WO2001079483A1

WO2001079483A1 - Process for producing alcohol from cellulose fiber

Info

Publication number: WO2001079483A1
Application number: PCT/JP2001/002429
Authority: WO
Inventors: Hideki Fukuda; Akihiko Kondo; Yasuya Fujita; Atsuo Tanaka; Mitsuyoshi Ueda; Hideo Noda
Original assignee: Kansai Chemical Engineering Co., Ltd.
Priority date: 2000-04-17
Filing date: 2001-03-26
Publication date: 2001-10-25
Also published as: JP4681199B2

Abstract

Cellulose fiber or paper, which can be hardly reclaimed as paper, is reacted with an enzyme or a microorganism capable of cleaving β-1,4-glucoside bond to produce glucose and then alcohol fermentation is effected to thereby effectively utilize paper resources having been dumped. Glucose and, in its turn, alcohol can be particularly effectively produced by using a microorganism expressing in the cell surface layer at least one enzyme selected from the group consisting of endo-β-1,4-glucanase, cellobiohydrolase and β-glucosidase.

Description

Description Method for producing alcohol from cellulose fiber

Technical field

The present invention relates to a method for producing a useful substance from waste generated from a paper recycling process. More specifically, the present invention relates to a method for producing alcoholic alcohol from cellulose fiber or paper which is difficult to regenerate as paper.

Background art

In recent years, environmental issues have been greatly highlighted. In particular, the use of paper has increased sharply with the recent conversion to consumers, and the destruction of forests due to the cutting of timber, resulting in a drastic change in the environment, has become a problem, and paper recycling is being considered. In Japan as well, according to data from fiscal 1996, about 30 million tons of paper were consumed, and about half, 15 million tons, were recovered as waste paper.

The recovered recovered paper is used as recycled paper. As the paper is recycled many times, the fibers become brittle, and some recycled paper is not used as papermaking raw material. Still, shredded paper has short fibers and is hardly a raw material in most cases. According to the above-mentioned materials, such non-recycled papers are discarded or incinerated in an amount of about ΙδΟΟ10,000 tons, which is one of the causes of environmental destruction. Therefore, not only the recycling of paper but also how to use this discarded waste paper or paper is an important issue in reusing resources and solving environmental problems.

By the way, although research on the recycling of waste paper has been widely conducted, there has been little research on the use of waste generated during the recovery of the recovered paper. At present, there is a demand for an effective use of used paper Kashiwa, which is not recycled paper.

Disclosure of the invention

The present inventors have found that glucose can be produced from cellulose fibers or papers that have been conventionally discarded and are difficult to regenerate, and that alcohol can be produced from this glucose. Is completed.

That is, the present invention relates to a method for producing glucose, which comprises a step of reacting cellulose fibers or papers, which are difficult to regenerate as paper, with an enzyme or a microorganism capable of cleaving a 1,4-darcoside bond.

In a preferred embodiment, the cellulose fibers or papers that are difficult to regenerate as paper are cellulose fibers or papers that are decomposed batchwise or continuously by a noncatalytic hydrothermal method.

In a preferred embodiment, the cellulose fiber that is difficult to regenerate as paper is a cellulose fiber decomposed by a non-catalytic hydrothermal method at 120 to 300 ° C.

In another preferred embodiment, the microorganism is capable of expressing at least one selected from the group consisting of endo] 31,4-glucanase, cellohydrolase, and j3-dalcosidase on a cell surface. One or more recombinant microorganisms.

In a further preferred embodiment, the microorganism comprises:

(A) β-Darcosidase;

(Β) endo] 3 1,4-glucanase; and

(C) Microorganisms that have been transformed to express an enzyme selected from the group consisting of a combination of endo i31,4-glucanase and β-gnorecosidase on the cell surface.

In a further preferred embodiment, the microorganism is yeast.

The present invention also provides cellulose fibers or papers that are difficult to recycle as paper, reacting an enzyme or a microorganism capable of cleaving a β 1,4-darcoside bond to produce dulcose; and reacting the obtained glucose with a microorganism B having alcohol fermentation ability. And alcohol production methods.

In a preferred embodiment, the cellulose fibers or papers which are difficult to regenerate as paper are cellulosic fibers or papers which are decomposed batchwise or continuously by a non-catalytic hydrothermal method.

In another preferred embodiment, the microorganism A may express at least one member selected from the group consisting of endo] 31,4-glucanase, cellohydrolase, and J3-dalcosidase on a cell surface. One or more recombinant microorganisms.

In a further preferred embodiment, said microorganism A comprises:

(A)] 3-Darcosidase;

(B) endo 31,4-dalcanase; and

(C) A microorganism that has been recombined to express on the cell surface an enzyme selected from the group consisting of endo / 31,4-glucanase and a combination of 3-darcosidase.

In a further preferred embodiment, the microorganism A and the microorganism B are the same microorganism.

In a more preferred embodiment, the microorganism A and the microorganism B are the same yeast.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing the decomposition of waste cake by cellulase. FIG. 2 is a schematic diagram of the construction of plasmid pICASl.

FIG. 3 is a schematic diagram of the construction of plasmid pBG211.

FIG. 4 is a schematic diagram of the construction of plasmid pEG19. FIG. 5 is a schematic diagram of the construction of plasmid pEG23u21.

FIG. 6 is a diagram showing the degradation of] 3-glucan using a yeast transformed with the plasmid pEG23u21 and expressing endo-1,4-glucanase on the cell surface.

FIG. 7 is a diagram showing the use of cellobiose by yeast expressing jS-dalcosidase 1 on the cell surface.

FIG. 8 is a schematic diagram showing the production of alcohol using a] 3-glucan as a substrate by using a yeast expressing β-darcosidase 1 and endo i3 1,4-gluca ^ ”-IIase II on the cell surface. .

BEST MODE FOR CARRYING OUT THE INVENTION

In the present specification, “cellulose fibers that are difficult to recycle as paper” refer to wastes containing cellulose fibers generated in a paper manufacturing process or a paper recycling process, and include papermaking lees, waste paper kashiwa, and the like.

Generally, paper is affected by heat, humidity, light, etc. during use, and undergoes processing steps such as beating, heating, drying, and mechanical pressure in the regeneration process. Deterioration and deterioration such as damage, shortening, and hardness. For this reason, a large amount of cellulose fibers that are not made during papermaking as recycled paper are generated, and waste paper Kashiwa is generated. Cellulose fibers that are difficult to recycle as such paper are used in the present invention.

“Paper that is difficult to recycle as paper” includes, for example, paper with shortened cellulose fibers, such as shredded paper. “Paper” is a concept that includes not only paper but also cardboard and other paperboard. In the present invention, cellulose fibers or papers that are difficult to regenerate as the paper (hereinafter, simply referred to as “raw material cellulose fibers”), cellulose fibers which are decomposed batchwise or continuously by a non-catalytic hydrothermal method, or Paper is also preferred. By subjecting the raw cellulose fiber to a non-catalytic hydrothermal treatment, for example, it is treated so as to form an appropriately long cellulose unit or oligosaccharide, or cross-linking between fibers (for example, between cellulose) is released, and cellulose content is reduced. It is considered that the cellulose was changed so that the decomposing enzyme could easily act. The raw material cellulose fiber that has been subjected to this treatment can be used as it is as a substrate for glucose production or alcohol fermentation.

Various non-catalytic hydrothermal methods have been studied for the purpose of decomposing cellulose. For example, Fukuoka University, Faculty of Engineering, Journal No. 61 (September 1998) states that cellulose is hydrolyzed by batch processing with hot water at 578-678K (that is, 305-405 ° C). Is described. The present inventors have found that although alcohol is fermented using the cellulose digest obtained by this treatment, alcohol fermentation hardly progresses for some reason. Furthermore, in the non-catalytic hydrothermal method described in the above-mentioned literature, there is a problem that the reaction time is extremely short, so that a cellulose decomposition product which is appropriately and stably treated cannot be obtained.

Therefore, as a result of studying a batch (batch) type non-catalytic hydrothermal method at a lower temperature, it depends on the concentration to be treated. It has been found that a decomposition product suitable for alcoholic fermentation can be obtained by treating at preferably 150 to 280 ° C, more preferably 180 to 250 ° C. Generally, the processing time is preferably in the range of 1 hour to 15 seconds.

Further, the hydrolysis of the raw material cellulose fiber by the non-catalytic hydrothermal method can also be performed by a continuous method. In the case of the continuous method, a non-catalytic hydrothermal method with a slightly higher temperature may be used due to the heat history time. About 10% by weight of raw material cellulose The fiber is run at 120-373 ° C, preferably 150-320 ° C, preferably for 1 hour to 1 second. By this continuous non-catalytic hydrothermal method, the raw material cellulose fiber can be converted into a decomposition product suitable for alcohol fermentation.

Further, the raw material cellulose fiber is decomposed by adding, for example, sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, or the like to an acidic, preferably weakly acidic condition using a non-catalytic hydrothermal method to obtain a raw material. The cellulose fiber of the family can be used as a decomposition product suitable for alcohol fermentation.

The “enzyme capable of cleaving a 31,4-darcoside bond” used in the present invention is not particularly limited as long as it is an enzyme capable of cleaving this β1,4-darcoside bond. Endo] 31,4-glucanase, cellobiohydrolase, β-darcosidase, carboxymethylcellulase and the like are used.

Of these enzymes, only one of the [3-dalcosidases] can degrade cellulose fibers to produce glucose. In addition, these enzymes can be combined to degrade the raw material cellulose fiber (including the above-mentioned cellulose fiber or papers obtained by non-catalytic hydrothermal treatment; the same applies hereinafter) to produce glucose, which indicates that glucose can be produced. It was first discovered in the invention.

It is more preferable to use a combination of two or more enzymes. For example, (1) the ability to combine end j3 1,4-glucanase and β-darcosidase \ (2) the ability to combine cellopiohydrolase and β-glucosidase (3) Endo j3 1,4-glucanase, cellopiohydrolase and] 3-glucosidase can be combined. It is most preferred to include | 3-dalcosidase to produce glucose. By these combinations, cellulose fiber is efficiently decomposed and glucose is produced.

Microorganisms that produce enzymes that can cleave β1,4-darcoside bonds are also preferably used. Such microorganisms include so-called cellulase producing bacteria. When referring to "cellulase", it is generally end.) 3 1, 4- Dalcanase refers to a group of enzymes that produce glucose from cellulose by cleaving the 1,4-darcoside bond produced together with endo] 31,4-dalcanase (eg, cellobiohydrolase, -dalcosidase). ) Is sometimes called cellulase. ,

Representative examples of the cellulase-producing bacteria include microorganisms belonging to the genus Trichoderma, the genus Closteridium, the genus Cellus Monas, the genus Pseudomonas, and the like.

Also, it goes without saying that microorganisms that produce endo / 31 / 4-glucanase, cellobiohydrolase, and jS-darcosidase alone can also be used. In the present invention, a microorganism expressing dalcosidase on the cell surface, endo] 31,

A microorganism that has been modified to express 4-Dulcanase on the cell surface or a microorganism that has been modified to express mouth piohydrolase on the cell surface may be used alone or in combination. Examples of the combination include: a combination of endo j31,4-glucanase and β-glucosidase; and Saccharobiohydrolase; a combination of 3-dalcosidase; Endo] 31,4-glucanase; Lase and glucosidase combinations.

Further, the following (1) to (3):

(1) Combination of endo 1,4-gluca "l-zetase with β-glucosidase;

(2) a combination of cellobiohydrolase and 3-darcosidase;

(3) endo] 3 1,4-glucanase, cellohydrolase and jS-glucosidase in combination;

Microorganisms that are recombinant so as to express a plurality of enzymes on the cell surface, such as, for example, are also preferably used.

Further preferred microorganisms are the following (A) to (C):

(A)-dalcosidase;

(B) End J3 1,4-gluca "I; (C) Combination of endo j31,4-glucanase and j3-dalcosidase A microorganism that has been recombined to express on the cell surface an enzyme selected from the group consisting of force.

Such a microorganism is created by applying a cell surface engineering technology using a so-called genetic recombination technology.

Murai et al., Applied and Environmental Microbiology, vol. 63, 1362-1366 (1997) are examples of cell surface engineering. According to this document, a darcoamylase gene was linked to a gene encoding 320 amino acids of the C-terminal region of α-gluture of the yeast, and dalcoamylase was immobilized on the cell surface. Disassembly.

Microorganisms having an enzyme that cleaves 1,4-darcoside bonds of the present invention, or host microorganisms for expressing such enzymes include, but are not limited to, filamentous fungi, bacteria, and enzymes. . Yeast is preferable in consideration of handling and the like.

An enzyme that cleaves a β 1,4-darcoside bond, a microorganism that produces such an enzyme, or a microorganism that expresses such an enzyme on the cell surface is preferably immobilized on a carrier. This enables reuse.

As the carrier and the method for immobilizing the enzyme, those skilled in the art can use carriers and methods commonly used, and examples thereof include a carrier binding method, an entrapment method, and a cross-linking method.

A porous body is preferably used as a carrier for immobilizing microorganisms. For example, a foam or resin such as polyvinyl alcohol, polyurethane foam, polystyrene foam, polyacrylamide, porous polyformal resin, and silicon foam is preferable. The size of the opening of the porous body may be determined in consideration of the size of the microorganism used. In the case of yeast, 50 to 1000 μιη is preferred.

The shape of the carrier is not limited. Considering the strength of the carrier, cultivation efficiency, etc., Spherical or cubic are preferred. The size may be determined depending on the microorganism to be used. In general, the diameter is preferably 2 to 50 m;

Glucose is produced when the above enzymes, microorganisms, immobilized enzymes, and immobilized microorganism are reacted with the cellulosic fiber of the raw material. The concentration of a raw material (cellulose fiber as raw material) serving as a substrate for the enzyme is not particularly limited. The reaction is carried out at an appropriate temperature (for example, generally 10 to 70 ° C) for an appropriate time depending on the enzyme used. When using a thermostable enzyme using a hyperthermostable enzyme, the reaction can proceed at 90 ° C. or higher.

This reaction can be a continuous reaction using a column when immobilized enzymes or microorganisms are used. Endo 1,4-Dulcanase, cellohydrohydrolase, and 3-Darcosidase treatments can be performed in this order by multistage column treatment. The obtained glucose is isolated by a conventional method.

Production of alcohol from glucose is also one of the present inventions. Using the obtained glucose as a substrate, a microorganism capable of alcohol fermentation may be reacted.

The microorganism capable of alcohol fermentation is not particularly limited, but yeast is preferably used. The yeast capable of alcohol fermentation is not particularly limited, and includes yeasts conventionally used in the fermentation industry, such as sake yeast, brewer's yeast, wine yeast, and baker's yeast.

In alcoholic fermentation, a two-step reaction is generally considered, in which darcos is first produced from the raw material cellulose fiber, and yeast having alcoholic fermentation ability is added thereto.

Another method is to perform alcohol fermentation directly from the raw cellulose fiber. This includes (i) a method using a decomposition solution obtained by decomposing the raw material cellulose fiber by a non-catalytic hydrothermal method, and (ii) decomposition of the raw material cellulose fiber and alcohol. And (iii) simultaneous decomposition of cellulose fibers in a decomposition solution obtained by decomposing the raw material cellulose fibers by a non-catalytic hydrothermal method and alcohol fermentation.

In the method (i), the decomposition liquid obtained by decomposing the raw material cellulose fiber by the non-catalytic hydrothermal method can be used as it is as the raw material for alcohol fermentation. It is performed by adding yeast having a function.

The method (ii) is carried out by coexisting an enzyme capable of decomposing cellulose fibers as a raw material or a (yet-replaced) microorganism and a microorganism having an alcohol fermentation ability. Alternatively, a yeast having an alcohol fermentation ability can be used as a host, for example, by using a yeast that has been modified to express β-glucosidase and Ζ or end 3,4-glucanase on the cell surface. , Done. When this recombinant yeast is used, the steps of decomposing the cellulose fibers to produce glucose and the step of fermenting the produced glucose with alcohol are performed simultaneously, which is effective.

The method (iii) is a combination of the methods (i) and (ii), and is a method for producing alcohol by further effectively utilizing the raw cellulose fiber.

Such a microorganism having an alcohol fermentation ability (including a recombinant yeast) may be immobilized as described above for glucose production. Example

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

Example 1: Glucose production from cellulose fiber that is difficult to regenerate

The waste liquid from the recycled paper manufacturing process was collected, and the unregenerated cellulose fibers were recovered. The recovered cellulose fibers are hereinafter referred to as used paper meal. Separately, crystalline Abysse (Avicel) and Filter paper powder C were prepared. These are dispersed in a 0.1 M sodium acetate buffer to a concentration of lOg / L, and reacted at 30 ° C using cellulase derived from Trichoderma reesei (manufactured by Funakoshi Co., Ltd.) to produce glucose. Quantification was performed using CII Test II Co. (Wako Pure Chemical Industries, Ltd.). The results are shown in Figure 1.

In Fig. 1, ▲ represents waste paper meal, ■ represents Avicel, and · represents Filter paper powder r C, respectively. Figure 1 shows for the first time that waste paper meal is degraded much more efficiently than other cellulosic materials, indicating that waste paper kashiwa is extremely promising as a raw material for glucose production. I have.

Example 2: Alcohol production from waste paper meal using yeast expressing β-darcosidase 1 and / or end 1,4-glucanase I on the cell surface

2-1] Preparation of yeast expressing 3-dalcosidase 1 on cell surface

FIGS. 2 and 3 show schematic diagrams for preparing a plasmid that expresses j3-dalcosidase 1 on the cell surface. Figure 2 is a schematic diagram of the creation of the plasmid pICASl, which is the material for the target plasmid, pBG211.

A plasmid pYGA2269 (Ashikari et al., Appl. Microbiol. Biotechnol. 30: 515-520 (1989)) having a sequence in which a DAP-coamylase derived from Rhizopus oryzae is connected to GAPDH promoter ^{1 to} goo from baccharomyces cerevisiae Two oligonucleotides 5, -ccgagctcaccagttctcaccggaaca-3

(Rooster system IJ number 1 no and b — gcccgcggcagaaacgagcaaagaaaa-3 (Rooster system number 2), amplified by PCR, cut with Sacl and SacII, GAPDH promoter and glucoamylase secretion signal sequence derived from Rhizopus oryzae A Sacl-SacII DNA fragment having the following sequence was prepared (fragment I).

Separately, the two oligonucleotides 5′-ggagatctccatggc-3 ′ (SEQ ID NO: 3) and 5′-tcgagccatggagatctccgc-3 ′ (Rooster column no. This produced a SacII-XhoI DNA fragment (fragment II).

Furthermore, plasmid pGAll (Murai et al., Appl Environ Microbiol 63:.. . 1362- 1366 (1997)) cut out Xhol- Kpnl fragment from, DNA fragment containing the C-terminal 320 amino acids and 44 _[rho flanking region of a single Aguruchun shed (Fragment II I) was obtained.

On the other hand, plasmid pRS404 (Sikorski et al., Genetics 122: 19-17 (1989)) was cut with Sacl-Kpnl, and the above fragments I to [II were mixed to produce plasmid pICAS1. This plasmid has a sequence containing the GAPDH promoter, a secretory sequence, the C-terminal 320 amino acids of a-agglutinin, and a 446 bp flanking region in this order.

Figure 3 shows the procedure for creating the desired plasmid pBG211 using this plasmid pICASl. Plasmid pICASl was digested with Bglll-Xhol, and the jS-darcosidase 1 gene from Aspergillus acule atus of plasmid pABG7 (Kawaguchi et al., Gene 173: 287-288 (1996)) was inserted into this site. That is, DNA was obtained by PCR using plasmid pABG7 as a template, 5, _gtcgagatctctga1: gaactggcgttctct-3, (distribution system's J number 5 no and -ttcactcgagccttgcaccttcgggagcgccg-3 '(measure <1> lj ^ "^ 6) as a primer. This fragment was digested with Bglll-Xhol, and this fragment was introduced into the Bglll-Xho cleavage site of the plasmid pICASl to obtain a plasmid pCS.This plasmid pMHCS contains the GAPDH promoter and secretory signal. [J, i3 -. Darukoshidaze structural gene, alpha-Aguruchun has a gene sequence comprising the C-terminal region 320 amino acids and 44 ⁶ bp flanking regions pMHCS was digested with BssH II and, GAPDH promoter, a secretory signal sequence BssHII-BssHII DNA fragment (320 amino acids and 446 bp flanking region) of the α-agglutin C-terminal region and the β-dalcosidase structural gene Segment IV) was isolate.

On the other hand, plasmid pMT34 (+3) containing 2 imDNA (Tajima et al., Yeast 1: 67 7 7 (1985)), and inserted into the Aatll site of plasmid pRS403 (Sikorski et al., Genetics 122: 19-17 (1989)) to produce multicopy plasmid pMHl. This plasmid pffll was digested with BssHII, and fragment IV was inserted therein to obtain a plasmid pBG211 expressing -dalcosidase on the cell surface.

Fuhus ¾> pBG21 丄 · ¾Γ¾3 (ϊ (; η3Γοιηγοθ3 cerevisiae MT81-1 (MATa ura3 trpl a de leu2 his3)) was introduced by the lithium acetate method using Yeast Maker (manufactured by CLONTEC). Medium (6.7 g / L yeast nitrogen base w / h amino acids, 20 g / L gnorecose, 0.03 g / L leucine, 0.02 g / L tryptophan, 0.02 g / L adenine, 0.02 g / L peracil). and the transformant was designated as MT8- 1 / _P BG211.

2-2 Preparation of yeast that expresses endo jS 1,4-dalcanase I (endglucanase I) on the cell surface

Figure 4 shows a schematic diagram of the construction of plasmid pEG19 that expresses endoglucanase I on the cell surface.

The EcoRI DNA fragment of plasmid pWI3 with 2 mDNA (Kanai et al., Appl.Microbiol. Biotechnol. 44: 759-765 (1996)) was converted to the plasmid pRS405 (Sikorski et al., Genetics 122: 19-17 (1989)). The plasmid was introduced into the Aatll site to generate plasmid pRS405 + 2.

On the other hand, GAPDH promoter of the plasmid Picas l, secretion signal sequence, a DNA fragment containing the C-terminal 320 Amino acid and 446bp flanking region of α- Aguruchinin was amplified by PCR, was cut with EcoRV, a plasmid _P RS405 + The plasmid was introduced into the Pvul I-Pvu II site of No. 2 to prepare a plasmid pMLCS5. The primer used for amplification was 5-ggaaacagctatgaccatgatatcgccaagcgcgcaa.tta-3 '(Rooster column number 7)

<±; Χ ΐ) -ttgtaaaacgatatccagtgagcgcgcgtaatacgactca-3 '(ΰ system' J number ^ "8) there were.

The endo j31,4-glucanase gene derived from Tricoderma reesei was inserted into the Bglll site of this plasmid pMLCS5. That is, as the 1st strana cDNA unplate prepared from the mRNA of T. reesei, & -gctcgagatctcccagcaaccggg taccagcacccccgag-3 '(rooster G column 9) ■ and -ggagatctggaaggcattgcgagt agtagtcgttgctatact-3' (SEQ ID NO: 10) Was amplified by PCR and cut with Bglll to obtain a Bglll DNA fragment of cDNA encoding endoglucanase I, which was introduced into the Bglll site of plasmid pMLCS5 to obtain plasmid pEG19.

The obtained plasmid pEG19 was transformed with the yeast Saccharomyces cerevisiae MT—8 in the same manner as in 2-1 to obtain SDi¾ "i-ya (6.7 g / L yeast nitrogen base w / o amino acids, 20 g / L (Gnorecose, 0.02 g / L tryptophan, 0.02 g / L histidine, 0.02 g / L adenine, 0.02 g / L peracil) The obtained transformant was designated as MT8-l / pEG19.

2-3 Preparation of yeast expressing β-dalcosidase 1 and endodalcanase I on cell surface

Using the transformant MT8-l / pBG211 as a host, plasmid pEG19 was transformed in the same manner as 2-1.SDJ¾-¾ (6.7 g / L yeast nitrogen base w / o amino acids, 20 g / L gnorecose, .02 g / L tryptophan, 0.02 g / L adenine, and 0.02 g / L peracil). The resulting transformant was designated as MT8_1 / pBG211 + pEG19.

2-4 Recall production from waste paper cake

3 - transformants the Darukoshidaze 1 expressed on the cell surface (MT8-1 / _P BG211), transformant end dull mosquito endoglucanase I expressed on the cell surface (M'T8- 1 / _P EG19) Contact And] A transformant (MT8-l / pBG211 + pEG19) that expresses 3-dalcosidase 1 and endoglucanase I on the cell surface is suspended in the SD medium (liquid medium) used for each selection, and then suspended at 30 ° C. C, and cultured for 48 hours to obtain a pre-culture solution. This pre-cultured solution was used as a 2 L jar armmenter (BMJ-02PIAb le) containing 1 L of YPD medium (10 g / L yeast extract, 20 g / L polypeptide, 5 g / L of glucose) containing 40 g / L waste paper meal. ) And cultured under microaerobic conditions of 0.01 to 0.03 ppm. All transformants grew despite the small amount of glucose added. This is probably because glucose was produced by the enzyme expressed on the cell surface, and it grew using the produced darcose. / 3 - Darukoshidaze growth 1 and Endodaru 'Kanaze I and a transformant that expressed on the cell surface _{(MT8-l / P BG211 +} pEG19) was the best.

When the amount of the dried cells reached about 15 g / L, the culture was terminated, the cells were collected by centrifugation, and 5 g / LYPD medium containing 70 g / L waste paper meal was used at 30 ° C, pH 6.0, Fermentation was performed under microaerobic conditions of 0.01 to 0.03 ppm. After culturing for 100 hours, the results of measuring the ethanol in the culture broth showed that the transformant (MT8-l / pBG211), the transformant (MT8-1 / PEG19) and the transformant (MT8-l / pBG211 + pEG19) ) Produced 3 g / L, 4 g / L and 6 g / L ethanol, respectively.

2-5 Alcohol production from waste paper meal decomposed by non-catalytic hydrothermal method

Attach a heater to a pressure-resistant container with an internal volume of 35 m1, an inner diameter of 16 mm, and a height of 200 mm, treat a 70 g / L wastepaper suspension under the conditions shown in Table 1, and use the resulting decomposition solution as it is as a carbon source. Was used for alcohol fermentation. The transformants used were a transformant expressing β-dalcosidase 1 on the cell surface (ΜΤ8-l / pBG21 1) and a transformant expressing -dalcosidase 1 and endoglucanase I on the cell surface (MT8-l / pBG211 + pEG19).

5g / LYPD medium with a concentration of treated waste paper of 70g / L at 30 ° C, pH 6.0, 0. Fermentation was performed under microaerobic conditions of 01-0.03 ppm. Table 1 shows the results of measuring ethanol in the culture solution after culturing for 100 hours.

table 1

The numbers represent the alcohol concentration (g / s) .The results in Table 1 show that when the waste paper treated at 400 ° C for 15 seconds is added to the culture medium compared to when untreated waste paper is added to the culture medium, the alcohol fermentation The degree was small, and it was even lower when mashed at 400 ° C for 30 minutes. The degree of alcohol fermentation was much greater when waste paper treated under milder conditions was added than without. It is probable that alcohol fermentation did not proceed due to factors such as the decomposition of cellulose to produce the necessary sugars or the formation of substances that hinder the reaction due to the treatment of waste paper under harsh conditions. On the other hand, it is probable that the treatment of the used paper under milder conditions decomposed the used paper to an appropriate degree and made it easier for enzymes to work on the used paper (cellulose). Example 3: Construction of yeast expressing endo / 3 1,4-glucanase II on cell surface and degradation of glucan

Preparation of yeast that expresses 3-1 endo | 31,4-glucanase II (hereinafter referred to as endoglucanase II) on the cell surface

Figure 5 shows the procedure for preparing plasmid pEG23u31 that expresses endoglucanase II on the cell surface.

Using the plasmid pICASl created in 2-1 as a template, SEQ ID NOs: 7 and Amplification by PCR was performed using the sequence represented by ぴ 8 as a probe. The obtained fragment was digested with EcoRV to obtain an EcoRV-EcoRV fragment. This fragment contains the GAPDH promoter, a secretory signal sequence, the C-terminal 320 amino acids of α-agglutinin and a 446 bp flanking region.

On the other hand, the plasmid pURI24 created by the present inventors, Tanaka and Ueda, was cut with PvuII and BamHI to obtain blunt ends. An EcoRV-EcoRV fragment was inserted into this blunt-ended site to create a plasmid pMUCS.

Next, PCR was carried out using the mRNA of Trichoderma reesei as a template and the prepared 1st strand cDNA. The primers used were 5, -cggcgagatctcacagcaga ctgtctgggg-3 '(rooster column number) and D -gacagctcgagggctttcttgcgagacacg-3' (SEQ ID NO: 12). The PCR product was cut with Bglll and Xhol, and A Bgll I-Xhol fragment having a sequence encoding 4-gunolecanase II (glucanase II) was obtained, and the Bglll-Xhol fragment was inserted into the Bglll-Xhol site of plasmid pMUCS to obtain the desired plasmid pEG23u31. .

The obtained plasmid pEG23u31 was introduced into the yeast Saccharomyces cerevisiae MT-8 in the same manner as 2-1 and SD ± ya (6.7 g / L yeast nitrogen base w / o ami no acids, 20 g / L glucose). , 0.02 g / L tryptophan, 0.02 g / L histidine, 0.02 g / L adenine, and 0.03 g / L leucine. The resulting transformant was designated as MT8-l / pEG23u31.

3-3 / 3-G / Recan Decomposition

Suspension of 10-g / L] 3-glucan was suspended in 50 mM acetate buffer (pH 5.0), and yeast MT8-l / pEG23u31, which expresses endoglucanase II on the cell surface, was added to the suspension. Allowed to react for hours. The amount of reducing sugars released was measured as the glucose equivalent by the Somogyi-Nelson method. As a control, MT8-1 (MT8-1 / pMUCS) with pMUCS used for integration of endoglucanase II gene was used. Using. Fig. 6 shows the results.

The results in FIG. 6 indicate that yeast having endoglucanase II on the cell surface efficiently degrades β-daltin. Example 4: Preparation of yeast expressing 3 dalcosidase 1 and Ζ or endoglucanase II on the cell surface

4-1 Preparation of yeast expressing β-dalcosidase 1 on cell surface

Here, yeast transformed with a chromosomal integration plasmid was prepared.

As in the case of 3-1, EcoRV-EcoRV fragments were prepared using plasmid pICASl as a template. This fragment was inserted into the PvuII site of a chromosome-integrated plasmid pRS403 (Sikorski, RS and P. Hieter, (1989) Genetics, 122: 19-27) to generate plasmid pIHCS.

On the other hand, PCR was performed using pBG211 prepared in 2-1 as a template to obtain a PCR product having a sequence encoding β-glucosidase 1. The primers used were D-gatctccatggctgatgaactggcgttctctcctcctttc-3 (Rokki No. 13) and 5-tggcgctcgagccttgcaccttcgggagcgccgcgtgaag-3 '(Rokki U number 14). The obtained PCR product was digested with Ncol and Xhol, introduced into the Ncol-Xhoi site of plasmid pIHCS, and the desired plasmid,] 3-dalcosidase 1 surface expression, chromosome-integrated plasmid pIBG13 was prepared. .

The obtained plasmid PIBG13 is cut into single-stranded DNA by cutting with Nhel, and then introduced into yeast Saccharomyces cerevisiae MT-8 in the same manner as in 2-1 to obtain an SD medium (6.7 g / h yeast). nitrogen base w / o amino acids, 20 g / L knorecose, 0.03 g / L leucine, 0.02 g / L tryptophan, 0.02 g / L adenine, 0.02 g / L peracil) The transformant was designated as MT8-1 / pIBG13.

4- 2 of yeast co-expressing jS-darcosidase 1 and endoglucanase II Create

The yeast MT8-1 / PEG23u31 that expresses endoglucanase II on the cell surface obtained in 3-1 and the plasmid pIBG13 (chromosome) that expresses & -darcosidase 1 obtained in 4-1 on the cell surface (Integrated type) to prepare a transformant MT8-l / pEG23u31pIBG13.

It was confirmed by plate assay that this transformant expressed β-darcosidase 1 and endoglucanase II.

After incubating the j3-dalcosidase 1 at 48 ° C for 30 hours at 30 ° C on an SD selection plate, overlay 7.5 g / L softlager containing lOmM synthetic substrate (4-methylumbellifery-; 3-D-glucoside). Incubation was further performed at 30 ° C for 12 hours. The plate was irradiated with ultraviolet light at a wavelength of 300 nm, and the fluorescence of the fluorescent substance generated by decomposition of the substrate was observed. The fluorescence was observed, and the expression was confirmed.

In addition, expression of Endalcanase II was performed by incubating at 30 ° C for 48 hours on an SD selection plate containing lg / L-glucan and staining with lg / L Congo Red to confirm halo. Was.

From the above, it was confirmed that MT8_l / pEG23u31pIBG13 expressed] 3-darcosidase 1 and endo-dalcanase II. 4.3 Utilization of cellobiose by yeast expressing 3-Darcosidase 1 on cell surface

Using MT8-l / pIBG13 and MT8_l / pEG23u31pIBG13, which express β-gnorecosidase 1 on the cell surface, cellobiose was cultured as a single carbon source, and the growth of yeast was observed. The MT8-1 strain was used as a control. 6. 7g / L yeast nitrogen base w / o amino acids, lOg / L cellobiose, 0.03g / L leucine, 0.02g / L tri'ptophan, 0.02g / L adenine, 0.02g / L peracil) Using the medium of 3 ^: The results of culturing at 0 ° C are shown in FIG.

This result indicates that the yeast expressing / 3-dalcosidase 1 on the cell surface was able to degrade cellobiose, which is a disaccharide originally incapable of being assimilated by the yeast, and was able to grow.

4-4] Ethanol production from soluble long-chain cellulose by yeast expressing 3-dalcosidase 1 and endoglucanase II

The transformant MT8-l / pEG23u31pIBG13 was transferred to an SD medium (6.7 g / L yeast nitrogen base w / o amino acids, 20 g / L gnorecose, 0.03 g / L leucine, 0.02 g / L triptofan, 0.02 g / L-Adenine, pre-cultured in a medium of 0.02 g / L peracil, and cultured on SD medium (6.7 g / L yeast nitrogen base w / o amino acids ヽ lOg / L j3-glucan,

The cells were cultured with 03 g / L leucine, 0.02 g / L tryptophan, 0.02 g / L adenine, and 0.02 g / L ゥ racil), and the amount of ethanol formed was measured by gas chromatography. Total sugars were measured by the sorbazole-sulfuric acid method. Fig. 8 shows the results. This result indicates that] -glucan was completely saccharified and alcohol fermentation was performed. Industrial applicability

By the method of the present invention, cellulose fibers that are not recycled as paper are effectively reused as resources. In particular, a microorganism that has been subjected to an enzyme capable of cleaving a 1,4-darcoside bond is preferably used.

Claims

The scope of the claims

1. A method for producing darcose, comprising a step of reacting a cellulose fiber or paper which is difficult to regenerate as paper with an enzyme or a microorganism capable of cleaving a 1,4-darcoside bond.

2. The method according to claim 1, wherein the cellulose fibers or papers which are difficult to regenerate as paper are cellulose fibers or papers which are decomposed batchwise or continuously by a non-catalytic hydrothermal method.

3. One or more recombinant microorganisms, wherein the microorganism has been modified to express at least one selected from the group consisting of endo-1,4-glucanase, cellobiohydrolase, and] 3-darcosidase on the cell surface. The method according to claim 1, wherein the microorganism is a microorganism.

4. The microorganism is any of the following (II) to (C):

(Α) β-Darcosidase;

(Β) End] 3 1,4-gluca ^ "I;

The method according to claim 3, wherein the (C) endo is a microorganism that has been recombined to express an enzyme selected from the group consisting of a combination of 31,4-glucanase and β-darcosidase on the cell surface. .

5. The method according to claim 3, wherein the microorganism is a yeast.

6. Glucose is produced by reacting cellulose fibers or papers, which are difficult to regenerate as paper, with enzymes or microorganisms that can cleave] 3 1,4-darcoside bonds. And a step of reacting the obtained glucose with a microorganism B having alcohol fermentation ability.

7. The method according to claim 6, wherein the cellulose fibers or papers which are difficult to regenerate as paper are cellulose fibers or papers which are decomposed batchwise or continuously by a non-catalytic hydrothermal method.

8. The microorganism A has been recombined to express at least one selected from the group consisting of endo] 31,4-glucanase, cellohydrolase, and] 3-darcosidase on the cell surface. 7. The method according to claim 6, which is a microorganism as described above.

9. The microorganism A has the following (A) to (C):

(A)] 3-Gnorecosidase;

(B) endo] 31,4-glucanase; and

(C) The microorganism according to any one of claims 6 to 8, which is a microorganism that has been recombinantly expressed so as to express an enzyme selected from the group consisting of a combination of endo 1,4-glucanase and j3-glucosidase on the cell surface. The method described in the section.

10. The method according to any one of claims 6 to 9, wherein the microorganism A and the microorganism B are the same microorganism.

11. The method according to claim 10, wherein the microorganism is a yeast.