention is circumscribed-β-1, and 4-dextranase/inscribe-β-1,4-zytase Nucleotide full length sequence or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention relevant nucleotide sequence, especially open reading frame sequence designs primer, and with prepared according to a conventional method Fushou spiral shell cDNA storehouse as template, amplification and must be about sequence.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention by chemosynthesis.
The present invention also relates to comprise the carrier of polynucleotide of the present invention, and with carrier of the present invention or circumscribed-β-1,4-dextranase/inscribe-β-1,4-zytase encoding sequence transformed host cells, and the method that produces polypeptide of the present invention through recombinant technology.
Recombinant DNA technology (Science, 1984 by routine; 224:1431), can utilize polymerized nucleoside acid sequence of the present invention to can be used to express or produce the circumscribed-β-1 of reorganization, 4-dextranase/inscribe-β-1,4-zytase.In general following steps are arranged:
(1). with coding of the present invention circumscribed-β-1,4-dextranase/inscribe-β-1, polynucleotide of 4-zytase (or varient), or transform or the transduction proper host cell with the recombinant expression vector that contains these polynucleotide;
(2). the host cell of in suitable medium, cultivating;
(3). separation, protein purification from substratum or cell.
Among the present invention, circumscribed-β-1,4-dextranase/inscribe-β-1,4-zytase polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to bacterial plasmid well known in the art, phage, yeast plasmid or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: and the expression vector based on T7 of in bacterium, expressing (Rosenberg, et al.Gene, 1987,56:125).In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used for making up and contains circumscribed-β-1,4-dextranase/inscribe-β-1,4-zytase DNA sequences encoding and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promotor; Lambda particles phage PL promotor; Eukaryotic promoter comprises LTRs and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus of CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor, retrovirus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate the Tetrahydrofolate dehydrogenase and the neomycin resistance of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin, kantlex or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells.Be preferably intestinal bacteria.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast etc.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.
The extracellular can be expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating by various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
Enzyme of the present invention also comprises the immobilized enzyme that is fixed on the solid phase carrier.Various enzyme immobilization technology well known in the art all can be used for preparing of the present invention immobilized circumscribed-β-1,4-dextranase/inscribe-β-1,4-zytase.
In addition, the invention provides circumscribed-β-1,4-dextranase/inscribe-β-1, the purposes of 4-zytase, its encoding sequence, carrier or host cell, they are used to the production simple sugars, especially the technology of glucose.Described simple sugars comprises cellobiose, simple pentose, glucose and composition thereof.
The technology of production simple sugars of the present invention comprises step: (a) with the above-mentioned circumscribed-β-1 of the present invention, and 4-dextranase/inscribe-β-1, the host cell of 4-zytase or conversion or transduction is handled cellulose materials, thereby produces simple sugars; (b) isolate described simple sugars.Enzyme of the present invention can directly produce simple sugars such as cellobiose, simple pentose.Preferred technology also comprises other enzymes of adding, and for example beta-glucosidase (cellobiose can be hydrolyzed to glucose) so just can directly produce glucose.
A kind of representational technology comprises step: (a) will cut with scissors cutted straw and the 0.05M pH5.2 acetate buffer solution that contains 0.1M sodium-chlor with machinery and mix (by weight/volume) at 45 ℃ of preheating 10-15 minutes by 20-30%.(b) add then 0.05-0.2% circumscribed-β-1,4-dextranase/inscribe-β-1,4-zytase and beta-glucosidase slowly stir, and at 45 ℃ of water-bath 12-24 hours, collect 90% supernatant part of natural subsidence.Evaporate to dryness obtains the glucose crude product.(C) precipitation part add contain 0.1M sodium-chlor 0.05M pH5.2 acetate buffer solution to original volume, slowly stir 45 ℃ of water-baths 24 hours, collect 90% supernatant part of natural subsidence.Evaporate to dryness obtains the glucose crude product.
The major advantage of dextran excision enzyme of the present invention is:
(1) active high.Of the present invention circumscribed-β-1,4-dextranase/inscribe-β-1, the 4-zytase has one of the Mierocrystalline cellulose excision enzyme (hydrolyzing microcrystalline cellulose) of the highest vigor and hemicellulase (the ratio vigor of two kinds of enzymes is every milligram of protein 37 and 1300 international unit).
(2) single this enzyme just can hydrolysis generate simple sugars without any chemically treated straw, and this character then is that the cellulase of having reported is not available, and this character has business development and is worth on the hydrolysis natural cellulose.
(3) when chlorion exists, has the dual vigor of hydrocellulose or hemicellulose; When not having chlorion to exist, a hydrolyzed hemicellulose.Be that chlorion can carry out the switched attenuator switched-mode adjusting to this cellulase to the selectivity of the catalyzed reaction of substrate.This helps to enlarge its range of application.
(4) the cellulolytic vigor of excision enzyme is not suppressed by product (cellobiose), and this is to improving the transformation efficiency of natural cellulose saccharogenesis, and it all is very favourable reducing cost.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1:
Fushou spiral shell is circumscribed-β-1, and 4-dextranase/inscribe-β-1,4-zytase purifying:
Fushou spiral shell extensively is distributed in Fujian China, Guangdong, Guangxi, Zhejiang, provinces such as Jiangsu, Fushou spiral shell is that 20 years previous crops are introduced from South America for edible, its happiness food rice shoot, even water quality is not good, Fushou spiral shell also can be bred growth at double, has become one in river course and the rice terrace to damage greatly.With the Fushou spiral shell buied on the market as experiment material.
(1) extracts Fushou spiral shell gastric juice, with 0.5 saturation ratio ammonium sulfate precipitation.
(2) collecting precipitation is with 10mM phosphoric acid buffer (containing 0.1M sodium-chlor and 1mMEDTA) dissolving, dialysis.
(3) go up DEAE-Sephadex A-50 column chromatography purification, collect and pass the peak.
(4) go up Bio-gel P-100 column chromatography purification, collect pNPC (the hydrolysis vigor peak of p-Nitrophenyl β-D-cellobioside).
(5) through phenyl-Sepharose CL-4B column chromatography purification, collect to merge pNPC hydrolysis vigor peak, promptly obtain Fushou spiral shell circumscribed-β-1,4-dextranase/inscribe-β-1, the pure zymin of 4-zytase.
By above step, the Fushou spiral shell that can obtain the SDS-PAGE homogeneous is circumscribed-β-1,4-dextranase/inscribe-β-1,4-zytase.
Embodiment 2
Circumscribed-β-1,4-dextranase/inscribe-β-1,4-zytase determined amino acid sequence
2.1 main agents and instrument
Pvdf membrane is available from Millipore company.Inscribe-Glu C proteolytic enzyme is available from Promega company.DTT is available from Boehringer company.Iodoacetic acid (ICH
2COOH) available from Sigma company.Electricity changes the film system and selects the 2117-250 NOVABLOT Electrophoretic Transfer Kit of LKB company for use.
2.2 circumscribed-β-1,4-dextranase/inscribe-β-1,4-zytase natural N-terminal determined amino acid sequence
Circumscribed-β-1,4-dextranase/inscribe-β-1, sample carries out SDS-PAGE on the pure zymin of 4-zytase, wherein concentrates glue 5%, separation gel 10%, sample 70ug on every hole, electrophoretic voltage 70V 30 minutes, 125V is 90 minutes again.
Electrophoresis changes film with separation gel with semidrying after finishing.Change film and finish the back,, cut circumscribed-β-1,4-dextranase/inscribe-β-1,4-zytase protein band with the distilled water decolouring with 0.2% Ponceau S (being dissolved in 3% trichoroacetic acid(TCA)) dyeing pvdf membrane 1 minute.Entrust the biochemical institute in Shanghai to carry out the Argine Monohydrochloride sequencing.
Record circumscribed-β-1,4-dextranase/inscribe-β-1,4-zytase natural N-terminal aminoacid sequence is:
NH
2-Ala-Ala-Gly-Ala-Gly-Val-Thr-Ser-Glu-Ile(SEQ?ID?NO：3)
2.3 contain circumscribed-β-1,4-dextranase/inscribe-β-1, the protease hydrolysis fragment of 4-zytase-terminal amino acid sequence is measured
Inscribe-Glu C protease hydrolysis is circumscribed-β-1, and 4-dextranase/inscribe-β-1, the 4-zytase, SDS-PAGE electrophoretic separation proteolysis product (Fig. 1), electrophoresis changes film with separation gel with semidrying after finishing.With 0.2mA/cm
2Current transfer 12 hours.Change film and finish the back,, cut component I shown in Fig. 1 arrow, carry out Argine Monohydrochloride sequencing (entrusting the biochemical institute in Shanghai) with the distilled water decolouring with 0.2% Ponceau S (being dissolved in 3% trichoroacetic acid(TCA)) dyeing pvdf membrane 1 minute.
Recording component I-terminal amino acid sequence is: NH
2-Leu-Phe-Arg-I1e-Ala-His-Ala-Ala-Asp-Pro (SEQ ID NO:4)
Embodiment 3
Circumscribed-β-1,4-dextranase/inscribe-β-1, the cDNA clone of 4-zytase
3.1 instrument
The PCR instrument is selected the SingleBlock of U.S. Ericomp company for use
TMSystem.
3.2 RNA extracting and reverse transcription
The stomach-tissue of getting fresh Fushou spiral shell is with the Trizol extracted total RNA, and with oligo (dT)
18For the primer reverse transcription generates cDNA as pcr template.
3.3 the first step PCR
According to two sections aminoacid sequences of protein sequencing gained, synthetic following PCR degenerate primer:
Primer?1A：
5′-GCA(T/G/C)??GCA(T/G/C)??GGA(T/G/C)??GCA(T/G/C)??GGA(T/G/C)
GTA(T/G/C)??AC-3′(SEQ?ID?NO：5)
Primer?1B：
5′-GG??A(G)TC??A(T/G/C)GC??A(T/G/C)GC??A(G)TG??A(T/G/C)GCA(G)AT-3′(SEQ?ID?NO：6)
With reverse transcription gained cDNA is template, carries out dna amplification reaction, obtains the DNA product of a treaty 600bp length.With this DNA product pBluescript II SK (+) T-vector that packs into, the clone is order-checking also, obtains the middle part fragment of this gene.
3.4 the second step PCR
According to the gene fragment that has got, the synthetic nest-type PRC primer of design,
Primer 2 (outer primer):
5′-ACC?AGC?ATC?AAC?TGA?ATG-3′(SEQ?ID?NO：7)
Primer 3 (inner primer):
5′-ATG?ACA?ATG?GCT?ACA?AC-3′(SEQ?ID?NO：8)
The nest-type PRC the first step is with outer primer Primer 2 and oligo (dT)
18Right for primer, second step is with inner primer Primer 3 and oligo (dT)
18Right for primer, can obtain the dna fragmentation of an about 900bp at last.With this DNA product pBluescript II SK (+) T-vector that packs into, the clone is order-checking also, obtains 3 ' end fragment of this gene.
3.5 the 3rd step PCR
According to gene fragment design synthetic following 5 '-RACE reverse transcription primer and nest-type PRC primer:
GSP1：
5′-ACA?TCC?CAG?TGC?T-3′(SEQ?ID?NO：9)
GSP2：
5′-GAG?CCT?TGA?CCC?AGT?TCT?G-3′(SEQ?ID?NO：10)
GSP3：
5′-ACC?GCC?CAG?TTG?TAG?TGC?TGG?T-3′(SEQ?ID?NO：11)
Anchor：
5′-GGC?CTG?CAG?TCG?ACT?AGT?ACT?TTT?TTT?TTT?TTT?TTT?T-3′(SEQ?IDNO：12)
UAP：
5′-GGC?CTG?CAG?TCG?ACT?AGT?AC-3′(SEQ?ID?NO：13)
With GSP1 is the total RNA of primer reverse transcription Fushou spiral shell stomach-tissue, generate the cDNA template, after RNaseH hydrolysis, cDNA3 ' end add poly (dA) tail, with this cDNA is that template is carried out the nest-type PRC amplification, obtain target DNA fragment, with this dna fragmentation pBluescript II SK (+) T-vector that packs into, the clone is order-checking also.This target DNA fragment is promptly circumscribed-β-1, and 4-dextranase/inscribe-β-1,5 ' end fragment of 4-xylanase gene.
3.6 the 4th step PCR
According to the circumscribed-β-1 that has got, 4-dextranase/inscribe-β-1,5 ' end fragment of 4-xylanase gene and 3 ' end fragment, synthetic following primer:
Primer?4A：
5′-CAG?GCT?GAC?CAG?AAT?CCA?CTA-3′(SEQ?ID?NO：14)
Primer?4B：
5′-TTC?AAC?TTT?ATT?GCC?CTC?TG-3′(SEQ?ID?NO：1?5)
With oligo (dT)
18Be the total RNA of primer reverse transcription Fushou spiral shell stomach-tissue, generate the cDNA template, choosing Primer 4A and Primer 4B is that primer amplification obtains this full length gene fragment, and with this dna fragmentation pBluescript II SK (+) T-vector that packs into, the clone is order-checking also.Gained fragment total length 1293bp (SEQID NO:1) wherein comprises open reading frame 1188bp, 5 ' non-translational region 77bp and 3 ' non-translational region 30bp, and coding contains the dextran excision enzyme (SEQ ID NO:2) of 395 amino-acid residues.
Sequence alignment shows, circumscribed-the β-1 of Fushou spiral shell, 4-dextranase/inscribe-β-1, the Mierocrystalline cellulose excision enzyme/zytase (exo-glucanase/xylanase) among the cDNA of 4-zytase and the amino-acid sequence of deriving thus and the aerobic bacteria cellulomonas fimi all has very big-difference.
Embodiment 4
Fushou spiral shell is circumscribed-β-1, and 4-dextranase/inscribe-β-1, the expression of 4-zytase cDNA in methanol yeast
4.1 make up the methanol yeast expression strain
With the circumscribed-β-1 that obtains among the embodiment 3,4-dextranase/inscribe-β-1, the 4-xylanase gene, after EcoRI and NotI enzyme are cut, among the methanol yeast secretion type expression plasmid pPIC9K that the same enzyme of packing into is cut, and transformed into escherichia coli E.coli DH12S, obtain containing the mono-clonal bacterial strain of secretor type recombinant expression plasmid pPIC9K-EGX.
Extracting secretor type recombinant expression plasmid pPIC9K-EGX, and get 5 μ g with restriction enzyme Bpu1102I simple tangentization.
Get 5ug linearization plasmid pPIC9K-EGX electricity and transform methanol yeast GS115 cell, electric transformant screens His on the MD flat board
+The clone.
A picking 20-30 His
+The clone, extracting DNA, and contain circumscribed-β-1,4-dextranase/inscribe-β-1, the clone of 4-xylanase gene with PCR method conclusive evidence.
To contain circumscribed-β-1,4-dextranase/inscribe-β-1, Mut is screened in being cloned on the MM flat board of 4-xylanase gene
+Phenotype.
4.2. the expression in the methanol yeast expression strain liquid medium within
To contain circumscribed-β-1,4-dextranase/inscribe-β-1, the clone of 4-xylanase gene is inoculated in the 1ml BMG substratum, and 30 ℃ of 220rpm cultivated 24 hours.
MG is cultivated bacterium liquid be inoculated into respectively in the 5ml BMGY substratum with 1: 200 ratio, 30 ℃ of 220rpm are cultured to OD
600=2-6 transfers to thalline in the BMMY substratum, makes bacterial concentration reach OD
600=1.0,30 ℃ of 220rpm cultivated 7 days, added methyl alcohol to 0.5% in per 24 hours.
Centrifugal collection thalline, measure in the cell and extracellular circumscribed-β-1,4-dextranase/inscribe-β-1,4-xylanase activity.
4.3. the expression of methanol yeast expression strain in solid medium
To contain circumscribed-β-1,4-dextranase/inscribe-β-1, the clone of 4-xylanase gene is inoculated in 1ml MG substratum (the basic nitrogenous source of 1.34% yeast, 0.1% glycerine, 0.1M pH6.0 phosphoric acid buffer, 20mM sodium-chlor, vitamin H 4 * 10
-5%), 30 ℃ of 220rpm cultivated 24 hours.
MG is cultivated bacterium liquid be inoculated into 5ml BMGY substratum (the basic nitrogenous source of 1.34% yeast, 0.1% glycerine, 0.1M pH6.0 phosphoric acid buffer, 20mM sodium-chlor, vitamin H 4 * 10 respectively with 1: 200 ratio
-5%), 30 ℃ of 220rpm are cultured to OD
600=2-6 respectively gets 0.5 μ gl and cultivates object point on the BMMY plate culture medium, cultivates 7 days, adds 100 μ l methyl alcohol in the loam cake of culture dish in per 24 hours for 30 ℃.
BMMY plate culture medium (the basic nitrogenous source of 1.34% yeast, 0.1M pH6.0 phosphoric acid buffer, 20mM sodium-chlor, 0.1% soluble xylan, 2% agar powder, vitamin H 4 * 10 are removed in the distilled water flushing
-5%, methyl alcohol 0.5%) surperficial methanol yeast bacterium colony, add 10ml 0.1% congo red staining 40 minutes, add 1N sodium chloride solution decolouring again, show golden yellow hydrolysis spot and show that promptly circumscribed-β-1 is arranged, 4-dextranase/inscribe-β-1,4-xylanase hydrolysis vigor produces.
The result as shown in Figure 2, blotch is the methanol yeast expression strain that contains cellulase, its process of growth discharges cellulase to the extracellular, makes the Mierocrystalline cellulose in the solid medium be hydrolyzed into simple sugars.The contrast bacterium that does not change Fushou spiral shell dextran excision enzyme gene over to does not have tangible spot and produces.
4.4. the separation and purification of reorganization dextran excision enzyme
1) 7 days medium centrifugal supernatant part of 4.2. fermentation, with 0.5 saturation ratio ammonium sulfate precipitation, its precipitation promptly is the raw product of this recombinase.
(2) raw product is dissolved dialysis with 10mM phosphoric acid buffer (containing 0.1M sodium-chlor and 1mMEDTA).
(3) go up DEAE-Sephadex A-50 column chromatography purification, collect and pass the peak.
(4) go up Bio-gel P-100 column chromatography purification, collect pNPC (the hydrolysis vigor peak of p-Nitrophenyl β-D-cellobioside).
(5) through phenyl-Sepharose CL-4B column chromatography purification, collect to merge pNPC hydrolysis vigor peak, promptly obtain Fushou spiral shell circumscribed-β-1,4-dextranase/inscribe-β-1, the pure zymin of 4-zytase.
By above step, the Fushou spiral shell that can obtain the SDS-PAGE homogeneous is circumscribed-β-1,4-dextranase/inscribe-β-1,4-zytase.
Embodiment 5
Circumscribed-β-1,4-dextranase/inscribe-β-1, the 4-Xylanase activity is measured
5.1 p-nitrophenol cellobioside (pNPC) hydrolysis vigor (Mierocrystalline cellulose excision enzyme) is measured
450 μ l 1mg/ml pNPC (being dissolved in the 0.1M pH5.2 acetate buffer solution that contains 0.1M sodium-chlor)+50 μ l 10mg/ml D-Gluconi Acid Lactone (being dissolved in the 0.1M pH5.2 the acetate buffer solution) → an amount of enzyme liquid of 5 minutes → adding of 45 ℃ of water-baths → 8 minutes → adding of 45 ℃ of water-baths, 525 μ l 2%Na
2CO
3Termination reaction, and the 410nm place absorbance value that develops the color → read.Optical extinction coefficient is 1.76 * 10
-5PM
-1
PNPC hydrolysis vigor is defined as under these conditions, and it is 1 unit (unit) that the per minute hydrolysis generates the required enzyme amount of 1pmol p-nitrophenol (pNP).
5.2 Microcrystalline Cellulose (Sigmacell) hydrolysis vitality test
500 μ l, 1% Sigamcell (being suspended in the 0.1M pH5.2 acetate buffer solution that contains 0.1M the sodium-chlor) → an amount of enzyme liquid of 5 minutes → adding of 45 ℃ of water-bath preheatings, DNS reagent 0.5ml → boiling water bath that 45 ℃ of water-baths 8 minutes → add contain 40 μ g/ml glucose 5 minutes, cold water cooling mends 0.5ml distilled water → read 540nm place absorbance value.
Microcrystalline Cellulose (Sigmacell) hydrolysis vigor is defined as under these conditions, and it is 1 unit (unit) that the per minute hydrolysis generates the required enzyme amount of 1 μ mol glucose reducing equivalent.
5.3 xylan hydrolysis vitality test
It is substrate that the xylan hydrolysis vitality test adopts soluble xylan.After getting xylan and adding water and stir fully, obtain the suspension liquid of xylan, this suspension liquid is centrifugal, get the supernatant lyophilize, promptly obtain soluble xylan.
500 μ l, 1% soluble xylan (being dissolved in the 0.1M pH5.2 acetate buffer solution that contains 0.1M sodium-chlor)? the an amount of enzyme liquid of 5 minutes → adding of 45 ℃ of water-bath preheatings, DNS reagent 0.5ml → boiling water bath that 45 ℃ of water-baths 8 minutes → add contain 40 μ g/ml glucose 5 minutes, cold water cooling mends 0.5ml distilled water → read 540nm place absorbance value.
The xylan hydrolysis vigor is defined as under these conditions, and it is 1 unit (unit) that the per minute hydrolysis generates the required enzyme amount of 1 μ mol glucose reducing equivalent.
5.4 endo-beta-1,4-glucanase hydrolysis vitality test
200 μ l, 1% Xylo-Mucine (being dissolved in the 0.05M pH5.2 acetate buffer solution that contains 0.1M the sodium-chlor) → an amount of enzyme liquid of 5 minutes → adding of 45 ℃ of water-bath preheatings, DNS reagent 0.5ml → boiling water bath that 45 ℃ of water-baths 8 minutes → add contain 40 μ g/ml glucose 5 minutes, cold water cooling mends 0.5ml distilled water → read 540nm place absorbance value.
5.5 hydrolysis dry rice straw vitality test
500 μ l dry rice straw suspension (contain dry rice straw 150mg, be suspended in the 0.05MpH5.2 acetate buffer solution that contains 0.1M sodium-chlor) → 45 ℃ of water-bath preheatings 5 minutes → add, 18 μ g are circumscribed-β-1,4-dextranase/inscribe-β-1, the 4-zytase, DNS reagent 0.5ml → boiling water bath that 45 ℃ of water-baths 40 minutes → add contain 40 μ g/ml glucose 5 minutes, the cold water cooling is mended the 0.5ml distilled water and is read 540nm place absorbance value.Calculate the reducing sugar growing amount then.
In the above conditions, be that the ratio vigor of this enzyme of substrate is 3.18 international unit with the straw.
Test result is summarized in table 1.
Table 1: the vigor of the excision enzyme of Fushou spiral shell Mierocrystalline cellulose excision enzyme and bibliographical information relatively
| The source | Excision enzyme | The vigor definition | Concentration of substrate | Than live (u/mg) |
| T.reesei | ?CBHI | μ mol glucose | 1％Avicel | ?0.0175 |
| ?CBHII | μ mol glucose | 1％Avicel | ?0.0391 | |
| P.F.var. cellulosa | ?Exo-I | μ mol glucose | 1％Avicel | ?------ |
| T.viride | ?Exo-I | μ mol glucose | 1％Avicel | ?0.008 |
| ?Exo-II | μ mol glucose | 1％Avicel | ?0.0032 | |
| ?Exo-III | μ mol glucose | 1％Avicel | ?0.0039 | |
| Irpex?lacteus | Circumscribed cellulase | μ mol glucose | 1％Avicel | ?0.0571 |
| ?Huwicola?insolens | ?CBH | μ mol glucose | 1％Avicel | ?1.60 |
| ?C.cellulolytium | ?CelA | μ mol glucose | 0.8％Avicel | ?5.4 |
| ?CelC | μ mol glucose | 0.8％Avicel | ?0.8 | |
| Fushou spiral shell | Circumscribed-β-1,4-dextranase/inscribe-β-1, the 4-zytase | μ mol glucose | 1％Sigmacel 1## | ?37.2 |
##Sigmacell and Avicel are the Microcrystalline Cellulose trade(brand)names of Sigma company.Avicel no longer appears in the goods catalogue after 98 years, has only Sigmacell
The # definition: it is 1 U that per minute hydrolyzing microcrystalline cellulose (Sigmacell) or polyxylose (birchwood) produce 1 μ mol simple sugars.
Embodiment 6
Chlorion is to circumscribed-β-1,4-dextranase/inscribe-β-1, the influence of 4-Xylanase activity
In the reaction system that contains the different concns chlorion, measure circumscribed-β-1,4-dextranase/inscribe-β-1, the 4-Xylanase activity is studied chlorion to circumscribed-β-1,4-dextranase/inscribe-β-1, the influence of 4-Xylanase activity.
The result as shown in Figure 3, chlorion is to the inscribe-β-1 of enzyme, the 4-Xylanase activity does not have obvious influence, and to circumscribed-β-1,4-dextranase vigor has tangible activation, and is circumscribed-β-1,4-dextranase vigor institute is essential.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table＜110〉Chinese Sciences Academy Biochemistry And Cell Biology Institute＜120 new circumscribed-β-1; 4-dextranase/inscribe-β-Isosorbide-5-Nitrae-zytase and application thereof＜130〉023315＜160〉15＜170〉PatentIn version 3.1＜210〉1＜211〉1293＜212〉DNA＜213〉Pomacea canaliculata (Ampullarium Crossean)＜220〉＜221〉CDS＜222〉(77) .. (1261)＜223〉＜400〉1caggctgacc agaatccact accaaggacc agaggagggc atcagttttg tcgtcgacga 60cgcttcagtc aagcgc atg ccc tct ggt gct gct ggt gct ggg gtg acc agc 112
Met?Pro?Ser?Gly?Ala?Ala?Gly?Ala?Gly?Val?Thr?Ser
1???????????????5??????????????????10gag?atc?gac?aga?ctg?aga?aga?agc?gac?ata?acg?gtt?cac?gtg?aat?gtt??????160Glu?Ile?Asp?Arg?Leu?Arg?Arg?Ser?Asp?Ile?Thr?Val?His?Val?Asn?Val
15?????????????????20??????????????????25ggt?ggt?aac?atc?aac?cac?ggt?caa?gtg?agc?att?cga?gtg?tta?caa?aag??????208Gly?Gly?Asn?Ile?Asn?His?Gly?Gln?Val?Ser?Ile?Arg?Val?Leu?Gln?Lys
30?????????????????35??????????????????40aga?aag?gca?ttc?ccg?ttc?ggg?aca?tgt?gtg?gcc?gcc?tgg?gcc?tac?aac??????256Arg?Lys?Ala?Phe?Pro?Phe?Gly?Thr?Cys?Val?Ala?Ala?Trp?Ala?Tyr?Asn45??????????????????50?????????????????55??????????????????60gat?ggg?tcc?aaa?gga?gca?tac?cgg?gat?ttc?atc?cac?cag?cac?tac?aac??????304Asp?Gly?Ser?Lys?Gly?Ala?Tyr?Arg?Asp?Phe?Ile?His?Gln?His?Tyr?Asn
65?????????????????70??????????????????75tgg?gcg?gtg?cca?gaa?aac?tca?ctc?aag?tgg?gct?agc?atc?gaa?cct?aac??????352Trp?Ala?Val?Pro?Glu?Asn?Ser?Leu?Lys?Trp?Ala?Ser?Ile?Glu?Pro?Asn
80?????????????????85???????????????????90agg?gga?caa?aag?aac?tat?cag?cct?ggc?cta?aac?atg?ctt?cac?gga?ctg??????400Arg?Gly?Gln?Lys?Asn?Tyr?Gln?Pro?Gly?Leu?Asn?Met?Leu?His?Gly?Leu
95??????????????????100????????????????105aga?aat?cac?ggg?att?aag?gtg?aga?ggt?cac?aac?ctg?gtg?tgg?tct?gtc??????448Arg?Asn?His?Gly?Ile?Lys?Val?Arg?Gly?His?Asn?Leu?Val?Trp?Ser?Val
110?????????????????115????????????????120gac?aat?acg?gtg?cag?aac?tgg?gtc?aag?gct?ctg?cat?ggg?gat?gag?ctt??????496Asp?Asn?Thr?Val?Gln?Asn?Trp?Val?Lys?Ala?Leu?His?Gly?Asp?Glu?Leu125?????????????????130?????????????????135?????????????????140cga?aag?gtt?gtc?cat?gac?cac?att?gtg?gaa?acc?atc?aac?aca?ttt?aag??????544Arg?Lys?Val?Val?His?Asp?His?Ile?Val?Glu?Thr?Ile?Asn?Thr?Phe?Lys
145????????????????150?????????????????155gga?tta?gtg?gag?cac?tgg?gat?gtg?aac?aac?gag?aac?ctg?cat?ggc?cag??????592Gly?Leu?Val?Glu?His?Trp?Asp?Val?Asn?Asn?Glu?Asn?Leu?His?Gly?Gln
160????????????????165?????????????????170tgg?tac?cag?cat?caa?ctg?aat?gac?aat?ggc?tac?aac?ctg?gaa?ctg?ttc??????640Trp?Tyr?Gln?His?Gln?Leu?Asn?Asp?Asn?Gly?Tyr?Asn?Leu?Glu?Leu?Phe
175?????????????????180????????????????185cgt?atc?gca?cac?gcc?gcc?gac?ccc?aac?gtc?aaa?ctc?ttc?ctc?aac?gac??????688Arg?Ile?Ala?His?Ala?Ala?Asp?Pro?Asn?Val?Lys?Leu?Phe?Leu?Asn?Asp
190?????????????????195????????????????200tac?aac?gtt?gtg?tcc?aac?agt?tat?tca?aca?aac?gac?tat?ctt?cga?caa??????736Tyr?Asn?Val?Val?Ser?Asn?Ser?Tyr?Ser?Thr?Asn?Asp?Tyr?Leu?Arg?Gln205?????????????????210????????????????215?????????????????220ggt?caa?cag?ttt?aag?gcc?gct?aat?gtg?ggt?ctt?tac?ggt?ttg?ggt?gct??????784Gly?Gln?Gln?Phe?Lys?Ala?Ala?Asn?Val?Gly?Leu?Tyr?Gly?Leu?Gly?Ala
225????????????????230?????????????????235cag?tgc?cac?ttt?ggc?gac?gaa?agc?gac?cca?gaa?ccc?ggt?act?aag?caa??????832Gln?Cys?His?Phe?Gly?Asp?Glu?Ser?Asp?Pro?Glu?Pro?Gly?Thr?Lys?Gln
240?????????????????245????????????????250cgt?ctg?gat?act?tta?gct?caa?gtg?ggc?gtg?ccc?atc?tgg?gcc?act?gag??????880Arg?Leu?Asp?Thr?Leu?Ala?Gln?Val?Gly?Val?Pro?Ile?Trp?Ala?Thr?Glu
255?????????????????260????????????????265ttg?gat?gtg?gta?gct?tcg?gat?gag?aac?aga?cga?gcg?gac?ttc?tac?gag??????928Leu?Asp?Val?Val?Ala?Ser?Asp?Glu?Asn?Arg?Arg?Ala?Asp?Phe?Tyr?Glu
270?????????????????275????????????????280cac?gcg?ctg?aca?gtc?ctg?tac?ggc?cat?cat?gcc?gtg?gag?ggc?atc?ctc??????976His?Ala?Leu?Thr?Val?Leu?Tyr?Gly?His?His?Ala?Val?Glu?Gly?Ile?Leu285?????????????????290????????????????295?????????????????300atg?tgg?ggc?ttc?tgg?gac?aag?gcc?cac?tgg?cgt?ggt?gcc?aga?gct?gct?????1024Met?Trp?Gly?Phe?Trp?Asp?Lys?Ala?His?Trp?Arg?Gly?Ala?Arg?Ala?Ala
305?????????????????310????????????????315ctt?gtt?gtc?gga?gac?aac?ctg?cag?ctg?acg?gcg?gcc?gga?cgt?cgc?gtg?????1072Leu?Val?Val?Gly?Asp?Asn?Leu?Gln?Leu?Thr?Ala?Ala?Gly?Arg?Arg?Val
320????????????????325?????????????????330ctg?gag?ctc?ttt?gag?cac?agg?tgg?atg?aca?gac?gag?acg?cac?aac?ctg?????1120Leu?Glu?Leu?Phe?Glu?His?Arg?Trp?Met?Thr?Asp?Glu?Thr?His?Asn?Leu
335?????????????????340????????????????345gca?gcg?ggc?act?cag?ttc?aca?gta?cgc?ggt?ttc?cat?ggc?gac?tac?gag?????1168Ala?Ala?Gly?Thr?Gln?Phe?Thr?Val?Arg?Gly?Phe?His?Gly?Asp?Tyr?Glu
350?????????????????355????????????????360gtg?caa?gtc?atc?gtc?cag?ggt?caa?gag?cac?acc?aac?ctg?agg?cag?aca?????1216Val?Gln?Val?Ile?Val?Gln?Gly?Gln?Glu?His?Thr?Asn?Leu?Arg?Gln?Thr365?????????????????370????????????????375?????????????????380ttc?tcg?ttg?ggc?aac?ggt?ccc?cac?acc?gtc?aac?att?aat?gtt?agc?????????1261Phe?Ser?Leu?Gly?Asn?Gly?Pro?His?Thr?Val?Asn?Ile?Asn?Val?Ser
385 390 395tagagcgaca ctcagagggc aataaagttg aa, 1293＜210〉2＜211〉395＜212〉PRT＜213〉Pomacea canaliculata (Ampullarium Crossean)＜400〉2Met Pro Ser Gly Ala Ala Gly Ala Gly Val Thr Ser Glu Ile Asp Arg1,5 10 15Leu Arg Arg Ser Asp Ile Thr Val His Val Asn Val Gly Gly Asn Ile
20?????????????????25??????????????????30Asn?His?Gly?Gln?Val?Ser?Ile?Arg?Val?Leu?Gln?Lys?Arg?Lys?Ala?Phe
35?????????????????40??????????????????45Pro?Phe?Gly?Thr?Cys?Val?Ala?Ala?Trp?Ala?Tyr?Asn?Asp?Gly?Ser?Lys
50?????????????????55??????????????????60Gly?Ala?Tyr?Arg?Asp?Phe?Ile?His?Gln?His?Tyr?Asn?Trp?Ala?Val?Pro65??????????????????70?????????????????75??????????????????80Glu?Asn?Ser?Leu?Lys?Trp?Ala?Ser?Ile?Glu?Pro?Asn?Arg?Gly?Gln?Lys
85?????????????????90??????????????????95Asn?Tyr?Gln?Pro?Gly?Leu?Asn?Met?Leu?His?Gly?Leu?Arg?Asn?His?Gly
100????????????????105?????????????????110Ile?Lys?Val?Arg?Gly?His?Asn?Leu?Val?Trp?Ser?Val?Asp?Asn?Thr?Val
115????????????????120?????????????????125Gln?Asn?Trp?Val?Lys?Ala?Leu?His?Gly?Asp?Glu?Leu?Arg?Lys?Val?Val
130????????????????135?????????????????140His?Asp?His?Ile?Val?Glu?Thr?Ile?Asn?Thr?Phe?Lys?Gly?Leu?Val?Glu145?????????????????150????????????????155?????????????????160His?Trp?Asp?Val?Asn?Asn?Glu?Asn?Leu?His?Gly?Gln?Trp?Tyr?Gln?His
165????????????????170?????????????????175Gln?Leu?Asn?Asp?Asn?Gly?Tyr?Asn?Leu?Glu?Leu?Phe?Arg?Ile?Ala?His
180????????????????185?????????????????190Ala?Ala?Asp?Pro?Asn?Val?Lys?Leu?Phe?Leu?Asn?Asp?Tyr?Asn?Val?Val
195????????????????200?????????????????205Ser?Asn?Ser?Tyr?Ser?Thr?Asn?Asp?Tyr?Leu?Arg?Gln?Gly?Gln?Gln?Phe
210?????????????????215????????????????220Lys?Ala?Ala?Asn?Val?Gly?Leu?Tyr?Gly?Leu?Gly?Ala?Gln?Cys?His?Phe225?????????????????230????????????????235?????????????????240Gly?Asp?Glu?Ser?Asp?Pro?Glu?Pro?Gly?Thr?Lys?Gln?Arg?Leu?Asp?Thr
245????????????????250?????????????????255Leu?Ala?Gln?Val?Gly?Val?Pro?Ile?Trp?Ala?Thr?Glu?Leu?Asp?Val?Val
260????????????????265?????????????????270Ala?Ser?Asp?Glu?Asn?Arg?Arg?Ala?Asp?Phe?Tyr?Glu?His?Ala?Leu?Thr
275?????????????????280????????????????285Val?Leu?Tyr?Gly?His?His?Ala?Val?Glu?Gly?Ile?Leu?Met?Trp?Gly?Phe
290?????????????????295????????????????300Trp?Asp?Lys?Ala?His?Trp?Arg?Gly?Ala?Arg?Ala?Ala?Leu?Val?Val?Gly305?????????????????310????????????????315?????????????????320Asp?Asn?Leu?Gln?Leu?Thr?Ala?Ala?Gly?Arg?Arg?Val?Leu?Glu?Leu?Phe
325????????????????330?????????????????335Glu?His?Arg?Trp?Met?Thr?Asp?Glu?Thr?His?Asn?Leu?Ala?Ala?Gly?Thr
340????????????????345?????????????????350Gln?Phe?Thr?Val?Arg?Gly?Phe?His?Gly?Asp?Tyr?Glu?Val?Gln?Val?Ile
355????????????????360?????????????????365Val?Gln?Gly?Gln?Glu?His?Thr?Asn?Leu?Arg?Gln?Thr?Phe?Ser?Leu?Gly
370 375 380Asn Gly Pro His Thr Val Asn Ile Asn Val Ser385,390 395＜210〉3＜211〉10＜212〉PRT＜213〉Pomacea canaliculata (Ampullarium Crossean)＜400〉3Ala Ala Gly Ala Gly Val Thr Ser Glu Ile1,5 10＜210〉4＜211〉10＜212〉PRT＜213〉Pomacea canaliculata (Ampullarium Crossean)＜400〉4Leu Phe Arg Ile Ala His Ala Ala Asp Pro1 5 10＜210〉5＜211〉20＜212〉DNA＜213〉primer＜220〉＜221〉misc_feature＜222〉(3) .. (3)＜223〉n=a; T; C; G＜220〉＜221〉misc_feature＜222〉(6) .. (6)＜223〉n=a; T; C; G＜220〉＜221〉misc_feature＜222〉(9) .. (9)＜223〉n=a; T; C; G＜220〉＜221〉misc_feature＜222〉(12) .. (12)＜223〉n=a; T; C; G＜220〉＜221〉misc_feature＜222〉(15) .. (15)＜223〉n=a; T; C; G＜220〉＜221〉misc_feature＜222〉(18) .. (18)＜223〉n=a; T; C; G＜400〉5gcngcnggng cnggngtnac 20＜210〉6＜211〉20＜212〉DNA＜213〉primer＜220〉＜221〉misc_feature＜222〉(6) .. (6)＜223〉n=a; T; C; G＜220〉＜221〉misc_feature＜222〉(9) .. (9)＜223〉n=a; T; C; G＜220〉＜221〉misc_feature＜222〉(15) .. (15)＜223〉n=a; T; c,g＜400〉6ggrtcngcng crtgngcrat 20＜210〉7＜211〉18＜212〉DNA＜213〉＜400〉7accagcatca actgaatg 18＜210〉8＜211〉17＜212〉DNA＜213〉＜400〉8atgacaatgg ctacaac 17＜210〉9＜211〉13＜212〉DNA＜213〉＜400〉9acatcccagt gct 13＜210〉10＜211〉19＜212〉DNA＜213〉＜400〉10gagccttgac ccagttctg 19＜210〉11＜211〉22＜212〉DNA＜213〉＜400〉11accgcccagt tgtagtgctg gt 22＜210〉12＜211〉37＜212〉DNA＜213〉＜400〉12ggcctgcagt cgactagtac tttttttttt ttttttt 37＜210〉13＜211〉20＜212〉DNA＜213〉＜400〉13ggcctgcagt cgactagtac 20＜210〉14＜211〉21＜212〉DNA＜213〉＜400〉14caggctgacc agaatccact a 21＜210〉15＜211〉20＜212〉DNA＜213〉＜400〉15ttcaacttta ttgccctctg 20。
Claims (10)
- One kind isolating circumscribed-β-1,4-dextranase/inscribe-β-1, the 4-zytase is characterized in that, it comprises: have the polypeptide of SEQ ID NO:2 aminoacid sequence or its conservative property variation polypeptide or its active fragments or its reactive derivative.
- 2. as claimed in claim 1 circumscribed-β-1,4-dextranase/inscribe-β-1, the 4-zytase is characterized in that, this enzyme is selected from down group:(a) has the polypeptide of SEQ ID N0:2 aminoacid sequence;(b) SEQ ID NO:2 aminoacid sequence is formed through replacement, disappearance or the interpolation of one or more amino-acid residues, and have the circumscribed and/or xylan inscribe function of dextran by (a) polypeptides derived.
- 3. isolating polynucleotide is characterized in that, it comprises a nucleotide sequence, and this nucleotide sequence is shown at least 70% homogeny with a kind of nucleotides sequence that is selected from down group:(a) coding is as the polynucleotide of polypeptide as described in claim 1 and 2;(b) with polynucleotide (a) complementary polynucleotide.
- 4. polynucleotide as claimed in claim 3 is characterized in that, these polynucleotide are selected from down group:(a) coding has the polynucleotide of aminoacid sequence shown in the SEQ ID NO:2;(b) has the nucleotide sequence of 77-1261 position among the SEQ ID NO:3;(c) has the nucleotide sequence of 1-1293 position among the SEQ ID NO:3.
- 5. a carrier is characterized in that, it contains the described polynucleotide of claim 3.
- 6. a transformed host cells is characterized in that, it contains the described carrier of claim 6.
- 7. circumscribed-β-1,4-dextranase/inscribe-β-1, the preparation method of 4-zytase is characterized in that, this method comprises:(a) be fit to express circumscribed-β-1,4-dextranase/inscribe-β-1 under the condition of 4-zytase, is cultivated the described host cell of claim 7;(b) from culture, isolate circumscribed-β-1,4-dextranase/inscribe-β-1,4-zytase.
- 8. as claimed in claim 1 circumscribed-β-1,4-dextranase/inscribe-β-1, the purposes of 4-zytase or the described host cell of claim 6 is characterized in that, is used for the technology of production simple sugars or glucose.
- 9. a method of producing simple sugars is characterized in that, comprises step:(a) with claim 1 described circumscribed-β-1,4-dextranase/inscribe-β-1, the described host cell of 4-zytase or claim 6 is handled cellulose materials, thereby produces simple sugars;(b) will isolate described glucose.
- 10. method as claimed in claim 9 is characterized in that, described cellulose materials is the cellulose materials without any Chemical Pretreatment, and described simple sugars comprises cellobiose, simple pentose, glucose and composition thereof.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101058794B (en) * | 2007-04-09 | 2010-08-04 | 重庆理工大学 | Method of constructing transfer cellulose enzyme gene saccharomyces cerevisiae |
| CN101310017B (en) * | 2005-11-16 | 2013-03-20 | 诺维信公司 | Polypeptides having endoglucanase activity and polynucleotides encoding same |
| CN105531364A (en) * | 2013-08-20 | 2016-04-27 | 韩国生命工学研究院 | Novel alkali-resistant glycoside hydrolase family 10 xylanase produced from micobacterium sp. HY-17 strain |
| CN107988186A (en) * | 2017-12-06 | 2018-05-04 | 南京林业大学 | A kind of Cold tolerance endo beta-1,4-glucanase and its expressing gene and application |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101250512B (en) * | 2008-04-17 | 2010-04-14 | 上海交通大学 | Bionic compatible purification method for endo-xylanase |
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2002
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101310017B (en) * | 2005-11-16 | 2013-03-20 | 诺维信公司 | Polypeptides having endoglucanase activity and polynucleotides encoding same |
| CN101058794B (en) * | 2007-04-09 | 2010-08-04 | 重庆理工大学 | Method of constructing transfer cellulose enzyme gene saccharomyces cerevisiae |
| CN105531364A (en) * | 2013-08-20 | 2016-04-27 | 韩国生命工学研究院 | Novel alkali-resistant glycoside hydrolase family 10 xylanase produced from micobacterium sp. HY-17 strain |
| CN105531364B (en) * | 2013-08-20 | 2019-08-23 | 韩国生命工学研究院 | New the 10th family's zytase of alkali resistance glycoside hydrolase produced from Microbacterium HY-17 bacterial strain |
| CN107988186A (en) * | 2017-12-06 | 2018-05-04 | 南京林业大学 | A kind of Cold tolerance endo beta-1,4-glucanase and its expressing gene and application |
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