JP2010098951A - Method for simply collecting and reusing cellulose-saccharifying amylase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To collect and reuse an amylase by a simple method. <P>SOLUTION: The batch or semibatch-type method of saccharification of a lignocellulose for obtaining glucose of a hexose or a pentose by adding a cellulose-saccharifying amylase to a lignocellulose raw material subjected to a pretreatment so that the amylase may act thereon includes reusing an unhydrolyzed lignocellulose residue having the added amylase attached thereto for the enzymatic saccharification of the next time. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a technique for saccharifying lignocellulosic biomass with an enzyme to produce glucose or the like, which is a raw material for fermentation of lactic acid, which is a raw material for ethanol and biodegradable plastics. In particular, the present invention relates to an industrial method in which lignocellulosic material is used as a substrate and saccharification enzyme once used can be recovered and reused by a simple method.

  There are roughly two methods for hydrolyzing cellulose into glucose, which is a constituent sugar. One is an acid saccharification method using a mineral acid such as sulfuric acid, and the other is an enzyme saccharification method using an enzyme and a microorganism that produces the enzyme. Although the former has been technically completed, there are still problems in neutralization and recovery of sulfuric acid in addition to the problem of overdecomposition. Regarding the latter, in recent years, the development led by the United States has accelerated and the enzyme price has fallen, but further reduction of the enzyme price is indispensable for practical use.

  As a countermeasure, a method of reducing the amount of enzyme added or a method of collecting and reusing an enzyme once used has been proposed.

  For example, Wood et al. Was able to halve the amount of enzyme used by irradiating ultrasonic waves at a rate of 15 minutes every 240 minutes when an enzyme preparation was added to mixed waste office paper to perform parallel saccharification and fermentation. Reporting. In addition, it is considered that not only the fibers are relaxed by ultrasonic waves, but also that the enzyme is adsorbed on the cellulose fibers and peels off, and is transferred to a new domain and acts again (non-patent document). 1, 2).

  In Non-Patent Document 3, steamed and crushed birch wood is added to a saccharification tank at a concentration of 5%, 20,000 units of cellulase is added, and the sugar solution and the enzyme solution are separated by ultrafiltration. While collecting and reusing the enzyme, 630 g of monosaccharide was obtained from 2 kg of birch wood in 8 days. It has been reported that this method saved 20% of the amount of enzyme used. However, a 20% saving does not lead to practical use.

  Furthermore, in Non-Patent Document 4, 90% of the enzyme is recovered from a low-concentration sugar solution obtained by saccharifying bagasse pretreated with alkali with cellulase using an ultrafiltration membrane having a fractional molecular weight of 10,000 to 20,000. The addition rate of the enzyme is 30 to 200 units per mL of the reaction solution, and the addition rate for 1 g of the substrate is 1,000 to 2,800 units in CMCase. The filter paper decomposition activity is considered to be 45 to 128 units (calculated when the CMCase is 720 units and the filter paper decomposition activity is 33 units). Under these conditions, the saccharification rate was 80%. On the other hand, in the case of a high-concentration sugar solution, it is reported that the amount of saccharification residue increases and cellulase is adsorbed to this residue, resulting in an enzyme recovery amount of 75-80%. From the above viewpoints, methods for recovering and reusing enzymes with ultrafiltration membranes have also been studied in designing saccharification apparatuses (Patent Documents 1 to 3).

  In Patent Document 4, the copy paper is suspended in 7 L volume of slurry so that the copy paper is 1.25% (w / w, absolutely dry), stirred at a stirring speed of 180 rpm, and this substrate is continuously fed to the saccharification reaction tank. Send it out.

The liquid amount in the saccharification reaction tank is 3 kg, the copy paper concentration is 0.25% (w / w, absolutely dry), the enzyme addition amount is 260 units per gram of copy paper, the residence time is 12 hours, 50 ° C., 250-300 rpm. Driving under conditions. The reaction solution is sent to the reaction solution storage tank, and the enzyme recovered by the ultrafiltration device is returned to the saccharification reaction tank. During the continuous operation for 1,000 hours, the saccharification rate was maintained at 98% or more. It has been reported that it is possible to repeatedly collect and reuse without adding a new enzyme by keeping the accumulated amount of residue with respect to the substrate to be added at 5% or less.
“Wood, BE”, Aldrich, HC, Ingram, LO, “Biotechnol. Prog.”, 1997, Vol. 13, p. 232-237 Tom P, Warren, AJ, Miller, TCJ, Kilburn, DG, Gilkes M.R. (Gilkes, MR), “Enzymatic Degradation of Insoluble Carbohydrates”, JNSaddler Edition), ACS Symposium Ser, ACS, San Diego, CA, 1995, 618, p. 145-163 Ishihara, M. et al., “Biotechnol. Bioeng.”, 1991, vol. 37, p. 948-954 Yasuto Akira, Journal of the Wood Society, 1989, Vol. 35, No. 12, p. 1067-1072 Japanese Patent Laid-Open No. 61-260875 JP 61-234790 A JP-A-63-87994 JP 2006-87319 A

  In order to remove the enzyme from the substrate by irradiating ultrasonic waves, transfer it to a new reaction domain and act again, or to collect and reuse the enzyme by ultrafiltration membrane on an industrial scale, the former has a great deal of initials. In the latter case, maintenance work such as membrane regeneration is expected. In proceeding with commercialization, it is desirable to collect and reuse enzymes by a simpler method.

  In order to solve the above-mentioned problems, the lignocellulose batch or semi-batch saccharification method of the present invention adds a cellulose hydrolyzing enzyme to a lignocellulose raw material that has been pretreated so that the enzyme acts, thereby producing a C6 sugar. This is a batch or semi-batch saccharification method of lignocellulose to obtain glucose or C5 sugar, and the lignocellulose residue that is not hydrolyzed and to which the added enzyme is attached is reused for the next enzymatic saccharification. It is characterized by.

  The lignocellulose raw material used in the present invention is, for example, timber such as softwood, hardwood, etc. (thinned wood, sawdust at sawing, building waste from concrete formwork or demolished house), southern ocean centering on palm Agricultural residues such as timber (trunk and fruit shells), rice straw, wheat straw, bagasse, corn stover, and plant tissues such as macroalgae and microalgae.

  And “pretreated so that the enzyme acts” means the strength of the plant tissue, and lignin (occupies 20 to 30% of lignocellulose) existing so as to embed cellulose and hemicellulose. It means removing by steaming or steam explosion without adding them under conditions where alkali or acid is added, and also relaxing the crystal structure of strong cellulose. In addition, examples of the raw material that has been subjected to such pretreatment include the above-mentioned papers derived from conifers and hardwoods.

  Paper is obtained by mechanically or chemically removing fibers from wood and peeling them into thin sheets. In particular, chemical pulp is substantially free of lignin embedding cellulose fibers, and can be regarded as a lignocellulose raw material suitable for enzymatic saccharification. Also, paper (fiber) can be easily shredded, and it is the flow limit that is prepared in a 10% suspension (slurry) with an absolute dry weight, which is higher than that of other lignocellulose raw materials. Concentration. For example, hard wood not only requires a great deal of cost for pulverization but also cannot be made into a slurry with a higher concentration than paper even if pulverized. For this reason, there is a disadvantage that a thick saccharified solution cannot be obtained regardless of the continuous or batch type enzymatic saccharification method, but paper that can compensate for this disadvantage to some extent is a suitable raw material.

  Although mechanical pulp has been established as a waste paper recovery route and is effectively reused at a high recovery rate, chemical pulp is used for office paper such as printing paper and copy paper, packaging paper, and office paper. Often discarded as garbage. As a paper resource that is not included in waste paper recycling, the percentage of paper waste (mainly offices) that is brought into municipal waste incineration facilities and incinerated is about 40% in urban areas. In addition, there is no need to build and maintain a new collection system, and a stable supply can be expected. In addition, high-quality paper such as copy paper contains white calcium carbonate as a filler in addition to chemical pulp in order to give whiteness and surface smoothness (Printopia, http: //www.jfpi.or .jp / printpia / kami / story08.htm), the solubility increases in the vicinity of strong acidity (pH 4.8). Since the pH at the time of enzymatic saccharification is lower, it is considered that it is almost dissolved and released into the saccharified solution.

  Preferably, lignocellulose pretreated so that the enzyme acts is adjusted to a slurry of 2 to 10% [dry wt / wt], and 200 to 1000 units of saccharifying enzyme per 1% of dry weight of lignocellulose is added to the slurry. And saccharify for 24-72 hours.

  Preferably, lignocellulose saccharified solution is suitable for ethanol fermentation by performing vacuum distillation or azeotropic addition of alcohol having 2 to 4 carbon atoms (for example, ethanol, butanol, etc.) to lignocellulose saccharified solution. Concentrate to a concentration of 10-15%.

  Preferably, the lignocellulose saccharified solution is concentrated using an ultrafiltration membrane.

  Preferably, the lignocellulose saccharified solution is recovered by allowing the lignocellulose saccharified solution to stand, and the whole or a part of the recovered lignocellulose residue is added as a reusable enzyme to the next enzymatic saccharification of lignocellulose.

  Preferably, reuse of the lignocellulose residue for the next treatment is repeated a plurality of times.

  Lignocellulose that is not solubilized in one saccharification treatment and remains as a residue with the enzyme attached is converted to solubilized and constituent sugars in the next saccharification treatment, and the amount of unreacted residue can be reduced.

  Preferably, the lignocellulose raw material is not included in a conventional waste paper collection system, but is derived from paper waste that has been incinerated at a municipal waste incineration plant, more preferably from office paper waste. .

  According to the present invention, the enzyme adhering to the residue can be reused together with the residue without being peeled off by using pH adjustment or ultrasonic waves, and saccharification treatment can be easily performed.

  On the other hand, taking the above-mentioned Patent Document 4 relating to the continuous enzymatic saccharification method on the premise of enzyme recovery / reuse as an example, there are the following problems. For example, the cellulose content in copy paper derived from chemical pulp is not constant depending on differences in raw materials (tree species), production method, lots, etc., but is considered to be generally in the range of 50 to 60%. Assuming that the cellulose ratio is 50%, the glucose solution concentration obtained from the 0.25% concentration paper slurry described in Patent Document 4 is 0.25 × 0.5 × 1. It is about 111 = 0.138 (%), and it is not efficient to apply this extremely diluted sugar solution as it is to ethanol fermentation. Application after concentration to an at least 10-15% glucose solution with an ultrafiltration membrane or the like is desirable. Considering practical application, the glucose concentration in the saccharified solution obtained by enzymatic treatment of lignocellulose is desirably as high as possible. Even if the concentration does not reach 10 to 15%, it is not 0.138% but 5% glucose solution. Obviously, it is cheaper to concentrate. In this patent, a large amount of enzyme is added to the reaction system per copy paper weight so as to show a saccharification rate of 98% or more in about 12 hours. The risk of enzyme cost loss is considered to be extremely high when the enzyme is deactivated due to (eg, deactivation of the enzyme, loss of the reaction liquid due to breakage).

  In contrast, in the present invention, the lignocellulose saccharified solution is suitable for ethanol fermentation by distilling under reduced pressure or azeotropically adding an alcohol having 2 to 4 carbon atoms. By concentrating to a concentration of ˜15% or by using an ultrafiltration membrane, the above problems are solved.

(Method of obtaining a thick saccharified solution)
In order to obtain a concentrated sugar solution by enzymatic saccharification of lignocellulose, it is a major premise that at least the lignocellulose in the treated suspension is at a concentration that can achieve this. As stated in the previous section, the paper has a flow limit of about 10% suspension. However, since paper is solubilized by an enzyme and its viscosity decreases, it is also possible to add paper in stages to make a suspension with a concentration higher than 10%. However, in that case, it is known that the absolute amount of glucose or the dimer cellobiose in the saccharified solution increases in the saccharified solution, thereby inhibiting the enzyme activity and decreasing the saccharification rate. Based on the verification experiment based on the above points, it was determined that the 10% paper suspension is the most efficient at the present time. A specific example of batch-type enzymatic saccharification treatment with this as the upper limit concentration will be described below.

  First, saccharification enzyme (GC220 Genencor) is added to a paper slurry prepared as a 5-10% suspension, and stirred at an optimum temperature (50-65 ° C.) and pH (4.0-5.5). Then, saccharification is performed for 24 to 72 hours. The saccharification rate varies depending on the amount of enzyme added per paper weight, but if the amount of enzyme added is sufficient, the saccharification rate is expected to be 90% or more. Assuming that the cellulose content of the fine paper to be tested is 55% and the saccharification rate is 90%, the glucose solution concentrations obtained by enzymatic saccharification treatment from 5 and 10% paper slurries are as follows.

(1) 5% paper slurry: 5 × 0.55 × 0.9 = 2.47 (% glucose solution)
(2) 10% paper slurry: 10 × 0.55 × 0.9 = 4.95 (% glucose solution)
By the above method, a glucose solution of a little less than 5% can be obtained. When concentrating to 10 to 15% suitable for ethanol fermentation, it is possible to concentrate about 2 to 3 times, so it is possible to remove moisture by distillation under reduced pressure without using an ultrafiltration membrane, If reusable ethanol or butanol is mixed and azeotroped in the saccharified solution, it can be concentrated more efficiently. Or it is also possible to apply to ethanol fermentation with 5% concentration. When the glucose concentration in the saccharified solution is about 0.14% as in Patent Document 4, these methods cannot be established.

(Enzyme recovery / reuse method)
Unreacted residues are also suspended in the paper saccharified solution (glucose solution) obtained by the above method, which precipitates in about 2 hours if the reaction vessel is stopped and allowed to stand. Solid-liquid separation is also easy. Cellulases, which are saccharifying enzymes, are roughly classified into three types, cellobiohydrase (CBH), endoglucanase (EG), and β-glycosidase (BGL). Among them, the reaction mechanism of CBH is the conventional enzyme. A new concept of processivity (one of the products remaining after the completion of the reaction is not released and becomes the next substrate) has been issued (by Yasushi Morikawa, “Enzymatic saccharification of cellulosic biomass”, Chemical Engineering, 71 (12), p.27-30 (2007)). Most cellulases are modular enzymes having a carbohydrate binding module (CBM) such as a cellulose binding domain (CBD) (Cazy (Carbohydrate-active Enzymes), http: // www .cazy.org /). That is, since cellulase functions in a state where it binds to cellulose as a substrate, a large amount of enzyme remains bound to the precipitated unreacted residue. By reusing the residue collected by a simple method of precipitation as an enzyme for the next saccharification treatment, the enzyme can be saved.

  On the other hand, additional costs and maintenance are anticipated in the incidental process of removing the enzyme from the residue by pH adjustment or ultrasonic treatment, and further using an ultrafiltration membrane. In Patent Document 4, care should be taken not to generate a residue to which the enzyme adheres in the reaction system by adding enough enzyme to a small amount of substrate to solubilize and saccharify all the substrates in a short time. The main focus is on draining a thin saccharified solution from the saccharification reaction tank and recovering and reusing the released enzyme in the ultrafiltration membrane, but it depends on the ultrafiltration membrane. It is still a system.

  The present invention is a stance in which an enzyme adhering to a residue can be easily reused together with the residue without causing pH adjustment or ultrasonic waves to be peeled off. As described above, the paper has a flow limit of 10% slurry, but the solubilization proceeds immediately after the addition of cellulase, and the fluidity improves and becomes smoother. Although the total amount of saccharification residue for the previous time when the viscosity has decreased is larger than that of the enzyme preparation having the same saccharification power, there is no significant problem in fluidity even if this is added to the new paper slurry for the next time. If there is a slight problem with fluidity, it can be solved by introducing new paper in stages.

  However, when concentrating the saccharified solution excluding the residue, when using the ultrafiltration membrane method instead of the vacuum distillation method, the enzyme released in the saccharified solution can be recovered and re-used as in Patent Documents 1 to 4. It is desirable to combine use, and the enzyme can be saved more. Needless to say, if the saccharifying power of the reusable enzyme is insufficient, it is necessary to replenish a new enzyme.

(Example 1: Production of concentrated saccharified solution)
As a standard paper sample, the same lot of high-quality OA paper (copy paper) derived from commercially available chemical pulp was purchased together, shredded with a shredder, and then dried in a dryer for all tests. . This was weighed and suspended in distilled water to a concentration of 2.5 to 10%, and applied to the enzymatic saccharification test as a slurry of 100 mL capacity. The enzyme preparation used was GC220 (liquid) derived from Trichoderma reesei, manufactured by Genencor, with a titer of 6,200 units / g as CMC (carboxymethylcellulose) saccharification activity and a specific gravity of 1.1 to 1.2. It was a display. This enzyme was added so as to be 1 to 20% (wt / wt) based on the total weight of the paper, and was treated in the experimental system shown in FIG. 1 for 32 to 72 hours under the conditions of 50 ° C., pH 4.0, and 200 rpm. . Lignocellulose slurry at the time of enzymatic saccharification experiment has been reported to be converted to 50 mM acetic acid buffer solution, but considering practical use, pH was adjusted only with sulfuric acid with a lower unit price (the difference in saccharification rate due to the difference between acetic acid and sulfuric acid is I was not able to admit).

  As shown in FIG. 2, in the basic experiment for enzymatic saccharification using 5% paper slurry, the heat treatment with the addition of dilute sulfuric acid had no particular effect as a pretreatment, and the saccharification rate was the same as that of no treatment. In the case of paper, the result shows that the saccharification rate depends only on the amount of enzyme added and there is no pretreatment effect. In the case of the enzyme GC220, it was found that a satisfactory sugar yield cannot be obtained unless at least 5% [wt / wt] is added per absolute dry weight of paper. From the perspective of the enzyme unit price and the profitability, the upper limit of the amount added per absolute dry weight of paper was estimated to be 5% [wt / wt], and the subsequent experiments were carried out based on this amount added. When the specific gravity of GC220 is 1.1 and the above addition amount of 5% [wt / wt] is converted into a titer, the CMC saccharification activity is 340 units per 1 g of paper.

  FIG. 3 shows the transition of glucose yield when the paper slurry concentration is 2.5, 5, and 10%, and 5% [wt / wt] of enzyme is added per absolute dry weight of paper. Although the glucose yield increased in proportion to the absolute amount of paper, the order was reversed when converted to glucose using the paper concentration as the denominator, and the lowest for the 10% paper slurry (FIG. 4). This was considered to be because 10% slurry had a large absolute amount of paper, so that more glucose and cellobiose were produced in the saccharified solution, and the enzyme activity was inhibited. In particular, as shown in FIG. 5, the production of cellobiose was significantly higher than those of 2.5 and 5% slurries. Therefore, 8 and 24 hours after adding GC220 5% [wt / wt], cellulase derived from Aspergillus niger (manufactured by Yakult Pharmaceutical Co., Ltd .: Y-2NC, cellulase 65%, dextrin 35% freeze-dried powder with CMC titer An additional test was performed in which 0.5% [wt / wt] of the indicated 60,000 units / g) was added.

  As shown in FIG. 6, with GC220 alone, the glucose concentration, which was about 2.5% at 32 hours, improved to 4%, and reached less than 5% at 72 hours. From the above results, it was found that a 10% paper slurry yielded a glucose solution of less than 5% in the treatment for at least 72 hours. However, the reaction could be terminated in about 32 hours when the glucose concentration reached 4%. I think it is desirable for efficiency. The cellulose content of the co-tested standard paper is 53.6% (Table 1), and the saccharification rate is about 90% as calculated below based on this calculation.

After 8 hours, 0.5% [wt / wt] of Y-2NC was added: 4.8 / (10 × 0.536) × 100 = 89.5 (%)
In addition, when cellobiose accumulates in the reaction solution, the enzyme activity is inhibited. As a countermeasure, it is a well-known enzyme reaction that, when used together with cellulase derived from A. niger that is rich in β-glycosidase that hydrolyzes cellobiose to glucose, it is eliminated and the saccharification rate is improved.

(Example 2: Recovery and reuse of residue 1)
As described in the previous section, in the case of the enzyme GC220, a satisfactory sugar yield cannot be obtained unless at least 5% [wt / wt] is added per dry weight of paper. In terms of titer, 340 units were required as CMC saccharification activity per gram of paper. However, considering practical use, this addition amount is set as the upper limit, and a sufficient sugar yield can be obtained while further reducing the amount of enzyme. desirable.

  Based on this point of view, the residue obtained by adding 5, 10, 20% [wt / wt] enzyme per 5% paper slurry to the absolute dry weight of the paper and treating for 48 hours is collected. The saccharification rate when added as an enzyme to the paper slurry was verified. FIG. 7 shows the amount of each residue after 48 hours of enzymatic saccharification. It settled in about 2 hours after standing, and the decrease in the apparent volume almost stopped after 24 hours. The larger the amount of enzyme added, the smaller the apparent volume was 8 mL vol at 20% addition, 12 mL vol at 10% addition, and 28 mL vol at 5% addition.

  In FIG. 8, these supernatants are gently removed, and the total amount of the residue containing a slight amount of saccharified solution is added to 5 g of each new paper, and finally made up to a volume of 100% 5% paper slurry. The results of treating these for 32 hours under the conditions of 50 ° C. and pH 4.0 are shown (a pattern in which 5% [wt / wt] of a new enzyme was added to the same 5% slurry as a control was also performed in parallel). In either case, glucose of about 0.5% is detected at the start (0 hour) by bringing in the saccharified solution for the previous time slightly, but after 32 hours, the glucose concentration is 1.2 to 1.3%. Reached. Subtracting the starting glucose concentration is 0.7-0.8%, which is the result of adding 1% enzyme per paper weight as shown in FIG. The saccharification power was higher than The yield of each sugar is as shown in FIG. 8, but visual solubilization of OA paper progressed greatly in 2 to 3 hours from the start of the treatment in any case where the residue was added, and the viscosity of the slurry. And the stirring efficiency was greatly improved. In particular, since the production of cellobiose is not inferior to that of the control, it is surmised that CBH and EG remained sufficiently in the residue.

  On the other hand, regarding the point that the saccharification power of the various residues in the next time is almost the same, the amount of the residue decreases (28, 12, 20%, inversely proportional to the increase of the enzyme addition amount (5, 10, 20% [wt / wt]). 8 mL vol), but because the absolute amount of enzyme per residue vol is different, it is presumed that the total amount of enzyme was the same overall. That is, from the viewpoint of reusing the enzyme that is still adsorbed on the residue, an enzyme addition amount of 5% [wt / wt] is considered economical.

  Based on the above results, the residue generated by adding 5% [wt / wt] enzyme per absolute dry weight of paper remains at least as high as saccharification power at least 1% [wt / wt] new enzyme. It was thought that

(Example 3: Recovery / reuse of residue 2)
As described in the previous section, the saccharification residue for one batch generated by adding 5% [wt / wt] enzyme to the 5% OA paper slurry and treating it for 48 hours is at least 1% [wt / wt]. It was thought that saccharification power equal to or better than the new enzyme of [wt] remained. Based on this viewpoint, the saccharification rate when a new enzyme was added to a 5% OA paper slurry in order to supplement the residue and saccharification power was verified.

  FIG. 9 shows the glucose yield after enzymatic saccharification for 32 hours (as a control, a pattern in which 5% [wt / wt] of a new enzyme was added to the same 5% OA paper slurry was also performed in parallel). As in Example 2, a little saccharified solution for the previous time was brought in, so that about 0.5% of glucose was detected at the start (0 hour). A glucose yield equal to or higher than that of the control was obtained when the enzyme was used in an amount of 3% [wt / wt] or more. In other words, when OA paper slurry with a concentration of 5% is the target, the amount of enzyme that conventionally required 5% [wt / wt] is reduced to at least 3% [wt / wt] by applying the residue from the previous time to the next time. The result that it can reduce was obtained.

  The saccharifying enzyme is not particularly limited as long as it can hydrolyze cellulose to glucose, but it is an enzyme mainly composed of cellulase produced by bacteria such as Trichoderma genus, Aspergillus genus, Rhizopus genus, or commercial. Enzyme preparations that are mass-produced are used alone or in combination. From the viewpoint of enzyme reuse, an enzyme that has been subjected to modification of a constituent protein or the like is more desirable in order to increase stability against time and heat. In addition, titers such as CMC saccharification activity and filter paper disintegration power displayed on commercially available enzyme preparations are only reference values, and are optimal additions to obtain a satisfactory sugar yield from the lignocellulose raw material to be treated. It is important to verify the quantity for each raw material.

  As described above, examples have been introduced for paper that has already been delignified, but these methods, particularly the enzyme recycling method, are not only paper, but also other lignocellulosic resources, such as wood such as conifers and hardwoods, It can also be applied to agricultural saccharification such as southern seawood (trunk and fruit hulls), mainly rice palm, rice straw, wheat straw, bagasse and corn stover, and enzymatic saccharification of macroalgae and microalgae. .

  According to the present invention, the amount of enzyme used can be reduced as compared with the conventional case, and as a result, the economics of producing saccharides from the lignocellulose raw material can be improved. This not only enhances the economics of the industry as a whole, producing and supplying ethanol, lactic acid and other chemicals using sugar as a fermentation substrate, but also applies foods such as molasses and starch to the aforementioned uses. It leads to avoiding moral problems.

It is a figure explaining the batch type saccharification process of Example 1. FIG. 2 is a graph showing changes in glucose concentration in each reactor in the saccharification treatment of Example 1. FIG. It is a graph which shows a time-dependent change of glucose concentration. It is a graph which shows the time-dependent change of the conversion rate (%) to glucose. It is a graph which shows a time-dependent change of a cellobiose density | concentration. It is a graph which shows a time-dependent change of the glucose concentration at the time of adding A enzyme. It is a photograph which shows the state after performing the enzymatic saccharification process for 48 hours. It is a graph which shows the time-dependent change of various saccharide | sugar when performing a saccharification process by a saccharified liquid residue, (a) shows cellobiose concentration, (b) shows glucose concentration, (c) shows time-dependent change of xylose concentration, respectively. . 6 is a graph showing changes with time in glucose concentration in Example 3.

Claims (8)

A lignocellulose batch or semi-batch saccharification method in which a cellulose hydrolase is added to a lignocellulose raw material that has been pretreated so that an enzyme acts to obtain glucose or C5 sugar as C6 sugar,
A lignocellulose batch or semi-batch saccharification method, wherein the lignocellulose residue that is not hydrolyzed and to which the added enzyme is attached is reused for the next enzymatic saccharification.   The lignocellulose pretreated so that the enzyme acts is adjusted to a slurry of 2 to 10% [dry wt / wt], and 200 to 1000 units of saccharifying enzyme is added to the slurry per 1 g of dry weight of lignocellulose. 24. The batch or semi-batch saccharification method of lignocellulose according to claim 1, wherein the saccharification treatment is performed for 24 to 72 hours.   Concentrating the lignocellulose saccharified solution to a concentration of 10 to 15% suitable for ethanol fermentation by performing vacuum distillation or azeotropic addition of an alcohol having 2 to 4 carbon atoms to the lignocellulose saccharified solution, The method according to claim 1 or 2.   The method for saccharification of lignocellulose according to claim 1 or 2, wherein the lignocellulose saccharified solution is concentrated using an ultrafiltration membrane.   The lignocellulose residue precipitated by collecting the lignocellulose saccharified solution is recovered, and the whole or a part of the recovered lignocellulose is added as a reusable enzyme to the next enzymatic saccharification of lignocellulose. Lignocellulose batch or semi-batch saccharification method.   The batch-type or semi-batch-type saccharification method of lignocellulose according to any one of claims 1 to 5, wherein the reuse of the lignocellulose residue is repeatedly performed a plurality of times.   The said lignocellulose raw material is not contained in the conventional waste paper collection | recovery system, but is derived from the paper waste incinerated at the municipal waste incineration plant, The lignocellulose as described in any one of Claims 1-6 Batch or semi-batch saccharification method.   The batch-type or semi-batch-type saccharification method of lignocellulose according to claim 7, wherein the lignocellulose raw material is derived from office paper waste.

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