CN1244701C - Cellulases, genes encoding them and uses thereof - Google Patents

Abstract

Genes encoding cellulases, and a gene encoding a protein that facilitates the action of such cellulases, the cellulases and a protein that facilitates the action of such cellulases, and enzyme preparations containing such proteins are described. The native hosts and the culture medium of said hosts containing said cellulases are also disclosed. These proteins are especially useful in the textile and detergent industry and in pulp and paper industry.

Description

Cellulase, its encoding gene and application thereof

Background of the present invention

The field of the invention:

The present invention relates to encode new neutral cellulase gene and contain the composition of new neutral cellulase. These compositions are at textile, and are particularly useful in detergent and paper pulp and the paper industry.

Correlation technique:

Cellulose is the line style polysaccharide by the glucose residue of β-Isosorbide-5-Nitrae key connection. At nature, cellulose usually and lignin and link such as the hemicellulose of xylan and glucomannans. The characteristic of known fiber element enzyme has hindered the practical application of cellulase, and these cellulases normally have the mixture of the cellulase of various activity and substrate specificity. Therefore, need to identify the source of the cellulase that can obtain only to have required activity.

Be known in the art the cellulase of numerous kinds, wherein great majority are acidic cellulases. Yet, also identified some neutrality and alkali cellulose enzyme. Celluzyme^Commercially available cellulase goods from Humicola insolens (Novo Nordisk, A/S). GB2,075,028 and EP406,314 described Humicola insolens cellulase as the enzyme additive in the washing detergent to reduce the roughness (stiffness index) of cotton-containing fabrics. Contain from Humicola insolens the endoglucanase activity cellulase be cloned in WO 93/11249 and EP531, describe in 372. EP510,091 has described the cellulase from bacillus NCIMB 40250 useful in detergent compositions. EP220,016, described as the useful cellulase of fining agent of yarn dyed fabric. WO94/07998 has described has the cellulase that improves the active modification of alkalescence. WO95/02675 has described the detergent compositions that contains two kinds of different cellulases: the first cellulase plays the effect of dust removal grain in catalysis; The second cellulase plays the color clarification in catalysis. WO92/18599 has described the detergent goods of cellulase and protease. Cellulase is also stuck with paste thickener and unnecessary dyestuff (EP576,526) industrial for the printing and dyeing after helping to remove fabrics printing and dyeing.

EP383,828 have described the graininess detergent compositions, and it contains surfactant, fabric softening clay material, and cellulase particle calciferous. US5433750 has described and has contained surfactant, increases the material system of cleaning action, softening clay, clay flocculating agent and high active cellulase, the detergent compositions of preferred Humicola insolens cellulase. The graininess detergent compositions that US5520838 describes comprises surfactant, increase material and the cellulase of cleaning action, preferred Humicola insolens cellulase, said composition is a kind of closely form, has quite high density and contains a small amount of inorganic fill salt.

Also be used for the industry of numerous species except industrial textile outer fiber element enzyme. For example, cellulase is in industrial deinking for newspaper and magazine (EP521,999). For improvement of the sewage (WO91/14822, WO91/17243) of paper pulp and as the processing to animal feed.

The unique property of each cellulase makes some cellulase more be applicable to certain purpose than other. Although this enzyme distinguishes in many ways, most important difference be the pH amount just when. Neutral cellulase has useful cellulase activity in pH scope 6-8, alkali cellulose enzyme has useful cellulase activity in pH scope 7.5-10. Acidic cellulase has activity in the pH4.5-6 scope, but has very little cellulase activity when higher pH value.

Neutrality and acidic cellulase are particularly useful in industrial textile. Klahorst, S. etc., textile chemist and colorist, 26:13-18,1994; Nilsson, T.E., the meeting of Aachen textile, DWI reports 114:85-88 (1995); Videbrek, T etc., ITB dyeing/printing and dyeing/polishing, in January, 1994,25-29 page or leaf; Klahorst, S, etc., AATCC international conference and exhibition, 4-7 day in October, 1992, the 243rd page, Atlanta, GA; Kochavi, D etc., american dye information, September nineteen ninety, 26-28 page or leaf; Tyndall, R.Michad, textile chemist and colorist 24:23 (1992); Lange, N.K, the procceedings of the TRICEL symposium second time of Trichoderma reesei cellulase and other hydrolase, Espoo, Finland, 1993, editor. P.Suominen etc., biotechnology and industrial fermentation WARF, the 8th volume, 1993, the 263-272 pages or leaves. When for the treatment of fabric, cellulase is attacked the cellulose molecular chain that forms cotton fiber, thereby affects the characteristic of fabric.

Traditionally, in " wearing away ", float stone is used for changing the characteristic of fabric. Little by little, cellulase has replaced float stone, and float stone also makes fabric produce required FINAL APPEARANCE, but it can cause to machinery the damage of clothes and sewage processing unit (plant). US4832,864, US4912056, US5006126, US5122159, US5213581 and EP307564 disclose the application of cellulase in biology is worn away.

Cellulase is particularly useful for the denim goods of wearing away with indigo dyeing, because these dyestuff great majority are positioned at the surface of yarn and do not penetrate fiber holes. When for the treatment of bafta, the neutral cellulase wash time longer than acid cellulose enzyme require in general. Yet obtainable neutral cellulase has less offensiveness (activity) than acidic cellulase to cotton fabric, and it is reported and do not resemble the such easy damage fabric intensity of acidic cellulase. Neutral cellulase has wider pH scope, and it is very little that the pH that therefore occurs during biology is worn away increases the impact of centering enzymatic activity.

Since acidic cellulase promote fabric redye (back staining) and frangible tendency has hindered its application. In addition, pH must be transferred in the scope that is suitable for acidic cellulase performance function. Therefore, clearly need a kind ofly not cause that fabric is redyed or frangible neutral cellulase goods.

Although in industrial textile, use cellulase to become more and more general, change simply employed cellulase mixture and can produce different polish results. These problems will more and more be concentrated in the research to the repeatably cellulase mixture with required feature. Therefore, very clear need to have activity especially when neutral and alkaline pH are planted in textile and detergent industry, do not damage fabric intensity, has the new cellulose enzyme of cleaning preferably and/or fabric protection and reduction roughness characteristic.

Brief summary of the present invention

Since recognized identify the textile biopolishing and biological wear away and in detergent applications the importance of useful enzyme, the inventor has screened in the fungal species neutrality with enzymatic feature and alkali cellulose enzyme useful in this technology.

These researchs have produced from myceliophthora, Myriococcum, Melanocarpus, the new cellulose enzyme of Sporothrix and Chaetomium.

The invention further relates to from produce this new cellulose enzyme the culture medium of using or the enzyme preparation of natural host preparation.

The invention further relates to the application in textile and detergent industry and in paper pulp and paper industry of this culture medium or this enzyme preparation.

These researchs further cause having identified useful especially 3 kinds of new cellulases in textile and detergent industry. Disclosed a kind of cellulase (this paper called after " 20K-cellulase ") with 20kDa of endoglucanase activity from the purifying goods of Melanocarpus kind or Myriococcum kind, a kind of 50kDa cellulase (" 50K-cellulase "), and the second 50kDa cellulase (" 50K-cellulase B "). Also disclose the new gene outcome that has high homology with cellulase family, this paper is called " protein with CBD " (wherein CBD refers to " cellulose binding ").

An object of the present invention is to provide and contain one or more new cellulose enzymes of the present invention, particularly 20K-cellulase, 50K-cellulase, 50K cellulase B and/or have the enzyme preparation of the protein of CBD.

Another object of the present invention provides the polishing of using these goods to be used for textile. Particularly the denim goods biology method of wearing away uses said goods to be used for the method for detergent compositions, particularly uses the 20K-cellulase, 50K cellulase, 50K-cellulase B and/or have the method for protein of CBD.

The invention still further relates to other neutrality and/or alkali cellulose enzyme with one or more amino acid sequences as herein described.

The invention still further relates to coding 20K-cellulase, 50K-cellulase, 50K-cellulase B and the gene with protein of CBD.

The invention still further relates to the new expression vector that contains this gene and relate to the new host who transforms with this carrier, particularly can express the host by the protein of this gene code high-levelly.

The invention still further relates to the host's of this conversion the culture medium of using, contain new 20K-cellulase, the 50K-cellulase, 50K cellulase B and/or have the culture medium of the protein of CBD, or contain from the enzymatic compositions (enzyme preparation) of one or more these protein of this culture medium preparation.

The invention further relates to and use this culture medium or the application of this enzyme preparation in textile and the industry that depollutes and in paper pulp and paper industry.

Brief description of the drawings

Fig. 1 (A and B) has shown ALKO 4179, the pH (Figure 1A) of the endoglucanase activity of CBS 689.95 and temperature (Figure 1B) dependence.

Fig. 2 (A and B) has shown ALKO 4124, the pH of the endoglucanase activity of CBS 687.95 (Fig. 2 A) and temperature (Fig. 2 B) dependence.

Fig. 3 (A and B) has shown ALKO 4237, the pH of the endoglucanase activity of CBS 685.95 (Fig. 3 A) and temperature (Fig. 3 B) dependence.

Fig. 4 (A and B) has shown ALKO 4265, the pH of the endoglucanase activity of CBS 730.95 (Fig. 4 A) and temperature (Fig. 4 B) dependence.

Fig. 5 (A and B) has shown ALKO 4125, the pH of the endoglucanase activity of CBS 688.95 (Fig. 5 A) and temperature (Fig. 5 B) dependence.

Fig. 6 (A and B) has shown with the washing effect of neutral cellulase and has redyed (Fig. 6 A) and dye orchid (Fig. 6 B).

Fig. 7 (A and B) has shown with the clean result of the Ecostone L that increases gradually enzyme dosage and has redyed (Fig. 7 A) and dye orchid (Fig. 7 B). 1 * corresponding to the enzyme dosage of neutral cellulase among Fig. 6 A and the 6B.

Fig. 8 has shown with SP-Sepharose^TMChromatography is from peak II purifying 20K-cellulase. The sample that contains 11.7g protein and 576,000ECU is crossed the 4.5 * 31cm post by embodiment 9 described formation. Collect the 15ml cut. In the peak of cut 148-161 the endoglucanase activity underestimated, because be used for crystallization occurring before the test at abundant this enzyme of dilution. The crystalline material of these cuts contains 486,000ECU.

Fig. 9 (A and B) has shown that the SDS-PAGE of 20K cellulase analyzes. The molecular weight of standard items shows with kDa.

A. from active S-Sepharose^TMThe partially crystallizable material (swimming lane 1) of cut precipitation.

B. on G50Sephadex, the partially crystallizable material is carried out the fraction of chromatogram. The cut that shows in swimming lane 19 and 25 does not contain the endoglucanase activity. For other cut, the live vol (with ECU) that adds to gel is as follows: cut 27,0.4; 29,2.4 (as 3.0 μ g protein); 3.0,2.1; 31,1.9; 33,0.46; With 35,1.1.

Figure 10 has shown through SP-Sepharose^TMChromatogram is to the 50K cellulase of peak III/IV and separating of 50K cellulase B. The sample that contains 200mg protein and 14,800ECU is crossed the 2.5 * 11cm post by embodiment 9 described formation. Collect the 6.8ml cut. Before the NaCl gradient wash-out a small amount of 50K-cellulase, and most of 50K-cellulase is at about 50mM NaCl place wash-out. Find that 50K-cellulase B is in the major protein mass peak of about 80mM NaCl.

Figure 11 has shown that the 50K-cellulase (11A) of purifying and the SDS-PAGE of 50K-cellulase B (11B) analyze. The swimming Taoist monastic name has shown the cut (3.3ml) from the Phenyl-Sepharose wash-out. For cut 36-41,2.5 each cut of μ l are crossed gel. For other cut, 2 μ l are crossed gel. In cut 37-38, find 50K-cellulase peak (11A) (contain respectively 780 and 880ECU/ml). 50K cellulase B peak (11B) in cut 30 and 31, it contains insignificant activity (not enough 4ECU/ml).

Figure 12 has shown the temperature dependency of 50K-cellulase endoglucanase activity within the reaction time of pH7.0 and 60 minutes.

Figure 13 has shown the pH dependence of 50K-cellulase endoglucanase activity when 50 ℃ (◆) and 70 ℃ ().

Figure 14 has shown that the Western that uses 20K-cellulase antiserum to make probe analyzes. Swimming lane 1,2 and 3 contains respectively from DEAE-Sepharose peak I, the 25 μ g protein of III and IV. Swimming lane 4 and 5 contains the pure 50K-cellulase of 2.0 and 0.2 μ g, and swimming lane 6 contains the pure 50K-cellulase of 0.6 μ g B. Swimming lane 7 and 8 contains respectively the about 25 μ g protein from the full growth medium of ALKO 4237 and ALKO 4124.

Figure 15 has shown the temperature dependency of 20K-cellulase in the endoglucanase activity of pH7 (10 minute reaction time).

Figure 16 (A and B) has shown the pH dependence of 20K-cellulase in the endoglucanase activity in 10 minutes (a) or 60 minutes (b) reaction time.

Figure 17 has shown the amino acid sequence data that the 20K-cellulase described in order-checking this paper illustrative material obtains. Sequence 429 is from the N end of protein, and other sequence is from the peptide of inner trypsinization.

Figure 18 has shown and is carrying the plasmid pALK1221 of 20K-cellulose enzyme gene, the restriction map of Melanocarpus albomyces DNA among pALK1222 and the pALK1223.

Figure 19 has shown the dna sequence dna of 20K cellulose enzyme gene. Arrow represents the signal peptidase Processing position predicted.

Figure 20 has shown and is carrying the plasmid pALK1233 of 50K-cellulose enzyme gene, pALK1234, the restriction map of Melanocarpus albomyces DNA among pALK1226 and the pALK1277.

Figure 21 (A and B) has shown the dna sequence dna of 50K-cellulose enzyme gene. Arrow represents the signal peptidase Processing position predicted.

Figure 22 has shown the restriction map of Melanocarpus albomyces DNA in the plasmid pALK1229 that carries 50K-cellulase B gene and pALK1236.

Figure 23 (A and B) has shown the dna sequence dna of 50K-cellulase B gene. Arrow represents the signal peptidase Processing position predicted.

Figure 24 has shown the plasmid map of pTTc0l.

Figure 25 has shown the plasmid map of pMS2.

Figure 26 has shown the restriction map of Melanocarpus albomyces DNA in carrying coding and have the plasmid pALK1230 of protein DNA of CBD. The sequence that provides in Figure 27 is used the arrow mark in Figure 26.

Figure 27 has shown the dna sequence dna of the cellulose enzyme gene of the protein with CBD in pALK1230.

Figure 28 has shown the plasmid map of pALK1231.

Figure 29 has shown the plasmid map of pALK1235.

Figure 30 has shown that the Western that uses 20K-cellulase antiserum to make probe analyzes. Swimming lane 1 and 2 contains the about 10 μ g protein from the full growth medium of transformant ALKO 3620/pALK1235/49 and ALKO 3620/ pALKl235/40. Swimming lane 3 contains the about 10 μ g protein from the full growth medium of ALKO3620. Swimming lane 4 and 5 contains the about 10 μ g protein from transformant ALKO 3620/pALK1231/16 and the full growth medium of ALKO 3620/pALK1231/14. Swimming lane 6 contains the pure 20K cellulase of 100ng.

Figure 31 has shown the plasmid map of pALK1238.

Figure 32 has shown the plasmid map of pALK1240.

Preserved material

ALKO 4179 Myceliophthora thermophila in October 12 nineteen ninety-five at Centraalbureau voor Schimmelcultures, P.O.Box 273,3740 AG BAARN are as CBS 689.95 preservations.

ALKO 4124, Myriococcum sp. in October 12 nineteen ninety-five at Centraalbureau voor Schimmelcultures, P.O.Box 273,3740 AG BAARN are as CBS 687.95 preservations.

ALKO 4237 Melanocarpus albomyces (=Myriococcum albomyces=Thielavia albomyces; Guarro etc., 1996, Mycol.Res.100 (1): 75) on October 11st, 1995 at Centraalbureau voor Schimmelcultures, P.O.Box 273,3740 AG BAARN are with CBS 685.95 preservations.

ALKO 4125, Sporotrichum thermophile in October 12 nineteen ninety-five at Centraalbureau voor Schimmelcultures, P.O.Box 273,3740 AG BAARN are as CBS 688.95 preservations.

ALKO 4265, Chaetomium thermophilum La Touche in November 8 nineteen ninety-five at Centraalbureau voor Schimmelcultures, P.O.Box 273,3740 AG BAARN are as CBS 730.95 preservations.

Plasmid pALK1221 on June 21st, 1996 with the DSM11024 preservation, λ 4237/5.1 on June 21st, 1996 with the DSM11012 preservation, all be preserved in German Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1B, D-38124 Braunschweig. Two plasmids all contain the 20K cellulose enzyme gene from Melanocarpus albomyces CBS 685.95.

At German Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1B, D-38124 Braunschweig, plasmid pALK1227 on June 21st, 1996 with DSM 11025 preservations, λ 4237/35 on June 21st, 1996 with DSM 11014 preservations. Both all contain the 50K cellulose enzyme gene from Melanocarpus albomyces CBS 685.95.

At German Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1B, D-38124 Braunschweig, plasmid pALK1229 on June 21st, 1996 with DSM 11026 preservations. λ 4237/3 on June 21st, 1996 with the DSM11011 preservation. PALK1229 contains the DNA of coding 50K-cellulase B, and λ 4237/3 and λ 4237/18 contain the 50K cellulase B gene from Melanocarpus albomyces CBS 685.95.

Plasmid pALK1230 is deposited in German Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH on June 21st, 1996 with DSM 11027, Mascheroder Weg 1B, and D-38124Braunschwei. PALK 1230 contains the gene from the protein with CBD of Melanocarpus albomyces CBS 685.95.

Detailed description of the preferred embodiments

In the following description, employed many terms in the industrial textile technology have been widely used. In order to provide specification and claims, comprise the clear and consistent understanding by the given scope of these terms, the definition below providing.

Biology is worn away: " biology is worn away " of fabric or clothes refers to use enzyme to replace using float stone or conduct to use interpolation step process fabric or the clothes, particularly denim goods of float stone.

Biopolishing: " biopolishing " refers to that enzyme is used for the controlled hydrolysis of cellulose fibre to change fabric or yam surface, make it prevent permanent balling-up, improve the fiber feel, such as flexibility and slickness, the clean surface structure through having reduced fine hair, cause the color clarification, improve the drapability of fabric, improve water vapor absorption power and also can improve dyeability.

Redye: the dyestuff of release has the tendency on the surface that again deposits to fabric fibre, and this effect is called " redying ".

Detergent: detergent refers to a kind of cleaning agent, it can contain surfactant (anion, nonionic, cation and amphoteric surfactant), increase material and other optional composition of cleaning action, such as antiredeposition and soil suspending agent, brightener, bleaching agent, dyestuff and pigment and hydrolase. Suitable being described in the U.S. Patent number 5433750 of detergent composition provides, and suitable being described in the U.S. Patent number 3664961 of surfactant provides.

Enzyme preparation, " enzyme preparation " refers to contain enzymatic compositions. Preferably extract (partially or completely purifying) this enzyme from microorganism or the culture medium that is used for this microorganism of growth. " from ... middle extraction " refers to separate required enzyme from cell mass. The available any method of this purpose that reaches is carried out this operation, comprises smudge cells, also can take out culture medium simply from the cell that consumes. Therefore, term " enzyme preparation " comprises any enzyme that contains former culture medium for the cultivation desired microorganisms and be discharged into this culture medium from microbial cell during cultivation or Downstream processing step.

The host of " basically can not " synthetic one or more enzymes refers to compare with wild type, and the activity of one or more described enzymes is suppressed, the host of defectiveness or shortage.

Amino acid sequence as specific amino acid sequence " equivalent " refers to not identical with the specific amino acid sequence, but contain at least some amino acid changes (disappearance of comparing the BA that does not basically affect this protein when being useful on required purpose with the similar activity of specific amino acid sequence, replace, put upside down, insert etc.). The BA of cellulase refers to the ability of its catalytic activity and/or its cellulose-binding material. The BA of 50K-cellulase B also comprises the ability of itself and cellulase synergistic effect. Preferably, " equivalence " amino acid sequence and specific amino acid sequence contain at least homogeny of 80%-99% at amino acid levels, most preferably at least 90%, in special highly preferred embodiment, have 95% homogeny at least at amino acid levels.

Cloning vector, cloning vector are a kind of plasmid or phage DNA or other dna sequence dna (such as linear DNA). It provides suitable nucleic acid carrier environment for genes of interest being shifted into host cell. Cloning vector of the present invention can be designed to self-replicating in protokaryon and eucaryon host. In the fungal host such as Trichoderma, cloning vector generally can not self-replicating and is replaced only to provide genes of interest is transported into Trichoderma host in order to insert subsequently the genomic carrier of Trichoderma. Cloning vector also can be identified with one or minority endonuclease recognition site, can be in this site cut this dna sequence dna with measurable mode enzyme and do not lose the basic biological function of carrier, and to copy and clone this DNA and the DNA montage can be entered this site in order to produce. Cloning vector also can contain the mark that is applicable to identify the cell that transforms with cloning vector. For example, mark is antibiotic resistance. In addition, this mark can provide at cloning vector, and separates with the genes of interest that provides. Term " carrier " is used for " cloning vector " sometimes.

Expression vector. Expression vector is a kind of cloning vector or similar in appearance to the carrier of cloning vector, but it can express genes of interest after transforming required host. When using fungal host, preferably genes of interest is offered fungal host as the part of clone or expression vector, this carrier is integrated into fungi chromosome, or makes genes of interest be integrated into host chromosome. Sequence as cloning vector or an expression vector part also can be integrated with genes of interest in integration process. In T.reesei, the site that can instruct genes of interest to integrate comprises cbh and/or egl locus. Most preferably, genes of interest relates to the gene that replaces the unwanted feature of one or more codings.

Genes of interest also preferably is placed under the control of some control sequence such as promoter sequence that carrier provides (integrating with genes of interest) (that is, with its effective connection). In addition, control sequence can be those sequences in insertion point.

To express the expression control sequenc (for example, shuttle vector can be provided in bacterial host, selecting gene) that certain gene can change expression vector at protokaryon or in eucaryon host according to carrier design is become. Expression control sequenc can contain transcription regulatory element, as, promoter, enhancer element, and transcription terminator, and/or translation adjusting element. For example, translation initiation and termination site.

According to the present invention, neutrality and alkali cellulose enzyme are provided, and for the production of processing this required useful neutrality of textile material and the method for alkali cellulose enzyme.

The natural host that produces protein of the present invention has:

1) ALKO 4179, Myceliophthora thermophila; At Centraalbureau voor Schimmelcultures, P.O.Box 273,3740 AG+BAARN are with CBS 689.95 preservations.

2) ALKO 4124, Myriococcum sp.; With CBS 687.95 preservations;

3) ALKO 4237, and Melanocarpus albomyces is with CBS 685.95 preservations;

4) ALKO 4125, and Sporotrichum thermophila is with CBS 688.95 preservations; And

5) ALKO 4265, and Chaetomium thermophilum La Touche is with CBS 730.95 preservations;

The culture medium of using that a particularly preferred embodiment of the present invention is natural host or the enzyme preparation for preparing from culture medium.

In a particularly preferred embodiment of the present invention, provide the 20K cellulase of purifying, 50K-cellulase, 50K-cellulase B and/or have the protein of CBD. For example, can by this paper, particularly obtain these protein from Melanocarpus kind or Myriococum kind described in the embodiment 9.

Produced the amino acid sequence data from cellulase as herein described. Therefore, the invention still further relates to neutrality or the alkali cellulose enzyme that contains amino acid sequence shown in one or more this paper. Therefore, the present invention plans to relate in function to be equivalent to the 20K-cellulase, the 50K-cellulase, 50K cellulase B and/or have the protein of CBD and have one or more amino acid sequences as herein described, or any neutrality or the alkali cellulose enzyme of substantially the same sequence. This neutrality or alkali cellulose enzyme can be from other bacterial strains of identical type or from the divergent evolution biology.

In another preferred embodiment, being used for the material (for example, the DEAE-Sepharose amalgamation liquid I in this paper Table VIII, III or IV) of the detached peaks that forms during the comfortable illustrative purifying procedure provides the 20K-cellulase. In also having an embodiment, can be separately or be combined with other oroteins in Melanocarpus albomyces ALKO 4237 culture mediums with other this proteinoid.

In another preferred embodiment, the material of the detached peaks that forms during the next comfortable illustrative purifying procedure provides the 50K-cellulase. In another embodiment, can be separately or be combined with other oroteins in ALKO 4237 culture mediums with other this proteinoid.

In another preferred embodiment, the material of the detached peaks that forms during the next comfortable illustrative purifying procedure provides 50K-cellulase B. In another embodiment, can be separately or be combined with other oroteins in ALKO 4237 culture mediums with other this proteinoid.

As described herein, with the ALKO 4265 of CBS 730.95 preservations, as the example of neutral cellulase, it is worn away in the method at biology of the present invention is not preferred to Chaetomium thermophilum La Touche, redyes because it causes in this article. Yet, evidence suggests that it is useful in other application (for example, at detergent).

Genetic sequence by the clones coding desired protein and help method for genetic modification host of the present invention by expressing this genetic sequence. Nucleic acid molecules (preferred DNA) attempted to refer in term used herein " genetic sequence ". The genetic sequence of coding desired protein is from various sources. These sources comprise genomic DNA, cDNA, synthetic DNA and combination thereof. Can use the carrier system generation for the production of the host of enzyme preparation of the present invention. This vector construction body (a) can further provide a kind of vector construction body (b) of separation, the required gene of construct (b) coding host genome at least a to be integrated into and with (a) or (b) (c) selected marker of coupling. In addition, the carrier of separation can be used for mark.

If the nucleic acid molecules such as DNA contains expression control sequenc, then be called " can express " polypeptide, wherein this expression control sequenc contains the nucleotides sequence that transcriptional regulatory information and this sequence " be effectively connected to " coded polypeptide and lists.

Effectively connect expression that to be a certain sequence make this sequence with a connected mode of regulating sequence (or a plurality of adjusting sequence) being connected in the impact of this adjusting sequence or under controlling. If if promoter function induce the sequence mRNA that causes coded protein to transcribe and two dna sequence dnas between connection character can (1) not cause importing frameshift mutation, (2) disturb expression to regulate sequence-directed mRNA, the ability of antisense RNA or protein expression or (3) are disturbed and are activated the template ability that the subregion sequence is transcribed, and then claim two dna sequence dnas (such as protein coding sequence and the promoter region sequence that is connected coded sequence 5 ' end) effectively to connect. Therefore, this promoter region effectively is connected with dna sequence dna if promoter can affect transcribing of dna sequence dna.

The definite character of the adjusting sequence that gene expression is required changes between kind or cell type, but generally must comprise and relate separately to 5 ' non-transcribed and 5 ' untranslated (non-coding) sequence of transcribing with translation initiation. Protein expression need to use the control band that function is arranged in this host in transforming the host. Can adopt many various adjusting sequences of transcribing and translate. In eucaryote, do not link to each other if transcribe with translating, whether contain this methionine according to clone's sequence, this control zone can provide or not provide initial methionine (AUG) password. In general, this zone comprises is enough to instruct the synthetic promoter region of host cell starting RNA.

As everyone knows, the translation of eukaryotic mrna starts at the codon place of coding the first methionine. Therefore, guarantee that preferably the bonding pad between the DNA sequence of eukaryotic promoter and coded protein or its functional derivatives does not contain any codon that interleaves of the methionine of encoding. The existence of this codon causes forming fusion (if the dna sequence dna of AUG codon and coded protein is in same reading frame) or frameshift mutation (if the sequence of AUG codon and coded protein is not in same reading frame).

In preferred embodiments, desired protein is owing to existing secretory signal sequence to secrete in culture medium on every side. If desired protein is not that burst itself is arranged, if or this burst can not in the host, bring into play well function, the sequence of coded protein can be effectively connected to so on the burst with host's homology or allos. Required coded sequence can link to each other with any burst that allows this protein of secretion from the host. This burst can be designed to contain or do not contain the specific protease site, and this signal peptide sequence is removed easily subsequently. In addition, can use protein is leaked to host in the culture medium, for example, have the host of sudden change at its film.

If necessary, can obtain the sequence 3 of coded protein ' do not transcribe and/or untranslated region by above-mentioned cloning process. Can keep 3 '-tanscription termination of nontranscribed domain regulates sequential element; Can keep 3 '-translation termination of non-translational region regulates sequential element or it instructs those elements of polyadenylation in eukaryotic.

Carrier of the present invention also can comprise other regulating element such as enhancer sequence that effectively connects.

In preferred embodiments, make up the upper stable transformant of heredity, with this required protein dna is integrated in the host chromosome. The coded sequence of desired protein can be from any source. From the beginning this integration can occur in cell, perhaps, and in most of preferred embodiments, help this integration through transforming with carrier, this carrier inserts host chromosome with himself on function, for example, have the DNA element that promotes that dna sequence dna is integrated in chromosome.

Also advance its chromosomal cell through importing the DNA stable integration that the one or more marks allow to be chosen in the host cell that contains expression vector in the chromosome select to import, for example, this mark can provide the biocide resistance, for example, to antibiotic, or heavy metal, such as the resistance of copper etc. Selectable marker gene can be directly links to each other with DNA gene order to be expressed or imports in the same cell through cotransformation.

Important factor comprises in selecting certain plasmid or viral vectors: identify and select to contain the convenience of the recipient cell of carrier with it from carrier-free those recipient cells; Required carrier copy number in specific host; Whether need this carrier that between different types of host cell, " to shuttle back and forth ".

In case prepared the construct that is used for the carrier of expressing or contains dna sequence dna, available various arbitrarily suitable methods comprise that aforesaid conversion imports suitable host cell with DNA construct. After importing carrier, growth receptors cell in the selection culture medium of selecting transformed cell growth. The fragment that the expression of clone's gene order causes producing required protein or produces this protein. Can in transformant, carry out this expression in a continuous manner or in the mode of control.

Therefore, the sequence of coded protein as herein described can be effectively connected on the required arbitrarily carrier and transform among the selected host, so that the expression of this protein in the host to be provided.

Theme material of the present invention also has coding to have the nucleic acid molecules of protein of cellulase BA and as follows with molecule or its restriction of any making nucleic acid molecular hybridization recited above:

Coding has the nucleic acid molecules of polypeptide of the enzymatic activity of cellulase, is selected from as follows:

(a) coding contains the nucleic acid molecules of peptide more than Figure 19 or the 21 described amino acid sequences;

(b) coding contains the nucleic acid molecules of peptide more than Figure 23 or the 27 described amino acid sequences:

(c) contain the nucleic acid molecules of the coded sequence of Figure 19 or 21 described nucleotide sequences;

(d) contain the nucleic acid molecules of the coded sequence of Figure 23 or 27 described nucleotide sequences;

(e) coding contains by DSM11024, DSM11012, the nucleic acid molecules of the polypeptide of the amino acid sequence of the DNA Insert Fragment coding that DSM11025 or DSM11014 are contained;

(f) coding contains by DSM11026, DSM11011, the nucleic acid molecules of the polypeptide of the amino acid sequence of contained DNA Insert Fragment coding among DSM11O13 or the DSM11027;

(g) contain DSM11024, DSM11012, the nucleic acid molecules of the coded sequence of the DNA Insert Fragment that DSM11025 or DSM11014 are contained;

(h) contain DSM11026, DSM11011, the nucleic acid molecules of the coded sequence of contained DNA Insert Fragment among DSM11013 or the DSM11027;

(i) with (a), (c), (e) or the nucleic acid molecules of any one molecular hyridization (g);

(j) because its coded sequence of degeneracy of genetic code and (a) the distinguishing nucleic acid molecules of coded sequence of arbitrary nucleic acid molecules in (i);

(k) coding has cellulase activity and has and Figure 19, and 21,23 or 27 described sequences demonstrate the nucleic acid molecules of polypeptide of the amino acid sequence of at least 80% homogeny.

Term herein: " hybridization " refers under the conventional hybridization condition, and the preferably hybridization under stringent condition is as by described such as Sambrook etc. (1989, molecular cloning, laboratory manual, second edition, cold spring harbor laboratory publishes, cold spring port, New York). In theory can be from any biology with this nucleic acid molecules with these nucleic acid molecules according to making nucleic acid molecular hybridization of the present invention. Preferably, they are from fungi, namely from Melanocarpus, and Myriococcum, Sporotrichum, those fungies that Myceliophthora and Chaltomium belong to. Can be from for example with the nucleic acid molecules of making nucleic acid molecular hybridization of the present invention, various biologies namely separate in the genomic library of fungi or the cDNA library.

For example, can identify and separate these nucleic acid molecules with the reverse complementary sequence of the fragment of nucleic acid molecules of the present invention or these molecules or these molecules through using according to the hybridization of standard technique (seeing Sambrook etc., (1989)).

As hybridization probe, for example, can use the nucleic acid molecules that has with the fragment of nucleotide sequence complete or substantially the same shown in the accompanying drawing or said sequence. Fragment as hybridization probe also can be with the synthetic fragment of conventional synthetic technology acquisition and the sequence substantially the same with nucleic acid molecules according to the present invention. In case identify and separated gene with making nucleic acid molecular hybridization of the present invention, must measure its sequence and analyze feature by the protein of said sequential coding.

Term " hybrid dna molecule " comprises fragment, derivative and the allelic variant of the above-mentioned nucleic acid molecules of the above-mentioned protein of coding or its biological active fragment.

Fragment is interpreted as that length is enough to the part nucleic acid molecules of encoding said proteins or its biological active fragment. Herein term " derivative " refer to that the sequence of the nucleotide sequence of these molecules and above-mentioned nucleic acid molecules is had any different in one or more positions and with said sequence height homology. Homology is interpreted as the sequence homogeny of finger at least 40%, particularly at least 60% homogeny, preferably surpasses 80 %, and more preferably surpasses 90%. The derivative of above-mentioned nucleic acid molecules can be disappearance, replaces, and inserts the result who adds or make up.

The protein that homology also refers to each nucleotide sequence or coding on the function and/or on the structure quite. With nucleic acid molecules above-mentioned nucleic acid molecules homology and that derived by the said nucleic acid molecules variant form of said molecule normally, its representative has the modification of identical biological function. They can be abiogenous variations, such as other biological or suddenly change sequence. But these sudden change naturally-occurrings or can realize through specific mutagenesis. And these variants can be the sequences that produces through synthetic. Allelic variant can be variant and the synthetic variant of processing that produce or hereditary of natural appearance.

Share specific common characteristic by the protein that the various variants of nucleic acid molecules of the present invention are encoded, such as enzymatic activity, molecular weight, immunological response, conformation etc., and physical features, such as electrophoretic mobility, chromatographic behavior, sedimenting system, solubility, spectral characteristic, stability, pH just when, temperature just when, etc. For example, can connect the enzymatic activity of the 11st page and embodiment 1 and 25 described detection fibers element enzymes.

The invention still further relates to the nucleic acid molecules of the protein with cellulase BA because the sequence of its sequence of degeneracy of genetic code and the molecule identified is above had any different and encoded.

Nucleic acid molecules of the present invention is RNA or dna molecular preferably, is more preferably genomic DNA or cDNA.

The invention still further relates to and identify specifically according to the antibody of one of above-mentioned protein of the present invention and the antibody with this characteristic. Fragment. These antibody can be monoclonals or polyclonal. Its production method is well known in the art and at for example Harlow and Lane " antibody, laboratory manual ", CSH publishes, and describes in detail in the cold spring harbor laboratory (1988).

And, the present invention relates to nucleic acid molecules according to the present invention or with the oligonucleotides of the complementary strand specific hybrid of this nucleic acid molecules. In this respect, term " specific hybrid " refers to that this oligonucleotides hybridizes specifically with nucleic acid molecules of the present invention under stringent hybridization condition, and does not show crisscrossing with the sequence of other polypeptide of coding under this condition. Preferably, this oligonucleotides has the length of at least 10 nucleotides, more preferably at least 15 nucleotides, most preferably at least 30 nucleotides. They preferably are no more than 100 nucleotides, more preferably no more than 80 nucleotides, are most preferably not exceeding 60 nucleotides. Hybridize specifically in order to guarantee they and nucleic acid molecules of the present invention, this oligonucleotides shows at least 80% homogeny with the corresponding nucleotide sequence of nucleic acid molecules of the present invention on its total length, preferred at least 95% homogeny, most preferably at least 99% homogeny. Can use these oligonucleotides, for example, as the probe of the sequence of screening coding cellulase in genome or cDNA library or as the PCR primer.

The sequence of coded protein as herein described can merge to make up the DNA of encoding fusion protein matter with other sequence in reading frame. For example, can be by top described preparation coding 50K-cellulase, the 20K-cellulase, 50K-cellulase B or have the recombinant vector of gene of the protein of CBD, sequence and T.reesei cellulase except this protein of encoding, hemicellulase or mannase, or this cellulase, the sequence of at least one functional areas of hemicellulase or mannase merges, as at US5298405, WO93/24622 and described in the GenBank submission L25310, this paper quotes each document for your guidance. Specifically, cellulase, hemicellulase or mannase are selected from CBHI, CBHII, EGI, EGII, XYLI, XYLII and MANI or its functional domain are such as secretion signal or core sequence. Mannase has identical functional domain structure with cellulase: contain the core space of avtive spot, contain the twisting district of serine-threonine enrichment region and contain the tail of land.

Can make up and contain the mannase that merges with the sequence of coding desired protein of the present invention or the biological hydrolase of cellulose or endoglucanase or zytase core space or from the core of this enzyme and the fusogenic peptide in twisting district. The result is that this protein contains mannase or the biological hydrolase of cellulose or endoglucanase or zytase core or core and twisting district, and the 50K-cellulase, 20K-cellulase, 50K-cellulase B or the protein with CBD sequence. Mannase or the biological hydrolase of cellulose or endoglucanase or zytase and the 50K-cellulase that provides in fusion constructs is provided this fused protein, the 20K-cellulase, 50K-cellulase B or have each zone of the protein of CBD activity.

But also construction of fusion protein matter, make to comprise and be placed on the 50K cellulase, the 20K cellulase, 50K cellulase B or have mannase or the biological hydrolase of cellulose or endoglucanase or zytase tailer sequence or its required fragment before the protein of CBD sequence, particularly so that allow to use the nonspecific protease site conduct in this tailer sequence from the fused protein of expressing, to reclaim the 50K-cellulase, the 20K-cellulase, 50K-cellulase B or have the protease site of the protein of CBD sequence. But construction of fusion protein matter provides protease site in catenation sequence in addition, and this catenation sequence is positioned at the 50K-cellulase, the 20K-cellulase, and 50K-cellulase B or have before the protein of CBD sequence contains or does not contain tailer sequence.

Through will preferably merging the new features that can produce 20K and 50K-cellulase and 50K-cellulase B with its joint and protein of the present invention such as the zone of cellulose binding (CBD). Preferably, this CBD and joint are the Trichoderma cellulases, and mannase or Melanocarpus albomyces have corresponding CBD and the joint area of the protein of CBS.

The invention provides the method that produces enzyme preparation, the unwanted cellulose lytic activity that this enzyme preparation has a defective partially or completely (namely, the ability of degraded cellulose) and be rich in textile or detergent industry or paper pulp and the required 50K-cellulase of paper conversion, the 20K-cellulase, 50K-cellulase B or have the protein of cbd protein. " cellulose lytic activity defective " refer to reduce, the ability of the oligosaccharide with cellulose degradation Cheng Gengxiao reduction or downtrod. These cellulose lytic activity defective goods reach and with recombinant DNA method the production of these goods are described in US 5298405; This paper quotes as a reference. Preferably, these goods are the active defective of EG activity and/or CBHI.

As described herein, available host of the present invention directly provides the 50K-cellulase, 20K-cellulase, 50K-cellulase B or have the protein of CBD. In addition, also can use growth host's the culture medium of using or from the 50K-cellulase of its purifying, 20K-cellulase, 50K-cellulase B or have the protein of CBD. In addition, if required activity is present in more than one the recombinant host, can from suitable host, separate these goods and combination before being used for method of the present invention.

In order to obtain enzyme preparation of the present invention, under suitable condition, cultivate the above-mentioned natural or recombinant host with required feature and (can express the required 50K-cellulase of viable economically amount, the 20K-cellulase, 50K-cellulase B or host and optional those with protein of CBD can not be expressed the host of one or more other unwanted cellulase basically), required enzyme is secreted into culture medium from the host, reclaims this enzyme preparation with methods known in the art from said culture medium.

Can produce enzyme preparation of the present invention (for example, at embodiment 1 or shown in the embodiment 30) through cultivation recombinant host or natural bacterial strain in the suitable growth medium of fermentation tank.

Enzyme preparation can be the culture medium that contains or do not contain the host cell of natural or conversion, or uses the enzyme preparation that method well known in the art reclaims from this culture medium. Yet, because 50K-cellulase, the 20K-cellulase, 50K-cellulase B secretes in culture medium and under the environmental condition of cellulose splitting water solution and shows activity, and therefore advantage of the present invention is that enzyme preparation of the present invention can need not to be further purified and directly utilize from culture medium. If necessary, this goods freeze-drying or concentrated its enzymatic activity and/or stabilisation can be convenient to store. Provide and use enzyme preparation of the present invention very economical, because (1), this enzyme can the semifinished product form use: needn't separate concrete enzyme from culture medium, (2) only need to reclaim culture medium and just can obtain required enzyme preparation because this enzyme secretion advances in the culture medium; Do not need from the host, to extract enzyme. Preferably, the host who is used for this production is Trichoderma, particularly T.reesei.

Enzyme preparation of the present invention can be used as solution or provides as solid, for example, and with dry powder or particle or solution form, particularly dustless particle, or stable liquid, maybe can concentrate or the stabilized enzyme goods, be convenient to store or use. Can imagine that the enzyme preparation that contains one or more neutral cellulases of the present invention can or be formed in partially or completely defective of the active aspect of certain enzyme by further enrichment, in order to satisfy in various application, such as the needs of special-purpose in industrial textile. Selection is used at particular industry by the enzymatic activity mixture of host and particularly fungus secretion, has advantage in wearing away such as biology.

The enzyme preparation of adjustable invention to be to satisfy textile, depollute or paper pulp and the various application of paper industry in the requirement of specific needs.

Can with not exclusively by other large molecule of identical host's secretion (for example, such as endoglucanase, protease, lipase, peroxidase, oxidizing ferment or amylase) maybe can strengthen effect, stability or the chemicals that cushions required enzyme preparation prepare mixture. Dustless particle can be coated. Can be according to established method, through adding the polyalcohol such as propane diols, carbohydrate or sugar alcohol, lactic acid or boric acid stable liquid enzyme preparation. Liquid detergents generally contains up to 90% water and the organic solvent of 0-20%. Can be by the protection form of the described preparation of EP238216 enzyme of the present invention.

Particularly when as detergent compositions, enzyme preparation of the present invention can contain surfactant, and this surfactant can be anionic, and nonionic is cationic, the mixture of both sexes or these types. Useful detergent compositions is for example, and WO94/07998 describes in the US patent No. 5443750 and the US patent No. 3664961.

If necessary, condition is further purified required enzyme routinely, as extracting precipitation, chromatogram, affinity chromatography, electrophoresis etc.

Enzyme preparation of the present invention is in industrial textile, preferably wears away and in biopolishing or particularly useful in detergent industry at biology. Other useful field is paper pulp and paper industry.

Non-enzymatic is worn away has 3 steps: destarch, friction and post processing. The first step, destarch relates to amylase and removes starch covering, or derivatives thereof. Second step, friction when carrying out without cellulase, is generally carried out with float stone washing denim goods, and is bleached when needs are lightened. Friction effect is not only float stone, and also has the result of the effect that the denim goods fiber rubs together. Friction generally then was the 3rd step, washing step, and washing step to remove unnecessary dyestuff, can add softening agent or brightener therebetween.

Wear away or during biology wears away, remove wholly or in part with the float stone friction at enzymatic, add cellulase and help to grind off indigo dye from cellulose surface. After this is processed, remove cellulase is not existed by enzyme lastingly with the mechanical strength that guarantees fiber infringement with the detergent washing. Can replace fully processing (for example, every 100kg float stone replaces with the 1kg commercial enzyme) with float stone with the cellulase processing. Yet, cellulase can be processed with float stone as a result the time and processed combination when needs produce heavy wear. Through also realizing peachiness fur effect in conjunction with neutral cellulase and float stone washing, wherein produce the tiny hair-like covering that stretches out. Cellulase of the present invention is particularly for reducing to redye in biology is worn away and to strengthen shinny (wearing and tearing) useful.

Biology is worn away preferably approximately carrying out under the pH4.5-9.5, most preferably between pH6.0-8.5. Range of reaction temperature is about 40-80 ℃, between preferred 50-70 ℃, most preferably between 50-60 ℃. Liquor ratio (ratio of the liquid volume that every fabric is heavy) can change preferred 4: 1-10 between about 2: 1 to 20: 1: 1, most preferably 4: 1-7: 1. The enzyme dosage range is about 25-1500nkat/g fabric, preferred 50-500nkat/g fabric, and 75 300nkat/g fabrics most preferably.

Cellulase of the present invention is used for the biopolishing of fabric or clothes in industrial textile, for example, depilation, lint, the color clarification, roughness reduces, and produces different result (for example, " peachiness fur ", " wear out " " sand is washed ", or " old outward appearance " effect) and the biopolishing of yarn (for example, reduce roughness, increase slickness). Cellulase of the present invention can be used for the biopolishing of acid and neutrallty condition.

Cellulase of the present invention for increasing the textile cleaning effect, for example, except soil, increases the fabric protection feature through reducing the textile roughness in detergent compositions, cellulase also has suede and color clarification and recovery Effects.

The fiber of available including natural fibers element or contain artificial cellulose's fiber or textile material that its mixture production is processed with enzyme preparation of the present invention. The example of native cellulose is cotton, flax, hemp, jute, and ramie. Artificial cellulose's example is viscose rayon, cellulose acetate, cellulose iii acetic acid, artificial silk, cupro and lyocell. Above-mentioned cellulose also can be used as the mixture of synthetic fibers, such as polyester, and polyamide or acrylic fiber. Textile material can be yarn or braiding or weaving or form with any alternate manner.

Except particularly useful to the processing fabric, cellulase of the present invention is generally useful in needing any field of cellulase activity. In paper pulp and paper industry, can use neutral cellulase, for example be used for different recyclable papers and the deinking with cardboard of neutrality or alkaline pH, for improvement of fiber quality, or the draining in the production of improvement paper. Other example removes trace cream thickener and unnecessary dyestuff after being included in fabrics printing and dyeing, and as the processing to animal feed. For example, if required application is the mechanical strength that increases paper pulp, 50K-cellulase so of the present invention, 20K-cellulase, 50K-cellulase B or the protein articles with CBD can provide one or more these proteinoid to strengthen or to be conducive to the ability that cellulose fibre combines. In a similar manner, in paper pulp refiner is used, 50K-cellulase of the present invention, the 20K-cellulase, 50K-cellulase B or the protein articles with CBD can provide one or more these proteinoid of certain level to expand to strengthen or to be beneficial to this class.

In more detailed mode the present invention is described among the embodiment below. These embodiment have only shown concrete application more of the present invention. It is the evidence that those skilled in the art produces some similar application. Therefore, these embodiment should not be construed as and dwindled scope of the present invention, and only are to be the application of the present invention that gets across.

Embodiment

Embodiment 1

Shaking flask and fermentation tank culture

In order to keep, with strains A LKO 4179, ALKO 4124, ALKO 4237, AlKO 4265 and ALKO 4125 line sporogenesis agar (ATCC culture medium 5, American type culture collection, filamentous fungi catalogue, the 18th edition, S.C.Jong and M.J.Edwards edit, (1991): 1 liter contains the 1g yeast extract, the 1g beef extract, the 2g tryptone, trace FeSO₄, 10g glucose and 15g agar; PH is 7.2) on. Agar slant was cultivated 3-6 days at 45 ℃.

For the application test (embodiment 3 and 4) of ALKO 4237, with colony inoculation in the following mineral medium of 500ml (Moloney, A.P. etc., biotechnology and bioengineering, 25:1169 (1983)): 1 liter contains 15g KH₂PO ₄，15g(NH ₄) ₂SO ₄，2.4ml 1M MgSO ₄×7H ₂O， 5.4ml 1M CaCl ₂, 20g SOlKa floc, the 15g corn flour, 1g yeast extract and 10ml 100 * trace element solution 1, wherein 1 liter of 100 * trace element solution contains 0.5g FeSO₄×7H ₂O， 0.156g MnSO ₄×H ₂O，0.14g ZnSO ₄×7H ₂O and 0.49g CoSO₄×7H ₂O; PH is adjusted to pH6.5. Cultivation was carried out 3 days under 45 ℃ in shaking table (250rpm). Obtained the endoglucanase activity of about 20-25nkat/ml.

According to Bailey, M.J. etc., the enzyme microbiological technique, 3:153 (1981) uses 1% (w/v) hydroxyethylcellulose, and HEC (Fluka AG#54290) makes substrate and measures routinely cellulase activity with the endoglucanase activity. Except as otherwise noted, this experimental condition is pH7.0 and 50 ℃ of lower reactions 10 minutes. A glucose restriction endonuclease unit (1nkat 1ECU) is defined as under condition determination within 1 second from HEC and produces enzyme amount corresponding to the reduced sugar with reducing power of 1 nanomole glucose. Yet, during with the described purifying enzyme of embodiment 9-12, Bailey etc., the condition determination of enzyme microbiological technique 3:152 (1981) has exceeded the line style scope, therefore presses embodiment 10 these determination methods of described modification. To each bacterial strain, filter paper activity is measured (measure cellulosic total hydrolysis and show and have the biological hydrolytic enzyme activities of cellulose) or under believable detectable limit, or extremely low.

In order to measure pH and temperature dependency (embodiment 2) and in order to carry out strains A LKO 4179, ALKO 4124, the application of AlKO 4265 and ALKO 4125 detects (embodiment 3 and 4), with the hot culture medium B (G.Szakacs of colony inoculation in the 500ml improvement, Hungary, Budapesti Muszaki Egyetem): 1 liter contains 6g Solka floc, the wheat that the 6g distiller expends, 3g oat spelt xylan, 2g CaCO₃, 1.5g soy meal, 1.5g (NH₄) ₂HPO ₄, the large wheat bran of 1g, 0.5g KH₂PO ₄，0.5g MgSO ₄×7H ₂O, 0.5g NaCl, (1 liter contains 1.6g MnSO to 0.5ml trace element solution 1₂，3.45g ZnSO ₄×7H ₂O, and 2.0g CoCl₄×6H ₂O) and 0.5ml trace element solution 2 (1 liter contains: 5.0g FeSO₄×7H ₂O and 2 dense H₂SO ₄); PH transfers to pH6.5. Cultivation in shaking table (250rpm) 45 ℃ carried out 3 days. Because strains A LKO 4179 in hot culture medium B, the endoglucanase activity of ALKO 4124 and ALKO 4237 is about 5nkat/ml, uses about 10 times of the concentrated culture filtrate of the Amicon inspissator of holding back 30kDa. The endoglucanase activity that obtains with ALKO 4265 is about 20nkat/ml, is 30-40nkat/ml with ALKO 4125.

Carry out below 1 liter of fermentation tank culture of ALKO 4179 in the culture medium: 1 liter contains 10g Solka floc, 3g cellobiose, 4g corn flour, 1.5g (NH₄) ₂HPO ₄，0.3g MgSO ₄×7H ₂O，0.5g NaCl，2g CaCO ₃, 0.5ml trace element solution 1 and 0.5ml trace element solution 2,0.5g KNO₃，0.3g CaCl ₂, 1g Tween 80; PH is adjusted to pH 6.5.

Carry out 1 liter of fermentation tank culture of ALKO 4124 in the hot culture medium B of improvement: 1 liter contains: 10g Solka floc, 1g Roth ' s xylan, 40g whey, 30g soy meal, 2g CaCO₃，5g(NH ₄) ₂SO ₄，0.5g KH ₂PO ₄，1.0g MgSO ₄×7H ₂O, 1.0g NaCl, 1g anti-foaming agent, 0.5ml trace element solution 1 and 0.5ml trace element solution 2.

Carry out 1 liter of fermentation tank culture of ALKO 4237 in the above in the described mineral medium. Use 10% (v/v) inoculum. Through adding ammonia [12.5% (V/V)] and phosphoric acid [1.7% (V/V)] pH is maintained pH6.5 ± 0.4. Fermentation temperature is 45 ℃. Fermentation tank (Biostat M.B.Braun, Melsungen, Germany) take 400rpm stir and air-flow as 1vvm. The endoglucanase activity of gained is as follows: ALKO 4179 is about 40nkat/ml, and ALKO 4124 is about 90nkat/ml, and ALKO 4237 is about 30nkat/ml. ALKO 4265 and ALKO 4125 do not cultivate in fermentation tank.

ALKO 4179, and ALKO 4124, and ALKO 4237 and ALKO 4125 are cultivating in 100 liters of pilot scale fermentation tanks under above-mentioned culture medium and the condition. The endoglucanase activity that obtains is about 40nkat/ml for ALKO 4179 and ALKO 4237, and ALKO 4124 is about 90 nkat/ml, and ALKO 4125 is about 100nkat/ml. Use is held back 10kDa and in Millipore PUF 100 milipore filters and Pellicon US boxlike inspissator culture filtrate is concentrated 10-20 doubly.

Embodiment 2

In culture filtrate, measure endoglucanase

Active pH and temperature dependency

In order to measure pH and temperature dependency, with strains A LKO 4179, ALKO 4124, and ALKO 4237, and ALKO 4265 and ALKO 4125 grow in the hot culture medium B of improvement. The sample (culture filtrate) that to cultivate from shaking flask is 5.0mM McI lvain ' s buffer solution (the 50mM citric acid-100mM Na of 4.5-8.5 in the pH scope₂HPO ₄) middle dilution. For strains A LKO 4179, the final pH value of culture filtrate buffer solution mixture is 4.3,5.4,6.3,7.3,8.1 and 8.7, and be 4.3,5.4,6.4,7.3 to strains A LKO 4124,8.1 and 8.5; Be 4.4,5.3 for strains A LKO 4237,, 6.2,7.1,8.0 and 8.5; Be 4.3,5.4,6.3,7.2 for strains A LKO 4265,8.1 and 8.5, be 4.3,5.4,6.4,7.3 for strains A LKO 4125,8.1 and 8.5. Adding BSA is 100 μ g/ml as protein carrier to concentration. As protease inhibitors, add pepsin inhibitor A and phenylmethylsulfonyl fluoride (PMSF) and be respectively 1.0 μ g/ml and 174 μ g/ml. Under each pH, it is active to measure endoglucanase under 50 ℃ of 60 minute reaction time. In the pH scope of about 4.5-7.5, ALKO 4179 shows active above the endoglucanase of its maximum 90%, detects maximum activity (Figure 1A) at about pH5.4-6.3. ALKO 4124 shows the endoglucanase activity above its maximum activity 80% in the pH scope of about 5.5-7.5, is approximately detecting maximum activity (Fig. 2 A) under the pH6.4. ALKO 4265 shows the endoglucanase activity above its maximum activity 80% in the pH scope of about 4.5-7.0, detects maximum activity (Fig. 4 A) when about pH5.5-6.5. ALKO 4237 shows the endoglucanase activity above its maximum activity 80% in the pH scope of about 4.5-6.0, detects maximum activity (Fig. 3 A) when about pH5.3. ALKO 4125 shows the endoglucanase activity above its maximum activity 90% in the pH scope of about 4.5-7.5, detects maximum activity (Fig. 5 A) when about pH6.5.

For the temperature dependency of carrying out the endoglucanase activity is measured. , dilute among the pH7.0 at 50mM McI lvain ' s buffer solution from the sample of culture filtrate. Adding BSA is 100 μ g/ml as protein carrier to concentration. Add pepsin inhibitor A and phenylmethylsulfonyl fluoride (PMSF) as protease inhibitors to being respectively 10 μ g/ml and 174 μ gml. The final pH value of culture filtrate buffer solution mixture is 7.3 (ALKO 4179, ALKO 4124 and ALKO 4125) and 7.2 (ALKO 4237 and ALKO 4265). Sample was cultivated 60 minutes for 50 ℃ and 60 at 40 ℃. Active at 50 ℃ and the 60 ℃ maximum endoglucanases that detect ALKO 4179, keep this activity (Figure 1B) of about 30% at 40 ℃. Active at 60 ℃ of maximum endoglucanases that detect ALKO 4124, keep about 70% activity at 50 ℃, keep 30% (Fig. 2 B) at 40 ℃. Active at 60 ℃ of maximum endoglucanases that detect ALKO 4237, keep about 60% activity at 50 ℃, keep 40% (Fig. 3 B) at 40 ℃. Active at 60 ℃ of maximum endoglucanases that detect ALKO 4265, keep about 50% activity at 50 ℃, keep 30% (Fig. 4 B) at 40 ℃. Active at 60 ℃ of maximum endoglucanases that detect ALKO 4125, keep about 80% activity at 50 ℃, keep 70% (Fig. 5 B) at 40 ℃.

Embodiment 3

The release of indigo dye under neutrallty condition

Check is from strains A LKO 4179, ALKO 4124, ALKO 4237, the cellulase goods of ALKO 4265 and ALKO 4125 (embodiment 1 and 2) under neutrallty condition from containing of indigo dyeing cotton denim goods fabric released dye wear away the ability of outward appearance with generation. Commercial acidic cellulase product Ecostone L (Primalco Ltd, Biotec, Finland) is with comparing.

The denim goods fabric obtains from Lauffenmuehl (Germany). The test fabric at 60 ℃ with Ecostone A 200 (1ml/ liter, Primalco Ltd., Biotec, Finland) prewashing 10 minutes. Then fabric is cut into the swatch of 12 * 12cm. With Minolta (Osaka, Japan) Chroma Meter CM 1000 R L^*a ^*The color on b systematic survey fabric cloth specimen two sides is as response value.

Carrying out as follows cellulase in LP-2 Launder-Ometer (Atlas, Illinois, USA) processes. With pack into the salt buffer of 0.05M citric acid/phosphoric acid of containing 200ml pH7 of about 7g denim goods cloth specimen, or in 1.2 liters of containers of the 0.05M citrate buffer solution of pH5.2, add 0.06ml 10%Berolo8 (Berol Nobel AS, Sweden) as surfactant.

A certain amount of steel ball is added in each container to help to remove fiber. Add cellulase solution after last in the container as endoglucanase active unit (embodiment 1). Then seal this container and put into 50 ℃ of Launder-Ometer water-baths. Move Launder-Ometer 2 hours with 42rpm.

After taking out cloth specimen from container, they were soaked in 200ml 0.01NaOH 10 minutes and with cold rinse 10 minutes. Cloth specimen spends the night 105 ℃ of lower dryings 1 hour and at air drying. Measure the color on cloth specimen two sides with Minolta Chroma Meter. The color measuring result of the denim goods fabric of processing shows in Table I.

Table I. the color measured values of the denim goods of processing with different cellulase goods

Enzyme source	Fabric	Fabric face			Fabric backing
								L	b	ΔE	L	b	ΔE
		pH7 *
--	--									2.3	0.8	3.1	1.5	0.1	0.9
		ALKO 4237	200	6.4	3.3	7.6	2.4	1.7	3.2
	400									7.7	3.8	8.1	2.5	1.8	3.0
		ALKO 4179	200	5.5	2.4	6.4	2.8	1.9	3.0
	400									4.6	2.8	5.1	2.2	1.5	3.0
		ALKO 4124	200	4.8	2.8	6.1	3.3	1.2	2.5
	400									ND	ND	ND	ND	ND	ND
		ALKO 4125	200	4.0	2.7	5.6	2.3	1.5	2.3
	400									ND	ND	ND	ND	ND	ND
		ALKO 4265	200	2.2	3.6	5.1	-4.9	6.6	9.2
	400									ND	ND	ND	ND	ND	ND
		Ecostone L
	200		1.6	0.7	1.6	0	1.7	1.6
									400	1.6	0.9	1.8	-1.9	2.2	2.8
pH5.2 **
								Ecostone L	200	2.10	2.33	3.30	-   2.74	4.35	4.71
	400	3.19	2.76	4.35	-   2.56	4.83	6.71
								L: the fabric light units subtracts the front fabric light units of processing after processing. B: the blue color unit of fabric subtracts the blue color unit of the front fabric of processing after processing. Δ E: between color sample and the target color (target fabric=untreated denim goods fabric) with L*a *b *The color distortion of color interval. The ND=undetermined*The ECU activity is measured under pH7.0.**The ECU activity is measured under pH4.8.

With the FINAL APPEARANCE of denim goods fabric after the different cellulase goods washings, measure the color of fabric (reverse side and front) for relatively. As seen using ALKO 4179 from the result shown in the Table I, ALKO 4124, and the brightness of clothes front lighting and blue color unit that ALKO 4237 and ALKO 4125 cellulase goods were washed obviously increase, and show and wear away preferably effect. Also increase but light units does not increase at the blue color unit of the inferior face of the fabric washed with ALKO 4265 goods. This may be to cause that simultaneously some redye because this enzyme works under this pH. With commodity acid product Ecostone L under the pH7 when this ECU is active to fabric without wearing away effect.

In this test, in the index of redying degree as fabric face of redying of fabric backing. In order quantitatively to redye level, before cellulase is processed with the color of rear measurement fabric backing. Shown in Table I, when ECU measures when identical, compare with the fabric of processing with ALKO 4265 or Ecostone L (pH5.2 and 7) goods, with ALKO 4179, ALKO 4124, on the fabric that ALKO 4237 and ALKO 4125 goods are processed in fact without redying.

Embodiment 4

Dyestuff under neutrallty condition during without Berol discharges

Except not using the Berol, this experimental program as described in example 3 above. The color measuring result of the denim goods fabric of processing is shown in the Table II.

Table II. with the color measuring of the denim goods fabric of different cellulase goods-process without Berol.

Enzyme source	Fabric	Fabric face			Fabric backing
								L	b	ΔE	L	b	ΔE
		pH7 *
--	--									2.1	0.5	2.2	1.7	-1.1	2.0
		ALKO 4237	200	5.5	3.1	7.0	1.8	2.3	3.5
ALKO 4179	200									4.4	3.2	5.6	1.4	2.2	2.7
		ALKO 4124	200	4.2	2.9	5.0	1.1	2.0	2.4
ALKO 4125	200									3.5	2.6	4.4	1.6	1.4	2.5
		ALKO 4265	200	3.3	3.3	5.3	-5.7	6.6	10.0
Ecostone L	200									14	0.9	1.7	0.3	1.4	1.8
		400	1.4	0.8	1.7	-0.1	1.7	1.8
	pH5.2 **
Ecostone L									200	2.0	2.1	2.9	-4.0	4.8	5.4
	L: the fabric light units subtracts the front fabric light units of processing after processing. B: the blue color unit of fabric subtracts the blue color unit of the front fabric of processing after processing. Δ E: between color sample and the target color (target fabric=untreated denim goods fabric) with L*a *b *The color distortion of color interval. The ND=undetermined*The ECU activity is measured under pH7.0.**The ECU activity is measured under pH4.8.

Compare with the result (embodiment 3) who contains the Berol acquisition, the data of Table II show uses ALKO 4179 without assistant Berol the time, and ALKO 4124, and ALKO 4237 and ALKO 4125 cellulase goods can reach the almost identical effect of wearing away.

Embodiment 5

With different cellulases at twill

Redying in the garrha washing

In this manual, reported to redye and depended on pH and/or enzyme type. Yet, as shown here, find to redye only letter and connect and depend on pH (Fig. 6 A and 6B and 7A and 7B).

In small-scale denim goods washing, under pH5 and pH7, study from ALKO 4237 with from 2 kinds of neutral cellulase goods and the acidic cellulase product Ecostone L of ALKO 4265 with the enzyme dosage that equates. Determine to wear away effect through the enhancing of measuring brightness and Lan Se as the catoptry in fiber front, redye (the again deposition of fiber surface indigo) and be determined as the blue look enhancing of reverse side and brightness decline. When pH7, the neutral cellulase of ALKO 4237 causes front face brightness and blue obviously enhancing and does not observe and redye (Fig. 6 A and 6B). When pH5, find similar wear away effect but slight redying arranged. When pH7, another neutral cellulase ALKO 4265 strengthens positive blueness but strongly redyes at reverse side. When pH5, observe similar effect with ALKO 4265 with ALKO 4237 goods. When pH7, acidic cellulase is not redyed or is reduced front face brightness (when using the endoglucanase active dose similar with ALKO 4265 and ALKO 4237, Fig. 7 A and 7B, 1 * dosage), is likely because of it does not play a role under this pH. On the other hand, when pH5, positive brightness and Lan Se strengthen and observe significantly at reverse side and redye. According to these results, under two kinds of pH values, can occur redying according to used cellulase goods.

Embodiment 6

The enzyme preparation that contains neutral cellulase is containing cotton

Application in the biopolishing of yarn fabric

In Launder-Ometer, process 100% cotton-spinning fabric with ALKO 4237 (embodiment 1) and ALKO 4467 cellulases. ALKO 4467 has the more UV-mutant of high-cellulose enzymatic activity from ALKO 4125.

Press embodiment 7 preliminary treatment 100% cotton-spinning fabric (from Pirkanmaan Vusi Varjaamo Ltd). The cellulase treatment conditions as described in Example 3, except not using Berol, and liquor ratio is 1: 15 (liquid volume of every part of fabric weight). With ECU active unit (embodiment 1) basis weight of fiber element enzyme.

Following method is for estimating the effect of enzyme preparation at the biopolishing of bafta: the loss in weight of the fabric of processing is defined as before the test and the percentage (before weighing, fabric is controlled in the atmosphere of 2 ℃ of 2H and 50+5%RH) of rear fabric weight. Formed by 3 people the one group evaluation of carrying out the cleaning surfaces effect of the fabric that enzyme processes. Fabric is classified with 1 to 5 score, wherein 5 surfaces that produce cleaning. Martindale rubbing method (SFS-4328) is used for estimating balling-up. Estimate balling-up (the many balls of 1=, not balling-up of 5=) with one group after 200 to 2000 circulations that rub.

Table III shows with ALKO 4237 and ALKO 4467 cellulase goods to be processed bafta at pH5 and all produces preferably cleaning surfaces and the remarkable proclivity that reduced for 7 times.

Table III. the loss in weight of the bafta of processing with neutral cellulase in Launder-Ometer, the cleaning surfaces effect has reached proclivity

Goods	Dosage ECU/ g	Time h	pH	Loss in weight %	The cleaning surfaces effect	Balling-up 200 circulations	2000 circulations
								-   ALKO4237   ALKO4237   ALKO4467   ALKO4467   -   ALKO4237   ALKO4237   ALKO4467   ALKO4467   -   ALKO4237   ALKO4237   ALKO4467   ALKO4467   -   ALKO4237   ALKO4237   ALKO4467   ALKO4467	-   200   400   200   400   -   200   400   200   400   -   200   400   200   400   -   200   400   200   400	1   1   1   1   1   2   2   2   2   2   1   1   1   1   1   2   2   2   2   2	5   5   5   5   5   5   5   5   5   5   7   7   7   7   7   7   7   7   7   7	0   2.3   3.2   1.2   1.9   0.1   4.4   6.0   3.0   4.0   0   2.5   3.8   0.8   14   0.1   4.8   6.0   2.2   3.0	1.0   3.5   3.5   2.5   2.8   1.0   4.0   4.3   3.5   3.8   1.0   3.0   4.0   2.0   2.0   1.0   4.0   4.3   2.5   3.3	1.0   4.0   4.0   3.7   3.7   1.0   4.2   4.2   4.0   4.0   1.0   3.7   4.0   3.5   3.6   1.2   3.8   4.0   4.0   3.8	1.0   3.8   3.8   3.4   3.4   1.0   4.1   4.3   3.8   3.9   1.0   3.5   3.9   3.3   3.7   1.1   4.0   4.3   3.4   3.7

Embodiment 7

In biopolishing, use of the present invention

The enzyme preparation that contains neutral cellulase

7a. the application of enzyme preparation in the biopolishing of textile and knitting.

In half industrial barrel washing machine (Esteri 20 HS-P), process 100% cotton-spinning fabric or 100% cotton knitting with cellulase of the present invention (embodiment 1). Treatment conditions are as follows:

A. preliminary treatment (only to yarn fabric).

60 ℃, 10 minutes, Ecostone A 200 (Primalco Ltd.Biotec, Finland) 1ml/l water.

B. enzyme is processed.

Temperature 50-60 ℃, pH7;

Liquor ratio 5-20: 1 (liquid volume of every part of fabric weight);

Processing time 20-90 minute, preferred 30-60 minute;

Enzyme dosage 50-900hKat/g fabric or knitting, preferred 200-600nKat/g fabric or knitting.

C. " after the washing " processing

40 ℃, 10 minutes, alkaline detergent,

D. dry processing:

Following standard method is used for estimating the cleaning surfaces effect of enzyme preparation: Martindale rubbing manipulation (SFS-4328) and washable power test (SFS-3378). Cause producing the cleaning surfaces effect with cellulase goods processing of the present invention, fabric and knitting flexibility and slickness strengthen and have reduced a proclivity.

7b. the application of enzyme preparation in the polishing of lyocell fabric and braid.

Cellulase goods of the present invention can be used in the different finishing methods of fibrillation formation control and 100%lyocell fabric and knitting and composition thereof. For produce at the lyocell fabric peachiness effect can be in half industrial barrel washing machine (Esteri 20HS-P) treatment conditions below the use:

A. sodium carbonate 2.5g/l, 60 ℃, 60 minutes processing time;

B. rinsing;

C. enzyme is processed: temperature 50-60 ℃, and pH7, liquor ratio 5-20: 1, processing time 40-120 minute, preferred 40-90 minute, enzyme dosage was the 100-1500nKat/g fabric, preferred 400-800nKat/g fabric;

D. post processing: 40 ℃ of alkaline detergents washed 10 minutes:

E. rinsing;

F. dry.

The result produces peachiness fur effect.

Embodiment 8

Wear away middle use enzyme preparation at biology

Processing the denim goods clothes with neutral cellulase goods (embodiment 1) in half industrial barrel washing machine (Esteri 20 HS-P) makes this clothes generation wear away outward appearance. Each machine burden is used about 1.0kg denim goods clothes (containing 2 kinds of dissimilar fabrics).

Treatment conditions are as follows:

A. destarch. 100 premium on currency, 60 ℃, 10 minutes; 100ml Ecostone A200 (Primalco Ltd., Biotec, Finland).

B. cellulase is processed. 100 premium on currency, 50 ℃, 45 minutes; 10g Berol 08 (Berol Nobel AS, Sweden); 30g citric acid+128g Na₂HPO ₄×2H ₂O makes to reach pH7.

With the quantitative neutral cellulase goods of endoglucanase active unit (ECU, embodiment 1):

1.ALKO 4237,260ECU/g clothes,

2.ALKO 4179,260ECU/g clothes,

3.ALKO 4124,300ECU/g clothes,

4.ALKO 4125,250ECU/g clothes,

C. afterwards washing, alkaline detergent washing, 40 ℃, 10 minutes.

D. dry.

With the naked eyes outward appearance of clothes with through using Minolta Chroma Meter CM 100R L^*a ^*b ^*Color (Table IV) evaluation result is measured with reflected value by system. The clothes that all these cellulases are processed obtain to wear away preferably effect. In the clothes that these cellulases are processed, have no through naked eyes and redye (checking the clothes inner face).

As seen the color measuring result who shows from Table IV obviously increases with the brightness of clothes outer surface and the blue color unit of the washing of neutral cellulase goods, demonstrates and wears away preferably effect.

Table IV. the color measuring of the denim goods clothes of processing with different cellulase goods

Enzyme source	The clothes outer surface		The clothes inner surface
					L	b	L	b
	A. fabric
1
					Be untreated	24.1	-8.5	57.1	0.17
The washing of cellulose-less enzyme	21.4	-14.0	54.5	-4.3
					ALKO4237	26.7	-17.3	56.5	-4.9
ALKO4179	26.8	-17.0	56.3	-4.5
					ALKO4125	28.0	-17.4	57.8	-4.1
ALKO4124	26.4	-17.5	57.1	-4.8
					B. fabric 2
Be untreated	22.5	-8.3	57.6	0.66
					ALKO4237	25.0	-16.3	56.1	-4.3
ALKO4179	25.0	-15.8	55.4	-4.4
					ALKO4125	26.7	-17.0	56.8	-4.0
ALKO4124	25.6	-17.0	56.4	-4.0

The brill of clothes after L=processes (be worth higher, clothes are brighter).

The blue color unit (negative value is larger, and clothes are more blue) of clothes after b=processes.

Embodiment 9

The purifying of neutral cellulase

Be used among the 25mM Tris/HCl pH7.2 from the line style gradient separations of 0 to the 0.5M NaCl concentrated growth medium from ALKO 4237 at 7 ℃ of DEAE agarose CL6B. Endoglucanase Peak Activity when finding 4 pH4.8. Peak I, the ECU that contains about 10% recovery, at about 150mM NaCl place wash-out, peak II (about 30% ECU) is at 230mM NaCl, peak III (about 20% ECU) is at 270mM NaCl place, and peak IV (about 40% ECU) is at 320mM NaCl place.

Table V has shown the result who tests its application in the biology of neutral pH and 50 ℃ is worn away of these peaks.

These results show that according to ECU and gross protein, peak II is than any other peak or more effective than unsegregated concentrate. The peak I that respectively contains the 70ECU/g denim goods and the mixture of II have also been tested. L (front) value is that 7.3, b (reverse side) is 2.5 as a result. Therefore, this mixture is more effective than arbitrary independent peak.

Increase purifying procedure to obtain the homogeneity sample of some desired proteins in these peaks. Separate concentrated ALKO 4237 growth mediums (4.5 liters) with ammonium sulfate. Every 100ml concentrate is suspended among 0.9 liter of 25mM Tris/HCl pH7.2 that contains 0.25mM EDTA with the protein of 17g to 42g ammonium sulfate precipitation, then is diluted with water to conductivity and is 4ms/cm and with 1M NaOH furnishing pH8.0. The solution of gained (about 45 liters) is at room temperature crossed 6.3 liters DEAE-Sepharose FF with the 150ml/ wheel cylinder^TMPost. The not combination of endoglucanase activity of peak I under this class condition. Be used among 20 liters of 25mM Tris/HCl pH7.7 that contain 0.25mM EDTA from the line style gradient of 0.0 to the 0.5M NaCl protein with 110ml/ minute elution of bound. The endoglucanase of peak II is at about 14ms/cm place wash-out. In refrigerating chamber, in DEAE, as seen be called the simple spike of peak III/IV with the small lot goods at about 25ms/cm place wash-out, replace the peak III and the IV that separate.

Table V. under neutrallty condition with DEAE-Sepharose^TMAmalgamation liquid discharges indigo dye

	Add
											Nothing	Concentrate		Peak I	Peak II		Peak III		Peak IV
	ECU/g	0	100	200	310	185	340	97	260	95											190
mg/g											0	10	20	41	9	26	24	46	5	10
	L (front)	2.9	5.2	7.0	5.5	7.3	10.3	4.4	5.8	3.9											4.3
B (reverse side)											0.4	2.6	3.5	3.5	2.9	25	0.9	1.4	0.1	0.5

The brightness in the blue look denim goods of parameter L (front) expression front, the blue look (that is, redying) of b (reverse side) expression reverse side. Fabric washs in LP-2 Launder-Ometer, then except not using Berol and used buffer solution (to see Biochemical Research as 0.05M McIlVaine pH7, Dawson, R, Deng editor, 1969, the data of Oxford University Press) outside, it is described with Minolta ChromaMeter measurement to press embodiment 3. Dose form is shown ECU/g denim goods and mg protein/g denim goods.

With the protein among ammonium sulfate (450g/ liter) the precipitation peak II (3.5 liters) and be suspended among the 25mM PIPES/KOH pH6.0 that 170ml contains 1mM EDTA. Process is at G25 Sephadex ^TM5 * 29cm post on gel filtration (rough) this material of part is converted at 25mM and contains among the sodium acetate pH4.0 of 1mM EDTA, then at SP-Sepharose^TMUpper classification separates. Fig. 8 has shown the SP-Sepharose that is loaded to the 25mM sodium acetate pH4.0 that contains 1mM EDTA of 150ml/h when this protein of 11.7g^TM4.5 * 31cm post on and the result that obtains of the post that forms with 75ml/l from the 0.0 line style gradient that obtains 0.4M NaCl of having in 3.4 liters of same buffer. Elute most of endoglucanase at 0.2M NaCl place. Use is measured in the improvement described in the embodiment 10. When active fractions is stored in 7 ℃, crystalline deposit occurs therein and almost contain whole endoglucanases activity. Crystallization slowly active fractions (15ml) is induced the formation crystal through control oneself 30 μ l suspensions of the cut that contains crystallization of inoculation. After 2 to 3 days, through centrifugal collection crystal, use the 25mM PIPES/KOH pH6.0 washing that contains 1mM EDTA and be dissolved in the 25mM Tris/HCl pH7.2 that contains 0.25mM EDTA. Analyze to show that through SDS-PAGE the crystallization of washing contains apparent molecular weight near the basically homogeneous protein of 20kDa (error of the SDS-PAGE of molecular weight in estimating be at least ± 10%, may be larger for unusual protein). This protein is called the 20K-cellulase. Through the G50Sephadex in containing the 50mM PIPES/KOH pH6.0 of 1mM EDTA^TMUpper gel filtration also can be removed contaminating protein matter. Its example shows in Fig. 9, wherein through the unwashed crystal of gel filtration purifying. Endoglucanase activity with the common wash-out of 20kDa protein behind cromoci (11.2kDa) volume shows this 20kDa protein process and Sephadex^TMInteract and exception deferral.

With the protein among the ammonium sulfate precipitation peak III/IV and with II protein described identical mode in peak is converted to the 25mM sodium acetate pH4.0 that contains 1mM EDTA. When converting 25mM sodium acetate pH4.0 to, a large amount of precipitations of formation are also discarded. Active supernatant is at SP-Sepharose^TMUpper classification separates. Be loaded on 2.5 * 11cm post with epsilon protein (for example 200mg protein) as shown in figure 10, most of endoglucanase activity are incorporated on the post and with the NaCl gradient elution of about 50mM NaCl. This Peak Activity then is the second peak of inactive protein matter.

SDS-PAGE analyze to show active and the non-activity peak all contains some protein, comprises that the apparent molecular weight that can not distinguish mutually with SDS-PAGE is near the protein of 50kDa. With at Phenyl Sepharose^TMOn chromatogram be further purified two peaks.

Merge active fractions (cut 15 to 18 among Figure 10), transfer to 50mM PIPES/KOHpH6.0 (through adding 0.25M PIPES/KOH pH6.0) and 15g% ammonium sulfate (through adding solid ammonium sulfate) and be loaded to the Phenyl Sepharose that uses the 25mM PIPES/KOH pH6.0 balance that contains 1mM EDTA and 15g% ammonium sulfate^TM1.5 * 8.5cm post on. The line style gradient from 15 to 0g% ammonium sulfate that is used among the 104ml 25mM PIPES/KOH pH6.0 is crossed post. When gradient finishes, with the further column scrubber of 25mM PIPES/KOH pH6.0. Wash out two protein peaks in gradient, first is the small peak of inactive protein matter, then is the main peak that contains most of endoglucanase activity. SDS-PAGE analyzes (Figure 11 A and B) and shows that two peaks contain apparent molecular weight near the basically homogeneous protein of 50kDa (that is, they are that the BioRad of the 47kDa ovalbumin standard items migration of dying in advance is slightly slow than apparent molecular weight). Can not distinguish this two kinds of protein through the inventor's SDS-PAGE analysis, can not distinguish even walk together electrophoresis when them as mixture. Protein in the Peak Activity is called the 50K cellulase, and the protein in the nonactive peak is called the 50K-PROTEIN B. Through separation by top to the described identical mode of active fractions at Phenyl Sepharose^TMUpper from SP-Sepharose^TMSecond (nonactive) peak (cut 19 to 23 among Figure 10) of wash-out obtains more substantial 50K-cellulase B.

Through loading SP-Sepharose^TMPost helps to produce more substantial 50K-cellulase and 50K-cellulase B. For example, be loaded to 4.5 * 31cm SP-Sepharose when 15g protein^TMWhen upper, the 50K-cellulase is obviously replaced by the protein of stronger combination and is eluted before not being incorporated in the NaCl gradient with the post knot. This material is by highly purified, and through as mentioned above at Phenyl Sepharose^TMUpper chromatography is therefrom isolated homogeneous 50K-cellulase.

In order to accelerate the large-scale purification of 50K-cellulase, put upside down SP-Sepharos and Phenyl Sepharose post. Regulate ammonium sulfate concentrations to about 15g%, the protein that will precipitate in peak III/IV is loaded on the Phenyl Sepharose as previously mentioned. When applied sample amount is higher (for example, 17g protein is loaded on 3.2 * 25cm Phenyl Sepharose post), most of gross protein flows through post, but 50K-cellulase (it is active to contain most of endoglucanases) combination, and the 15 line style gradient end wash-outs to 0g% ammonium sulfate in 25mM PIPES/KOH pH6.0. Western with the rabbit anti-serum of identifying 50K-cellulase B the analysis showed that 50K cellulase B is wash-out before the 50K-cellulase just. Through realizing being further purified by noted earlier the separation at SP-Sepharose. In the situation of this SP-Sepharose and Phenyl Sepharose reversed order, can will directly be loaded in next purification step without desalting with the protein among the peak III/IV of ammonium sulfate precipitation. The a large amount of protein precipitations that occur in the time of so also can avoiding among the peak III/IV concentrated protein to be directly changed into 25ml sodium acetate pH4.0 for SP-Sepharose. Because the 50K-cellulase is only in conjunction with SP-Spharose, the obvious interference of front gross protein of loading on the separation that Phenyl Sepharose carries out significantly reduces SP-Sepharose.

In Launder-Ometer, test respectively 50K-cellulase and 50K-cellulase B and whether be responsible for the beneficial effect of the peak IV that reported among the embodiment 10 to observe them. Find that two kinds of protein all have beneficial effect (Table VI). When using low concentration in this experiment, they itself do not increase indigo dye from the release of denim goods outer surface (namely, the L front does not increase), but they effectively reduce dyestuff redye the inner surface of denim goods (the L reverse side become more on the occasion of and the b reverse side become less), particularly when using with the 20K-cellulase.

20K-cellulase effect is better when pH5 reaches at pH7 in the Launder-Ometer test. When pH5, the 20K-cellulase of every gram denim goods 0.2mg is increased to 5.2 with the L front from 3.2. Add the 50K-cellulase with the 20K cellulase with the amount of the every gram denim goods of 0.1mg and also reduce redying when the pH5 (L reverse side and b reverse side 0.0 and 2.6 1.3 and 1.5 when becoming respectively the mixture of usefulness 20K-and 50K-cellulase from only with the 20K-cellulase time).

Table VI. use the 20K-cellulase, 50K-cellulase and 50K cellulase B discharge indigo dye.

Condition is identical with Table V, and dosage represents with the mg albumen prime number of every gram denim goods.

Sample	Dosage   (mg/g)	L Positive	L Reverse side	b Reverse side
					Only has buffer solution 20K-cellulase 50K-cellulase 50K-cellulase B 20K-cellulase+50K-cellulase 20K-cellulase+50K-cellulase B	-   0.18   0.09   0.15   0.075   0.31   0.15   0.18+0.075   0.09+0.075   0.18+0.15	2.8   5.6   4.8   2.6   3.0   2.8   2.7   5.6   5.1   4.7	-0.6   -1.0   -1.5   -0.3   0.4   1.3   1.5   0.3   0.3   0.0	1.6   4.0   3.3   1.0   1.3   0.8   0.5   2.5   2.1   3.0

Embodiment 10

The feature of 20K-cellulase

Although (called after resists-EGI the polyclonal antibody for preparing for the cellulase from Trichoderma reesei purifying, anti--CBHI and anti--CBH II antibody) identify the protein in ALKO 4237 growth mediums, but with pure 20K-cellulase extremely weak cross reaction is only arranged in the Western engram analysis under the same conditions.

When the antiserum for pure 20K cellulase that produces in rabbit is surveyed from the growth medium of ALKO 4237 in Western analyzes, except the strong band of also observing at about 35kDa place of 20kDa band. It is active that this 35kDa protein is not detected obvious endoglucanase. In addition, near the 20kDa band, see weak band (Figure 14).

ALKO 4124 produces almost the banding pattern identical with ALKO 4237, shows that it and other fungi probably contain the cellulase closely similar with 20K-cellulase of the present invention.

Amino acid sequence from 20K-cellulase pancreatin peptide shows in Figure 17.

The 20K-cellulase of purifying is in the biology of neutral pH is worn away when not adding other enzymatic activity, and effect is better, as shown in Table VII.

Table VII, carry out biology with the 20K-cellulase of purifying and wear away:

Condition is as in the experiment as shown in the Table V. Dose form is shown mg protein/g denim goods fabric. " full culture medium " refers to the growth medium that unsegregated ALKO 4237 concentrates.

Add	Dosage	L Positive	b Positive	L Reverse side	b Reverse side
						Buffer solution	0.0	3.6	0.1	0.5	0.6
20K	0.72	8.9	2.9	-1.1	4.7
						20K	0.25	60	2.3	-0.5	3.6
20K	0.07	55.3	1.7	-0.4	2.9
						Full culture medium	20	6.1	2.8	-2.9	5.5

Compare with unsegregated culture medium, the 20K-cellulase produces brightness (the L front=6.0-6.1) of same degree when 1/80th protein dosage. And the less fabric backing (L reverse side=-0.5 is with respect to-2.9, b reverse side=3.6, with respect to 5.5) of redying. The fabric of processing with the 20K cellulase has satisfied tender texture.

Although the 20K-cellulase without other additive produces wonderful good effect, as 20K and DEAE-Sepharose amalgamation liquid I, III or IV produce better appearance of fabrics and quality (Table VIII) when using together.

Table VIII .20K cellulase with from the synergy of endoglucanase amalgamation liquid biology is worn away of DEAE-Sepharose wash-out.

Condition is identical with Table V

Add	Dosage	L Positive	b Positive	L Reverse side	b Reverse side
						Buffer solution	0.0	3.8	0.2	-0.7	1.5
20K	0.18	5.8	2.3	-2.2	5.5
						Amalgamation liquid I	15	51	1.9	-3.1	5.7
Amalgamation liquid III	47	5.2	1.6	-0.1	2.6
						Amalgamation liquid IV	14	5.6	0.9	0.4	1.8
20K+ amalgamation liquid I	15.18	7.1	2.8	0.7	3.3
						20K+ amalgamation liquid III	47.18	7.6	3.1	-1.7	5.3
20K+ amalgamation liquid IV	14.18	8.6	2.6	0.8	3.2
						Full culture medium	20	5.7	2.4	-4.1	5.9

20K-cellulase and amalgamation liquid I, the mixture of III and IV produce the outward appearance of brighter (it is positive to increase L) than arbitrary independent composition. At least for the combination of 20K-cellulase and amalgamation liquid IV, very clear is owing to synergy is not only the effect of increase. And, with redying in fact than only redying little (L reverse side corrigendum, the b face is less) with what the 20K-cellulase was observed of all mixtures. The combination of 20K and amalgamation liquid IV is effective especially. Amalgamation liquid IV contains numerous protein, and one of them (50kDa polypeptide) is combined at Sephadex G100 and S-Sepharose during the chromatogram of liquid IV and the active common purifying of endoglucanase. Although only realized that with the 20K-cellulase biology is worn away preferably, add that with the 20K cellulase one or more protein from amalgamation liquid IV purifying probably produce better result. The biology that carries out with 20K cellulase and mixture from the 50K-cellulase of amalgamation liquid III/IV purifying and 50K-cellulase B is worn away (the Table VI of embodiment 9) is provided. Therefore, the invention is not restricted to only use the 20K-cellulase. Other oroteins in ALKO 4237 culture mediums also is useful separately or with suitable combination.

Standard endoglucanase in descriptions such as Bailey measures (1981, loc.cit) in, select the enzyme amount, it produces about 0.6mM reduction homologue from 1% hydroxyethylcellulose enzyme under 10 minutes and pH4.8 (0.05M Na-citrate buffer solution), causing final absorptance to change (Δ A540) is 0.2 to 0.25. This head and shoulders above the amount proportional band of A540 and 20K-cellulase.

Therefore, by following update routine. Use enough enzymes in 0.05M HEPES buffer solution (pH7.0), to produce about 0.2mM reduction homologue in 10 minutes. In order to reach the critical concentration of reduction homologue, in the DNS system, forming color on this critical concentration, in the DNS reagent of storing, newly add 0.12mM glucose. When the endoglucanase of identifying 20K cellulase and 50K-cellulase is active, use the method (being called " improvement " method). Make substrate with 1% hydroxyethylcellulose, the amount proportional band of Δ A540 and 20K-and 50K-cellulase is rather narrow, and therefore 2% carboxymethyl cellulose is as alternative substrate. During with 2% carboxymethyl cellulose, the scope ratio that Δ A540 is relevant with 50K cellulase amount line style with 20K is wide with 1% hydroxyethylcellulose. The endoglucanase activity of measuring with 2% carboxymethyl cellulose with measure with 1% hydroxyethylcellulose compare for the 20K cellulase to be about 8-10 times, to be approximately 50 times for the 50K-cellulase.

Can't detect the 20K-cellulase activity for the characteristic substrate 4-methylumbelliferyl-β of the biological hydrolase of cellulose-D-lactoside. Activity to filter paper is also extremely low, but can detect.

The 20K cellulase has sizable heat endurance. With 7 μ g/ml at 25mM Tris-HCl, among the 0.2mM EDTA 100 ℃ to its insulation 30 or 60 minutes, then measure in that pH7.0 and 50 ℃ are lower. When pH7.2, keep respectively 52% and 35% of endoglucanase activity, when pH8.8, keep respectively 40% and 22%. (at room temperature measure these pH values; Actual pH at 100 ℃ is lower). At 80 ℃, during pH7.2, keep 70% activity in 60 minutes.

These results show that this enzyme is suitable for the application that enzyme wherein may (for example, accidental) be heated up. Owing to advancing at high temperature has resistance to irreversible inactivation, it is 70 ℃ (Figure 15) that this enzyme shows optimum temperature at 10 minutes duration of test of pH7.0. Being higher than that 70 ℃ of activity of observing reduce mainly is because the modulation of enzyme conformation, and this enzyme recovers that it is most of active when returning to 50 ℃.

In the time of 50 ℃, the 20K cellulase shows 80 ℃ or its larger maximum activity in 4 to 9 pH scope, be almost 50% when pH10. All like this in the mensuration of 10 minutes (Figure 16 A) and 60 minutes (Figure 16 B). These figure have also shown the pH dependence of this enzyme in the time of 70 ℃. In 10 minutes measured, in the scope of pH4.5 to 8, this enzyme had greater activity at 70 ℃ of ratios at 50 ℃, but when pH10 approximately active equal (Figure 16 A). In 60 minutes mensuration (that is, near commercial condition), between pH5.5 and 7.5, this enzyme has greater activity at 70 ℃ of ratios at 50 ℃. Yet during pH10, this enzyme is only lower slightly than activity 50 ℃ the time in the time of 70 ℃. In fact, this means that this enzyme effect of use is good equally in larger pH scope and under the temperature up at least 70 ℃.

Embodiment 11

The feature of 50K-cellulase

Pure 50K-cellulase has the biological hydrolytic enzyme activities of endoglucanase activity (to hydroxyethylcellulose) and cellulose (to 4-methylumblliferyl-β-D-lactoside, basically press van Tilbeurgh etc., at Enzymology method [1988] vol.160, measure described in the pp45-59). To be 1.8 pure enzyme sample contain 2030ECU/ml and 300PCU/ml (1PCU is the live vol that per second discharges the 1nmol methyl umbelliferone) to A280 when pH7.0 and 50 ℃.

In Western analyzes, the 50K-cellulase is identified strongly by the antiserum (KH1057) that produces for T.reesei endoglucanase I, but only by the faint identification of antiserum (being respectively KH1050 and KH1053) of the biological hydrolase I of anti-T.reesei cellulose and II. This enzyme of antiserum nonrecognition (Figure 14) for the generation of 20K cellulase. When using the rabbit anti-serum that produces for 50K cellulase itself in Western analyzes, to survey the growth medium of ALKO 4237, significantly be with except only the band that approximately 50kDa place is very strong, also seeing one (molecular weight 33 and 47kDa between).

When this protein was processed with Endoglucanases Hf, the apparent molecular weight of the 50K-cellulase of measuring through SDS-PAGE reduced about 2 to 5kDa, showed that this enzyme contains the sugar that can be removed by this Endoglucanases.

The 50K-cellulase has unusual resistance to trypsinization, shows that it has unusual rock-steady structure. Yet it can be through processing and cracking with hydrogen bromide, and then the fragment of gained can digest with pancreatin or lysyl inscribe PEPC C. The sequence of some peptides that so obtain shows in Table I X.

Table I X

Peptide sequence (uncertain residue represents with lowercase) from the separation of 50K-cellulase

#507  VYLLDETEHR

#509  XXLNPGGAYYGT

#563  MsEGAECEYDGVCDKDG

#565  NPYRVXITDYYGNS

#603  DPTGARSELNPGGAYYGTGYXDAQ

#605  XXVPDYhQHGVda

#610  NEMDIXEANSRA

#611  LPXGMNSALYLSEMDPTGARSELNP

#612  VEPSPEVTYSNLRXGEIXgXF

#619  DGCGWNPYRVvITtDYYnN

#620  LPCGMXSALY

#621  ADGCQPRTNYIVLDdLIHPXXQ

The 50K-cellulase is a kind of stable enzyme, reaches in larger pH value scope and at high temperature shows the endoglucanase activity, therefore, is applicable to many industrial conditions. When the reaction time of pH7.0 and 60 minutes. Optimum temperature is 65 to 70 ℃, even with reaction time of such length, it still shows the activity (Figure 12) at 50 ℃ viewed 50% in the time of 75 ℃.

Under 60 minute reaction time, pH has the activity of substantial constant just when extremely wide between pH4.4 to 7.0 in the time of 50 ℃, 50% and 30% the when activity pH9 and 10 o'clock equals pH7.0 respectively. In the time of 70 ℃, have clear and definite pH6 just when, between pH5 and 7, this activity (having 60 minute reaction time) is high 3 times or larger during than 50 ℃. Yet, be higher than at 8 o'clock at pH4.4 and pH value, this activity 50 ℃ 70 ℃ higher (in 60 minutes measure), show that the enzyme stability of pH scope 5 to 7.5 both sides in the time of 70 ℃ descends. The pH dependence represents in Figure 13.

Embodiment 12

The feature of 50K-cellulase B

Measure less than detectable endoglucanase active with hydroxyethylcellulose or carboxymethyl cellulose to 50K-cellulase B (being called in the past the 50K-PROTEIN B). When acid pH, 50K-cellulase B has the biological hydrolytic enzyme activities of low cellulose, and its (measuring with 4-methylumbelliferyl-β-D-lactoside) is no more than 0.1% of 50K cellulase when pH5. In addition, 50K cellulase B is to the filter paper of pH4.8 be used for the pH5 of enzyme assay and amorphous Scolca Floc-cellulose that 7 o'clock acidity expands has detectable activity.

In Western analyzes, the strong identification of antiserum (KH1050) that 50K-cellulase B is produced for the biological hydrolase I of T.reesei cellulose, but only by the faint identification of antiserum of the biological hydrolase II of anti-T.reesei cellulose or endoglucanase I or anti-50K-cellulase. Can not be by the antiserum of anti-20K-cellulase identification (Figure 14). Table X has shown the peptide sequence that separates from 50K-cellulase B.

Table X

Peptide sequence (uncertain residue represents with lowercase) from 50K-cellulase B separation

#534  vGNPDFYGK

#535  FGPIGSTY

#631  LSQYFIQDGeRK

#632  FTVVSRFEENK

#636  HEYGTNVGSRFYLMNGPDK

Embodiment 13

The stability of neutral cellulase in different detergents

In 3 kinds of different detergent solutions, measured the stability of neutral cellulase goods. Detergent solution is OMO^Total (or OMO^ Neste，Lever UK)，OMO ^Color (Lever S.A) and Colour Detergen Liqmd (Unilever, The Netherlands). The cellulase goods of test are ALKO 4125, and ALKO 4179, and the concentrated culture filtered fluid of ALKO 4237 and ALKO 4265 (embodiment 1) reaches 20K-and the 50K-cellulase (embodiment 9) from ALKO 4237 bacterial strain purifying.

The cellulase goods are cultivated in 0.25% detergent solution at 40 ℃. Cultivate sampling and measuring after 5-30 minute (pH7,50 ℃) to the activity (ECU/ml embodiment 1) of hydroxyethylcellulose enzyme.

The goods of test are as follows: the culture filtered fluid:

ALKO 4125：780ECU/ml(pH7，50℃)

ALKO 4179：830ECU/ml

ALKO 4265：760ECU/ml

ALKO 4237：650ECU/ml

Protein purification:

20K-cellulase: 9423ECU/ml

50K-cellulase: 10100ECU/ml

The result shows in Table X I-XIII.

ALKO 4179 in the detergent of all 3 kinds of tests, and ALKO 4265 and ALKO 4237 cellulase goods and 20K and 50K cellulase almost kept the stability of 100 % in 30 minutes 40 ℃ of insulations. At Colour Detergent Liquid with at OMO ^30 minutes ALKO 4125 of 40 ℃ of insulations have stability among the Neste.

The stability (pH7.5-7.9) of the different cellulases of Table X I. in 0.25%Colour Detergent Liquid

Goods	Enzyme preparation % (ml)	pH *	The % residual activity
								0′	5′	10′	20′	30′
			The culture filtered fluid:
ALKO4125   ALKO4179   ALKO4265   ALKO4237	6   6   6   6	7.3   7.1   7.2   7.1									100   100   100   100	97   99   100   100	98   100   100   82	98   100   100   95	99   10   100   100
			The protein of purifying from ALKO4237:
20K-cellulase 50K-cellulase	1   1	7.8   7.6									100   100	98   100	99   100	97   100	100   100

^*The pH of insulation 30 ' rear 0.25% detergent+enzyme solutions

The different cellulases of Table X II. are O.25%OMO^Total (or OMO^Neste pH8.5) stability in

Goods	Enzyme preparation % (ml)	pH *	The % residual activity
								0′	5′	10′	20′	30′
			The culture filtered fluid:
ALKO4125 ALKO4179 ALKO4265 ALKO4265 ALKO4237 ALKO4237	6     6     6     4     4     2	7.8     7.3     7.1     7.8     7.8     7.3									100     100     100     100     100     100	98     98     100     99     100     99	96     96     100     97     100     97	86     96     100     100     99     99	87     99     100     100     100     99
			The protein of purifying from ALKO4237:
20K-cellulase 50K-cellulase	1     1	8.2     7.8									100     100	100     95	99     92	93     95	100     94

^*The pH of insulation 30 ' rear 0.25% detergent+enzyme solutions

The different cellulases of Table X III. are at 0.25%OMO^Stability among the Colour (pH9.6-10)

Goods	Enzyme preparation % (ml)	pH *	The % residual activity
								0′	5′	10′	20′	30′
			The culture filtered fluid:
ALKO4125 ALKO4179 ALKO4265 ALKO4265 ALKO4237 ALKO4237	6     6     6     4     4     2	9.6     8.3     9.1     8.5     8.5     9.1									100   100   100   100   100   100	(15)   97   100   93   98   93	(15)   100   100   95   96   95	(13)   97   100   99   96   99	(14)   99   100   98   99   98
			The protein of purifying from ALKO4237:
20K-cellulase 50K-cellulase	1     1	9.8     8.9									100   100	99   100	100   100	100   100	100   100

^*The pH of insulation 30 ' rear 0.25% detergent+enzyme solutions

Embodiment 14

Neutral cellulase in detergent to the effect of HEC substrate

Use hydroxyethylcellulose (HEC) to measure the effect of different neutral cellulases in detergent as substrate. The cellulase goods of test are that ALKO 4265 and ALKO 4237 concentrated culture filtrates reach 20K-and the 50K-cellulase from ALKO 4237 bacterial strain purifying. Through 1%HEC being dissolved in preparation HEC substrate in 0.25% detergent solution. Described in the activity (ECU/ml) of 40 ℃ of each cellulase goods of measurement to HEC through using these substrates to press embodiment 1. The detergent that uses in these experiments and cellulase goods are being implemented to fall and are being described in 13.

The pH of substrate

HEC/ pH of buffer 7

HEC/Colour Detergent Liquid      pH7.5

HEC/OMO ^Total                  pH7.8

HEC/OMO ^Color                  pH9.7

Table X IV. is at the ECU of different detergent cellulase goods (relatively being the active % of the ECU that measures in the pH7 buffer solution)

Goods	Active % ECU/ buffer solution	ECU/     col.det.liquid	ECU/   OMO Total	ECU/ OMO Color
					Culture filtrate:
The protein of ALK-4265 ALK-4237 purifying: 20K-cellulase 50K-cellulase	100  100  100  100	89     97     100     92	96     95     93     79	59   40   81   46

When using HEC to make substrate, ALKO 4237 and ALKO 4265 cellulase goods and 20K-and 50K-cellulase have function in the detergent of all 3 kinds of tests.

Embodiment 15

Use at detergent at wollen fabrics

In neutral cellulase

Described in this experiment neutral cellulase as fabric softener and prevented that thereby napping minimizing bafta behind the detergent repeated washing from playing the ability of proclivity. The cellulase goods of test are that ALKO 4237 concentrated culture filtrates reach 20K-and the 50K-cellulase (embodiment 1 and 9) from ALKO 4237 bacterial strain purifying.

Wash experiment with Launder-Ometer LP-2 (Atlas, Illinois, USA). With (embodiment 3) unbleached cotton-spinning fabric sheet of about 10g prewashing pack into contain that 150ml has or 1.2 liters of containers of 0.25% detergent solution of cellulose-less enzyme in. Cellulase dosage is take albumen quality as the basis. Detergent solution is OMO^Total (Lever, UK) and Colour Detergent Liquid (Unilever, The Netherlands). In each container, add a certain amount of steel ball to increase mechanism. Launder Ometer moves 0.5 or 1 hour at 40 ℃ with 42rpm. With the rinsing at interval and dry laundering of textile fabrics 4 times.

The loss in weight (with embodiment 6) is used for describing the suede amount of removing from fabric face.

The loss in weight of fabric after Table X V. washs in detergent for the first time with neutral cellulase.

Sample number	Goods	The enzyme dosage that represents with protein/g fabric	Time h	The % loss in weight
					At Colour Detergent Liquid:
1   2   3   4   5   6   7     8     9   10	-ALK04237 ALK04237 20K-cellulase 20K-cellulase 20K-cellulase 50K-cellulase 50K-cellulase-20K-cellulase	-   11   22   2   5   8   2     5     -   8	-   1   1   1   1   1   1     1     0.5   0.5	0.05   0.3   0.7   0.1   0.5   1.0   0.1     0.2     0.2   0.5
					At OMOTotal：
11   12   13   14	-20K-cellulase-20K-cellulase	-   8   -   8	1   1   0.5   0.5	0.03   1.1   0.1   0.7

In Table X V, shown that the fabric of processing with cellulase after the washing first time is than only the obvious increase of fabric weight loss with detergent-treatment is more in Launder-Ometer. And the loss in weight increases with the effect of cellulase dosage, with the 20K cellulase loss in weight further increased when wash time extended to 1 hour from 0.5 hour. The 20K-cellulase is at Colour Detergent Liquid with at OMO^Effect is equally good among the Total. These results show that especially 20K-cellulase and ALKO 4237 cellulase goods serve as the suede agent in detergent after washing once.

With sample 1,2, after 4 and 7 (Table X V) washed 3 times again, one group that is comprised of 3 people was carried out fabric evaluation. Require the group member by pliability and the naked eyes outward appearance of the fabric of following Evaluation Division reason.

The pliability of fabric:

A. more not soft with the fabric that cellulase is processed with the fabric ratio of cellulase processing.

B. equally soft with the fabric that cellulase is processed with the fabric ratio of cellulase processing.

C. the fabric of processing with cellulase is harder than the fabric of not processing with cellulase.

The result represents in Table X VI.

Score with from 1 to 5 classifies to estimate the naked eyes outward appearance of fabric to fabric. 5 points of lint-free or balls of expression and fabric quality are more obvious. 1 submeter is shown with many balls and suede. Calculate the total points of each fabric and divided by group member's number. The average of the naked eyes outward appearance of each fabric shows in Table X VI.

Table X VI. is with neutral cellulase pliability and naked eyes outward appearance of fabric behind time repeated washing in detergent

Goods	The enzyme dosage that represents with protein/g fabric	Time h	Flexibility	The naked eyes outward appearance
					In Colour Detergent Liquid:
-ALKO 4237 20K-cellulase 50K-cellulases	-   11     2     2	1   1     1     1	100%: with cellulase more soft 100%: with cellulase more soft 100%: indistinction	1   3.2     3.7     1.7

After processing for 4 times, the fabric that cellulase is processed is than only using the fabric naked eyes outward appearance of detergent-treatment obviously better. Therefore the fabric of processing with these cellulases keeps preferably outward appearance, and compares with the fabric of not processing with cellulase and to repeat to prevent napping after clean. After washing 4 times, the fabric that ALKO 4237 and 20K-cellulase are processed is than only using the fabric of detergent-treatment more soft.

Embodiment 16

Use at detergent in the wool knitting

In neutral cellulase

Described in this experiment neutral cellulase as fabric softener and prevented that thereby napping from reducing in detergent behind the repeated washing tendency of balling-up on the wool knitting of dyeing. The cellulase goods of test are that ALKO 4237 concentrated culture filtrates reach the 20K-cellulase (embodiment 1 and 9) from ALKO 4237 bacterial strain purifying.

Green cotton wool pieces of fabric is containing or not Colour Liquid Detergent or the OMO of cellulase on Launder-Ometer by embodiment 15 is described^Washing is 1 hour 3 or 10 times among the Total.

The judging panel who is comprised of 3 people carries out the evaluation of knitting. Require the judging panel to press flexibility and the naked eyes outward appearance (obverse and reverse) of the knitting of embodiment 15 described evaluations processing. Press the loss in weight of embodiment 15 described mensuration knitting. The result shows in Table X VII.

After washing 3 times, the knitting that the 20K-cellulase is processed is than only using the double-edged naked eyes outward appearance of knitting of detergent-treatment better. Process 10 ratios of knitting with ALKO 4237 cellulase goods and only have obviously better naked eyes outward appearance and brighter green with the detergent-treatment knitting. Wash the knitting that has detected the cellulase processing after 1 time and have better naked eyes outward appearance (particularly at reverse side), and in the washing process that increases, further improve. Also more soft than the knitting of only using detergent-treatment with the knitting that cellulase is processed.

Table X VII. is with containing or not flexibility, the loss in weight and the naked eyes outward appearance of detergent woollen knitting behind 3 or 10 repeated washings of cellulase. Before the washing, the pH of 0.25%Colour Detergent solution is 7.9,0.25%OMO^Total solution is 8.4.

Goods	The enzyme dosage that represents with protein/g fabric	Wash time	PH after the washing	Loss in weight %	Flexibility	The naked eyes outward appearance
								Positive	Reverse side
						Colour Detereent Liquid
-  20K * -   A4237	-     5   -   20	3     3   10   10	7.9     7.4   8.0   7.9	0.46     0.88   1.46   2.80	33%: with cellulase more soft 100%: more soft with cellulase									1   1.5     1 2.5	1   2.7     1   2.8
						OMO  Total
-   A4237	-   20	10   10	8.3   8.2	0.57   1.57	100%: more soft with cellulase									1   2.3	1   2.8

^*=20K-cellulase

Embodiment 17

Use in outmoded cotton wool knitting

Neutral cellulase in the detergent

Describe in this experiment neutral cellulase and renewed ability with softening agent as fabric.

In the Cylinda washing machine with program 3 at 60 ℃, 10ml OMO^The green cotton wool knitting of washing is 10 times among the Colour (Lever, UK), and the centre has drying steps. This has simulated the washing of knitting in the reality.

After processing for 10 times, this outmoded knitting has the outward appearance of unappealing and brown, and some fine hair are arranged on the surface.

Through behind these 10 repeated washings, the lint knitting is used for using or not using the washing experiment of cellulase. By embodiment 15 described in Launder Ometer with Colour Liquid Detergent washing pieces of fabric 1 hour to 3 time and have middle rinsing and drying steps. The cellulase goods that use are the concentrated culture filtrate of ALKO 4237 and from purifying 20K-and the 50K-cellulase (embodiment 9) of ALKO 4237.

The judging panel who is comprised of 3 people carries out the evaluation of knitting. Require the judging panel to press flexibility and the naked eyes outward appearance (obverse and reverse) of the knitting of embodiment 15 described evaluations processing. Press the loss in weight of embodiment 15 described mensuration knitting. The result represents in Table X VII.

After the washing once, the knitting that ALKO 4237 and 20K-cellulase are processed has than the better naked eyes outward appearance of the knitting of only using detergent-treatment. After washing 2 to 3 times, the knitting that the 20K-cellulase is processed further forms preferably naked eyes outward appearance and more attractive outward appearance. After washing 2 times, compare with the knitting with detergent-treatment only, also improved the naked eyes outward appearance with the knitting that the 50K-cellulase is processed. In general, the knitting of processing with cellulase is obviously improved and is had noticeable outward appearance, and the outward appearance that the knitting of not processing with cellulase still has having no attraction and fades.

Table X VIII. is with containing or not flexibility, the loss in weight and the naked eyes outward appearance of outmoded lint knitting behind the detergent repeated washing 1 to 3 time of cellulase. Before the washing, the pH of 0.25%Colour Detergent solution is 7.9.

Goods	The enzyme dosage that represents with mg protein/g fabric	Washing times	PH after the washing	Loss in weight %	Flexibility	The naked eyes outward appearance
								Positive	Reverse side
						-   ALKO4237     20K *   -   20K *   50K *   50K *   -   20K *	-   20     5   -   5   5   15   -   5			1   1     1   2   2   2   2   3   3	ND   ND     ND   7.9   7.7   7.7   7.3   ND   ND	0.   0.61     0   0.10   0.46   0.26   0.49   0.31   0.88	100%: indistinction 100%: indistinction 100%: with cellulase more soft 100%: indistinction 100%: indistinction 100%: more soft with cellulase	1   1   1.5     1   2.5   1   1   1   3.0	1   1.5   1.5     1   2.2   1.2   1.3   1   2.2

The ND=undetermined

^*=20K-or 50K-cellulase

Embodiment 18

The separation of ALKO 4237 chromosomal DNAs

Structure with genomic library

Melanocarpus albomyces ALKO 4237 in potato glucose (PD, Difco, the USA) culture medium of diastatochromogenes 42 ℃, 250rpm growth 3 days. According to Raeder and Broda, the applied microbiology communication, 1:17-20 (1985) separates chromosomal DNA. Briefly, with 20mM EDTA washing mycelium and cracking in Extraction buffer (200mM Tris-HCl (pH8.5), 250mM NaCl, 25mM EDTA, 0.5%SDS). With phenol and chloroform: (24: mixture 1v/v) extracts DNA to isoamyl alcohol. With RNA enzymic digestion RNA.

Digest chromosomal DNA and process with calf intestine alkaline phosphatase with Sau3A (Boehringer Mannheim, Germany) part. The DNA that uses β-agarose (Boehringer Mannheim, Germany) to separate the 5-15Kb scope from Ago-Gel also is used for making up genome ALKO 4237 libraries.

Use predigested λ DASH^II BamHI support agent box (Stratagene, USA) makes up this library and is undertaken by shop instruction in all steps subsequently. Briefly, approximately the DNA of 200ng size separation connects into 1 μ g DASH^In the arm of II preparation, and pack with Gtgapack II packaging extract (Stratagene, USA). Through with the serial dilutions ehec infection XL1-Blue MRA (P2) of the bacteriophage of packing-cell and on the NZY flat board coated plate measure the titre in this library. This library in the SM-that contains 4% (v/v) chloroform is slow in the liquid in 40 ℃ of storages. It need not increase for screening.

Embodiment 19

With the degenerate primer amplification, the clone also

Order-checking 20K-cellulase

For with PCR (PCR) amplification 20K-cellulose enzyme gene, synthesized take the pair of degenerate primers of peptide sequence (Figure 17) as the basis. Primer 1 (429-32) is from the amino acid #8-14 (Figure 17) of N-end peptide #429, primer 2. (fr28-16) be designed to the antisense strand (Figure 17) of the amino acid #2-8 of peptide fr28. Add the fragment that another EcoRI restriction site is beneficial to clonal expansion at 5 ' end.

Primer 1 (429-32)

       EcoRI

5′-ATA GAATTC TA(C/T)TGG GA(C/T)TG(C/T)TG(C/T)AA(A/G)CC

                Y      W   D      C      C      K      P

Primer 2 (fr28-16)

       EcoRI

5′-ATA GAATTC TT(A/G)TC(A/C/GIT)GC(A/G)TT(C/T)TG(A/G)AA

               N      D          A      N      Q      F

CCA

 W

In the PCR reaction, ALKO 4237 genomic DNAs (embodiment 18) of 1 μ g purifying are as template. Use Dynazyme archaeal dna polymerase (Finnzymes Ltd, Finland) according to shop instruction.

Template DNA (0.7 μ g/ μ l) 1.4 μ l

Primer 1 (0.5 μ g/ μ l) 1 μ l

Primer 2 (0.5 μ g/ μ l) 1 μ l

dNTPs            (2mM)            5μl

10 * PCR buffer solution, 10 μ l

dH2O                              82μl

Dynazyme         (2U/μl)         1μl

Amount to 101.4 μ l

Under following condition, carry out the PCR reaction:

95 ℃ of steps 15 minutes

95 ℃ of steps 21 minute

56 ℃ of steps 31 minute

72 ℃ of steps 41 minute

Step 5 is carried out " step 2 " 29 times again

72 ℃ of steps 68 minutes

4 ℃ of steps 7 are kept

Analyze 10 μ l reactant mixtures with agarose gel electrophoresis, detect the long wall scroll band corresponding to about 600bp. Digest remaining PCR product and walk agarose electrophoresis with the EcoRI restriction endonuclease. Cutting-out contains the agar block of this dna fragmentation and uses Magic PCR Prps (Promega, USA) method according to the shop instruction purifying. The fragment of separating connects with pBluescript II SK+ (Stratagene, the USA) plasmid that cuts with EcoRI equally. With the Escherichia coli XL-Blue cell (Stratagene, USA) that connects the mixture transformed competence colibacillus. The DNA that separates some gained bacterium colonies with Magic Minipreps (Promega, USA) method according to shop instruction. With the agarose electrophoretic analysis DNA, has clone's called after pALK549 of expection feature.

ABI (the applying biological system USA) kit of use take fluorescently-labeled T3 and T7 primer or the sequence specific primers that contains fluorescently-labeled dideoxy nucleotide as the basis with Tag dyestuff primer cycle sequencing according to the Melanocarpus DNA of shop instruction order-checking from pALK549. Because the high GC content of Melanocarpus DNA under 58 ℃ annealing temperature, carries out sequencing reaction with 5% (v/v) DMSO. (applying biological system, USA) the upper analysis are used science of heredity calculation type sequence analysis software bag to sequencing reaction, identify the sequence that obtains for 7.2 editions at ABI 373A sequenator.

The peptide (Figure 17) that Insert Fragment (594bp) the coding most of 20K-cellulases of discovery in pALK549 produce. The DNA of pcr amplification (except primer) is corresponding to the nucleotides 175-716 of Figure 19.

Press the chromosomal DNA of embodiment 18 described separation Myriococcum kind ALKO 4124. The PCR reaction of making template with primer 429-32 and fr28-16 and ALKO 4124 chromosomal DNAs produce with from the big or small identical fragment of the DNA of ALKO 4237. Part this fragment that checks order, it is almost identical with ALKO 4237 sequences. The ALKO 4124 of deducibility Myriococcum kind has almost identical with the 20K-cellulase of Melanocarpus albomyces ALKO 4237 protein. This result and ALKO 423720K-cellulase specific antibody in Western analyzes are also identified from the observed result of the 20K protein belt (Figure 14) of ALKO 4124 growth mediums consistent. Enzyme from two bacterial strains is worn away the same good result of generation in the experiment (embodiment 3 and 4) at biology.

Embodiment 20

Cloning and sequencing Melanocarpus albomyces

ALKO 423720K cellulose enzyme gene

Growth Escherichia coli XL1-Blue MRA (P2)-cell (Stratagene, USA) and be diluted to OD600=0.5 in LB+0.2% maltose+10mM MgSO4. Also be coated on the NZY flat board with the top agarose of NZY in 15 minutes with 37 ℃ of infection cells of Melanocarpus albomyces ALKO 4237 genomic libraries (embodiment 18). Dull and stereotyped 37 ℃ of overnight incubation. Transfer on the nylon filter paper (Hybond, Amersham, UK) according to Stratagene specification money plaque.

According to Boehringer, DIG dna marker and the detection on-radiation application manual PCR fragment (embodiment 19) of foxalin mark purifying. Hybridize at 68 ℃. In SM buffer solution/chloroform, choose positive colony and screen purifying with second.

4 positive colonies have been found under these conditions. According to Sambrook etc. at molecular cloning, laboratory manual, second edition, publishing house of cold spring harbor laboratory, cold spring port, New York, 1989 described carrying out from cloning extensive separation phageλ DNA. Through analyzing phage DNA with some Restriction Enzyme dna digestions, with the DNA of PCR Probe Hybridization digestion. Isolate 3 hybridized fragments; About 2.6kb EcoRI-XhoI fragment, approximately 4.9kb XhoI fragment and approximately 3kb SocI fragment. They are inserted the pBluescript II Sk of same cutting⁺In the carrier (Stratagene, USA), produce respectively plasmid pALK1221, pALK1222 and pALK1223 (Figure 18).

Press the Melanocorpus albomyces DNA among the embodiment 19 described order-checking pALK1221. The dna sequence dna of coding Melanocarpus albomyces 20K-cellulase shows in Figure 19. The long 936bp of this sequence has coding 235 amino acid whose ORFs (ORF); This gene has 2 intrones. The signal peptide Processing position of inferring is after alanine-21, and the N of mature protein end is with alanine-22 beginning, as peptide sequencing result indicated (Figure 17, peptide #429). It is the full-length proteins precursor of the protein of 25.0kDa that this ORF expection produces molecular weight, and maturation protein is 22.9kD. Result (embodiment 10) quite well that this and protein purification work obtain. The different genes product of about 35kDa protein (embodiment 10) the right and wrong 20K-cellulase that detects with 20K-cellulase antiserum before these results also confirm.

As if the 20K-cellulase of Melanocarpus albomyces belong to cellulase K family and glucosyl group hydrolase 45 families (Henrissat and Bairoch, biochemical magazine, 293:781-788 (1993)). The 20K-cellulase demonstrates with Humicola insolens endoglucanase V (embl:a 23635) has homology (in 235 amino acid are overlapping about 76% homogeny being arranged), but the 20K-cellulase has wonderful feature, it is containing cellulose land (CBD) and its attachment not, this CBD and attachment are that (Schulein etc. 1993 for the feature of Humicola insolens endoglucanase V and other relevant endoglucanase,: Suominen and Reinikainen (editor), biotechnology and industrial fermentation Research foundation, Helsinki, the 8th volume, 109; Saloheimo etc., 1994, molecular microbiology, 13,219). This feature of 20K cellulase may be the reason of wearing away (embodiment 10) this enzyme generation excellent results in the experiment at biology.

Embodiment 21

With degenerate primer amplification, Cloning and sequencing

50K-cellulose enzyme dna

The peptide (table IX) that the 50K-cellulase produces has some homologys with Humicola grisea endoglucanase I (DDBJ:D63516). For with PCR (PCR) amplification 50K-cellulose enzyme gene, synthesized a pair of take the degenerate primer of peptide sequence (Table I X) as the basis. Primer 1 (507-128) is from the amino acid #5-10 of peptide #507, and primer 2 (509-rev) is designed to the reaction chain (Table I X) of the amino acid #4-9 of peptide 509. From with the protein of relatively deriving of Humicola grisea endoglucanase I order and the corresponding primer of two peptides justice-antisense feature is arranged.

Primer 1 (507-128)

5′-GA(C/T)GA(A/G)AC(A/C/G/F)GA(A/G)CA(C/T)(A/C)G

     D      E      T          E      H      R

Primer 2 (509-rev)

5′-TA(A/C/G/T)GC(A/C/G/T)CC(A/C/G/T)CC(A/C/G/T)GG(A/G)TT

     Y         A           G          G          P      N

In the PCR reaction, ALKO 4237 genomic DNAs (embodiment 18) of the purifying of 1.5 μ g are as template. Use Dynazyme archaeal dna polymerase (Finnzymes Ltd., Finland) according to shop instruction.

Template DNA (0.3 μ g/ μ l) 5 μ l

Primer 1 (0.5 μ g/ μ l) 1 μ l

Primer 2 (0.5 μ g/ μ l) 1 μ l

dNTPs                (2mM)            5μl

10 * PCR buffer solution, 10 μ l

dH2O                                  79μl

Dynazyme             (2U/μl)         1μl

Amount to 102 μ l

Under following condition, carry out the PCR reaction:

95 ℃ of steps 15 minutes

95 ℃ of steps 21 minute

56 ℃ of steps 31 minute

72 ℃ of steps 41 minute

Step 5 is carried out " step 2 " 29 times again

72 ℃ of steps 68 minutes

4 ℃ of steps 7 are kept

Analyze 10 μ l reactant mixtures with agarose gel electrophoresis, detect the wall scroll band that is approximately 160bp corresponding to length. Remaining PCR product is loaded on the Ago-Gel of electrophoresis, downcuts the Agarose plug that contains this dna fragmentation, according to producer purifying is described with Magic PCR Preps (Promega USA) method.

With the fragment of separating with EcoRV endonuclease enzymic digestion and press Holteon and Graham (1990), nucleic acids research, 19,1156. described pBluescript II Sk+ (Seratagene, USA) the plasmids connections that add the ddT tail. Connect the Escherichia coli XL-Blue cell (Stratagene, USA) of mixture transformed competence colibacillus with this. With Magic Minipres (Promega, USA) method according to shop instruction isolated plasmid dna from the bacterium colony of some gained. With the agarose electrophoretic analysis DNA, have one of feature clone of expection called after pALK1064.

Press the Insert Fragment (161bp) among the described order-checking of embodiment 19 pALK1064, and find to contain an ORF, expect that it is peptide with Hunicola grisea endoglucanase I (DDBJ:D63516) homology. This ORF also encodes from the peptide #612 (Table I X) of the 50K-cellulase of purifying. The DNA of pcr amplification (except primer) is corresponding to the nucleotides 404-530 among Figure 21.

With primer 507 and 590-rev and make PCR that template (embodiment 19) carries out with ALKO 4124 chromosomal DNAs and produce and fragment from the formed objects of ALKO 4237DNA. This shows that Myriococcum sp.ALKO 4124 has fairly similar in the protein of Melanocarpus albomyces ALKO 423750K-cellulase. Also support this point from the fact that the enzyme of two bacterial strains is worn away the similar good result of generation in the experiment at biology.

Embodiment 22

Cloning and sequencing Melanocarpus albomyces

ALKO 423750K-cellulose enzyme gene

Press the gene pool of embodiment 20 described Melanocarpus albomycws ALKO 4237 for the preparation of hybridizing. According to Boehringer, DIG dna marker and detection on-radiation, application manual carries the purifying PCR fragment (embodiment 21) of part 50K-cellulose enzyme gene with the foxalin mark. Hybridization is carried out at 68 ℃. In SM buffer solution/chloroform, choose positive colony, take turns screening with second and carry out purifying.

Find under these conditions 10 positive colonies. According to Sambrook etc., 1989 carry out the extensive phageλ DNA that separates from the clone. Through analyzing phage DNA with some Restriction Enzyme dna digestions, the DNA of digestion and 50K-cellulase specific PCR probe are hybridized. Isolate 4 hybridized fragments: about 2.8kb SacI-XhoI fragment, approximately 5kb SacI fragment, approximately 3.2kb Xho I fragment and about 2kb EcoRI fragment. They are inserted in the pBluescript II SK+ carrier (Stratagene, USA) that same enzyme cuts, produce respectively plasmid pALK 1234, pALK1233, pALK1226 and pALK1227 (Figure 20).

From above-mentioned 50K-cellulase specific plasmids order-checking Melanocarpus albomyces ALKO 4237 DNA. Its sequence measurement is described in embodiment 19.

The DNA of coding Melanocarpus albomyces 50K-cellulase shows in Figure 21 (A and B). This sequence has disclosed the ORF that a length is approximately 1363bp, is interrupted by an introne. ORF 428 amino acid of encoding. The protein molecular weight of expection is to be 44.8kDa after 46.8 kDa and the signal peptide cracking. All peptides among the Table I X are found in the protein sequence (Fig. 2) of expection, although identify that during peptide sequencing more uncertain amino acid are proved to be incorrect. This protein shows with Humicola grisea endoglucanase I (DDBJ:D63516) has homology.

Embodiment 23

With degenerate primer amplification, Cloning and sequencing

50K-cellulase B DNA

Peptide (Table X) from 50K-cellulase B has certain homology with the biological hydrolase I of Humicola grisea cellulose (DDBJ:D63515). For with PCR (PCR) amplification 50K-cellulase B gene, synthesized a pair of take the degenerate primer of peptide sequence (Table X) as the basis. Primer 1 (636) (guesses that first amino acid is lysine from the amino acid #1-5 (Table X) of peptide #636, because isolating this peptide with after the protease cracking digestion behind the lysine), primer 2 (534-rev) is designed to the antisense strand of the amino acid #3-8 (Table X) of peptide #534. Compare order and the corresponding primer of inferring 2 peptides in this protein with the biological hydrolase I of Humicola grisea cellulose justice-antisense character is arranged.

Primer 1 (636)

5′-AA(A/G)CA(C/T)GA(A/G)TA(C/T)GG(A/C/G/T)AC

     K     H      E      Y      G          T

Primer 2 (534-rev)

5′-CC(A/G)TA(A/G)AA(A/G)TC(A/C/G/T)GG(A/G)TT

     G      Y      F      D          P      N

In PCR reaction, with ALKO 4237 genomic DNAs (embodiment 18) of 1.5 μ g purifying as template. Use Dynazyme archaeal dna polymerase (Finnzyme Ltd., Finland) according to shop instruction

Template DNA (0.3 μ g/ μ l) 5 μ l

Primer 1 (0.3 μ g/ μ l) 1.7 μ l

Primer 2 (0.3 μ g/ μ l) 1.7 μ l

dNTPs                (2mM)            5μl

10 * PCR buffer solution, 10 μ l

dH2O                                  80μl

Dynazyme             (2U/μl)         1μl

Amount to 104.4 μ l

Under following condition, carry out the PCR reaction:

95 ℃ of steps 15 minutes

95 ℃ of steps 21 minute

48 ℃ of steps 31 minute

72 ℃ of steps 42 minutes

Step 5 is carried out " step 2 " 34 times again

72 ℃ of steps 68 minutes

4 ℃ of steps 7 are kept

Analyze 20 μ l reactant mixtures with agarose gel electrophoresis, detect some bands. Wherein a band apparent size is 700bp, and this size and expected in the same size when with the biological hydrolase gene comparison of Humicola grisea cellulose are if particularly this fragment contains 1 or a plurality of introne. With Magic PCR Preps (Promega, USA) method according to shop instruction purifying PCR product.

The fragment of separating with used the enzymic digestion of EcoRV endonuclease, and press Holton and Graham, described pBluescript II SK+ (Stratagene, USA) the plasmid connection that adds the ddT tail of nucleic acids research 19:1156 (1990). With the Escherichia coli XL-Blue cell (Stratagene, USA) that connects the mixture transformed competence colibacillus. The DNA that separates some gained bacterium colonies with Magic Minipreps (Promega, USA) method according to shop instruction. With the agarose electrophoretic analysis DNA, has clone's called after pALK1224 of about 700bp Insert Fragment.

Press the Insert Fragment among the described order-checking of embodiment 19 pALK1224, find that it contains coding from the ORF of the full peptide #636 (Table X) of 50K-cellulase B. ORF is contemplated to the peptide with biological hydrolase I (DDBJ:D63515) homology of Humicola grisea cellulose. The DNA (except primer) of PCR amplification is corresponding to the nucleotides 371-1023 among Figure 23.

Embodiment 24

Cloning and sequencing Melanocarpus albomyces

ALKO 423750K-cellulase B gene

Press the genomic library of embodiment 20 described Melanocarpus albomyces ALKO 4237 for the preparation of hybridizing. Through take out the Insert Fragment among the pALK1224 with restriction endonuclease EcoRI and Hind III digested plasmid. The DNA of digestion is walked agarose electrophoresis. Cutting-out contains the Agarose plug of about 700bp dna fragmentation, with Magic PCR Preps (Promega, USA) method according to the shop instruction purifying.

According to Boehringer, DIG dna marker and detect on-radiation, application manual is with the PCR fragment (embodiment 23) of foxalin mark from pALK 1224 purifying that carry part 50K cellulase B gene. Hybridize at 68 ℃. In SM buffer solution/chloroform, choose positive colony, and take turns the screening purifying with second.

Find under these conditions 3 positive colonies. According to Sambrook etc., at molecular cloning: laboratory manual, second edition, publishing house of cold spring harbor laboratory, the cold spring port, New York, 1989 carry out from cloning extensive separation phageλ DNA. Through analyzing phage DNA with some Restriction Enzyme dna digestions, with the DNA of 50K-cellulase B specific PCR Probe Hybridization digestion. Separate the 3.5kb NotI fragment of hybridization and insert in the pBluescript II SK+ carrier (Stratagene, USA) that same enzyme cuts, produce plasmid pALK1229 (Figure 22).

Through find with the 0.2kb NotI-PstI-fragment of pALK1229 and phage DNA hybridization this gene 5 ' hold. Separate the 2.4kb PstI-fragment of hybridization and insert in the pBluescript II SK+ carrier (Stratagene, USA) that same enzyme cuts, produce plasmid pALK1236 (Figure 22).

Press the partial insertion fragment among embodiment 19 described order-checking pALK1229 and the pALK1236. The DNA of coding Melanocarpus albomyces 50K-cellulase B represents in Figure 23 (A and B).

It is the ORF of 1734bp that this sequence has disclosed a length that is interrupted by 5 intrones. ORF 452 amino acid of encoding. The protein molecular weight of expection is to be 47.6kDa behind 49.9kDa and the cracking signal peptide. Having found all peptides in the Table X in the protein sequence (Figure 23 A and B) of expection, is wrong although some amino acid of identifying with uncertainty during peptide sequencing are proved to be. The protein of expection demonstrates with the biological hydrolase I (DDBJ:D63515) of Humicola grisea cellulose and the biological hydrolase of other cellulose has homology. Yet, 50K-cellulase B has wonderful feature, it is containing cellulose land (CBD) and its attachment not, and this CBD and attachment are the features of Humicola grisea cellulose biology hydrolase I and the biological hydrolases of many other celluloses.

Embodiment 25

Screen with Trichoderma reesei cellulose enzyme gene

Melanocarpus albomyces ALKO 4237 genomic libraries

Press the genomic library of embodiment 20 described Melanocarpus albomyces ALKO 4237 for the preparation of hybridizing.

Through cutting plasmid pTTcO1 (Figure 24) with restriction endonuclease HincII and behind electrophoresis, separating about 1.6kb fragment from Ago-Gel and separate the dna fragmentation that carries Trichoderma reesei cbh1 specific DNA. Separate about 1.5kb fragment and separate the dna fragmentation that carries Trichoderma reesei egl2 specific DNA through cut plasmid pMSZ (Figure 25) and the Ago-Gel behind the electrophoresis with restriction endonuclease BamHI and EcoRI. The cbh1 gene cloning is at Teeri etc., describe in the biology/technology 1:696-699 (1983) and this DNA sequence at Shoemaker etc., describe in the biology/technology 1:691-696 (1983). Gel2 (being called at first " egl3 ") gene is described among the gene 63:11-21 (1988) at Saloheimo etc.

According to Boehringer, DIG dna marker and detection on-radiation, this fragment of application manual foxalin mark. Also hybridize at 60 ℃ with the egl2 probe at 68 ℃ with the cbh1 probe. In SM buffer solution/chloroform, choose positive colony, and take turns the screening purifying with second.

Positive and 6 the egl2 positive colonies of 13 cbh1 have been found under these conditions. A clone and two kinds of probes are all hybridized. By top described from this clone and separate λ DNA. Through separating phage DNA with some Restriction Enzyme dna digestions, the DNA of digestion and cbh1 and egl2 Probe Hybridization. This clone also hybridizes with 20K-cellulase specific PCR fragment (embodiment 19). A clone (λ-16) is obviously positive, and 2 other clones (λ-8/1 and λ-5/2) are the faint positive; All these clones choose with the cbh1 probe at first.

Also insert the same pBluescript II SK+ that cuts with Trichoderma reesei cbh1 probe with the about 4kb EcoRI fragment from λ-16 that 20K-cellulase specific PCR fragment is all hybridized from the Ago-Gel separation behind the electrophoresis. The plasmid called after pALK1230 (Figure 26) of gained.

Press the partial insertion fragment among the embodiment 19 described order-checking pALK1230. This DNA 20K-cellulase of as if not encoding, but the protein of coding and some fibre element enzyme, particularly cellulose binding (CBD) homology. Therefore, this gene outcome probably has affinity with cellulosic material, so this gene outcome called after has the protein of CBD. This sequence shows in Figure 27.

Use the DNA that clones from 19 λ to make template and carry out the PCR reaction with primer 636 and 534-rev (embodiment 23). A λ clone, namely λ-3 produces the band of an about 700bp size, the situation when being used as template similar in appearance to ALKO 4237 chromosomal DNAs in embodiment 23. This clone chooses with Trichoderma cbh1 probe at first. Digest λ DNA with some restriction endonuclease, and hybridize with 50K-cellulase B specific probe. This clone demonstrate to embodiment 24 in 3 similar Restriction Enzyme collection of illustrative plates of clone. Deducibility λ-3 also carries 50K-cellulase B gene.

Embodiment 26

Fused protein

Merge through the sequence of at least one functional areas of sequence and Trichoderma reesei cellulase or hemicellulase or said cellulase or the hemicellulase of the cellulase of will encode and to prepare the 20K-cellulase of encoding, the recombinant vector of 50K-cellulase or 50K-cellulase B, as at US 5,298,405, WO 93/24621 and described in the Genbank registration number L25310, this paper quotes for your guidance. Specifically, this enzyme is selected from CBHI, CBHII, and EGI, EGII, XYLI, XYLII and MANI or its functional areas are such as secretion signal or core sequence.

But construction of fusion protein matter, it contains N end mannase or the biological hydrolase of cellulose or endoglucanase core space or its core and the twisting district of merging with a Melanocarpus cellulase sequence. The result is that this protein contains a N end mannase or the biological hydrolase of cellulose or endoglucanase core or core and twisting district, and a C end Melanocarpus cellulase. Each regional Trichoderma mannase or the biological hydrolase of cellulose or endoglucanase and the Melanocarpus cellulase activity that provides in fusion constructs is provided fused protein. In addition, in construct, can comprise the biological hydrolase of modification Trichoderma mannase or cellulose or the activity of endoglucanase or the sudden change of Melanocarpus cellulase activity. In this case, Trichoderma enzymatic activity and the Melanocarpus cellulase activity of the modification in each zone that provides in fusion constructs is provided fused protein.

But also construction of fusion protein matter, the biological hydrolase of mannase or cellulose or endoglucanase tail or its required fragment are placed on before the Melanocarpus cellulase sequence, particularly allow to use the protease site that from the fused protein of expressing, reclaims Melanocarpus cellulase part in the conduct of the nonspecific protease site of afterbody. In addition, but construction of fusion protein matter, and it provides and is placed on the Melanocarpus cellulase protease site that is arranged in synthetic attachment before, and contains or do not contain tailer sequence.

Embodiment 27

The host

By above-mentioned preparation encode required fused protein or Melanocarpus protein recombinant precursor and transform such as the aspergillus kind, in the filamentous fungi of preferred trichoderma kind.

Embodiment 28

The trichoderma background that is used for the 20K-cellulase production

Described in the present embodiment and worn away the only background of testing with the trichodermin cellulase that is identified for the 20K cellulase production. The purpose of these experiments is to cause and redye wearing away middle meeting under neutrallty condition in order to measure any trichoderma cellulase.

Select Trichoderma reesei strains A LKO 3620 (disappearance endoglucanase 2 genes) as the host of these experiments. In the former research, shown that trichoderma EGII (endoglucanase II) enzyme causes the adverse effect to cotton fiber structure, thereby the strength characteristics of weakening cotton-containing fabrics (: Miettinen-Oinonen etc.: cellulase is on the impact of cotton fiber and fabric,: the TIWC96 procceedings, 1996, Vol.1 (2) is among the pp.197).

Described at pH6.5 with wear away experiment 7 times except not using the Berol by embodiment 3.

The trichoderma cellulase goods of test:

ALKO3133 (egl2 and cbh2 disappearance)

ALKO3269 (egl2 and egL1 disappearance)

ALKO3268 (egl2 and cbh1 disappearance)

The dosage of trichoderma goods is the about 2.5mg of every g fabric (two low dosages, L) or approximately 5mg (two high doses, H) gross protein. Use when needed the 20K-cellulase of every g fabric 0.4mg purifying.

The color measuring result of the denim goods fabric of processing represents in Table X IX.

Wear away the result and show that ALKO 3269 (egl2 and egL1 disappearance) background causes less redying than ALKO 3268 (egl2 and cbh1 disappearance) or ALKO 3133 (egL2 and cbh2 disappearance) background under neutrallty condition. Therefore, the preferred host for biological 20K-cellulase production of wearing away is ALKO3269 one class bacterial strain. Although have higher 20K-cellulase concentration, the trichoderma background probably has minimum importance. ALKO3269 class background is probably the same good to the 20K-cellulase production with it with 50K-cellulase B production to the 50K-cellulase in biology is worn away.

Table X IX. is with containing (+) or not containing the color measuring of the denim goods fabric that the different trichoderma cellulase goods of (-) 20K-cellulase process

Goods/dosage	20K   +/-	pH	Positive		ΔE	Reverse side		ΔE
									L	b	L	b
			-   ALKO3620/L   ALKO3620/L   ALKO3133/L   ALKO3133/H   ALKO3133/L   ALKO3133/H   ALKO3269/L   ALKO3269/H   ALKO3269/L   ALKO3269/H   ALKO3268/L   ALKO3268/H   ALKO3268/L   ALKO3268/H   -   ALKO3620/L   ALKO3620/L   ALKO3133/L   ALKO3133/L   ALKO3269/L   ALKO3269/L   ALKO3268/L   ALKO3268/L	-   -   +   -   -   +   +   -   -   +   +   -   -   +   +   -   -   +   -   +   -   +   -   +		6.5   6.5   6.5   6.5   6.5   6.5   6.5   6.5   6.5   6.5   6.5   6.5   6.5   6.5   6.5   7.0   7.0   7.0   7.0   7.0   7.0   7.0   7.0   7.0	2.2   2.2   5.5   1.9   4.2   5.7   8.5   2.9   4.3   6.6   7.9   2.9   4.2   5.9   7.1   2.9   3.3   6.7   3.2   5.9   3.6   6.4   2.9   8.4						1.1   2.6   4.0   2.2   1.9   4.3   4.0   1.9   1.5   4.2   3.9   1.7   2.0   3.2   3.7   0.8   1.2   3.4   1.0   3.7   1.2   3.4   1.4   3.1	3.1   3.0   7.7   3.7   4.5   7.8   9.4   4.4   4.5   8.7   8.5   3.7   4.3   7.7   7.7   2.6   1.9   5.6   1.4   5.5   2.2   5.9   3.9   9.6	0.7   -0.7   -1.3   0.2   -1.5   0.3   -1.4   0.8   0.6   1.1   0.7   0.1   -0.7   -1.2   -2.0   0.7   1.7   1.1   0.6   0.1   1.3   1.2   0.5   1.1	0.1   2.6   5.0   1.6   3.3   4.5   5.9   0.8   1.3   4.0   3.7   1.8   3.4   4.5   5.8   0.5   0.3   3.2   0.6   4.3   -0.3   3.2   0.4   3.5	1.4   2.9   5.5   2.3   4.8   5.0   7.8   1.6   2.6   4.3   5.1   3.0   5.0   6.0   7.3   1.5   1.1   2.9   0.9   3.1   1.3   2.8   2.5   4.6

Embodiment 29

In T.reesei, produce Melanocarpus albomyces

ALKO 423720K-cellulase

Make up the production that Trichoderma reesei bacterial strain is used for Melanocarpus albomyces ALKO 4237 20K-cellulases. Bacterial strain produces Melanocarpus 20K cellulase and can not produce endoglucanase II and the biological hydrolase I of the cellulose territory endoglucanase I of T.reesei. The goods of this class trichoderma cellulose lytic activity defective, and produce these goods at US 5,298 with recombinant DNA method, 405 or Suominen etc. (1993). Step gene at Trichoderma reesei high frequency replaces. II. the loss effect of single cellulose enzyme gene. Common molecular genetics, 241:523, middle description, this paper quotes for your guidance.

In the structure of the bacterial strain of producing Melanocarpus albomyces 20K-cellulase, use the expression cassette (Figure 28 and 29) from plasmid pALK1231 or pALK1235 to transform parent Trichoderma reesei strains A LKO3620. In expression cassette, from strong cbh1 promoter expression 20K-cellulase. The integration of expression cassette causes replacing parent cbh1 (pALK 1231) or egl1 (pALK1235) gene.

In host strain ALKO3620, (Mattern etc. (1998) give the carrier that the aspergillus of phleomycin resistance transforms to use the recombinant DNA method described in the US 5298405 (this paper quotes for your guidance) to use 3.3kb XbaI-Bgl II fragment from the different wall bacterium of Hindustan chain ble gene to replace the egl2 gene. Genetic of fungi is learned communication, 35:25:Drocourt etc. (1990), be used for transforming low wait and higher eucaryote with the expression cassette of the different wall bacterium of the Hindustan chain ble gene of acquisition phleomycin resistance. Nucleic acids research, 18:4009).

Be used for to make up produce the plasmid pALK1231 of bacterial strain of Melanocarpus cellulase and pALK1235 about the cbh1 promoter, the 20K cellulose enzyme gene is identical mutually with the cbh1 terminator, and it is described below:

^*T.reesei cbh1 (the biological hydrolase 1 of cellulose) promoter: this promoter is from Trichoderma reesei VTT-D-80133 (Teeri etc., (1983), from the molecular cloning of the main cellulose enzyme gene of Trichoderma reesei. Biology/technology 1:696.). (Karhunen etc., (1993) are in the step gene replacement of Trichoderma reesei high frequency to use 2.2kb EcoRI-SacII fragment in this construct. I. the excessive production of endoglucanase I. The molecule General Genetics, 241:515). The sequence of the promoter region before ATG is by (1983) such as Shoemaker, from the molecular cloning of the biological hydrolase of circumscribed cellulose of Trichoderma reesei bacterial strain L27. Biology/technology 1.691). Last 15 nucleotides (SacII site underscoring) of T.reesei (2) cbh1 promoter areCCGCGGACTGCATC (Shoemaker etc., 1983). Cbh1 promoter from T.reesei bacterial strain VTT-D-80133 is sequenced in the ALKO research laboratory, and notices that a nucleotide difference is arranged in dna sequence dna in above-mentioned zone. In T.reesei bacterial strain VTT-D-80133, the sequence before ATG isCCGCGGACTG/C/GCATC (SacII site underscoring, the cytimidine that adds in dna sequence dna is between oblique line).

Add the nucleotides lost in the promoter (SacII after to ATG 10bps) and accurately promoter is fused to first ATG upper (below seeing) of Melanocarpus 20K-cellulase, process use PCR (PCR) method is accomplished this point. Order-checking fusions and PCR fragment are to guarantee mistake not occur in reaction. In pALK1231, promoter region is also as homologous dna (with cbh1 3 '-fragment, seeing below) and the performance function is integrated into the cbh1 locus with target with transforming DNA.

^*Melanocarpus albomyces 20K cellulose enzyme gene: the nucleotide sequence of the 20K-cellulose enzyme gene of coding 20kDa cellulase and the amino acid sequence of derivation provide (Figure 19) in embodiment 20. In two plasmids, use the 0.9kb fragment that begins from the ATG-codon.

^*T.reesei cbh1 terminator: (Karhunen etc., (1993) replace at a step gene of Trichoderma reesei high frequency 739bp AvaII fragment that will 113bp begins before cbh1 gene terminator codon. I. the excessive production of endoglucanase I. The molecule General Genetics 241:515) is added to behind the 20K-cellulose enzyme gene to guarantee tanscription termination.

Except above-mentioned substance, plasmid pALK1231 contains:

^*AmdS gene: separate this gene from aspergillus nidulans VH1-TRS * 6, its coding acetamidase (Hynes etc., (1983), contain separation and the application in the analysis of structure and regulating density thereof of the genomic clone of aspergillus nidulans amdS gene, molecular cytobiology, 3:1430). Acetamidase is so that this bacterial strain process uses acetamide to grow as only nitrogen source, and this feature is used for selecting transformant. 3.1kb fragment (SpeI-XbaI) (Kelly J. and Hynes M. (1985) are with the amdS genetic transformation black aspergillus of aspergillus nidulans, EMBO J. 4:475) from plasmid p3SR2 is used for this plasmid. This fragment contains the promoter region of the 1007bp of amdS gene, the terminator zone of the code area of 1897bp (comprising introne) and 183bp.

^*Cbh1 3 '-fragment: separate this fragment (1.7kb from T.reesei ALK02466 through using plasmid rescue, BamHI-EcoRI, initial 1.4kb after this gene terminator, Suominen etc., (1993), the step gene at Trichoderma reesei high frequency replaces. II. the loss effect of single cellulose enzyme gene. The molecule General Genetics, 241:523.). (Harkki etc. (1991), the genetic modification trichoderma has the bacterial strain of new cellulose enzyme feature to strains A LKO 2466 with generation from strains A LKO 233. The enzyme microbiological technique, 13:227.). 3 ' fragment is used with promoter region in order to through homologous recombination the 20K-cellulose enzyme gene is targeted to the cbh1 locus.

Plasmid pALK1235 contains:

^*The hph gene: the gene of coding HmB phosphotransferase is isolated (Yanish-Perron etc. at first from e. coli k-12 JM109, (1985), the nucleotide sequence of improved M13 phage clone carrier and host strain: M13mp18 and pUC19 carrier, gene 33:103) and it give resistance to HYG (HmB). Resistance (with the deactivation of HmB phosphotransferase phosphorylation) to hygromycin is used for selecting transformant. The hph gene is isolated (Mach etc. as 2.2kb NotI-PvuII fragment with pKi promoter and cbh2 terminator (below seeing) from plasmid pRLMex30, (1994), use the homology expression signal to transform Trichoderma reesei according to Hygromycin B resistant, General Genetics, 25:567).

^*PKi promoter: use T.reesei QM 9414DNA to make template through synthetic about 0.75kb pKi (pyruvate kinase) promoter (Schindler etc. (1993) that are used for expressing hph of PCR, the evaluation of the gene (pki1) of Trichoderma reesei coding pyruvate kinase, gene, 130:271).

^*The cbh2 terminator: cbh2 terminator sequence begins (from terminator codon to PvuII site 0.5kb after being right after cbh2 gene terminator codon; Mach etc. (1994) use the homology expression signal to transform Trichoderma reesei according to Hygromycin B resistant. General Genetics is 25:567) and from plasmid pRLMex 30.

^*Egl1 5 '-fragment: separate 1.8Kb egl1 5 '-fragment (ScaI-StuI) (Mandels and Reese (1957) from T.reesei QM6a, be subjected to the impact of carbon source and metal to induce cellulase among the Trichoderma viridae, the bacteriology magazine, 73:269). This fragment is arranged in egl1 upstream of coding region about 1.35kb place and it is integrated into the egl1 locus for target with transforming DNA.

^*Egl1 3 '-fragment: with 5 '-fragment is the same, separates 1.6kb egl1 3 '-fragment (ScaI-XhoI) from T.reesei QM6a. This fragment is positioned at 0.3kb place, egl1 gene downstream, and it enters the egl1 locus for the target transforming DNA.

In vector construction, use Sambrook etc., (1989).: molecular cloning, laboratory manual, second edition, publishing house of cold spring harbor laboratory, cold spring port, the described standard DNA method in New York. Restriction Enzyme, T₄Dna ligase, the Klenow fragment of dna polymerase i, T4 DNA polymerase, polynucleotide kinase and Taq polymerase are from Boehringer Mannheim (Germany) and New England's biology laboratory (USA). Use each enzyme according to shop instruction. Through using Qiagen post (Qiagen GmbH, Germany) or Promega Magic Minipreps (Promega, USA) according to the scheme isolated plasmid dna of producer. In PCR-reaction and the oligonucleotides that in sequencing reaction, uses (the applying biological system, USA) the 381A dna synthesizer is synthetic with ABI. Dna sequencing is by embodiment 19 described carrying out.

Process (New England's biology laboratory through β-agarase I, USA) or use QIAEX gel extraction kit (Qiagen GmbH, Germany) (the FMC biological product USA) separates the dna fragmentation that is used for the clone or transforms from the low melting-point agarose gel according to shop instruction.

Press Penttila etc., (1987), the filamentous fungi Trichoderma reesei multipurpose conversion system that is used for the cellulose cracking, gene, 61:155 is described and press Karhunen etc., and (1993) replace at one step of Trichoderma reesei medium-high frequency gene, I, the excessive production of endoglucanase I. The molecule General Genetics, the described modification of carrying out of 241:515 transforms T.reesei ALKO 3620. The T.reesei transformant is transferred on the selection culture medium and by the conidium purifying. Before the potato agar glucose forms spore, they are being selected inclined-plane growth 2 generation stable conversion.

Embodiment 30

           Melanocarpus albomyces ALKO4237

The 20K-cellulase produces the sign of transformant

Include 4% whey in shaking flask, 1.5% compound nitrogen source from cereal, 5%KH₂PO ₄With 0.5% (NH₄) ₂SO ₄Culture medium in the growth purifying transformant. At 30 ℃ with 250rpm grown culture 7 days.

With the Dot blot device with the direct trace of culture supernatant to nitrocellulose filter. Use the immunostaining of CBHI monoclonal antibody specific CI-258 and with monoclonal antibody specific EI-2 (Aho etc., (1991), the monoclonal antibody of the biological hydrolase I of anti-Trichoderma reesei cellulose and II and endoglucanase I core and cellulose binding, european journal of biological chemistry, EGI 200:643) and Proto Blot Western trace AP system (Promega, USA) detect CBHI according to producer's suggestion.

T.reesei strains A LKO 3620/pALK1231/14, ALKO3620/pALK1231/16, ALKO 3620/pALK 1231/20 and ALKO 3620/pALK 1231/59 do not contain the cbh1 gene. The cbh1 gene is replaced by the amds marker gene in pALK 1231 expression cassettes and 20K-cellulase construct. In Southern hybridization, confirm the replacement of cbh1 gene. T.reesei strains A LKO 3620/pALK 1235/40 and ALKO 3620/pALK 1235/49 do not contain the egl1 gene. The egl1 gene is replaced by the hph marker gene in pALK 1235 expression cassettes and 20K-cellulase construct. Confirmed that in Southern hybridization the egl1 gene replaces. The host strain ALKO 3620 that is used for transforming is egl2 gene defection type (being replaced by the ble gene (Mattern etc., 1988, Drocount etc., 1990) from the different wall bacterium of Hindustan chain). Therefore, these not production of bacterial strain trichoderma cellulase composition EGII and CBHI or EGI.

Sample from the culture supernatant is upward walked electrophoresis at the polyacrylamide plate gel that contains 0.1%SDS at Bio-Rad Mini Protean II electrophoresis system (U.S.). Polyclonal antibody for the preparation of the 20K-cellulase of purifying is used for detecting the protein that produces at the Western trace. In detection, use Promega ' s ProtoBlot^The AP system. Western result shows in Figure 30. Transformant ALKO 3620/pALK 1235/49, ALKO 3620/pALK 1235/40, ALKO 3620/pALK 1231/14 and ALKO 3620/pALK1231/16 ( swimming lane 1,2,4 and 5) produce the protein with the reaction of polyclone 20K-cellulase antiserum. The protein size that transformant produces is big or small identical with the 20K-cellulase (swimming lane 6) of purifying. ALKO 3620 (swimming lane 3) does not produce corresponding protein.

The endoglucanase of pressing embodiment 10 described mensuration transformants is active. When using 2% carboxymethyl cellulose (CMC) when making substrate, reaction temperature rises to 70 ℃, so the endoglucanase activity of ALKO 3620 is heat-inactivated. When using 1% hydroxyl when cellulose is made substrate, before measuring, enzymatic activity carries out hot deactivation. From the sample of growth medium at 0.05M HEPES, dilution and 70 ℃ of insulations 20 minutes in pH 7.0 buffer solutions. Almost completely hot deactivation endoglucanase I (remaining main endoglucanase in ALKO 3620. The activity of the negative transformant of egl1-descends approximately 30% in hot deactivation, shows the cellulosic hot deactivation of 20K-less. The endoglucanase activity provides in Table X X. When HEC was substrate, the 20K-cellulase activity was inferred as through the activity that will obtain after the heat treatment activity before divided by 0.7 heat treatment that obtains.

Table X X. produces the endoglucanase activity of the T.reesei transformant of Melanocarpus albomyces 20K-cellulase.

Substrate	CMC	70℃，pH7.0	20K-cellulase activity (50 ℃ of the HEC of artificial unit/ml), pH7.0
				ALKO4237   ALKO3620   ALKO3620/pALK1231/14   ALKO3620/pALK1231/16   ALKO3620/pALK1231/20   ALKO3620/pALK1231/59   ALKO3620/pALK1231/40   ALKO3620/pALK1231/49	- *   50 ***   2400   2600   6500   6800   2400   2100	100 **   38 ***   350   350   750   750   325   350

^*Do not measure

^**Not heat-inactivated, also contain the 50K-cellulase, 50K cellulase B and other cellulase activity.

^***Activity by the trichoderma cellulase generation.

The endoglucanase activity of T.reesei host strain ALKO 3620 is 70 ℃ of almost completely deactivations. The transformant that produces Melanocarpus albomyces 20K-cellulase produces in a large number relatively heat-staple 20K-cellulase. The endoglucanase level of production of transformant is than several times of 20K-cellulase parent strain ALKO 4237 height.

Embodiment 31

In T.reesei, produce Melanocarpus albomyces

ALKO 423750K cellulase

Make up Trichoderma reesei bacterial strain and be used for Melanocarpus albomyces ALKO 4237 50K-cellulase production. This bacterial strain is produced Melanocarpus 50K-cellulase and can not be produced T.reesei endoglucanase II and the biological hydrolase of cellulose or endoglucanase I. In the structure of the bacterial strain that produces Melanocarpus albomyces 50K-cellulase, use the expression cassette from plasmid pALK 1238 or pALK1240 (Figure 31 and 32) to transform parent Trichoderma reesei strains A LKO 3620. In expression cassette, the 50K cellulase is expressed under strong cbhl promoter. The integration of expression cassette causes replacing parent cbh1 (pALK 1238) or egl1 (pALK 1240) gene. Except replacing the 20K-cellulose enzyme gene with embodiment 22 described 50K-cellulose enzyme genes (1.7kb fragment from the ATG codon). Clone and transform by embodiment 29 is described. Then similar the apparent modification of those skilled in the art is identified the transformant that produces Melanocarpus albomyces 50K-cellulase to embodiment 30. Similar with 30 to embodiment 29, so that the apparent modification preparation of those skilled in the art is produced Melanocarpus albomyces 50K-cellulase B and the transformant with protein of CBD.

Owing to fully described the present invention now, it will be understood by those skilled in the art that and in condition under the prerequisite of the spirit or scope that do not affect the present invention or its any embodiment, to carry out the present invention in the extensive and suitable scope of parameter etc. All lists of references that this paper quotes fully are incorporated herein by reference.

Sequence table

(1) general information:

(i) applicant:

(A) title: Primalco Ltd

(B) street: Valta-akseli

(C) city: Rajamaki

(E) country: Finland

(F) postcode: (ZIP): 05200

(G) phone :+358 9 13311

(H) fax :+358 9 133 1236

(ii) invention exercise question: new cellulose enzyme, encode their group and application thereof

(iii) sequence number: 37

(iv) computer-reader form:

(A) medium type: Floppy disk

(B) computer: IBM PC compatible

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

(D) software: PatentIn Release #1.0, Version #1.30 (EPO)

(2) SEQ ID NO:1 information:

(i) sequence description:

(A) length: 30 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

(ii) molecule type: peptide

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

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(A) title/keyword: peptide

(B) position: 1 ... 30

(D) out of Memory :/mark=No_429

(xi) sequence description of SEQ ID NO:l:

Ala Asn Gly Gln Ser Thr Arg Tyr Trp Asp cys Cys Lys Pro Ser Cys

1               5                   10                  15

Gly Trp Arg Gly Lys Gly Pro Val Asn Gln Pro Val Tyr Ser

            20                  25                  30

(2) SEQ ID NO:2 information:

(i) sequence description:

(A) length: 7 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

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(A) biology: Melanocarpus albomyces

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(xi) sequence description of SEQ ID NO:2:

    Tyr Gly Gly Ile Ser Ser Arg

    1               5

(2) SEQ ID NO:3 information:

(i) sequence description:

(A) length: 4 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

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(A) biology: Melanocarpus albomyces

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(xi) sequence description of SEQ ID NO:3:

Cys Gly Trp Arg

l

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(xi) sequence description of SEQ ID NO:4:

   Pro Ser Cys Gly Trp Arg

   1               5

(2) SEQ ID NO:5 information:

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(A) length: 6 amino acid

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(C) chain:

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(xi) sequence description of SEQ ID NO:5:

 Tyr Trp Asp Cys Cys Lys

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(i) sequence description:

(A) length: 17 amino acid

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(A) biology: Melanocarpus albomyces

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(xi) sequence description of SEQ ID NO:6:

   Gln Glu Cys Asp Ser Phe Pro Glu Pro Leu Lys Pro Gly Cys Gln Trp

   1               5                   10                  15

   Arg

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(xi) sequence description of SEQ ID NO:7:

     Arg His Asp Asp Gly Gly Phe Ala

     l               5

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(xi) sequence description of SEQ ID NO:8:

   Tyr Trp Asp Cys Cys Lys Pro

   1               5

(2) SEQ ID NO:9 information:

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(xi) sequence description of SEQ ID NO:9:

    Gly Lys Gly Pro Val Asn Gln Pro Val Tyr Ser Cys Asp Ala Asn Phe

    1               5                   10                  15

    Gln Arg

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   Val Gln Cys pro Glu Glu Leu Val Ala Arg

   1               5                   10

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(xi) sequence description of SEQ ID NO:11:

Asp Trp Phe Gln Asn Ala Asp Asn Pro Ser Phe Thr Phe Glu Arg

1               5                   10                  15

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(A) biology: Melanocarpus albomyces

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(xi) sequence description of SEQ ID NO:12:

Thr Met Val Val Gln Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn

1               5                   10                  15

His Phe Asp Leu Asn Ile Pro Gly Gly Gly Val Gly Leu Phe

            20                  25                  30

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(D) topology: line style

(ii) molecule type: peptide

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: peptide

(B) position: 1 ... 10

(D) out of Memory :/mark=No_507

(xi) sequence description of SEQ ID NO:13:

Val Tyr Leu Leu Asp Glu Thr Glu His Arg

1               5                   10

(2) SEQ ID NO:14 information:

(i) sequence description:

(A) length: 12 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

(ii) molecule type: peptide

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: peptide

(B) position: 1 ... 12

(D) out of Memory :/mark=No_509

(xi) sequence description of SEQ ID NO:14:

Xaa Xaa Leu Asn Pro Gly Gly Ala Tyr Tyr Gly Thr

1               5                   10

(2) SEQ ID NO:15 information:

(i) sequence description:

(A) length: 17 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

(ii) molecule type: peptide

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: peptide

(B) position: 1 ... 17

(D) out of Memory :/mark=No_563

(xi) sequence description of SEQ ID NO:15:

Met Ser Glu Gly Ala Glu Cys Glu Tyr Asp Gly Val Cys Asp Lys Asp

1               5                   10                  15

Gly

(2) SEQ ID NO:16 information:

(i) sequence description:

(A) length: 14 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

(ii) molecule type: peptide

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: peptide

(B) position: 1 ... 14

(D) out of Memory :/mark=No_565

(xi) sequence description of SEQ ID NO:16:

Asn Pro Tyr Arg Val Xaa Ile Thr Asp Tyr Tyr Gly Asn Ser

1               5                       10

(2) SEQ ID NO:17 information:

(i) sequence description:

(A) length: 24 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

(ii) molecule type: peptide

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: peptide

(B) position: 1 ... 24

(D) out of Memory :/mark=No_603

(xi) sequence description of SEQ ID NO:17:

Asp Pro Thr Gly Ala Arg Ser Glu Leu Asn Pro Gly Gly Ala Tyr Tyr

1               5                   10                  15

Gly Thr Gly Tyr Xaa Asp Ala Gln

            20

(2) SEQ ID NO:18 information:

(i) sequence description:

(A) length: 13 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

(ii) molecule type: peptide

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: peptide

(B) position: 1 ... 13

(D) out of Memory :/mark=No_605

(xi) sequence description of SEQ ID NO:18:

Xaa Xaa Val Pro Asp Tyr His Gln His Gly Val Asp Ala

1               5                   10

(2) SEQ ID NO:19 information:

(i) sequence description:

(A) length: 12 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

(ii) molecule type: peptide

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: peptide

(B) position: 1 ... 12

(D) out of Memory :/mark=No_610

(xi) sequence description of SEQ ID NO:19:

Asn Glu Met Asp Ile Xaa Glu Ala Asn Ser Arg Ala

1               5                   10

(2) SEQ ID NO:20 information:

(i) sequence description:

(A) length: 25 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

(ii) molecule type: peptide

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: peptide

(B) position: 1 ... 25

(D) out of Memory :/mark=No_611

(xi) sequence description of SEQ ID NO:20:

Leu Pro Xaa Gly Met Asn Ser Ala Leu Tyr Leu Ser Glu Met Asp Pro

1               5                   10                         15

Thr Gly Ala Arg Ser Glu Leu Asn Pro

            20                  25

(2) SEQ ID NO:21 information:

(i) sequence description:

(A) length: 21 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

(ii) molecule type: peptide

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: peptide

(B) position: 1 ... 21

(D) out of Memory :/mark=No_612

(xi) sequence description of SEQ ID NO:21:

Val Glu Pro Ser Pro Glu Val Thr Tyr Ser Asn Leu Arg Xaa Gly Glu

1               5                   10                  15

Ile Xaa Gly Xaa Phe

            20

(2) SEQ ID NO:22 information:

(i) sequence description:

(A) length: 19 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

(ii) molecule type: peptide

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: peptide

(B) position: 1 ... 19

(D) out of Memory :/mark=No_619

(xi) sequence description of SEQ ID NO:22:

Asp Gly Cys Gly Trp Asn Pro Tyr Arg Val Val Ile Thr Thr Asp Tyr

1               5                   10                  15

Tyr Asn Asn

(2) SEQ ID NO:23 information:

(i) sequence description:

(A) length: 10 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

(ii) molecule type: peptide

(vi) primary source:

(A) biology: Melanocarpus albomvces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: peptide

(B) position: 1 ... 10

(D) out of Memory :/mark=No_620

(xi) sequence description of SEQ ID NO:23:

Leu Pro Cys Gly Met Xaa Ser Ala Leu Tyr

1               5                   10

(2) SEQ ID NO:24 information:

(i) sequence description:

(A) length: 22 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

(ii) molecule type: peptide

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: peptide

(B) position: 1 ... 22

(D) out of Memory :/mark=No_621

(xi) sequence description of SEQ ID NO:24:

Ala Asp Gly Cys Gln Pro Arg Thr Asn Tyr Ile Val Leu Asp Asp Leu

1               5                   10                  15

Leu His Pro Xaa Xaa Gln

            20

(2) SEQ ID NO:25 information:

(i) sequence description:

(A) length: 9 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

(ii) molecule type: peptide

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: peptide

(B) position: 1 ... 9

(D) out of Memory :/mark=No_534

(xi) sequence description of SEQ ID NO:25:

Val Gly Asn Pro Asp Phe Tyr Gly Lys

1               5

(2) SEQ ID NO:26 information:

(i) sequence description:

(A) length: 8 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

(ii) molecule type: peptide

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: peptide

(B) position: 1 ... 8

(D) out of Memory :/mark=No_535

(xi) sequence description of SEQ ID NO:26:

Phe Gly Pro Ile Gly Ser Thr Tyr

1               5

(2) SEQ ID NO:27 information:

(i) sequence description:

(A) length: 12 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

(ii) molecule type: peptide

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: peptide

(B) position: 1 ... 12

(D) out of Memory :/mark=No_631

(xi) sequence description of SEQ ID NO:27:

Leu Ser Gln Tyr Phe Ile Gln Asp Gly Glu Arg Lys

1               5                   10

(2) SEQ ID NO:28 information:

(i) sequence description:

(A) length: 11 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

(ii) molecule type: peptide

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: peptide

(B) position: 1 ... 11

(D) out of Memory :/mark=No_632

(xi) sequence description of SEQ ID NO:28:

Phe Thr Val Val Ser Arg Phe Glu Glu Asn Lys

1               5                   10

(2) SEQ ID NO:29 information:

(i) sequence description:

(A) length: 19 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

(ii) molecule type: peptide

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: peptide

(B) position: 1 ... 19

(D) out of Memory :/mark=No_636

(xi) sequence description of SEQ ID NO:29:

His Glu Tyr Gly Thr Asn Val Gly Ser Arg Phe Tyr Leu Met Asn Gly

1               5                   10                  15

   Pro Asp Lys

(2) SEQ ID NO:30 information:

(i) sequence signature:

(A) length: 936 base-pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: line style

(ii) molecule type: DNA (genome)

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: extron

(B) position: 33 ... 115

(D) out of Memory :/codon _ initial=33

/ product=" 20K-cellulase "

(ix) feature:

(A) title/keyword: extron

(B) position: 187 ... 435

(D) out of Memory :/product=" 20K-cellulase "

(ix) feature:

(A) title/keyword: extron

(B) position: 506 ... 881

(D) out of Memory :/product=" 20K-cellulase "

(xi) sequence description of SEQ ID NO:30:

TCGCCCCTAA CCGAGAACCA AAGACTCCAA GAATGCGCTC TACTCCCGTT CTCCGCGCCC   60

TCCTGGCCGC AGCATTGCCC CTCGGGGCCC TCGCCGCCAA CGGTCAGTCC ACGAGGTAAC  120

TGATCACCCG CCTCATTACG CGTGCCGACC GGACCGCGTT CAGGGCTCAC TGCTCACCGC  180

ATCCAGATAC TGGGACTGCT GCAAGCCGTC GTGCGGCTGG CGCGGAAAGG GCCCCGTGAA  240

CCAGCCCGTC TACTCGTGCG ACGCCAACTT CCAGCGCATC CACGACTTCG ATGCCGTCTC  300

GGGCTGCGAG GGCGGCCCCG CCTTCTCGTG CGCCGACCAC AGCCCCTGGG CCATTAATGA  360

CAACCTCTCG TACGGCTTCG CGGCGACTGC ACTCAGCGGC CAGACCGAGG AGTCGTGGTG  420

CTGTGCCTGC TACGCGTGAG TGTGCTTGGG CCCAACGTCG GTGATTCCGG AGTTCAGACC  480

ACTGACCCAG CGACCCGCTC GCCAGTCTGA CCTTTACATC GGGTCCCGTG GCCGGCAAGA  540

CCATGGTCGT CCAGTCGACC AGCACGGGCG GCGACCTCGG CAGCAACCAC TTCGACCTCA  600

ACATCCCCGG CGGCGGCGTC GGCCTCTTCG ACGGCTGCAC TCCCCAGTTC GGCGGCCTCC  660

CGGGCGCACG GTACGGCGGC ATCTCGTCGC GCCAGGAGTG CGACTCGTTC CCCGAGCCGC  720

TCAAGCCCGG CTGCCAGTGG CGCTTCGACT GGTTCCAGAA CGCCGACAAC CCGTCCTTTA  780

CCTTCGAGCG GGTCCAGTGC CCCGAGGAGC TGGTCGCTCG GACCGGCTGC AGGCGCCACG  840

ACGACGGCGG CTTCGCCGTC TTCAAGGCCC CCAGCGCCTG ATCCGTTTTT GGGCAGTGTC  900

CGTGTGACGG CAGCTACGTG GAACGACCTG GAGCTC                            936

(2) SEQ ID NO:31 information:

(i) sequence description:

(A) length: 235 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

(ii) molecule type: protein

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: protein

(B) position: 1 ... 235

(D) out of Memory :/mark=20K-cellulase

(xi) sequence description of SEQ ID NO:31:

Met Arg Ser Thr Pro Val Leu Arg Ala Leu Leu Ala Ala Ala Leu Pro

1               5                   10                  15

Leu Gly Ala Leu Ala Ala Asn Gly Gln Ser Thr Arg Tyr Trp Asp Cys

            20                  25                  30

Cys Lys Pro Ser Cys Gly Trp Arg Gly Lys Gly Pro Val Asn Gln Pro

        35                  40                  45

Val Tyr Ser Cys Asp Ala Asn Phe Gln Arg Ile His Asp Phe Asp Ala

    50                  55                  60

Val Ser Gly Cys Glu Gly Gly Pro Ala Phe Ser Cys Ala Asp His Ser

65                  70                  75                  80

Pro Trp Ala Ile Asn Asp Asn Leu Ser Tyr Gly Phe Ala Ala Thr Ala

                85                  90                  95

Leu Ser Gly Gln Thr Glu Glu Ser Trp Cys Cys Ala Cys Tyr Ala Leu

            100                 105                 110

Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Thr Met Val Val Gln Ser

        115                 120                 125

Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn Ile

    130                 135                 140

Pro Gly Gly Gly Val Gly Leu Phe Asp Gly Cys Thr Pro Gln Phe Gly

145                 150                 155                 160

Gly Leu Pro Gly Ala Arg Tyr Gly Gly Ile Ser Ser Arg Gln Glu Cys

                165                 170                 175

Asp Ser Phe Pro Glu Pro Leu Lys Pro Gly Cys Gln Trp Arg Phe Asp

            180                 185                 190

Trp Phe Gln Asn Ala Asp Asn Pro Ser Phe Thr Phe Glu Arg Val Gln

        195                 200                 205

Cys Pro Glu Glu Leu Val Ala Arg Thr Gly Cys Arg Arg His Asp Asp

    210                 215                 220

Gly Gly Phe Ala Val Phe Lys Ala Pro Ser Ala

225                 230                 235

(2) SEQ ID NO:32 information:

(i) sequence signature:

(A) length: 1894 base-pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: line style

(ii) molecule type: DNA (genome)

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: extron

(B) position: 233 ... 838

(D) out of Memory :/product=" 50K-cellulase "

(ix) feature:

(A) title/keyword: extron

(B) position: 916 ... 1596

(D) out of Memory :/product=" 50K-cellulase "

(xi) sequence description of SEQ ID NO:32:

GAATTCGGGG GTTGCCAGGG AGTCGTACAG GGGTGGGTGG AGGGGGATGG GGGATGGAAG    60

GGGGATGGAG AAGAAAGCAT ATATGGGACG TTTGTGCTCG CCGGCTCCCC TCTGCCACGT   120

TCCCTTGCCT CCTTGCCTGG GTTGTTGTTG GTCTTCCCTT CACCATCCGA CAAACCAACC   180

TGCTGCGGGT GAACTCGCAG AGCGCCTTCG GACGACGACA GACAGACGCA CCATGACTCG   240

CAACATCGCC CTGCTCGGCG CCGCGTCGGC GCTCCTGGGC CTCGCCCACG GCCAGAAGCC   300

GGGCGAGACG CCCGAGGTGC ACCCGCAGCT GACGACGTTC CGGTGCACCA AGGCGGACGG   360

GTGCCAGCCG CGGACCAACT ACATTGTGCT GGACTCGCTG TCGCACCCGG TGCACCAGGT   420

GGACAACGAC TACAACTGCG GCGACTGGGG GCAGAAGCCC AACGCGACGG CGTGCCCGGA   480

CGTCGAGTCG TGCGCGCGCA ACTGCATCAT GGAGGGCGTG CCCGACTACA GCCAGCACGG   540

CGTCACGACG AGCGACACGT CGCTGCGCCT GCAGCAGCTC GTCGACGGCC GCCTCGTCAC   600

GCCGCGCGTC TACCTGCTCG ACGAGACCGA GCACCGCTAC GAGATGATGC ACCTGACCGG   660

CCAGGAGTTC ACCTTTGAGG TCGACGCCAC CAAGCTGCCC TGCGGCATGA ACAGCGCCCT   720

CTACCTGTCC GAGATGGACC CGACCGGCGC CCGGAGCGAG CTCAACCCCG GCGGTGCCTA   780

CTACGGCACC GGCTACTGCG ACGCCCAGTG CTTCGTGACG CCATTCATCA ACGGCATTGT   840

GAGTGTTCCC CTTTGGCCCC CCCCCTGAAA ATAGATGTAC CTGGGTGCTA ACCCCGGGGT   900

GTCGCACCAA AACAGGGCAA CATCGAGGGC AAGGGCTCGT GCTGCAACGA GATGGACATC   960

TGGGAGGCCA ACTCGCGGGC GACGCACGTG GCGCCGCACA CGTGCAACCA GACGGGTCTG  1020

TACATGTGCG AGGGCGCCGA GTGCGAGTAC GACGGCGTGT GCGACAAGGA CGGGTGCGGG  1080

TGGAACCCGT ACCGGGTCAA CATCACCGAC TACTACGGCA ACTCGGACGC GTTCCGCGTC  1140

GACACGCGGC GGCCCTTCAC CGTGGTGACG CAGTTCCCGG CCGACGCCGA GGGCCGGCTC  1200

GAGAGCATCC ACCGGCTGTA CGTGCAGGAC GGCAAGGTGA TCGAGTCGTA CGTCGTCGAC  1260

GCGCCGGGCC TGCCCCGGAC CGACTCGCTC AACGACGAGT TCTGCGCCGC CACGGGCGCC  1320

GCGCGCTACC TCGACCTCGG CGGCACCGCG GGCATGGGCG ACGCCATGAC GCGCGGCATG  1380

GTGCTGGCCA TGAGCATCTG GTGGGACGAG TCCGGCTTCA TGAACTGGCT CGACAGCGGC  1440

GAGGCCGGCC CCTGCCTGCC CGACGAGGGC GACCCCAAGA ACATTGTCAA GGTCGAGCCC  1500

AGCCCCGAGG TCACCTACAG CAACCTGCGC TGGGGCGAGA TCGGGTCGAC CTTTGAGGCC  1560

GAGTCCGACG ACGACGGCGA CGGCGACGAC TGCTAGATAA CTAACTAGTG GGCGGAAAGG  1620

GCGGGGGATG CGTAACTTAC ATACAGCCCG GAGTTGTTTT GAGTGTAGAG TATTGAGCTT  1680

TCGATGTGTT AGTTGAGTGG AATGGAAAAT TCGCGTCTTT GCCCCGGTGG TTGCGATAAA  1740

CAATAGTCGG CTGGTGCATT TGTGACACTT CAATTGCGCT GTTGGCTTGG TGACAGACAC  1800

GGCAGCGTCG ATGACCCGAC ACCCAGAATA ATTCGCATGG TTGATTATGT TATTGTGCTT  1860

TAAATCGGAG GCTGATGCTC ATCTCTTCGA ATTC                              1894

(2) SEQ ID NO:33 information:

(i) sequence description:

(A) length: 428 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

(ii) molecule type: protein

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: protein

(B) position: 1 ... 428

(D) out of Memory :/mark=50K-cellulase

(xi) sequence description of SEQ ID NO:33:

Met Thr Arg Asn Ile Ala Leu Leu Gly Ala Ala Ser Ala Leu Leu Gly

1               5                   10                  15

Leu Ala His Gly Gln Lys Pro Gly Glu Thr Pro Glu Val His Pro Gln

            20                  25                  50

Leu Thr Thr Phe Arg Cys Thr Lys Ala Asp Gly Cys Gln Pro Arg Thr

        35                  40                  45

Asn Tyr Ile Val Leu Asp Ser Leu Ser His Pro val His Gln Val Asp

    50                  55                  60

Asn Asp Tyr Asn Cys Gly Asp Trp Gly Gln Lys Pro Asn Ala Thr Ala

65                  70                  75                  80

Cys Pro Asp Val Glu Ser Cys Ala Arg Asn Cys Ile Met Glu Gly Val

                85                  90                  95

Pro Asp Tyr Ser Gln His Gly Val Thr Thr Ser Asp Thr Ser Leu Arg

            100                 105                 110

Leu Gln Gln Leu Val Asp Gly Arg Leu Val Thr Pro Arg Val Tyr Leu

        115                 120                 125

Leu Asp Glu Thr Glu His Arg Tyr Glu Met Met His Leu Thr Gly Gln

    130                 135                 140

Glu Phe Thr Phe Glu Val Asp Ala Thr Lys Leu Pro Cys Gly Met Asn

145                 150                 155                 160

Ser Ala Leu Tyr Leu Ser Glu Met Asp Pro Thr Gly Ala Arg Ser Glu

                165                 170                 175

Leu Asn Pro Gly Gly Ala Tyr Tyr Gly Thr Gly Tyr Cys Asp Ala Gln

            180                 185                 190

Cys Phe Val Thr Pro Phe Ile Asn Gly Ile Gly Asn Ile Glu Gly Lys

        195                 200                 205

Gly Ser Cys Cys Asn Glu Met Asp Ile Trp Glu Ala Asn Ser Arg Ala

    210                 215                 220

Thr His Val Ala Pro His Thr Cys Asn Gln Thr Gly Leu Tyr Met Cys

225                 230                 235                 240

Glu Gly Ala Glu Cys Glu Tyr Asp Gly Val Cys Asp Lys Asp Gly Cys

                245                 250                 255

Gly Trp Asn Pro Tyr Arg Val Asn Ile Thr Asp Tyr Tyr Gly Asn Ser

            260                 265                 270

Asp Ala Phe Arg Val Asp Thr Arg Arg Pro Phe Thr Val Val Thr Gln

        275                 280                 285

Phe Pro Ala Asp Ala Glu Gly Arg Leu Glu Ser Ile His Arg Leu Tyr

    290                 295                 300

Val Gln Asp Gly Lys Val Ile Glu Ser Tyr Val Val Asp Ala Pro Gly

305                 310                 315                 320

Leu Pro Arg Thr Asp Ser Leu Asn Asp Glu Phe Cys Ala Ala Thr Gly

                325                 330                 335

Ala Ala Arg Tyr Leu Asp Leu Gly Gly Thr Ala Gly Met Gly Asp Ala

            340                 345                 350

Met Thr Arg Gly Met Val Leu Ala Met Ser Ile Trp Trp Asp Glu Ser

        355                 360                 365

Gly Phe Met Asn Trp Leu Asp Ser Gly Glu Ala Gly Pro Cys Leu Pro

    370                 375                 380

Asp Glu Gly Asp Pro Lys Asn Ile Val Lys Val Glu Pro Ser Pro Glu

385                 390                 395                 400

Val Thr Tyr Ser Asn Leu Arg Trp Gly Glu Ile Gly Ser Thr Phe Glu

                405                 410                 415

Ala Glu Ser Asp Asp Asp Gly Asp Gly Asp Asp Cys

            420                 425

(2) SEQ ID NO:34 information:

(i) sequence signature:

(A) length: 2000 base-pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: line style

(ii) molecule type: DNA (genome)

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: extron

(B) position: 154 ... 729

(D) out of Memory :/product=" 50K-cellulase B "

(ix) feature:

(A) title/keyword: extron

(B) position: 810 ... 946

(D) out of Memory :/product=" 50K-cellulase B "

(ix) feature:

(A) title/keyword: extron

(B) position: 1018 ... 1230

(D) out of Memory :/product=" 50K-cellulase B "

(ix) feature:

(A) title/keyword: extron

(B) position: 1308 ... 1551

(D) out of Memory :/product=" 50K-cellulase B "

(ix) feature:

(A) title/keyword: extron

(B) position: 1637 ... 1767

(D) out of Memory :/product=" 50K-cellulase B "

(ix) feature:

(A) title/keyword: extron

(B) position: 1831 ... 1888

(D) out of Memory :/product=" 50K-cellulase B "

(xi) sequence description of SEQ ID NO:34:

CCCGGTCTGG AGACGGGGAG CGCGCCAGCG ACGCAGGATA AGAAGGCGAC GACCGCGCCT    60

CCGAGCCAGG CCCAGGACAG CAGGAGAACT CGCCACGCGC AAGCAGCACG CCCGATCGAC   120

AGTGTCCCGC TCTGCCCACA GCACTCTGCA ACCATGATGA TGAAGCAGTA CCTCCAGTAC   180

CTCGCGGCCG CGCTGCCGCT CGTCGGCCTC GCCGCCGGCC AGCGCGCTGG TAACGAGACG   240

CCCGAGAACC ACCCCCCGCT CACCTGGCAG AGGTGCACGG CCCCGGGCAA CTGCCAGACC   300

GTGAACGCCG AGGTCGTCAT TGACGCCAAC TGGCGCTGGC TGCACGACGA CAACATGCAG   360

AACTGCTACG ACGGCAACCA GTGGACCAAC GCCTGCAGCA CCGCCACCGA CTGCGCTGAG   420

AAGTGCATGA TCGAGGGTGC CGGCGACTAC CTGGGCACCT ACGGCGCCTC GACCAGCGGC   480

GACGCCCTGA CGCTCAAGTT CGTCACCAAG CACGAGTACG GCACCAACGT CGGCTCGCGC   540

TTCTACCTCA TGAACGGCCC GGACAAGTAC CAGATGTTCA ACCTCATGGG CAACGAGCTT   600

GCCTTTGACG TCGACCTCTC GACCGTCGAG TGCGGCATCA ACAGCGCCCT GTACTTCGTC   660

GCCATGGAGG AGGACGGCGG CATGGCCAGC TACCCGAGCA ACCAGGCCGG CGCCCGGTAC   720

GGCACTGGGG TGAGTTGAGC TCCGCTTTGT TTCGAGTCGC AACGAGGCAC TTTCTGGGCG   780

CCGGCTAACT CTCTCGATTC CTCCGACAGT ACTGCGATGC CCAATGCGCT CGTGATCTCA   840

AGTTCGTTGG CGGCAAGGCC AACATTGAGG GCTGGAAGTC GTCCACCAGC GACCCCAACG   900

CTGGCGTCGG CCCGTACGGC AGCTGCTGCG CTGAGATCGA CGTCTGGTGA GTGCGAGACC   960

GTCCACCCAG GTTCGGATGC GGGGTGGAAA TTTCGCGGCT AACGGAGCAC CCCCCAGGGA  1020

GTCGAATGCC TATGCCTTCG CTTTCACGCC GCACGCGTGC ACGACCAACG AGTACCACGT  1080

CTGCGAGACC ACCAACTGCG GTGGCACCTA CTCGGAGGAC CGCTTCGCCG GCAAGTGCGA  1140

CGCCAACGGC TGCGACTACA ACCCCTACCG CATGGGCAAC CCCGACTTCT ACGGCAAGGG  1200

CAAGACGCTC GACACCAGCC GCAAGTTCAC GTGCGTGACC CCTTGTGGCG CAACCTTTCT  1260

CTGCCTGCCT GGACACACTG AAACTGACAC GTCGTTTTCG GCTGCAGCGT CGTCTCCCGC  1320

TTCGAGGAGA ACAAGCTCTC CCAGTACTTC ATCCAGGACG GCCGCAAGAT CGAGATCCCG  1380

CCGCCGACGT GGGAGGGCAT GCCCAACAGC AGCGAGATCA CCCCCGAGCT CTGCTCCACC  1440

ATGTTCGATG TGTTCAACGA CCGCAACCGC TTCGAGGAGG TCGGCGGCTT CGAGCAGCTG  1500

AACAACGCCC TCCGGGTTCC CATGGTCCTC GTCATGTCCA TCTGGGACGA CGTAAGTACC  1560

CGCCGACCTC CCTAGCCACA CAAGCCGCAT CCGGCGAGGC ACGCCATCGC TGCTGCTAAC  1620

ACGAGACCGT TCGTAGCACT ACGCCAACAT GCTCTGGCTC GACTCCATCT ACCCGCCCGA  1680

GAAGGAGGGC CAGCCCGGCG CCGCCCGTGG CGACTGCCCC ACGGACTCGG GTGTCCCCGC  1740

CGAGGTCGAG GCTCAGTTCC CCGACGCGTA AGACTTGCCC CCGACCCCAA GCTTCCACTT  1800

CTGGATGCCG AATGCTAACA CGCGAAACAG CCAGGTCGTC TGGTCCAACA TCCGCTTCGG  1860

CCCCATCGGC TCGACCTACG ACTTCTAAGC CGGTCCATGC ACTCGCAGCC CTGGGCCCGT  1920

CACGCCCGCC ACCTCCCCTC GCGGAAACTC TCCGTGCGTC GCGGGCTCCA AAGCATTTTG  1980

GCCTCAAGTT TTTTTCGTTC                                              2000

(2) SEQ ID NO:35 information:

(i) sequence description:

(A) length: 452 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

(ii) molecule type: protein

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: protein

(B) position: 1 ... 452

(D) out of Memory :/mark=50K-cellulase-B

(xi) sequence description of SEQ ID NO:35:

Met Met Met Lys Gln Tyr Leu Gln Tyr Leu Ala Ala Ala Leu Pro Leu

1               5                   10                  15

Val Gly Leu Ala Ala Gly Gln Arg Ala Gly Asn Glu Thr Pro Glu Asn

            20                  25                  30

His Pro Pro Leu Thr Trp Gln Arg Cys Thr Ala Pro Gly Asn Cys Gln

        35                  40                  45

Thr Val Asn Ala Glu Val Val Ile Asp Ala Asn Trp Arg Trp Leu His

    50                  55                  60

Asp Asp Asn Met Gln Asn Cys Tyr Asp Gly Asn Gln Trp Thr Asn Ala

65                  70                  75                  80

Cys Ser Thr Ala Thr Asp Cys Ala Glu Lys Cys Met Ile Glu Gly Ala

                85                  90                  95

Gly Asp Tyr Leu Gly Thr Tyr Gly Ala Ser Thr Ser Gly Asp Ala Leu

            100                 105                 110

Thr Leu Lys Phe Val Thr Lys His Glu Tyr Gly Thr Asn Val Gly Ser

        115                 120                 125

Arg Phe Tyr Leu Met Asn Gly Pro Asp Lys Tyr Gln Met Phe Asn Leu

    130                 135                 140

Met Gly Asn Glu Leu Ala Phe Asp Val Asp Leu Ser Thr Val Glu Cys

145                 150                 155                 160

Gly Ile Asn Ser Ala Leu Tyr Phe Val Ala Met Glu Glu Asp Gly Gly

                165                 170                 175

Met Ala Ser Tyr Pro Ser Asn Gln Ala Gly Ala Arg Tyr Gly Thr Gly

            180                 185                 190

Tyr Cys Asp Ala Gln Cys Ala Arg Asp Leu Lys Phe Val Gly Gly Lys

        195                 200                 205

Ala Asn Ile Glu Gly Trp Lys Ser Ser Thr Ser Asp Pro Asn Ala Gly

    210                 215                 220

Val Gly Pro Tyr Gly Ser Cys Cys Ala Glu Ile Asp Val Trp Glu Ser

225                 230                 235                 240

Asn Ala Tyr Ala Phe Ala Phe Thr Pro His Ala Cys Thr Thr Asn Glu

                245                 250                 255

Tyr His Val Cys Glu Thr Thr Asn Cys Gly Gly Thr Tyr Ser Glu Asp

            260                 265                 270

Arg Phe Ala Gly Lys Cys Asp Ala Asn Gly Cys Asp Tyr Asn Pro Tyr

        275                 280                 285

Arg Met Gly Asn Pro Asp Phe Tyr Gly Lys Gly Lys Thr Leu Asp Thr

    290                 295                 300

Ser Arg Lys Phe Thr Val Val Ser Arg Phe Glu Glu Asn Lys Leu Ser

305                 310                 315                 320

Gln Tyr Phe Ile Gln Asp Gly Arg Lys Ile Glu Ile Pro Pro Pro Thr

                325                 330                 335

Trp Glu Gly Met Pro Asn Ser Ser Glu Ile Thr Pro Glu Leu Cys Ser

            340                 345                 350

Thr Met Phe Asp Val Phe Asn Asp Arg Asn Arg Phe Glu Glu Val Gly

        355                 360                 365

Gly Phe Glu Gln Leu Asn Asn Ala Leu Arg Val Pro Met Val Leu Val

    370                 375                 380

Met Ser Ile Trp Asp Asp His Tyr Ala Asn Met Leu Trp Leu Asp Ser

385                 390                 395                 400

Ile Tyr Pro Pro Glu Lys Glu Gly Gln Pro Gly Ala Ala Arg Gly Asp

                405                 410                 415

Cys Pro Thr Asp Ser Gly Val Pro Ala Glu Val Glu Ala Gln Phe Pro

            420                 425                 430

Asp Ala Gln Val Val Trp Ser Asn Ile Arg Phe Gly Pro Ile Gly Ser

        435                 440                 445

Thr Tyr Asp Phe

    450

(2) SEQ ID NO:36 information:

(i) sequence signature:

(A) length: 887 base-pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: line style

(ii) molecule type: DNA (genome)

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: extron

(B) position: 351 ... 455

(D) out of Memory :/product=" protein-with-CBD "

(xi) sequence description of SEQ ID NO:36:

CCATGGACGC GAACTGCGAC GTCTTCTGCC CCGAGCTGAA GACCCAGAGC ATCCAGACCG   60

GCAACCAGTG CACCCAGGAG ATGAAGGTCT ACGAGAACAT TGACGGCTGG CTCGACAGCC  120

TGCCCGGCAA CGTCCCCATC ACCGGTCCGC AGCCCGGCTC TGGTAAGTCA AAGAGATGAT  180

GCCTACCTAC CTTCCCACCT TCCCACCCAG CCGCAAATAC CTTTCTCCCT CCCCGTGCCC  240

CGTATTCTTT CAACGCCCCG AGACTGACAG ACCCGCTCGT CCCAGGCGGC AACCCCGGCA  300

ACGGCGGCGG CAGCAACCCG GGCAACGGCG GCGGCGGCGG CTGCACCGTC CAGAAGTGGG  360

GCCAGTGCGG CGGCATCGGC TACTCGGGCT GCACCACCTG CAAGGCCGGC TCGACCTGCC  420

CGGCCCAGAA CGAGTACTAC TCGCAGTGCC TGTAAAGCGG CCGTGGGCTA GGTGGCCGAG  480

CGGGGGGGTT TCTTCATTGG TTGAGCAAAT AGAACAGGAT TTCCGGCTCG TTGGCAGCGG  540

CGCGCCGCGG GGATGGTGTT GTACAATTCA AGACCTCAGT ACCGAGGGAC CTGGAAAGGA  600

GTCAGTCTGC TTGTACGGAG GCTGGCTGCC CCGTGGCGGC GCTGGCAAGG TAGATAGCCC  660

TTCATTGCTG TAACTAGTAT GCTATATACC TCTGCACATT TGCAGCCCCA TGGTGTGAAC  720

AACAAGTGAC AAGGCTTCCA GTTCCAGCCT CGCGCAATTG TCACGATATC CTTGGTCCAT  780

CTATATGTAT GGGCATGAGC GAGTCGAGAA AATGTACCGC GAAAAATCGT AGTGACCTGC  840

GCACTGCGCC GTTCTACCAC CGTAGGATTG AAGTGAATCT CGAATTC                887

(2) SEQ ID NO:37 information:

(i) sequence description:

(A) length: 34 amino acid

(B) type: amino acid

(C) chain:

(D) topology: line style

(ii) molecule type: peptide

(vi) primary source:

(A) biology: Melanocarpus albomyces

(B) bacterial strain: ALKO4237

(ix) feature:

(A) title/keyword: protein

(B) position: 1 ... 34

(D) out of Memory :/mark=prot-with-CBD

(xi) sequence description of SEQ ID NO:37:

Gln Lys Trp Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Cys Thr Thr

1               5                   10                  15

Cys Lys Ala Gly Ser Thr Cys Pro Ala Gln Asn Glu Tyr Tyr Ser Gln

            20                  25                  30

Cys Leu

Claims (27)

1. coding has the nucleic acid molecules of peptide more than the enzymatic activity of cellulase, and it comprises and is selected from following nucleic acid molecules:

(a) coding has the nucleic acid molecules of the polypeptide of the described amino acid sequence of SEQ ID NO 31 or 33;

(b) coding has the nucleic acid molecules of the polypeptide of the described amino acid sequence of SEQ ID NO 35;

(c) has the nucleic acid molecules of the coded sequence of the described nucleotide sequence of SEQ ID NO 30 or 32;

(d) has the nucleic acid molecules of the coded sequence of the described nucleotide sequence of SEQ ID NO 34;

(e) to have by preserving number be the plasmid pALK1221 of DSM11024 to coding, preserving number is the λ 4237/5.1 of DSM11012, and preserving number is the nucleic acid molecules of polypeptide of the amino acid whose sequence of DNA Insert Fragment coded sequence coding contained among the pALK1227 of DSM11025 or the λ 4237/35 that preserving number is DSM11014;

(f) to have by preserving number be the plasmid pALK1229 of DSM11026 to coding, and preserving number is the nucleic acid molecules of polypeptide of the amino acid sequence of fungal DNA Insert Fragment coded sequence coding contained among the λ 4237/3 of DSM11011 or the λ 4237/18 that preserving number is DSM11013;

(g) has the plasmid pALK1221 that preserving number is DSM11024, preserving number is the λ 4237/5.1 of DSM11012, preserving number is the pALK1227 of DSM11025, and preserving number is the nucleic acid molecules of the coded sequence of fungal DNA Insert Fragment contained among the λ 4237/35 of DM11014;

(h) have the plasmid pALK1229 that preserving number is DSM11026, preserving number is the nucleic acid molecules of the coded sequence of fungal DNA Insert Fragment contained among the λ 4237/3 of DSM11011 or the λ 4237/18 that preserving number is DSM11013;

(i) because its coded sequence of degeneracy of genetic code and (a) the distinguishing nucleic acid molecules of coded sequence of each nucleic acid molecules in (h); Or

(j) coding has cellulase activity and has the No.31 with SEQ ID, 33 or 35 sequence demonstrates the nucleic acid molecules of polypeptide of the amino acid sequence of at least 80% homogeny, this nucleic acid molecules is by SEQ ID No.30, and 32 or 34 sequence obtains by lacking, replace, put upside down, insert to derive.

2. the nucleic acid molecules of claim 1, it is a kind of RNA.

3. the nucleic acid molecules of claim 1, it is a kind of DNA.

4. the DNA of claim 3, it is genomic DNA or cDNA.

5. carrier contains in the claim 1 to 4 each nucleic acid molecules.

6. the carrier of claim 5, wherein this nucleic acid molecules is connected on the expression control sequenc that allows to express in protokaryon or eukaryotic host cell effectively.

With in the claim 1 to 4 each nucleic acid molecules or with the host cell of the carrier conversion of claim 5 or 6.

8. the host cell of claim 7, it belongs to filamentous fungi.

9. the host cell of claim 7 to 8, it belongs to trichoderma or aspergillus.

10. the host cell of claim 9, it is Trichoderma reesei.

11. a production has the method for the polypeptide of cellulase activity, comprises the host cell of cultivating in the claim 7 to 10 each and the step that reclaims protein from culture medium.

12. one kind has cellulase activity, by each nucleic acid molecules in the claim 1 to 4, and claim 5 or 6 polypeptide vector encoded and that can be obtained by the method for claim 11.

13. identify specifically the antibody of the polypeptide of claim 12.

14. preparation contains the method for enzyme preparation of the polypeptide of claim 12, comprises the host cell of cultivating in the claim 7 to 10 each and reclaims polypeptide or isolated cell and obtain the step of supernatant from culture medium from cell.

15. with the obtainable enzyme preparation of the method for claim 14.

16. an enzyme preparation,

Comprise at least a cellulase that is separated by following fungal bacterial strain, this fungal bacterial strain is selected from: Sporotrichum thermophile CBS 688.95, Myceliophthora thermophila CBS 689.95, Melanocarpus albomyces or Myriococcum albomyces CBS 685.95, the Myriococcum kind of CBS 687.95 representatives, Chaetomium thermophilum CBS 730.95;

Or comprise at least a cellulase, this cellulase is from being selected from Melanocarpus, the genus of Myriococcum or kind, said cellulase have basically with from the identical biological characteristics of the cellulase of the CBS 685.95 of above-mentioned preservation or CBS 687.95 bacterial strains;

Or comprise at least a cellulase, this cellulase is from genus or the kind of Chaetomium, said cellulase have basically with from the identical biological characteristics of the cellulase of CBS 730.95 bacterial strains of above-mentioned preservation.

17. the enzyme preparation of claim 15 or 16, it is liquid.

18. the enzyme preparation of claim 15 or 16, it is dry.

19. the method that biology is worn away, it comprises the step that adds the goods of claim 15 or 16 in cotton-containing fabrics or the clothes.

20. the method for claim 19, wherein fabric or clothes are denim goods.

21. the method for a biopolishing, it comprises the step that adds the goods of claim 15 or 16 in the textile material that is selected from fabric, clothes and yarn.

22. the method for claim 21, wherein textile material is to be produced by the fiber of including natural fibers element or the fiber or its mixture that contain the artificial cellulose.

23. the method for claim 21, textile material wherein are the mixtures of the fiber of synthetic fibers and containing cellulose.

24. a detergent compositions comprises enzyme preparation and a kind of surfactant or the surface reactive material of claim 15 or 16.

25. a processing contains the method for the textile material of cellulose fibre, wherein said method comprises said textile material is mixed with the detergent compositions of claim 24.

26. a processing derives from wooden paper pulp or the method for fiber, comprises the step that adds the enzyme preparation of claim 15 or 16 in wooden machinery or chemical pulp or the secondary fiber to deriving from.

27. a method of improving the animal feed quality comprises with the enzyme preparation of claim 15 or 16 and processes vegetable material.

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