CN1294633A - Novel endo-xylogalacturonase - Google Patents

Abstract

Polypeptides possessing a novel activity, namely endo-xylogalacturonase activity, are disclosed. These polypeptides can degrade pectin found in plant extracts and plant materials, and in particular the ''hairy'' regions of pectin polymers. In particular, the polypeptides can cleave in a galacturonic acid polymer at internal glycocidic bonds. The novel enzyme XghA is disclosed, and its amino acid sequence and encoding DNA sequence are given. This polypeptide was expressed in yeast cells and has been used to treat vegetable material, in particular soy and fruit juice in the preparation of edible foodstuffs.

Description

New endo-xylogalacturonase

Invention field

The present invention relates to a kind of new endo-xylogalacturonase (endo-xylogalacturonase, XGH) and homologue.The invention still further relates to and in plant and pectous material working method, use endo-xylogalacturonase to produce fruit juice and other plant milk extract.

Background of invention

Zymin is through being usually used in the vegetable material course of processing, for example in the extraction and liquefaction step and their filtration and clarification steps of fruit and fruit juice.Commercial zymin comprises the enzyme mixture of depolymerized pectin polymkeric substance, and the pectin polymkeric substance is a kind of main component of plant cell wall.These enzymes comprise pectin lyase, polygalacturonase, Rohapect MPE, cellulase, xyloglucanase, Galactanase and arabanase (arabinanase).

Pectin is to occur as the moiety of higher plant cell wall in itself.They are present in blastema wall thin layer, and are embedded between the cellulosic fibre.The composition of pectin is different in different floristics, and relevant with the age and the ripening degree of fruit.The source of being rich in pectin is lemon and oranges and tangerines, and their polysaccharide content can be up to 30%.

Most of pectin polymkeric substance comprise the homotype polygalacturonic acid region and branched " hair " zone of " smooth "." slick " zone is made up of the homotype polygalacturonic acid skeleton of linearity." hair " zone of apple is made up of three kinds of different subunits: subunit's I is xylogalacturonase (the polygalacturonic acid skeleton has been replaced by wood sugar to a great extent); Subunit's II is short rhamnosyl polygalacturonic acid skeleton part, is rich in long araban, Polygalactan and/or arabogalactan side chain (" hair ") relatively; Subunit's III is a kind of rhamnosyl polygalacturonic acid oligomer, contains the skeleton that a staggered rhamnosyl and galacturonic acid residue are formed.

Many well-known polygalacturonases that are used for industrial food processing are " smooth " part of depolymerized pectin polymkeric substance only, and " hair " zone of not degrading.Therefore, be example with the Sucus Mali pumilae production process, because the not degraded part of pectin polymkeric substance causes the obstruction of filtration or ultra-filtration membrane, cause efficient filtration, cause production loss.

Existing report has several enzymes can degrade part hair district, for example the rhamnosyl polygalacturonic acid district of skeleton (subunit's III).These enzymes are known as rhamnosyl polygalacturonase (RGase), and wherein these enzymes have several types., the xylogalacturonase part (subunit's I) in " hair " zone digestion of still resisting enzyme so far, therefore, xylogalacturonase carries over as the inertia carbohydrate in the enzymatic inscribe digestive process of prior art.

Owing to other plant, resemble the existence of also finding xylogalacturonase in soybean and pea, watermelon, grape and the Pollen Pini as leguminous plants, the enzyme of these polymkeric substance of degrading will be useful in vegetable material processing.

Identified a kind of circumscribed polygalacturonase (42KDa, SDS-PAGE) ¹, it is not disturbed by single wood sugar side chain, with a kind of solvable " hair " pectin polysaccharide from soybean as substrate, the xylogalacturonase of can degrading.This kind of enzyme works in circumscribed mode, because the disaccharides that it produces galacturonic acid or is made up of wood sugar and galacturonic acid.This enzyme has been purified near homogeneous (component HTP2 and Q2), and has carried out partly characterizing.Compare (homotype of not degrading polygalacturonic acid) with known rhamnosyl polygalacturonase, this kind of enzyme is not very special for xylogalacturonase, and it also can act on pectic acid.In addition, this kind of enzyme can not digest the xylogalacturonase skeleton in mode at random, does not therefore also find to have the active enzyme of endo-xylogalacturonase so far.

Summary of the invention

The present invention comes from a kind of new endo-xylogalacturonase and encodes its separation and the sign of cDNA.Endo-xylogalacturonase cDNA sequence is seen SEQ.IDNo.1.Aminoacid sequence from the ORF of Nucleotide 98-1315 is seen SEQ.ID No.2.

That first part of the present invention provides is a kind of (for example separate and/or purifying) has the active polypeptide of endo-xylogalacturonase.A kind of polypeptide that comprises endo-xylogalacturonase also is provided, has for example a kind ofly comprised the described polypeptide of sequence of SEQ.ID No.2, or a kind of and its homologous polypeptide basically, perhaps had the fragment of the polypeptide of 5 amino acid whose SEQ.ID No.2 at least.

Preferably, polypeptide of the present invention has following one or more feature, that is: (1) has the endo-xylogalacturonase activity; (2) has from 2.5 to 6 optimum pH scope; (3) has optimum activity 50 ℃ to 70 ℃ temperature; And/or (4) have the molecular weight size from 40 to 50Kda.

" endo-xylogalacturonase activity " is defined as the ability that can cut the galacturonic acid polymer (as finding) that is replaced by wood sugar to small part in the internal sugar glycosidic bond in pectin.This activity allows to cut the adjacent polygalacturonic acid (the such unit of none is the end that is present in polymkeric substance, and this can cleavedly be relative with circumscribed active terminal units) of non-terminal units thus.Cutting preferably occur in [galacturonic acid (1,4) galacturonic acid] key.Preferably, polypeptide can not cut terminal xylose residues, for example [galacturonic acid (3-1) wood sugar] key in the galacturonic acid residue that wood sugar replaces.This polypeptide tends to cut between two adjacent non-wood sugar-substituted polygalacturonic acid units, can be replaced 40-80% by other glycosyl unit (as wood sugar) in the substrate polymer.

Two galacturonic acid residues of polypeptide cutting of the present invention can all be replaced by (wood sugar), perhaps have only a quilt (wood sugar) replacement or (preferably) all not to be replaced by (wood sugar).Selectively or in addition, two galacturonic acid residues can all be methylated, and perhaps have one can be methylated, perhaps (preferably) do not methylated.

Preferably, polypeptide of the present invention comes from microorganism, and it has the gene of the active a kind of enzyme of coding endo-xylogalacturonase.More preferably, this microorganism is a kind of microbe, and preferably fungi is filamentous fungus ideally.Preferred organism is: Aspergillus (Aspergillus), Trichoderma (Trichoderma), Penicillium (Penicillium), the mould genus of top spore (Acremonium), Fusarium (Fusarium), Humicola (Humicola), Neurospora (Neurospora), Mucor (Mucor), transparent little Scytalidium (Scytallidium), myceliophthora (Myceliophtora), Thielavia (Thielavia), blue stain fungi (Talaromyces), Thermomyces, Thermoascus, Chaetomium (Chaetomium), Sporotrichum (Sporotrichum), Corynascus, the kind of Calcarisporiella or Mycelia.Best organism is some kinds that come from aspergillus niger.(as Raper and Fennell definition, Aspergillus (Aspergillus), Williams﹠amp; Wilkins company, Baltimore, pp293-344,1965), include, but are not limited to aspergillus niger, Aspergillus awamori (Aspergillus awamori), Tabin aspergillus (Aspergillustubigensis), microorganism Aspergillus aculeatus (Aspergillus aculeatus), smelly aspergillus (Aspergillusfoetidus), aspergillus japonicus (Aspergillus japonicus) or Fructus Fici aspergillus (Aspergillusficuum) especially.

Second section of the present invention provides a kind of (for example separating and/or purifying), and code book is invented the polynucleotide of first part's polypeptide.For example, the invention provides a kind of polynucleotide of coding endo-xylogalacturonase, the aminoacid sequence of this xylogalacturonase enzyme is seen SEQ ID No2.Having the present invention further provides a kind of coding has and the SEQ ID No2 aminoacid sequence polynucleotide of the polypeptide of homologous aminoacid sequence basically.A kind of following polynucleotide that are selected from are also provided: the polynucleotide that (a) comprise nucleotide sequence shown in the SEQ ID No1 or its complementary sequence; (b) comprise can with the polynucleotide of the nucleotide sequence shown in the SEQ ID No1 or its fragment hybridization; (c) comprise can with the polynucleotide of nucleotide sequence shown in the SEQ ID No1 or its segmental complementary sequence hybridization; And/or (d) comprise can be with (a) and (b), (c) defined polynucleotide since the degeneracy of genetic code and with the polynucleotide of their degeneracys.

Polynucleotide of the present invention also comprise a kind of polynucleotide, its:

A. a kind of active polypeptide of endo-xylogalacturonase that has of encoding, these polynucleotide are: the encoding sequence of (1) SEQ ID No1; (2) can be optionally with (1) in the sequence of complementary sequence hybridization of sequence of definition; (3) with (1) or (2) in respectively the sequence of definition because genetic code and the sequence of degeneracy; Or

B. with (a) the middle polynucleotide complementary sequence that defines.

" can hybridize " speech meaning and be target polynucleotide of the present invention can with the nucleic acid that is used as probe (as the nucleotide sequence among the SEQ ID No1, or its fragment or its complementary sequence) to be significantly higher than the level hybridization of background.The hybridization that background level takes place may be because for example, and the existence of other polynucleotide (resembling dna molecular) is for example in screening-gene group or cDNA library.In this case, background is meant the level of the signal that interaction produces between the non-specific polynucleotide part that occurs in probe and the library, intensity is no more than with observed target polynucleotide special interactional 1/10, preferably is no more than 1/100.Interactional intensity can be measured, and for example, adopts the radiolabeled probe, as using ³²P.Will be described later suitable condition.

Preferably, polynucleotide of the present invention and polypeptide be from a kind of organism, for example the fungi of fungi, particularly Aspergillus.

The present invention also provides polynucleotide probes, and it comprises at least 15 nucleotide fragments of the invention described above polynucleotide.

In third part, the invention provides the carrier that comprises polynucleotide of the present invention, comprise cloning vector and expression vector, in the method for the 4th part growth, proper host cell is advanced in these carriers conversions or transfection, for example in can expressing polypeptide of the present invention or the present invention, carried out under the condition of sequence coded polypeptide.The 5th part provides the host cell that comprises polynucleotide of the present invention or carrier, and wherein the genome of these polynucleotide and host cell is allogenic." with the genome of host cell be allogenic " this speech meaning is in the natural genome that is not present in host cell of polynucleotide.Preferably, host cell is a yeast cell, the yeast cell of genus kluyveromyces (Kluyveromyces) or yeast belong (Saccharomyces), or a kind of fungal cell for example, for example Aspergillus.

Have the active polypeptide of the present invention of endo-xylogalacturonase and can in the 6th part, be used to handle the vegetable material that comprises plant pulp and plant milk extract.For example, they can be in order to handle apple pulp and/or Normal juice in the Sucus Mali pumilae production process.Polypeptide of the present invention and suitable carriers or comprise that the diluent of damping fluid combines are to produce a kind of composition/zymin.The present invention provides a kind of composition that comprises polypeptide of the present invention in the 7th part like this.This composition also can comprise other component, and such as one or more enzymes, for example polygalacturonase comprises inscribe arabanase and rhamnosyl polygalacturonase, cellulase and/or xylogalacturonase enzyme.

Therefore, polypeptide of the present invention and composition can be used in the vegetable material method for processing, degrade or the pectin fraction of modified plant material cell walls.Therefore in the 8th part, the invention provides the method for a kind of degraded or modified plant cell walls, this method comprises with polypeptide of the present invention or composition and contacting with plant cell wall.

The present invention also provides a kind of method of processing vegetable material, and this method comprises with polypeptide of the present invention or composition and contacting with vegetable material, with the pectin in degraded or the modified plant material.Preferably, vegetable material is plant pulp or plant milk extract.

Especially, degraded preferably includes the endo-type of the xylogalacturonase enzyme subunit of the pectin composition of plant cell wall is sheared.Preferably, vegetable material is fruit or vegetable and pulp, or fruit or vegetable extract, for example, and apple pulp or Sucus Mali pumilae.

It is a kind of by contact the vegetable material of the processing that obtains with vegetable material with polypeptide of the present invention or composition that the present invention also provides.Preferably, the vegetable material of processing is fruit or vegetables juice, for example Sucus Mali pumilae.

The present invention also provides a kind of minimizing plant milk extract method of viscosity, and this method comprises that polypeptide of the present invention or the composition with significant quantity contacts with plant milk extract, is contained in pectin in this plant milk extract with degraded.

Preferred characteristics of a part of the present invention and characteristic also are suitable for others.

Detailed Description A. polynucleotide

The invention provides a kind of polynucleotide, its:

A. a kind of active polypeptide of endo-xylogalacturonase that has of encoding, these polynucleotide are: the encoding sequence of (1) SEQ ID No.1; (2) optionally with (1) in the sequence of complementary sequence hybridization of sequence of definition; Or (3) are because the sequence of the nucleotide sequence degeneracy that defines in the degeneracy of genetic code and (1) or (2); Or

B. be one section with the polynucleotide complementary sequence described in (a).

Polynucleotide of the present invention also comprise the varient of the encoding sequence with the active SEQ ID of endo-xylogalacturonase No.1.Varient can be formed by interpolation, replacement and/or disappearance.Therefore these varients can have the ability in the internal shear galacturonic acid polymer.Common polynucleotide of the present invention comprise the successive nucleotide sequence, its under selective conditions can with the complementary sequence hybridization of the encoding sequence of SEQ ID No.1.

The complementary sequence of polynucleotide of the present invention and SEQ ID No1 encoding sequence can be to be significantly higher than the level hybridization of background.Background hybridization may take place, for example, because the existence of other cDNA in the cDNA library.Interact between the complementary sequence of polynucleotide of the present invention and SEQ ID No1 encoding sequence the signal level that produced generally than interact between other sequence and the SEQID No1 encoding sequence the signal that produces strong at least 10 times, preferably strong at least 100 times.Interactional intensity can be measured, for example adopt as ³²P mark radioactive probe.(for example generally can adopt low rigorous condition, 0.03M sodium-chlor, 0.03M Trisodium Citrate, at about 40 ℃), medium rigorous condition (for example, 0.03M sodium-chlor, 0.03M Trisodium Citrate, at about 50 ℃) and high rigorous condition is (for example, 0.03M sodium-chlor, the 0.03M Trisodium Citrate is at about 60 ℃) can realize optionally hybridizing.

One preferably can with the polynucleotide of the complementary sequence selective cross of the dna sequence dna of SEQ ID No1 and general at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% the homogeny that exists of encoding sequence of SEQ ID No1.The zone that covers has 20 at least, and preferably at least 30, for example at least 40, at least 50, at least 60 or preferred at least 100 successive Nucleotide perhaps most preferably are the total lengths that covers SEQ ID No1.

Combination between above-mentioned any different sequence homogeny and minimum length can be used for defining polynucleotide of the present invention, preferably adopt stricter combination (referring to that higher sequence homogeny covers longer length), for example above 25, the one section polynucleotide that preferably surpasses the sequence homogeny at least 90% of 30 Nucleotide perhaps surpass 40 Nucleotide and have the one section polynucleotide that reaches 95% sequence homogeny at least.

Can modify the encoding sequence of SEQ ID No1 by the method for nucleotide subsitution, for example from 1,2,3 to 10,25,50 or 100 nucleotide subsitutions.The polynucleotide of SEQID No1 can also be by one or more insertions and/or deletion (as identical with above-mentioned metathetical number) and/or in the extension of an end or two ends and modified.The general coding of adorned polynucleotide has the active polypeptide of endo-xylogalacturonase.After adorned sequence was translated, the displacement of degeneracy and/or displacement can cause the displacement of conserved amino acid, for example the part of relevant polypeptide in the 11st page table.

Polynucleotide of the present invention can comprise DNA or RNA.They may be strand or two strands.Their also inner polynucleotide that comprise the Nucleotide of synthetic or modification, prior art is known can carry out the modification of number of different types to polynucleotide, comprise methyl-phosphonate skeleton and thiophosphatephosphorothioate (phosphorothioate) skeleton, at 3 ' of molecule-and/or 5 '-terminal acridine or poly-lysine chain of increasing.Be to be understood that for the purposes of the present invention polynucleotide as described herein can be modified by the existing method in any this area.

Be appreciated that those skilled in the art can use conventional technology, do not influence the nucleotide subsitution of the peptide sequence of polynucleotide encoding of the present invention, to reflect any codon preference of waiting to express the host living beings of polypeptide of the present invention.

Polynucleotide of the present invention also can be used as primer (for example PCR primer), the primer that is used for other amplified reaction, be used as probe, for example adopt the conventional radioactivity or the demonstration probe of on-radiation method mark, perhaps these polynucleotide can be cloned in the carrier.

This class primer, probe and other segmental length is at least at 15, and preferably at least 20, for example at least 25,30 or 40 Nucleotide.Generally can reach the length of 40,50,60,70,100 or 150 Nucleotide.Probe and segmental length can surpass 150 Nucleotide, for example 200,300,400,500,600,700 length of nucleotides, even reach only than the short several Nucleotide (as 5 or 10 Nucleotide) of the encoding sequence of SEQ ID No1.

Polynucleotide of the present invention (resembling DNA) and primer can or pass through to produce in any methods such as those skilled in the art's available technology by reorganization, synthetic preparation.They also the technology of available standards clone.Polynucleotide provide with the form of separation and/or purifying usually.

Usually, primer will be produced by the synthetic mode, comprise the substep operation of the purpose nucleotide sequence of next Nucleotide.The method of finishing these tasks with automatic technology can get in the art.

Long polynucleotide with the method preparation of reorganization, are for example used PCR (polymerase chain reaction) clone technology usually.This according to a pair of primer of zone design of endo-xylogalacturonase gene to be cloned (for example relates to, about 15-30 Nucleotide a pair of), primer is contacted with mRNA that obtains from fungi, yeast, schizomycete or prokaryotic cell prokaryocyte or cDNA, under the condition that the amplification of purpose zone can take place, carry out the polymerase chain reaction, separate the fragment (for example, by purification reaction mixture on sepharose) of amplification and the DNA that reclaims amplification.Primer should be designed to contain suitable restriction enzyme site, so that the DNA of amplification can be cloned in the suitable cloning vector.

This technology can be used to obtain all or part of endo-xylogalacturonase sequence described herein.Corresponding to the cDNA of SEQ ID No.1 or the genomic clone of endo-xylogalacturonase gene, for example comprise that intron and promoter region are included within the present invention, its also available similar mode (as recombination method, PCR, clone technology) obtains from the genomic dna from fungi, yeast, schizomycete or prokaryotic cell prokaryocyte.

Although technology described herein is well known in the art, can be with reference to some documents especially Sambrook etc., molecular cloning, laboratory manual (Molecular Cloning, ALaboratory Manual) (1989) and Ausubel etc., modern molecular biology method (Current Protocols in Molecular Biology) (1995), John WileySons, Inc.

The polynucleotide that do not have 100% homogeny with SEQ ID No.1 but drop in the scope of the invention can obtain in many ways.Like this, the varient of endo-xylogalacturonase described herein can pass through, and such as the genome dna library of detecting from the certain limit organism, for example those methods as the organism in polypeptide of the present invention source obtain.In addition, also can obtain the protokaryon homologue of other fungi, plant or endo-xylogalacturonase, and these homologues and its fragment, it generally can be hybridized with SEQ ID No.1.These sequences can obtain by cDNA library or the genomic library of detecting from other kind, and in (for example wait until under the high rigorous condition, 0.03M sodium-chlor, the 0.03M Trisodium Citrate is at 50 ℃-60 ℃) detect these libraries with the probe that comprises all or part SEQ ID No.1.The nucleic acid probe that comprises all or part SEQ ID No.1 can be used to detect the cDNA library from other kind, for example the kind in the conduct of those descriptions polypeptide of the present invention source.

Plant the also available degenerate pcr of homologue and obtain, this PCR is with designing at the varient of coding conserved amino acid sequence and the primer of the target sequence between the homologue.This primer comprises one or more degeneracy sites, the low rigorous condition of used condition in the time of can using than the unique sequence primer cloned sequence that adopts at known array.

Perhaps, the polypeptide of this class can obtain by endo-xylogalacturonase or its varient are carried out site-directed mutagenesis method.For example, changing at the reticent codon of needs sequence, is useful in the codon preference with the particular host cell of this polynucleotide sequence of optimization expression.In order to introduce Restriction Enzyme inscribe site, perhaps, also need some other sequence to change in order to change by the character of the polypeptide of this polynucleotide encoding or function.

The present invention comprises the double-stranded polynucleotide that have polynucleotide of the present invention and complementary sequence thereof.

Polynucleotide of the present invention or primer can have a kind of show tags.Suitable mark comprise as ³²P and ³⁵The labelled with radioisotope of S, enzyme mark or other protein labeling are as vitamin H and DIG ^-Haptens.These marks can add on polynucleotide of the present invention or the primer, and can adopt the technology of knowing to detect.

The present invention also provides the polynucleotide of following code book invention polypeptide.Because these polynucleotide sequences can be used for recombinant production polypeptide of the present invention, therefore do not need these sequences to have the ability of hybridizing, though generally need this ability with SEQ ID No1.In addition, these polynucleotide can be labeled, and can as above make as needs.B. polypeptide

Polypeptide of the present invention comprises aminoacid sequence shown in the SEQ ID No2 or homologous sequence basically, the perhaps fragment of arbitrary sequence, and it can have the endo-xylogalacturonase activity.In general, the naturally occurring aminoacid sequence shown in the SEQ ID No2 is preferred.

Particularly, polypeptide of the present invention can comprise:

The described peptide sequence of a.SEQ ID No2;

Its homologous sequence between b. naturally occurring varient or different plant species; Perhaps

C. with (a) or (b) described sequence have the protein of at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence homogeny.

Varient can be, and is for example naturally occurring in fungi, bacterium, yeast or vegetable cell.It can bring into play function to go up identical mode substantially with the protein of SEQ ID No2.Equally, homologue should be a naturally occurring equivalent protein in another species between a kind of protein kind, and it has the endo-xylogalacturonase activity.

Homologue can for example be produced SEQ ID No2 polypeptide by following step in bacterium, yeast, fungi or the vegetable cell and obtain in suitable cell between varient and kind.Also can detect the clone that yeast, bacterium, fungi or vegetable cell library obtain to comprise homologue between varient and kind with above-mentioned probe.This clone can come by the reorganization known or synthetic technology to produce polypeptide of the present invention through routine operation.

Preferably, the protein of polypeptide of the present invention and SEQ ID No2 has at least 60% homogeny, more preferably is at least 70%, 80%, 90%, 95%, 97% or at least 99% sequence homogeny.The length that covers should reach 20 successive amino acid at least, and preferably at least 30, for example at least 40,60,100,200 or 300 successive amino acid perhaps reach the total length of SEQ ID No2.

Can provide polypeptide of the present invention by homologue between the sequence of SEQ ID No2 polypeptide and varient and kind is modified.Can carry out amino acid whose displacement, for example from 1,2 or 3 to 10,20 to 30 displacements.Also can carry out same number of insertion or disappearance.Polypeptide after the modification generally keeps the activity of endo-xylogalacturonase.

According to the displacement that following table can be guarded, be arranged in same district, table the second hurdle, in the preferred third column with between the amino acid of delegation can the phase double replacement.

Aliphatics	Nonpolar	GAP
			ILV
		Polarity-neutral		CSTM
	NQ
			Polarity-electrically charged	DE
	KR
		Aromatic series			HFWY
Other			NQDE

Polypeptide of the present invention also comprises the fragment of above-mentioned full-length polypeptide and varient thereof, comprises the fragment of sequence shown in the SEQ ID No2.This class fragment has generally kept the activity of endo-xylogalacturonase.Fragment can have 10,15,20,30,50,100 or 200 amino acid whose length at least.

Polypeptide of the present invention can be isolating form basically.Be appreciated that this polypeptide also can be considered to isolating basically when mixing with carrier that does not influence its specific purpose or diluent.Polypeptide of the present invention also can be in the form of purifying basically, and polypeptide of the present invention generally should comprise in the polypeptide of preparation and surpass 50%, for example surpasses the polypeptide of the present invention of 80%, 90%, 95%, 98% or 99% (by weight).Also can carry out chemically modified to polypeptide of the present invention, for example posttranslational modification for example can or comprise one or more adorned amino-acid residues by glycosylation (one or many).

Also can modify these polypeptide by adding histidine residues or T7 label, thus help to identify and purifying they, perhaps, will discuss below by increasing their secretions from cell of signal peptide sequence promotion.

If desired, polypeptide of the present invention can be produced by the synthetic method, although normally adopt recombination method manufacturing discussed below.

Particularly preferred polypeptide of the present invention comprises the polypeptide of being made up of the 19th～406 amino acid of aminoacid sequence shown in the SEQ ID No2, does not constitute the terminal signal peptide of the 1st～18 amino acid whose N-among the SEQ ID No2 because it does not contain.These polypeptide and fragment thereof can contain the defined amino acid in front and change.

When recombinant expressed production polypeptide of the present invention, use yeast and fungal host cells; expectation can provide the needed posttranslational modification of best biologic activity (for example, proteolysis processing, myristoylation, glycosylation, brachymemma and tyrosine, Serine or Threonine phosphorylation).C. reorganization aspect

Polynucleotide of the present invention can be incorporated into a reorganization reproducibility carrier, in for example a kind of clone or the expression vector.Carrier can be used to duplicate this nucleic acid molecule in suitable host cells.Therefore in yet another embodiment, the invention provides a kind of method for preparing polynucleotide of the present invention, comprise polynucleotide of the present invention are introduced a kind of reproducible carrier, carrier is imported a kind of consistency host cell, cultivate host cell under certain condition so that carrier duplicates.Can from host cell, reclaim carrier.Suitable host cells will be described in next section with expression vector.Expression vector

Preferably, polynucleotide of the present invention are connected effectively with a kind of regulating and controlling sequence in carrier, and this regulating and controlling sequence can make the host cell expression encoding sequence, and promptly carrier is an expression vector.The meaning of " effectively connect " speech is meant that described composition is adjacent and allow them to bring into play their function in a predefined manner on the position.A kind of regulating and controlling sequence, for example promotor, enhanser or other expression regulation signal " are connected " effectively with encoding sequence, place it in suitable position, make encoding sequence can with the matched condition of control sequence under realize to express.

Carrier can be, for example has replication origin, randomly expresses plasmid, virus or the phage vector in the promotor of polynucleotide of the present invention and promoter regulation zone randomly.

The dna sequence dna of this polypeptide of encoding can be imported into the part of proper host cell as expression construct, and wherein this dna sequence dna can be connected effectively with the expression signal that the dna sequence dna that can instruct in the host cell is expressed.The step of converting that this expression construct is transformed appropriate host is very familiar 3,4 to one skilled in the art.This expression construct can be used for transforming the host, as the part of the carrier that carries a kind of selected marker, perhaps this expression construct as independent molecule with the carrier cotransformation that carries a kind of selected marker.Carrier can comprise one or more selected markers.

Preferred selected marker 3,4 include but are not limited to: all if can be used to transform most of filamentous fungus and zymic common tags gene, as acetamidase gene or cDNA (from the amdS gene or the cDNA of Aspergillus nidulans (A.nidulans) or aspergillus oryzae (A.oryzae) or aspergillus niger) or provide opposing to resemble the gene of G418, Totomycin, phleomycin or F-1991 resistance (benA).In addition, can use more specificity selected marker, for example need the nutrient defect type mark of the host strain of corresponding sudden change: for example URA3 (from yeast saccharomyces cerevisiae or from the similar gene of other zymic), pyrG (from Aspergillus nidulans or aspergillus niger) or argB (from Aspergillus nidulans or aspergillus niger).In a kind of preferred embodiment,, expression construct is imported the back selected marker is deleted from transformed host cells, thereby acquisition can be produced the transformed host cell of the polypeptide that does not contain selected marker according to the method described in the EP-A-0635574.

Other mark comprises ATP synthetic enzyme, subunit 9 (oliC), Orotidine-5 '-'-phosphoric acid-decarboxylase (pvrA), (this also can be used for yeast to bacterium G418 resistant gene, but can not be used for fungi), ampicillin resistance gene (being used for intestinal bacteria), neomycin resistance gene (genus bacillus) and intestinal bacteria uidA gene, coding beta-Glucuronidase (GUS).Carrier can be used for external, for example is used to prepare RNA or is used for transfection or transformed host cell.

For most filamentous fungus and yeast, preferred expression construct is to be incorporated in the host cell gene group to obtain stable conversion.But, for some yeast, suitable episomal vector system is arranged also, wherein can mix this expression construct and be used for stable and high-caliber expression.Example wherein comprises respectively derived from 21 μ of yeast belong and genus kluyveromyces and the carrier of pKD1 plasmid.For the expression construct that is integrated in the host cell gene group, expression construct can be incorporated into genomic site at random, or utilizes the homologous recombination method to be integrated in predetermined target site, and preferred target site comprises a cance high-expression gene.Ding Yi a cance high-expression gene here, its mRNA for example should account for 0.05% (w/w) of the total mRNA amount of cell at least under inductive condition, perhaps its gene product accounts for 1% (w/w) of total protein of cell amount at least, perhaps, for the excretory gene product, the excretory level reaches 0.1g/L at least.The example of many suitable cance high-expression genes will be provided here subsequently.

For a kind of expression construct of given host cell, comprise usually following (according to respect to the coding strand 5 '-end of coding said polypeptide in the first part to 3 '-end direction) element that connects effectively: the promoter sequence that (1) can instruct the dna sequence dna of coding said polypeptide to transcribe in given host cell; (2) optional, one can be instructed described polypeptide to be secreted into signal peptide sequence in the substratum in given host cell; (3) dna sequence dna of encoding mature and preferred activity form polypeptide, and preferably (4) can stop the transcription termination region (terminator) of transcribing in the dna sequence dna downstream of coding said polypeptide.

By selecting allogenic control region, for example promotor, secreting signal peptide and terminator etc. are used to improve the zone of expression level, the enhancing of Nucleotide that can realize encoding more than the polypeptide of the present invention is expressed, if desired, also can improve the secretion level of target protein in the expressive host of having selected and/or the derivable control of expression of polypeptides of the present invention is provided.

Remove the natural promoter of coding polypeptide gene of the present invention, other promotor also can be used to instruct polypeptide expression of the present invention.Can be according in required expressive host, instructing polypeptide expression efficient of the present invention to select promotor.

Available multiple 3,4 can instruct the promotor of transcribing of polypeptide of the present invention in host cell.Preferably, promoter sequence comes from previously defined cance high-expression gene.Promotor from the cance high-expression gene example preferably from and/or be included in and be used for the predetermined target site that expression construct is integrated, include but are not limited to the gene of coding glycolytic ferment, as the gene of triose-phosphate isomerase (TPI), phosphoglyceraldehy-de dehydrogenase (GAPDH), phosphoglyceric kinase (PGK), pyruvate kinase (PYK), ethanol dehydrogenase (ADH) and coding amylase, glucoamylase, xylosidase, cellobiohydrolase, beta-galactosidase enzymes, ethanol (methyl alcohol) oxydase, elongation factor and ribosomal protein.The example of special suitable cance high-expression gene comprise from the LAC4 gene of the kind of for example genus kluyveromyces, respectively from the methanol oxidase gene (AOX and MOX) of the kind of Hansenula (Hansenula) and Pichia (Pichia), from glucoamylase (glaA) gene, aspergillus oryzae TAKA-amylase gene, the gpdA gene of Aspergillus nidulans and the cellobiose hydrolase gene of T.reesei of aspergillus niger and Aspergillus awamori.

The strong composing type that can use in the expressed in fungi host and/or the example of inducible expression promotor have xylosidase (xlnA), phytase, ATP synthetic enzyme, subunit 9 (oliC), triosephosphate isomerase (tpi), ethanol dehydrogenase (AdhA), α-Dian Fenmei (amy), amyloglucosidase (AG-derives from the glaA gene), acetamidase (amdS) and the glyceraldehyde-3-phosphate dehydrogenase promotors such as (gpd) available from fungal gene.

The example of strong Yeast promoter derives from ethanol dehydrogenase, lactalase, glycerol 3-phosphate acid kinase and the isogenic promotor of triosephosphate isomerase.

The example of strong bacterium promotor is the promotor of α-Dian Fenmei and SPO2, and from the promotor of extracellular protease gene.Host cell and expression

Preferably, polypeptide produces with a kind of form of secretory protein, and in the case, the dna sequence dna of the mature form of polynucleotide is connected with the dna sequence dna of coded signal sequence effectively in the coding schedule expression constructs.Preferably, signal sequence is natural (homologous) for the dna sequence dna of coded polypeptide.In addition, signal sequence is external (allogenic) for the dna sequence dna of coded polypeptide, and signal sequence is preferably endogenous for the host cell of expressible dna sequence therein in the case.The example of the signal sequence of suitable yeast host cell is the signal sequence from yeast α-factor gene.Similarly, a kind of signal sequence that is suitable for filamentous fungus is for example from filamentous fungus glucoamylase gene (as aspergillus niger glaA gene).This can use with amyloglucosidase (AG) promotor self, also can unite use with other promotor.The heterozygosis signal sequence also can be used on the row of scope of the present invention.

Preferred allos secretion homing sequence has amyloglucosidase (AG) gene that derives from fungi (glaA-18 and 24 amino acid whose versions, for example from Aspergillus), α-factor gene (for example yeast belong and genus kluyveromyces) or alpha-amylase gene (bacillus).

In the downstream of code book invention polypeptid DNA sequence, expression construct preferably comprises 3 '-non-translational region, wherein has one or more Transcription Termination sites, is also referred to as terminator.The source of terminator is not crucial.Terminator can be to be natural terminator for code book invention polypeptid DNA sequence.But preferably, in yeast host cell, use the yeast terminator to reach and in fungal host cells, use the fungi terminator.More preferably, terminator should be endogenous for the host cell of the dna sequence dna of expressing coding polypeptide of the present invention therein.

On the other hand, the invention provides the method for preparing polypeptide according to the present invention, be included under the condition of the vector expression that can make the encoding sequence that has the polypeptide of the present invention of encoding, cultivate, and reclaim polypeptide expressed by aforementioned expression vector transfection or transformed host cells.

On the other hand, the present invention also provides involved polynucleotide of the present invention or carrier transfection or transformed host cells.Preferably, polynucleotide of the present invention are carried in the carrier so that duplicate and express.Should select the cell with above-mentioned carrier consistency, for example prokaryotic cell prokaryocyte (as bacterium), fungi, yeast or vegetable cell.

According to the characteristics of the polynucleotide of code book invention polypeptide, and/or the needs of the further processing of expressing protein, preferred eukaryotic host cell is as yeast or fungi.In general, yeast cell is better than the fungal cell, because their easy handlings.But some protein are difficult to secrete from yeast cell, perhaps can not correctly process (for example excessive glycosylation in yeast cell) in some cases.In these cases, should select the fungal host body.

When polypeptide of the present invention will also can be selected heterologous host with the formal representation of essentially no other pectin degrading enzyme.Can reach this requirement by selecting the host who does not generally produce these enzymes, for example Kluyveromyces lactis (Kluyveromyces lactis).

The present invention includes the process that the method for the dna sequence dna by recombinant expressed coded polypeptide is produced polypeptide of the present invention.For this purpose, dna sequence dna of the present invention can be used for the exchange of gene amplification and/or expression signal, for example promotor, secretory signal sequence, thus in homologous or allogenic host cell, allow economic polypeptide production.Homology host cell herein is defined as a kind of and dna sequence dna institute deutero-kind for a kind of or with a kind of host cell of varient.

Proper host cell is prokaryotic micro-organisms bacterium or more preferably be most eukaryotes for example preferably, and fungi for example is such as yeast or filamentous fungus or vegetable cell.

Because it can enter nutrient solution by secretory protein, it is very suitable as heterologous host from the bacterium of the kind of bacillus.Other suitable bacterium as the host is that those belong to the bacterium of (Pseudomonas) from streptomyces (Streptomyces) and false unit cell.A kind of yeast host cell of dna sequence dna of preferred expression coded polypeptide is from yeast belong, genus kluyveromyces, Hansenula, Pichia, Yarrowia and Schizosaccharomyces (Schizosaccharomyces).More preferably yeast host cell is the kind that is selected from down dependent of dead military hero: yeast saccharomyces cerevisiae, Kluyveromyces lactis (being also referred to as Kluyveromyces marxianus lactic acid mutation (Kluyveromyces marxianus var.1actis)), Hansenula polymorpha (Hansenulapolymorpha), complete red saccharomyces pastorianus (Pichia pastoris), Yarrowia lipolytica and schizosaccharomyces pombe (Schizosaccharomyces pombe).

Yet most preferably express coded polypeptide dna sequence dna be filamentous fungal host cell.Preferred filamentous fungal host cell is selected from down the kind of dependent of dead military hero: Aspergillus, Trichoderma, Fusarium, Penicillium, the mould genus of top spore, Neurospora, Thermoascus, myceliophthora, Sporotrichum, Thielavia and Talaromyces.More preferably, filamentous fungal host cell is to belong to aspergillus oryzae, Aspergillus sojae (Aspergillus sojae), Aspergillus nidulans, from aspergillus niger group's kind (by Raper and Fennell definition, Aspergillus, Willians ﹠amp; Wilkins company, Baltimore, pp293-344,1965).This includes but are not limited to: aspergillus niger, Aspergillus awamori, Tabin aspergillus, microorganism Aspergillus aculeatus, smelly aspergillus, Aspergillus nidulans, aspergillus japonicus, aspergillus oryzae and Fructus Fici aspergillus, and further comprises Trichoderma reesei, fusarium graminaria (Fusariumgraminearum), Penicllium chrysogenum (Penicillium chrysogenum), Acremoniumalabamense, red arteries and veins spore mould (Neurospora crassa), Myceliophtorathermophilum, Sporotrichum cellulophilum and Thielavia terrestris.

The example of preferred expressive host is a fungi in the scope of the invention, for example the kind of kind of Aspergillus (being described in EP-A-184438 and EP-A-284603) and Trichoderma; Bacterium (being described in EP-A-134048 and EP-A253455) such as the kind of the shaft-like genus of gemma, for example, the kind of subtilis, Bacillus licheniformis (Bacillus licheniformis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Rhodopseudomonas; And yeast, for example kind of kind of genus kluyveromyces (being described in EP-A-096430, for example Kluyveromyces lactis and EP-A-301670) and yeast belong, for example yeast saccharomyces cerevisiae.Host cell is cultivated and recombinant production

According to the present invention, the production of polynucleotide of the present invention can be subjected to the influence of cultured microorganism expressive host, and this expressive host is transformed by one or more polynucleotide of the present invention, and cultivates at conventional nutrition fermention medium.

Can cultivate with step well known in the art according to recombinant host cell of the present invention.For the combination of each promotor and host cell, the culture condition that is of value to the dna sequence dna expression of coded polypeptide is an available.Tire and promptly stop to cultivate when reaching required cell density or polypeptide, and with known step recovery polypeptide.

Fermention medium can comprise the known substratum that contains carbon source (for example glucose, maltose, molasses etc.), nitrogenous source (for example ammonium sulfate, ammonium nitrate, ammonium chloride etc.), organic nitrogen source (for example yeast extract, malt extract, peptone etc.) and inorganic nutrients source (for example phosphoric acid, magnesium, sodium, zinc, iron etc.).Randomly, also can comprise inductor (for example apple MHR, pectin or xylogalacturonase).

Select the suitable medium can be according to the expression host cell of selecting and/or according to the regulation and control needs of expression construct.These substratum are well known to those skilled in the art.If desired, substratum can comprise additional component and resists other potential contaminative microorganism with the expressive host that helps transforming.

The time that fermentation can be carried out is 0.5-20 days, with in batches, the operation format of successive or batch feeding is suitable 0-45 ℃ temperature range, for example, the pH between 2 and 10.Preferred fermentation condition is the pH of temperature in 20 and 37 ℃ of scopes and/or between 3 and 9.Appropriate condition is normally according to expressive host and want expressed protein to select.

After the fermentation, can cell be removed from fermenting broth by centrifugal or filtration.After removing cell, polypeptide of the present invention is recyclable, if need, uses the ordinary method purifying and separates.D. plant or contain the working method of pectin material

Plant and pectous material comprise part and the plant milk extract of plant pulp, plant.In scope of the present invention, the extract of vegetable material is can be by any material that derives from vegetable material that extracts (machinery and/or chemical), processing or get by other isolation technique.Extract can be juice, nectar, base-material (base) or the enriched material that made by them.Vegetable material can comprise or from vegetables, for example, Radix Dauci Sativae, celery, onion, beanpod or bean (soybean, soya bean, pea) or fruit, for example, the operatic circle or seed fruit (apple, pears, Wen Bai etc.), grape, tomato, citrus (orange, lemon, bitter orange, mandarin orange), melon, plum class, cherry, Ribes nigrum L., red currant, raspberry, blackberry, blueberry, big fruit blueberry, pineapple and other tropical fruit, tree and its part (for example from pine tree pollen).According to the present invention, apple and Sucus Mali pumilae are particularly preferred.

Polypeptide of the present invention can be used to handle the vegetable material that comprises plant pulp or plant milk extract thus.For instance, it can be used for handling apple pulp and/or original fruit juice in the production process of Sucus Mali pumilae.They also can be used for handling fluent meterial or solid food or edible food ingredient.Usually, polypeptide of the present invention is with the carrier of suitable (solid or liquid) or comprise that the thinner of damping fluid uses, to produce a kind of composition or zymin.

Polypeptide is stably prepared with liquid or dry powder form usually.Usually, product is made a kind of composition that randomly comprises stabilizing buffer for example and/or sanitas.Composition can comprise that also other can digest the enzyme of vegetable material or pectin, and other polygalacturonase for example is such as inscribe-arabanase, rhamnosyl polygalacturonase and/or polygalacturonase.Some is used enzyme fixing or to combine or enter solid carrier particle with solid carrier particle be preferred on solid substrate.Composition also can comprise the enzyme of multiple other degrading plant material, for example cellulase and other polygalacturonase.

Therefore polypeptide of the present invention and composition can be used in the vegetable material working method, with the pectin composition of degraded or modified plant material cell walls ²

Usually, polypeptide of the present invention is used as aforesaid composition/zymin.This composition generally is added into as in the plant pulp that obtains with mechanical workout (resemble and pulverize or squeezing).Composition and plant are hatched together, carried out usually 10 minutes to 5 hours, such as 30 minutes to 2 hours, preferably about 1 hour.Preferably, processing temperature is 10-55 ℃, for example from 15-25 ℃, most suitably, and about 20 ℃, and material to be processed per ton can use 10-300g, 30-70g preferably, the most suitably about enzyme of 50g.All enzymes or its composition can be in order or are joined simultaneously in the plant pulp.According to the composition of zymin, vegetable material can at first be macerated (being the thick soup shape) or liquefaction.Output, the viscosity of extract and/or the quality of extract of using polypeptide machined parameters of the present invention for example to extract can be improved.

In addition, or remove the above, polypeptide of the present invention can join in the Normal juice that obtains from squeezing or liquefaction plant pulp.About dosage, temperature, hold-time aspect, the mode of handling Normal juice and processing plant pulp is similar.In addition, other enzyme is in for example the enzyme discussed of those fronts is also included within.Common incubation conditions is identical with the earlier paragraphs description.After Normal juice and polypeptide of the present invention are hatched together, fruit juice through centrifugal or (ultrafiltration) filter to produce finished product.

The composition that contains polypeptide of the present invention also can use in the preparation process of fruit or vegetables juice.

The end product of these steps is usually in for some time of 1 minute to 1 hour of 85 ℃ of thermal treatment, carries out under the condition of deactivation polypeptide of the present invention partially or completely.

Because to the high special effect of pectin, polypeptide of the present invention also can be used for preparing the pectin of the characteristic with modification, for example, is used for the modified gel ability of special applications.

Polypeptide of the present invention also can add the animal-feed that is rich in pectin or xylogalacturonase, in for example soy-containing food, with the fragmentation of improvement plant cell wall, thereby improves the utilization of animal to plant nutrition.If soak in advance or fluid food is preferred, polypeptide of the present invention can add in the feed of feed or storage.Advantageously, polypeptide of the present invention can continue the xylogalacturonase in the degradation in vivo food.The polypeptide of originated from fungus of the present invention generally has only lower pH, and can discharge important nutritive substance in the such sour environment of the stomach that resembles animal.Therefore the present invention has also conceived (as animal) feed or the grain that comprises one or more polypeptide of the present invention.

Polypeptide of the present invention also can be used in the process of the substitute (or surrogate) of producing milk from soybean.The substitute humans and animals of this milk all can use.A common high viscosity that problem is the soybean slurries in the process of these milk substitutes of preparation causes needs undesirable to the concentration of slurry dilution to dried solid 10-15%.The zymin that comprises polypeptide of the present invention can add, or adds in the course of processing of slurries, can make under the dried solid situation of a kind of higher concentration (40-50% usually) to process.Enzyme also can be used in the making of flavour product, for example makes of soybean.The detection of pectin degrading enzyme

New detection method described herein and substrate make can be identified and the activity of definite inscribe-xylogalacturonase enzyme.Yet these methods can be used for detecting the activity whether other pectin degrading enzyme has inscribe-xylogalacturonase enzyme.

The substrate that can be used for this kind detection method can comprise the tragakanta that has passed through strong acid treatment.Preferred acid is trifluoroacetic acid (TFA).Tragakanta can be randomly by saponification and/or its by alkaline purification, for example a kind of alkali metal hydroxide, for example sodium hydroxide.

Another part of the present invention relates to the detection method of identifying or detecting a kind of polypeptide of energy depolymerized pectin.Activity can be inscribe-xylogalacturonase enzyme or, maybe can be pectin lyase, polygalacturonase, esterase, cellulase, xylogalacturonase enzyme, galacturonic acid enzyme, arabanase and rhamnosyl polygalacturonase.This detection method comprises:

A. the substrate that provides earlier paragraphs to describe is as the substrate of candidate compound (being generally a peptide species); With

B. contact substrate with candidate compound, detect the minimizing that whether causes any carbohydrate.

The amount energy measurement of the minimizing of carbohydrate comes out.If necessary, they can be compared with the amount of carbohydrate in the control experiment that does not have candidate compound to exist then.

Measurement can comprise that BCA detects.This can comprise with the minimizing of the carbohydrate that exists measures the amount that Cu (II) is reduced to Cu (I).This can be by contacting with two cinchonic acids (BCA), and the amount that forms of definite BCA-Cu (I) mixture.

The present invention now is described about the following embodiment that only is for example but does not limit to.Embodiment is arranged in the drawings.

Fig. 1 is that (subunit's I is an xylogalacturonase to the imaginary structural representation of advantage group to a kind of apple MHR (the hair district of modification) that the highest molecular weight arranged, subunit's II is the skeleton that is rich in the arabinan side chain, and subunit's III is a rhamnosyl polygalacturonase oligomer.The distribution of ethanoyl does not provide, but major portion is thought to be positioned within subunit's III.Annotate: Gal A=galacturonic acid; The rham=rhamnosyl; The gal=semi-lactosi; The xyl=wood sugar; The ara=pectinose);

Fig. 2 is the collection of illustrative plates (structure is described in embodiment 1) of the carrier pCVlacK according to the present invention;

Fig. 3 is the HPAEC figure after xylogalacturonase enzyme (a kind of polypeptide of the present invention) degraded xylogalacturonase is used in explanation;

Fig. 4 is the figure of explanation with the HPSEC of before the xylogalacturonase enzyme liberating and degraded back xylogalacturonase;

Fig. 5 illustrates with degrade the fully Malsi-ToF mass spectrum of product of xylogalacturonase of xylogalacturonase enzyme;

Fig. 6 A-G shows to use separately respectively and common HPSEC elution profile with inscribe-arabanase, rhamnosyl polygalacturonase, xylogalacturonase enzyme liberating MHR-S;

Fig. 7 and 8 is respectively the figure of HPSEC and HPAEC, and type of elution shows with xylogalacturonase enzyme liberating soy pectin; And

Fig. 9 shows that the multiple sequence of PG, XghA and RHG sequence (amino acid identical with the XghA sequence replaces with a point) compares, for the breach that acquisition ideal sequence is relatively introduced is pointed out with (-).Conserved amino acid shadow representation in all plants, fungi and protokaryon PG.Annotate: the Atub=Tabin aspergillus; The Anig=aspergillus niger; The Aac=microorganism Aspergillus aculeatus.

The constructed embodiment 1.1 of embodiment 1 Tabin aspergillus cDNA expression library: the structure of expression vector

Initial vector pGBHSA20 (CBS997.96) comprises the promotor and the terminator of Kluyveromyces lactis lactase gene (lac4), a kind of G418 selection marker and the escherichia coli plasmid pTZ18r that is used for breeding this host.Kluyveromyces lactis KARSCEN box ¹⁷(the A.A doctor Winkier present of Dutch Leiden university's cytobiology and genetics system) is cloned into the single Sma I site of this carrier.Gained carrier called after pCVlacK (Fig. 2).Single Hind III and Xho I site lay respectively at lac4 promotor and terminator flank, can be used as the cloning site of synthetic cDNA from Tabin aspergillus poly (A) RNA.The separation of embodiment 1.2:poly (A) RNA and cDNA are synthetic

Conidial three increments of Tabin aspergillus are originally with 10 ⁶Spore/ml density is inoculated in the 300ml substratum, wherein contains (every liter) 6gNaNO ₃, 0.5gKCl, 1.5gKH ₂PO ₄, 0.5gMgSO ₄(pH6.5), 1ml 1000xTimberlake spore composition (every ml, 50mgEDTA, 22mgFeSO ₄.7H ₂O, 5mgMnCl ₂.2H ₂O, 22mgZnSO ₄.7H ₂O, 1.6mgCuSO ₄.5H ₂O, 1.7mgCoCl ₂.6H ₂O, 1.5mgNa ₂MoO ₄.2H ₂O, 11mgH ₃BO ₃, be adjusted to pH6.5) and the 10ml100xTimberlake VITAMIN (every ml, 0.2mg thiamines-HCl, 0.2mg riboflavin, the 0.2mg niacinamide, the 1mg benadon, 0.02mg pantothenic acid, 0.4 μ m vitamin H is adjusted to pH5-6), 1g yeast extract, 5gSoyoptim ^TM(soyflour from French Societe IndustridlleOleagineux degreasing, baking).Culture is hatched on 150 rev/mins rotary shaker in 28 ℃.A kind of mycelium of culture is gathered in the crops after 10 hours in inoculation, and the mycelium of other two kinds of cultures is gathered in the crops after 16 and 24 hours in inoculation.From the mycelium that 1g cleans and pushes, extract total RNA with RNAzol method (Cinna/Biotecx).Use Qiagen ^TMOligotex post (Westburg) separates Poly (A) RNA.Merge and be positioned at 10,16 and 24 hours equivalent Poly (A) RNA of time point.Use ZAP-cDNA synthetic agent box (Stratagene ^TM) synthesize cDNA with the method for modifying below: with synthetic first chain of Superscript II reversed transcriptive enzyme (GibcoBRL).Add to final volume 28.5 μ l to 7.5 μ g Poly (A) RNA, 2 μ l connecting joint-primers and the water that do not have a RNA enzyme.This mixture in 70 ℃ hatch 10 minutes after in cooled on ice.Add following component: the first chain damping fluid of 10 μ l 5x, 5 μ l 0.1M DTT, 3 μ l, the first chain methyl nucleoside acid mixture and 1 μ l RNA enzyme encapsulant.These were hatched 10 minutes in 25 ℃, hatched 2 minutes for 42 ℃ then.Then, add 2.5 μ l Superscript II reversed transcriptive enzymes (200U/ μ l), mixing was hatched 50 minutes in 42 ℃.Second improvement of present method is to connect Hind III joint to have replaced EcoR I joint.

The cDNA village, often used in village names separates size with Sephacryl S-500 post.Fraction under the wash-out does not contain any cDNA from the post at first, but second and the third stage divide and comprise maximum sized cDNA.Ensuing fraction probably contains a large amount of relatively non-full-length cDNAs, and is useless to making up the library.Use ClontechLigation Express ^TMTest kit with second and the cDNA that divides of the third stage be connected to the Hind III and the Xho I site of pCVlacK expression vector.Each connects mixture and is converted into electroreception attitude intestinal bacteria XL-Blue MRF in two batches ¹Cell.Four parts transform suspension bed board (LB+50 μ g/ml penbritin) on 32 agar plates.37 ℃ hatch 16 hours after, obtain 7366 transformants.By the 2.5mlLB substratum is poured on the flat board, scrape the cell harvesting bacterium then; The 0.5ml cell suspension is added in the glycerine in-80 ℃ of preservations; Remaining 2ml is used as DNA and separates (Qiagen ^TMSpin miniprep test kit).If find that the conversion subnumber on each flat board is lower, 2.5ml is forwarded on second, third or the 4th flat board.This produces 325 single transformants in 22 storehouses.The DNA that mixes each storehouse moderate is used as the conversion of Kluyveromyces lactis.Embodiment 1.3: expression library transforms Kluyveromyces lactis

Incubated overnight be grown in lactic acid yeast kluyveromyces strain CBS2359 on 30 ℃ the YPD (the Bacto-peptone of 10g/l yeast extract, 20g/l, the glucose of 20g/l) in the fresh YPD of 150ml, dilute 3000-, 600-, 300 and 100 times, and on 160 rev/mins rotary shaker, hatched 6 hours in 30 ℃.Optical density value is the cell of the culture of 0.7-1.0 with the electroreception attitude of preparing ⁶

Transform the cell of electroreception attitude with the intestinal bacteria library DNA of 1 μ g merging.Use BioradGenepulser ^TMSystem transform, setting is 1.4kV, 200 ohm and 25 μ F.The selection of transformant is with double-deck YPD flat board (YPD of the Bacto-agar of 20g/l): bottom contains 50 μ g/mlG418, and the upper strata is nonselective.The transformation mixture of 660 μ L is taped against on 80 double-layer plates.The equal portions liquid of drawing 1.5 and 15 μ L is to each flat board.Approximately obtain 10,000 transformants.

Embodiment 2 substrates prepare embodiment 2.1: prepare MHR-S from apple

After apple is handled with polygalacturonase, be separated as the filter membrane retentate from the hair district (MHR) of the modification of apple, MHR is formed MHR-S by saponification thereupon ⁸Embodiment 2.2: xylogalacturonase is synthetic in the tragakanta

5g tragakanta (Sigma, St.Louis, MO, the U.S.) is suspended in the ice-cold distilled water of 990ml.The 5M NaOH solution that 10ml is ice-cold joins in this solution.After 24 hours, saponified tragakanta (sGT) is fully dialysed with distilled water in 4 ℃, is concentrated into 1L under reduced pressure.To gentle acid cleavage, the trifluoroacetic acid (TFA) of 7.65ml is joined in this sGT solution.The final concentration of TFA is 0.1M in the solution.SGT/TFA solution is heated to boiling point in microwave oven, hatched in boiling water bath then 1 hour.Last hydrolysate is fully dialysed and freeze-drying to distilled water in 4 ℃.This step produces the material (further being called wood sugar-polygalacturonic acid) of 2.61g.Embodiment 2.3: the sign of wood sugar-polygalacturonic acid

In order to determine to be derived from the sugar component of tragakanta and wood sugar-polygalacturonic acid originally, sample 1M H ₂SO ₄(100 ℃, 3 hours) ⁸Hydrolysis, in order to use quantitatively monose of gas-chromatography (GC), neutral sugar is converted into their alditol acetate form.Carry out colorimetric with a hydroxyl hexichol and identify uronic acid composition in the hydrolyzed solution ⁸The table 1 of face more as follows of the molar percentage (mol/%) of the sugared composition in the tragakanta (GT) and xylogalacturonase (XG) originally.

Table 1

Sugar (mol/%]
								Substrate	Rhamnosyl (Rha)	Fucose (Fuc)	Pectinose (Ara)	Wood sugar (Xyl)	Semi-lactosi (Gal)	Glucose (Glc)	Polygalacturonic acid (GlaA)
GT	?2	?5	?26	?17	?7	?7	?36
								XG	?3	?0	?1	?25	?9	?6	?56

Mild acid hydrolysis has been removed Arabic glycosyl and fucosido effectively from polysaccharide, yet the ratio of GlaA:Xyl does not change basically.

The ethanoyl of tragakanta and methyl-esterified degree are measured with high-pressure liquid phase (HPLC) ⁷Tragakanta methylates and the degree of ethanoylization is respectively about 75% and 20% (calculating with mole methyl or acetyl group/every mole of GlaA).All methyl and acetyl group have been removed in the saponification of glue.The molecular weight distribution of polymkeric substance is carried out on three Bio-Gel TSK pillars (40XL, 30XL and 20XL) continuously with high pressure molecular exclusion chromatography (HPSEC) method ⁸, the gentle acidic hydrolysis of saponified tragakanta is accompanied by the minimizing of molecular weight, although the product that obtains still has high molecular weight.According to the elution profile of HPSEC, do reference compound with amylopectin, the estimation molecular weight of xylogalacturonase is approximately 1100kDa.

The xylogalacturonase proof can be resisted the inscribe-polygalacturonase of all tests and the enzyme degradation of rhamnosyl polygalacturonase effectively.

Embodiment 3 usefulness BCA methods screening library embodiment 3.1: the growth of transformant and zymin

About 10000 Kluyveromyces lactis that produce among the embodiment 1.3,3500 single clones of picking forward in each isolating hole of porous culture plate.Transformant in the porous culture plate is containing in the substratum I of 80ng/mL microbiotic G418 in 30 ℃ of growths 48 hours, contains in the water of the every 500ml of substratum (pH6.0): D-N.F,USP MANNITOL, 10.00g; NH ₄H ₂PO ₄, 1.50g; KH ₂PO ₄, 0.25g; (NH ₄) ₂SO ₄, 0.50g; CaCL ₂.2H ₂O, 0.01g; MgSO ₄.7H ₂O, 0.15g; Trace element H ₃BO ₃, 375 μ g; CuSO ₄.5H ₂O, 40l μ g; KI, 75 μ g; MnSO ₄.4H ₂O, 300 μ g; Na ₂MoO ₄, 150 μ g; ZnSO ₄.7H ₂O, 300 μ g; FeCl ₃.6H ₂O, 200 μ g) and the VITAMIN calcium pantothenate, 500 μ g; VitB1,500 μ g; Meso-inositol, 500 μ g; Pyridoxol, 500 μ g; Nicotinic acid, 500 μ g; Vitamin H, 5 μ g), and with 35 culture plates preserve with 15% glycerine storage liquid form.

These transformants are inoculated in a series of 35 porous culture plates, wherein contain the 200 identical μ L substratum of 80ng/mL microbiotic G418, do copy board simultaneously.The Kluyveromyces lactis transformant was grown two days in 30 ℃ incubator.At Hermle ^TM3000 rev/mins of centrifugation cells in the zk380 whizzer.Embodiment 3.2: degradation of substrates

25 μ L supernatant sucking-offs with every hole carefully forward in the new porous culture plate, add the substrate buffer solution (from MHR-S or xylogalacturonase or the sGT/TFA of embodiment 2) of 25 μ L0.2% or add 100mM sodium-acetate buffer, pH5.0.In 30 ℃ incubator, after the overnight incubation, detect the increase of recuding sugars with the BCA method.

Embodiment 3.3:BCA method

The BCA method is based on by recuding sugars monomer and oligomer and makes Cu (II) revert to the principle of Cu (I).Two cinchonic acids (BCA) and Cu (I) form a kind of mixture.This mixture presents intense violet color, and it can detect with spectrophotometer.Along with this color of the increase of concentration of reduced sugar can be deepened.The used method of the present invention is that the improvement 9 of currently known methods is used to screen purpose.

Step is included in the porous culture plate mixes 10 μ L and comprises reducing sugar and 90 μ L water and 100 μ LBCA reagent from embodiment 3.2 samples.Pass through to mix two kinds of solution every day, A and B, 1: 1 (v/v) prepares fresh BCA reagent.Solution A contains 54.28gNa at every liter of distilled water ₂CO ₃, 24.20gNaHCO ₃And 1.942gNa ₂BCA.Solution B contains 1.248gCuSO in every liter of distilled water ₄5H ₂O and 1.262g L-Serine.The culture plate cover lid that will contain sample, reagent and water was hatched 1 hour in 80 ℃ of incubators.The cooling culture plate is after 15 minutes, with microplate reader (SLT Laboratory Instruments, Australia; The EAR400 type) reads the absorption value of 550nm.The detection line of semi-lactosi shows that it is linear measuring in 0 to 125 μ M semi-lactosi scope.

The transformant that produces than blank high 0.1 unit absorption value in BCA detects is selected, and cultivates again, and adopts wood sugar-polygalacturonic acid to carry out BCA as substrate and detect, and checks the ability of its degraded xylogalacturonase.Find three transformants that produce the xylogalacturonase enzyme.

Embodiment 4 embodiment 4.1: the sign of the cDNA of coding xylogalacturonase enzyme

With found after the restriction enzyme digestion pattern of analyzing these insets the same, all plasmid insets of these three kinds of transformants are identical.Kluyveromyces lactis transformant 27E8 has the xylogalacturonase enzymic activity, is used as the granulated glass sphere method and separates the pCVlacK expression plasmid ¹⁰After plasmid transformation escherichia coli and the propagation, the cDNA inset is cut out from pCVlacK with Hind III/Xho I digestion.This digestion discharges 1.0 and the fragment of 0.4Kb, and reason is an inherent Hind III site that appears in the nucleotide sequence of back.The dna sequence dna of cDNA inset be used on the lac4 regulating and controlling sequence and based on 5 ' of cDNA sequence-and 3 '-Auele Specific Primer carried out double-stranded evaluation.The dna sequence dna of cDNA inset and the aminoacid sequence of inferring are showed in SEQ ID No.1.There is 5 '-non-translated sequence of 20 Nucleotide the upstream of ATG transcription initiation codon.The polyA tail is found to have in the non-translated sequence back of 130 Nucleotide in TAA termination codon downstream.A kind of 406 amino acid whose protein of the open reading frame of 1218 Nucleotide (xghA) coding are showed in SEQ ID No.2, called after XghA.According to (3 ,-1) rule indication, potential signal shearing site is between 18 and 19 positions ¹¹From therefore ATG codon initial sum is positioned at 5 '-non-coding region of 20 base pairs with TAA codon terminated ORF after, and there are 3 '-non-coding region of 130 base pairs and polyA tail to be connected on its downstream.Cover the sequence content of sequence TCATCATGGC very similar initial translation in higher eucaryotic cells of ATG initiator codon ¹⁴XghA cDNA is coded in N-terminal 18 amino acid whose apparent signal sequences, at Ala ¹⁸And Ala ¹⁹Between the shearing site of signal peptide is arranged.Find at Asn ¹⁷⁸-Ser-Thr and Asn ³⁰¹-Val-Thr has two potential N-glycosylation sites.

Show with albumen database comparing amino acid sequence, with the polygalacturonase sequence of protokaryon, fungi, plant and with the rhamnosyl polygalacturonase A of Aspergillus and the sequence homology of B.Use from the sequence of EMBL database aminoacid sequence and compare XghA.XghA and inscribe PG have the sequence similarity of 31-39%, with the circumscribed PG of Tabin aspergillus 44% sequence similarity are arranged.The sequence similarity of XghA and 2RGH-A has 30% (aspergillus niger) and 32% (microorganism Aspergillus aculeatus), and very limited with the similarity of the RGH-B of aspergillus niger.

Fig. 9 shows the multiple sequence analysis relatively of Xgh and PG and RGH-A.Multiple sequence comparison shows that four conservative amino acid structure territories, and this is the description of first polygalacturonase of plant, fungi and bacterial origin being carried out ¹⁵When all PG series arrangement that obtains from database retrieval are got up to compare, have only four little amino acid fragments to guard: NXD, DD, HG and RXK (shade among Fig. 9, wherein X represents a kind of variable amino acid).The key amino acid that relates in hydrolysis reaction is thought in the histidine residues of three asparagicacid residues of structural domain I and structural domain II and structural domain III.These structural domains are conservative fully in XghA.Supposing that the 4th structural domain contains relates to substrate bonded amino acid.The arginine residues of this structural domain is the glycine residue among the XghA.This structural domain is not too cautious on the RGH sequence, have only two to guard in three asparagicacid residues, and Histidine is replaced by glycine.Embodiment 4.2:Southern engram analysis

The Tabin aspergillus genomic dna of the copy number of XghA gene after with several enzymic digestions carries out Southern engram analysis (not having display result).Hybridize with the 1.0kbHind III fragment of xghA down in rigorous condition (65 ℃ and 0.2xSSC) and low rigorous condition (60 ℃ and 1xSSC), clearly illustrate that one hybridized fragment.This confirms that the xghA gene is that single copy exists in the Tabin aspergillus genome.

The expression of embodiment 5 enzymes

The Kluyveromyces lactis transformant of expressing the cDNA of endo-xylogalacturonase is relayed to the reagent pipe from porous culture plate glycerine storage liquid: the substratum (seeing embodiment 3.1) that adds the 1-2mL that contains 80ng/mLG418 in the glycerine storage liquid of 10 μ L.These cultures are in 30 ℃ of growths two days on 200 rev/mins rotary shaker, are used for being inoculated in containing in the Erlenmeyer culturing bottle that 20mL adds antibiotic same medium.For the production of extensive enzyme, culture is used for being inoculated in and contains in the culturing bottle of 1 liter of Erlenmeyer that 500mL adds antibiotic same medium.Cell was grown two days on 200 rev/mins rotary shaker in 30 ℃.Centrifugal culture is with sedimentation cell, and supernatant liquor is used for doing purifying.

Crude zyme preparation is at Hitrap ^TMCarry out pre-concentration on the Q ion exchange column (Pharmacia Biotech, Sweden), flow velocity is the 0.3mL/ branch.Wash-out in FPLC system carries out (Pharmacia Biotech, Sweden) from the initial damping fluid of piperazine (pH5.0) (buffer A) of 20mM to the 0.5MNaCl (buffer B) piperazine (pH5.0) solution of 20mM with a kind of salt gradient.Adopt following gradient: in 1 minute to 10% B damping fluid, in 19 minutes to 35% B, through 2 minutes to 100% B and continue 3 minutes.Adopt the method inspection activity of embodiment 3 descriptions and collect active fraction.With piperazine (pH5.0) damping fluid of 20mM with 3 times of diluted samples, last MiniQ post (Pharmacia Biotech, Sweden).The initial damping fluid of piperazine (pH5.0) of going up employing 20mM in Smart system (Pharmacia Biotech, Sweden) carries out wash-out to the linear pH gradient of the HCl of 10mM, and flow velocity is 0.4mL/ minute.Collect active fraction and adopt SDS-PAGE to check.The method that adopts silver to dye is found a protein band in the position of the about 60kDa of molecular weight.Infer with molecular weight based on the dna sequence dna prediction be that the difference of 45kDa (seeing embodiment 4) is owing to glycosylated reason.

Embodiment 6pH and temperature are to the influence of enzymic activity

The enzyme of the purifying that the method for describing as embodiment 5 obtains is used for the sign of enzyme.In the measurement of t=0 as blank.For the mensuration of pH stability, the McIlvaine damping fluid of pH scope from 2.5 to 8, will not add the enzyme preincubate 1 hour of the purifying of substrate.Enzyme-to-substrate was hatched 2 hours then, and the increase of reducing sugar is measured with embodiment 3 described methods.Enzyme is stable in the scope of pH from 3 to 6.

Be to measure the optimal conditions of pH and temperature, the enzyme of purifying and substrate were pH scope from 2.5 to 8 or from 20 to 80 ℃ of incubations of temperature range 2 hours.After this, the increase of reducing sugar is measured with embodiment 3 described methods.Temperature when being 60 ℃ and pH3.0 enzyme optimum activity is arranged.Show at the scope enzyme of pH2.5 to 5.0 and to be no more than 50% activity.When the activity during pH2.5 still has pH3.0 95% of maximum activity.There is not to measure the value when being lower than pH2.5.

The mode of action of embodiment 7 xylogalacturonase enzymes

Supernatant liquor with the Kluyveromyces lactis clone who produces the xylogalacturonase enzyme is monitored by high-efficiency anion displacement chromatography (HPAEC) and efficient molecular exclusion chromatography (HPSEC) the degraded of xylogalacturonase (tragakanta of modification, embodiment 2.2).

Dionex carbopack PA1 post with 4 * 250ml specification carries out HPAEC.Carry out wash-out with 0.1MNaOH (solution A) and the 1M NaOAc (solution B) in 0.1M NaOH.Adopt following gradient: from 0 to 62%B, to 100%B, 100%B continues 5 minutes then in 5 minutes in 50 minutes.Enzyme does not produce wood sugar (being expected at 5 minutes hold-time) and galacturonic acid (being expected at 15 minutes hold-time), even hatches after 8 hours and do not have yet.Only have oligomer to discharge, minimum oligomer was found in about 22 minutes hold-time: this is wood sugar-galacturonic acid dimer.(among Fig. 3, rolled off the production line center line (B) (A): t=1 hour: t=4 hour, top line (C): t=8 hour hatch).

Carry out HPSEC:Bio-Gel TSK 40 (300 * 7.5mm is from Biorad) with three successive posts, Bio-Gel TSK 30XL (300 * 7.5mm is from Biorad) and TSK GelG2500P XL (300 * 7.8mm is from TosoHaas).Fig. 4 illustrates that the high molecular part of xylogalacturonase is by degraded promptly (polymkeric substance before (B) representative degraded of reaching the standard grade, the polymkeric substance after (A) representative degraded of rolling off the production line).

When with Maldi-ToF mass spectrograph monitoring degraded product, identify that the product of finding is shown in table 2.Table 2

Oligomer	The composition of degraded product	Crest number (among Fig. 4)	Molecular weight (Da)
				Dimer	galAxy1	?1	?349.1
Tripolymer	GalA2xy1	?2	?525.1
				The tetramer	GalA2xy12 GalA3xy1	?3a,3b	?657.3,701.3
Pentamer	GalA3xy12 GalA4xy11	?4a,4b	?833.4,877.4
				Six aggressiveness	GalA4xy12	?5	?1009.5
Heptamer	GalA4xy13 GalA5xy12	?6a,6b	?1141.6,1185.5
				Eight aggressiveness	GalA6xy12	?7	?1361.5

When hatching, MHR-S and XghA form two kinds of products.These products even just appearance after hatching the very short time.

These results show that xylogalacturonase degraded with internal-cutting way by the xylogalacturonase enzyme.The figure that obtains with those from comparing that polygalacturonic acid digestion obtains, it is very clear that what do not form the MHR-S degraded product is the polygalacturonic acid oligomer.This shows that XghA produces the galacturonic acid oligomer that is replaced by wood sugar.

When the supernatant liquor of the Kluyveromyces lactis transformant that will produce the xylogalacturonase enzyme and polygalacturonic acid are hatched, do not find the degraded of this substrate.

Embodiment 8 xylogalacturonase enzymes are with the fully degraded of other enzyme to MHR-S

Degraded for research MHR-S, with 200 μ L in the NaOAc of 50mM pH5.0 damping fluid 0.3%MHR-S solution and the xylogalacturonase enzyme of 5 μ L purifying, 5 μ L inscribe arabanases, 5 μ L rhamnosyl polygalacturonases or hatch jointly with the combination of these enzymes, add in order or simultaneously.Not enzyme-added MHR-S is used as contrast.

Resemble as described in the embodiment 7, the degraded of MHR-S is monitored with HPSEC.The result shows in 6G at Fig. 6, (b) representative contrast of wherein reaching the standard grade, and (d) representative of rolling off the production line is hatched with enzyme.Hatch be with:

A: arabanase;

B: xylogalacturonase enzyme;

C: rhamnosyl polygalacturonase:

D: inscribe-arabanase and xylogalacturonase enzyme add in proper order;

E: inscribe-arabanase and xylogalacturonase enzyme add jointly;

F: inscribe-arabanase and rhamnosyl polygalacturonase add in proper order; And

G: inscribe-arabanase, rhamnosyl and xylogalacturonase enzyme add jointly.

Fig. 6 B shows the xylogalacturonase enzyme MHR-S that can degrade: can be observed to the lower molecular weight material and move.Inscribe-arabanase (Fig. 6 A) and rhamnosyl polygalacturonase (Fig. 6 C) also produce moving of number molecular weight., in once hatching, use two kinds of different enzymes can obtain preferably the result (Fig. 6 D, inscribe-arabanase and xylogalacturonase enzyme add in proper order; 6E, inscribe-arabanase and xylogalacturonase enzyme add jointly; Add in proper order with 6F inscribe-arabanase and rhamnosyl polygalacturonase).The difference of Fig. 6 D and 6E is significant: add inscribe arabanase and xylogalacturonase enzyme jointly and add much effective than order.Fashionable when three kinds of enzymes are added jointly, the almost completely degraded of high molecular weight material is possible (Fig. 6 G).

Embodiment 9: xylogalacturonase enzyme and other enzyme use jointly and improve filtering rate

See by experiment whether the xylogalacturonase enzyme can prevent in filtration procedure that filter membrane from stopping up.The apple MHR-S that adopts embodiment 2.1 described method preparations is as a kind of substrate.A kind of at 50mM acetic acid, 0.5% solution in the damping fluid of pH4.0 and the combination of three kinds of enzymes: the common incubation of inscribe-arabanase, rhamnosyl polygalacturonase and xylogalacturonase enzyme (ea/rg/xgh), combination with two kinds of enzymes: the common incubation of inscribe-arabanase and rhamnosyl polygalacturonase (ea/rg), with with the common incubation of xylogalacturonase enzyme (xgh), condition was hatched 17 hours respectively at 30 ℃.Solution filters under 2 bar pressures with the Amicon device that the 30KD filter membrane is housed.Filtered liquid weight increases in time.The results are shown in Fig. 5.

Embodiment 10 xylogalacturonase enzymes are to the degraded of soy pectin fraction

Separated and the sign of a kind of solvable fraction 1MASS of alkaline soyabeen grists (being rich in the material of pectin) ¹⁶A kind of at 50mM, 0.25% 1MASS solution of pH5 sodium-acetate buffer wherein contains 0.01% NaN ₃, hatch with the xylogalacturonase enzyme.With the distribution that HPSEC comes the analyzing molecules amount, study the release of oligomerization degraded product in 30 ℃ of Digestive systems that obtain after hatching 24 hours with HPSEC.Analyze as embodiment 7 described methods.

Fig. 7 shows the variation of the molecular weight distribution of measuring as HPSEC: at the material (curve c) that the xylogalacturonase enzyme is handled, peak value appears at about 20 minutes, represents high molecular weight material, be reduced to the initial substance value 70% (curve a).

In Fig. 8, shown the result that HPSEC analyzes.The material (curve b) that the xylogalacturonase enzyme is handled is compared with blank (curve (a)), the xylogalacturonase enzyme has caused the release of characteristic wood sugar galacturonic acid dimer (doing sign with an X) and other undetermined oligomer (peak on X right side) as can be seen, and the peak that occurs with Fig. 6 among the embodiment 7 is similar.

Reference

1.G.Beldman, L.A.M.van den Broeck, H.A.Schols, M.J.FSearle-van Leeuwen, K.M.J van Laere and A.G.J.Voragen (1996) biotechnology communication (Biotechnology Letters) 18:707-712

2.C.Grassin and P.Fauquembergue (1996) pectin and polygalacturonase, biotechnology progress (Progress in Biotechnology) 14:453-462

3.Goosen Deng the people., 1992, " conversion of filamentous fungus and gene regulating: summary " rolled up at applied mycology handbook (Handbook of Applied Mycology) the 4th: " fungal organism technology " (Fungal Biotechnology), D.K.

4.Romanos Deng the people, yeast (Yeast) 8:423-488 (1992)

5.Arora R.P.Elander and K.G.Mukerji compile Marcel Deker In., New York, 151-195 page or leaf

6．K.N.Faber,P.Haima,W.Harder,M。Veenhuis, G.AB (1994), modern genetics (Current Genetics) 25:305-310

7.A.G.J.Voragen, H.A.Schols and W.Pilnik, (1986) FoodHydrocoll1:65-70

8.H.A.Schols, M.A.Posthumus, A.G.J.Voragen (1990) carbohydrate research (Carbohydrate Research) 206:117-129

9.J.D.Fox, J.F.Robyt (1991) analytical biochemistry (Analytical Biochemistry) 195:93-96

10.M.A.Sobanski and Didlinson (1995) Measurement for Biochemistry (BiotechnologyTechniques) 9:225-230

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13.H.A.Schols, C.C., J.M.Geraerds, M.J.F.Searle van Leeuwen, F.J.M.Kormelink, A.G.J.Voragen (1990) carbohydrate research (CarbohydrateResearch) 206:105-115

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Sequence table (1) general information:

(ⅰ) applicant:

(A) name: GIST-BROCADES B.V.

(B) street: Wateringseweg 1

(C) city: Delft

(E) country: Holland

(F) postcode: 2611 XT

(ⅱ) denomination of invention: novel endo-xylogalacturonase

(ⅲ) sequence number: 2

(ⅳ) computer-reader form:

(A) media type: disk

(B) computer: IBM compatible

(C) operating system: PC-DOS/MS-DOS

(D) software: PatnetIn Release#1.0 Version#1.25 (EPO)

(ⅴ) current application information:

The application number: the information of N/A (2) SEQ.ID.NO.1:

(ⅰ) sequence feature:

(A) length: 1602 base-pairs

(B) type: nucleic acid

(C) chain: two strands

(D) topological structure: linearity

(ⅱ) molecule type: cDNA

(ⅲ) the false plan: non-

(ⅳ) antisense: no

(ⅵ) initial source

(A) biology: Tabin aspergillus

(ⅸ) feature

(A) title/keyword: CDS

(B) position: 98..1318 CTCGAG is the XhoI site

The sequence description of (ⅹ ⅰ) SEQ.ID.NO.1: GCTTGTGTTT CTTAGGAGAA TTATTATTCT TTTGTTATGT TGCGCTTGTA GTTGGAAAAG 60 GTGAAGAGAC AAAGCTTGAA TTCCGAAATC GCTCATC ATG GCG CTA TAT CGT AAC 115

                                     Met Ala Leu Tyr Arg Asn

                                       1               5 CTC TAC CTT CTG GCC AGC CTT GGG CTA AGC AGT GCT GCT CCC TCC AAG         163 Leu Tyr Leu Leu Ala Ser Leu Gly Leu Ser Ser Ala Ala Pro Ser Lys

     10                      15                  20 GTC CAG CGA GCC CCG GAT TCT TCC ATT CAT GCT CGC GCT GTC TGT ACC         211 Val Gln Arg Ala Pro Asp Ser Ser Ile His Ala Arg Ala Val Cys Thr

     25                  30                  35 CCG ACC GCA GGA GGC GAT TCG TCC ACC GAC GAT GTC CCC GCC ATC ACC         259 Pro Thr Ala Gly Gly Asp Ser Ser Thr Asp Asp Val Pro Ala Ile Thr

 40                  45                  50 GAG GCC CTC AGC TCG TGC GGA AAT GGT GGC ACC ATC GTC TTC CCC GAG         307 Glu Ala Leu Ser Ser Cys Gly Asn Gly Gly Thr Ile Val Phe Pro Glu  55                  60                  65                  70 GGC AGC ACC TAC TAC CTC AAC AGT GTG CTG GAC TTG GGC AGC TGC AGT         355 Gly Ser Thr Tyr Tyr Leu Asn Ser Val Leu Asp Leu Gly Ser Cys Ser

             75                  80                  85 GAT TGC GAC ATC CAG GTG GAA GGT CTT CTG AAG TTC GCC AGC GAT ACC         403 Asp Cys Asp Ile Gln Val Glu Gly Leu Leu Lys Phe Ala Ser Asp Thr

         90                  95                 100 GAT TAC TGG AGC GGT CGC ACT GCC ATG ATC AGT GTT TCC AAT GTA GAT         451 Asp Tyr Trp Ser Gly Arg Thr Ala Met Ile Ser Val Ser Asn Val Asp

    105                 110                 115 GGT TTG AAG CTG CGC TCA TTG ACT GGA TCT GGT GTC ATT GAT GGC AAT         499 Gly Leu Lys Leu Arg Ser Leu Thr Gly Ser Gly Val Ile Asp Gly Asn

120                 125                 130 GGC CAG GAT GCG TGG GAT CTC TTT GCT TCG GAC AGT AGT TAC TCA CGC         547 Gly Gln Asp Ala Trp Asp Leu Phe Ala Ser Asp Ser Ser Tyr Ser Arg 135                 140                 145                 150 CCG ACG CTC TTG TAC ATC ACT GGC GGC AGC AAC CTA GAA ATC TCC GGG         595 Pro Thr Leu Leu Tyr Ile Thr Gly Gly Ser Asn Leu Glu Ile Ser Gly

            155                 160                 165 CTG CGT CAA AAG AAT CCA CCT AAC GTG TTC AAC TCG GTC AAG GGT GGC         643 Leu Arg Gln Lys ASn Pro Pro Asn Val Phe ASn Ser Val Lys Gly Gly

        170                 175                 180 GCC ACT AAT GTC GTC TTC TCC AAC CTG AAG ATG GAT GCC AAC TCC AAG         691 Ala Thr ASn Val Val Phe Ser Asn Leu Lys Met Asp Ala ASn Ser Lys

    185                 190                 195 TCG GAC AAT CCG CCC AAG AAC ACT GAT GGG TTC GAC ATT GGC GAG AGT         739 Ser Asp Asn Pro Pro Lys Asn Thr Asp Gly Phe Asp Ile Gly Glu Ser

200                 205                 210 ACC TAT GTG ACC ATC ACC GAG GTC ACC GTA GTC AAC GAT GAC GAC TGT         787 Thr Tyr Val Thr Ile Thr Glu Val Thr Val Val Asn Asp Asp Asp Cys 215                 220                 225                 230 GTC GCC TTC AAG CCC AGT TCC AAC TAC GTG ACA GTG GAC ACG ATC AGC         835 Val Ala Phe Lys Pro Ser Ser Asn Tyr Val Thr Val Asp Thr Ile Ser

            235                 240                 245 TGC ACC GGC TCC CAT GGA ATT TCC GTG GGA TCA TTA GGA AAG TCG AGC         863 Cys Thr Gly Ser Mis Gly Ile Ser Val Gly Ser Leu Gly Lys Ser Ser

        250                 255                 260 GAC GAC TCG GTC AAG AAC ATT TAT GTC ACG GGC GCA ACT ATG ATC AAC         931 Asp ASp Ser Vel Lys Asn Ile Tyr Val Thr Gly Ala Thr Met Ile Asn

    265                 270                  275 TCC ACC AAA GCC GCC GGG ATC AAG ACT TAT CCG AGT GGA GGC GAC CAC         979 Ser Thr Lys Ala Ala Gly Ile Lys Thr Tyr Pro Ser Gly Gly Asp His

280                 285                 290 GGT ACC TCC ACG GTC AGC AAT GTG ACC TTC AAC GAT TTC ACT GTG GAC        1027 Gly Thr Ser Thr Val Ser Asn Val Thr Phe Asn Asp Phe Thr Val Asp 295                 300                 305                 310 AAC TCC GAC TAT GCC TTC CAG ATC CAG AGC TGC TAT GGC GAG GAC GAT        1075 ASn Ser Asp Tyr Ala Phe Gln Ile Gln Ser Cys Tyr Gly Glu Asp Asp

            315                 320                 325 GAC TAT TGC GAG GAA AAC CCG GGC AAC GCC AAA CTG ACT GAT ATA GTC        1123 Asp Tyr Cys Glu Glu Asn Pro Gly Asn Ala Lys Leu Thr Asp Ile Val

        330                 335                 340 GTG TCA AGC TTC AGT GGG ACA ACC AGT GAC AAG TAC GAT CCG GTC GTG        1171 Val Ser Ser Phe Ser Gly Thr Thr Ser Asp Lys Tyr Asp Pro Val Val

    345                 350                 355 GCC AAC CTC GAC TGC GGT GCG GAT GGA ACT TGT GGC ATC TCC ATC AGT        1219 Ala Asn Leu Asp Cys Gly Ala Asp Gly Thr Cys Gly Ile Ser Ile Ser

360                 365                 370 GGG TTC GAT GTC AAG GCG CCA TCG GGC AAG TCT GAA GTG TTG TGC GCC        1267 Gly Phe Asp Val Lys Ala Pro Ser Gly Lys Ser Glu Val Leu Cys Ala 375                 380                 385                 390 AAC ACC CCG TCT GAT TTG GGC GTC ACT TGC ACT TCG GGG GCT TCG GGC        1315 Asn Thr Pro Ser Asp Leu Gly Val Thr Cys Thr Ser Gly Ala Ser Gly

The information of 395 400 405 TAAATAGCTT TGGCCGGGTT GCTTTCTGAA TCCACTGAGT GGAGGTCTTC TTCGGGTTTG, 1375 ATATTTTGTA TGGTCGTGTG TATAGCAGAA TGTGACAATA GAATTAGTGA AATTGCCATT, 1435 CTTTTCGAAA GACAAAAAAA AAAAAAAAAA AAAAAAAAAA ACTCGAGAAT TTATACTTAG, 1495 ATAAGTATGT ACTTACAGGT ATATTTCTAT GAGATACTGA TGTATACATG CATGATAATA, 1555 TTTAAACGGT TATTAGTGCC GATTGTCTTG TGCGATAATG ACGTTCC, 1602 (2) SEQ.ID.NO.2:

(ⅰ) sequence feature:

(A) length: 406 amino acid

(B) type: amino acid

(D) topological structure: linear (ⅱ) molecule type: the sequence description of protein (ⅹ ⅰ) SEQ.ID.NO.2: Met Ala Leu Tyr Arg Asn Leu Tyr Leu Leu Ala Ser Leu Gly Leu Ser

1               5                  10                  15 Ser Ala Ala Pro Ser Lys Val Gln Arg Ala Pro Asp Ser Ser Ile His

         20                  25                  30 Ala Arg Ala Val Cys Thr Pro Thr Ala Gly Gly Asp Ser Ser Thr Asp

35??????????????????40??????????????????45Asp?Val?Pro?Ala?Ile?Thr?Glu?Ala?Leu?Ser?Ser?Cys?Gly?Asn?Gly?Gly

50??????????????????55??????????????????60Thr?Ile?Val?Phe?Pro?Glu?Gly?Ser?Thr?Tyr?Tyr?Leu?Asn?Ser?Val?Leu?65??????????????????70??????????????????75??????????????????80Asp?Leu?Gly?Ser?Cys?Ser?Asp?Cys?Asp?Ile?Gln?Val?Glu?Gly?Leu?Leu

85??????????????????90??????????????????95Lys?Phe?Ala?Ser?Asp?Thr?Asp?Tyr?Trp?Ser?Gly?Arg?Thr?Ala?Met?Ile

100?????????????????105?????????????????110Ser?Val?Ser?Asn?Val?Asp?Gly?Leu?Lys?Leu?Arg?Ser?Leu?Thr?Gly?Ser

115?????????????????120?????????????????125Gly?Val?Ile?Asp?Gly?Asn?Gly?Gln?Asp?Ala?Trp?Asp?Leu?Phe?Ala?Ser

130?????????????????135?????????????????140Asp?Ser?Ser?Tyr?Ser?Arg?Pro?Thr?Leu?Leu?Tyr?Ile?Thr?Gly?Gly?Ser145?????????????????150?????????????????155?????????????????160Asn?Leu?Glu?Ile?Ser?Gly?Leu?Arg?Gln?Lys?Asn?Pro?Pro?Asn?Val?Phe

165?????????????????170?????????????????175Asn?Ser?Val?Lys?Gly?Gly?Ala?Thr?Asn?Val?Val?Phe?Ser?Asn?Leu?Lys

180?????????????????185?????????????????190Met?Asp?Ala?Asn?Ser?Lys?Ser?Asp?Asn?Pro?Pro?Lys?Asn?Thr?Asp?Gly

195?????????????????200?????????????????205Phe?Asp?Ile?Gly?Glu?Ser?Thr?Tyr?Val?Thr?Ile?Thr?Glu?Val?Thr?Val

210?????????????????215?????????????????220Val?Asn?Asp?Asp?Asp?Cys?Val?Ala?Phe?Lys?Pro?Ser?Ser?Asn?Tyr?Val225?????????????????230?????????????????235?????????????????240Thr?Val?Asp?Thr?Ile?Ser?Cys?Thr?Gly?Ser?His?Gly?Ile?Ser?Val?Gly

245?????????????????250?????????????????255Ser?Leu?Gly?Lys?Ser?Ser?Asp?Asp?Ser?Val?Lys?Asn?Ile?Tyr?Val?Thr

260?????????????????265??????????????????270Gly?Ala?Thr?Met?Ile?Asn?Ser?Thr?Lys?Ala?Ala?Gly?Ile?Lys?Thr?Tyr

275?????????????????280?????????????????285Pro?Ser?Gly?Gly?Asp?His?Gly?Thr?Ser?Thr?Val?Ser?Asn?Val?Thr?Phe

290?????????????????295?????????????????300Asn?Asp?Phe?Thr?Val?Asp?Asn?Ser?Asp?Tyr?Ala?Phe?Gln?Ile?Gln?Ser305?????????????????310?????????????????315?????????????????320Cys?Tyr?Gly?Glu?Asp?Asp?Asp?Tyr?Cys?Glu?Glu?Asn?Pro?Gly?Asn?Ala

325?????????????????330?????????????????335Lys?Leu?Thr?Asp?Ile?Val?Val?Ser?Ser?Phe?Ser?Gly?Thr?Thr?Ser?Asp

340?????????????????345?????????????????350Lys?Tyr?Asp?Pro?Val?Val?Ala?Asn?Leu?Asp?Cys?Gly?Ala?Asp?Gly?Thr

355?????????????????360?????????????????365Cys?Gly?Ile?Ser?Ile?Ser?Gly?Phe?Asp?Val?Lys?Ala?Pro?Ser?Gly?Lys

370?????????????????375?????????????????380Ser?Glu?Val?Leu?Cys?Ala?Asn?Thr?Pro?Ser?Asp?Leu?Gly?Val?Thr?Cys385?????????????????390?????????????????395?????????????????400Thr?Ser?Gly?Ala?Ser?Gly

405

Claims (38)

1. one kind has the active polypeptide of endo-xylogalacturonase.

2. one kind obtains and has an active polypeptide with interior xylogalacturonase enzymic activity of endo-xylogalacturonase from fungi.

3. according to the polypeptide of claim 2, wherein this fungi is an Aspergillus.

4. according to the polypeptide of aforementioned arbitrary claim, it comprises the described sequence of SEQ.ID No.2, or a kind of and its fragment of homologous sequence or arbitrary sequence basically.

5. according to the polypeptide of claim 4, wherein this fragment has at least 5 amino acid or homologous sequence and SEQ.ID No.2 that at least 60% homogeny is arranged.

6. according to the polypeptide of claim 5, it comprises the 19-406 amino acid of the described aminoacid sequence of SEQ.ID No.2.

7. a coding is according to the polynucleotide of the polypeptide of arbitrary aforementioned claim.

8. polynucleotide comprise:

(a) the described polynucleotide sequence of SEQ.ID No.1, or its complementary sequence;

(b) can with the polynucleotide sequence of the described nucleotide sequence hybridization of SEQ.ID No.1, or its fragment;

(c) can with the polynucleotide sequence of the complementary sequence hybridization of the described polynucleotide sequence of SEQ.ID No.1, or its fragment;

(d) polynucleotide sequence, itself since the degeneracy of genetic code with (a) (b) or arbitrary polynucleotide of definition (c) be degeneracy.

9. polynucleotide according to Claim 8, its:

A. coding has the active polypeptide of endo-xylogalacturonase, and these polynucleotide are:

(1) encoding sequence of SEQ.ID No.1;

(2) sequence of the complementary sequence selective cross of sequence of definition in a kind of and (1); Or

(3) a kind of owing to the degeneracy of genetic code and the sequence of the sequence degeneracy of (1) or (2) middle definition, perhaps

B. one kind with (a) in the definition polynucleotide complementary sequence.

10. isolating polynucleotide that from fungi, obtain according to claim 7,8 or 9.

11. according to the polynucleotide of claim 10, wherein fungi is an Aspergillus.

12. segmental polynucleotide probes that comprises at least 15 Nucleotide of the polynucleotide of each definition in the claim 7 to 11.

13. carrier that comprises the polynucleotide of each definition in the claim 7 to 12.

14. an expression vector that comprises the polynucleotide of each definition in the claim 7 to 11, described polynucleotide are connected effectively with the regulating and controlling sequence that one or more can instruct polynucleotide to express in host cell.

15. one kind with transforming according to each carrier in the claim 13 to 14 or the host cell of transfection.

16. include or contain the host cell according to each polynucleotide in the claim 7 to 11, wherein the genome of these polynucleotide and host cell is allogenic.

17. according to the host cell of claim 15 or 16, it is a yeast cell.

18. a method for preparing according to each polypeptide in any claim 1 to 6 is included in and hatches or cultivate under the condition that allows expression of polypeptides according to each host cell in the claim 15 to 17, and purified polypeptide randomly.

19. one kind comprises or expresses that wherein this polypeptide and host cell are allogenic according to the host cell of each polypeptide in the claim 1 to 6.

20. composition that comprises according to each polypeptide in the claim 1 to 6.

21., wherein also comprise polypeptide with inscribe arabanase, rhamnosyl polygalacturonase or polygalacturonase activity according to the composition of claim 20.

22. a method of handling vegetable material, this method comprise use according in the claim 1 to 6 each polypeptide or contact vegetable material according to the composition of claim 20 or 21.

23., wherein handle the pectin that comprises in degraded or the modified plant material according to the method for claim 22.

24., be used for degraded or modified plant cell walls according to the method for claim 22.

25., wherein handle the endo-type shearing that comprises the xylogalacturonase subunit of the pectin composition in the material according to the method for claim 22 or 23.

26. according to each method in the claim 22 to 24, wherein material comprises plant, plant pulp, plant extraction liquid or edible food or composition wherein.

27. according to the method for claim 26, wherein material is fruit or vegetable and pulp, juice or extract.

28. the vegetable material of a processing, it is to use according to each polypeptide or contact with vegetable material according to the composition of claim 20 or 21 in any claim 1 to 6 to obtain, or by obtaining according to each method in the claim 22 to 26.

29. according to the vegetable material of the processing of claim 27, it is a kind of fruit or vegetables juice.

30. one kind is reduced the vegetable material method of viscosity, this method comprise use according in the claim 1 to 6 each polypeptide or contact vegetable material according to the composition of claim 20 or 21 with under the amount of pectin contained in effective degradable material and the condition.

31. according in any claim 1 to 6 each polypeptide or according to the composition of claim 20 or 21 purposes in the method for handling vegetable material.

32., wherein handle the xylogalacturonase substituting group that comprises pectin in the endo-type shearing vegetable material according to the purposes of claim 31.

33. according in the claim 1 to 6 each polypeptide or according to the composition of claim 20 or 21 purposes in the method for processing plant pulp, juice or extract, this method comprises with polypeptide or composition and pulp, juice or extract incubation down to the small part depolymerized pectin.

34. (animal) feed or food that comprises according to each polypeptide in the claim 1 to 6.

35. composition that comprises (randomly saponified) with the tragakanta (sGT) of strong acid treatment.

36. identify or detect to have the detection method of the active polypeptide of pectin degrading, comprising for one kind:

A. as the substrate of candidate compound, provide (randomly saponified) tragakanta of handling with strong acid (sGT/TFA), and

B. whether contact sGT/TFA with candidate compound, detecting has any recuding sugars to produce.

37. according to the detection method of claim 35, wherein measured the amount of recuding sugars, and randomly with the contrast that does not have candidate compound in the carbohydrate that produces amount relatively.

38. according to the detection method of claim 35 or 36, it comprises by measuring by carbohydrate reduction Cu (II) and be the amount of Cu (I), randomly by contact the amount of BCA-Cu (I) mixture of definite formation with two cinchonic acids (BCA).

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