KR101883628B1 - Method for enhancing detoxification activity of laccase using secondary biomass substrate - Google Patents

Abstract

The present invention relates to a method for enhancing the detoxification activity of a lacase by adding a small amount of a secondary biomass substrate containing a natural mediator such as p-coumaric acid in a biodegradation process of biomass using a lacase, Simultaneous pretreatment and saccharification processes (SPS). According to the present invention, by adding a small amount of a low-cost secondary biomass substrate, the detoxification of the lacase, which removes toxic substances that may be generated in the decomposition of the biomass, by a natural mediator contained in the secondary substrate The secondary substrate used is low in environmental toxicity and therefore does not require any additional cleaning steps and is used as it is without purification. Therefore, it is economical, environmentally friendly, low cost, and environmentally friendly, and simultaneously pretreatment and saccharification SPS) process, it is possible to produce the saccharide from the biomass with excellent saccharification yield quickly, economically and environmentally.

Description

[0001] The present invention relates to a method for enhancing the detoxification activity of laccase using a secondary biomass substrate,

The present invention relates to a method for enhancing the detoxification activity of a lacase by adding a small amount of a secondary biomass substrate containing a natural mediator such as p-coumaric acid in a biodegradation process of biomass using a lacase, Simultaneous pretreatment and saccharification processes (SPS).

With the depletion of fossil fuels and global efforts to reduce CO ₂ , interest in renewable energy, especially biomass-based energy production, is increasing.

Biomass is a new and renewable energy producing biofuels, electricity, and heat using vegetable raw materials, and it is attracting attention as a possible alternative in terms of environmental friendliness, economical efficiency and technical feasibility.

Of these biomass resources, lignocellulosic biomass has the greatest amount and is free from ethical problems regardless of food. Accordingly, the production and development of a second-generation bioavailable product using lignocellulose as a woody resource is being studied.

Production of biofuels using woody biomass generally requires pretreatment, saccharification, and fermentation. The dual preprocessing process refers to a process that improves the rate and yield of enzymatic hydrolysis reaction. It decomposes complexes of lignin, cellulose and hemicellulose, which are degradable aromatic polymers, It is necessary to increase the accessibility of the enzyme and increase the specific surface area of the biomass to increase the amount of the effective enzyme. Thus, since the production cost of the biofuel is determined according to the efficiency of the pretreatment process, the pretreatment process is important for producing biofuel from the biomass It is one of the elements.

The pretreatment process for promoting the fermentation of such woody biomass includes physical and chemical methods. This is a useful fermentable saccharide that can be used by the microorganism from the biomass material. In order to obtain monosaccharide of pentose or hexose, A hydrolysis process is required.

However, toxic substances such as phenol compounds are induced during hydrolysis pretreatment or saccharification of woody biomass. These toxic substances inhibit the growth and fermentation of microorganisms and reduce the efficiency of the fermentation process thereafter. Therefore, the production efficiency of organic acids and alcohols Is lowered. Therefore, there is a need for a detoxification process that removes toxic substances generated after hydrolysis pretreatment and saccharification.

Conventionally, as shown in FIG. 1, sequential pretreatment and saccharification (SqPS), in which pretreatment and saccharification are separated and proceed sequentially, are mainly used. In this case, And the biomass used as the raw material in the washing process is often lost, resulting in lowering of the actual energy efficiency, the possibility of environmental pollution due to the generation of waste water by a large amount of water used, This causes a problem that the process cost is increased.

Therefore, there is a need to develop a simple but efficient system that can overcome these problems.

As an alternative to solve the above problems, the present inventors have proposed Simultaneous Pretreatment and Saccharification (SPS) methodology that simultaneously performs pre-treatment and saccharification in Korean Patent Application No. 2015-0056209. The SPS process simultaneously pretreats and glycosylates the biomass immersed in an enzyme produced by inoculating a fungal consortium into a culture medium, and the toxicity of toxic substances, especially phenolic compounds, which may be generated in degradation of the biomass, ), It is possible to omit the washing step included in the sequential preprocessing and saccharification step (SqPS), thereby reducing the loss of biomass by the washing step and improving the saccharification yield.

However, since the lacase alone does not exhibit a satisfactory detoxifying activity, the role of mediator is important to broaden the substrate specificity of the enzyme. The use of an organic mediator improves the redox potential of the lacase and thus improves the rate of catalysis. Accordingly, studies on the enhancement of toxicity of laccase through the Laccase mediator system (LMS) have been under way [U. Fillat and M. B. Roncero, Biochem. Eng. J., 2009, 4, 193-198.].

However, until now all commercially available vehicles are highly environmentally toxic and not suitable for environmentally friendly reactions. In addition, after application of an organic commercial mediator such as 1-hydroxybenzotriazole, very reactive free radical species have been generated and any residual free radicals are sufficient to denature the hydrolytic enzymes used in the later stage , Intermittent washing after final hydrolysis was required to remove the generated free radicals. The need for extensive cleaning with distilled water increases the overall cost of the process and also creates many environmental toxic effluents. Also, there is a problem in that the residual mediator can not be completely removed only by such a washing step.

Therefore, it is required to develop an efficient, economical and environmentally friendly pretreatment and saccharification process in woody biomass.

Therefore, the inventors of the present invention have conducted research for an efficient, economical and environmentally friendly method for promoting the detoxification activity of lacase, and a method of adding a low-cost secondary biomass substrate having a natural mediator to lacase, It is possible to increase the detoxification activity of the lacase by supplying natural intermediates to the biosubstance, and the secondary biomass substrate is used as it is without purification, and thus it is an economical and environmentally friendly method with low cost and environmental toxicity. .

Accordingly, an object of the present invention is to provide a method for reducing toxic substances generated in a biomass in a pretreatment process or a saccharification process by reducing the toxic substance to lacase, eco-friendly to improve toxic removal efficiency, .

It is another object of the present invention to provide a simultaneous pretreatment and saccharification method of biomass with improved toxicity removal efficiency of the detoxification process and improved saccharification yield.

Another object of the present invention is to provide a simultaneous pretreatment and saccharification composition capable of obtaining a saccharide from a biomass and improving toxic removal efficiency.

In order to accomplish the above object, the present invention provides a method for reducing toxic substances produced in a biomass during a pretreatment or saccharification process to a lacquer in a detoxification process, which is different from the target biomass, A method for promoting the detoxification activity of a lacase in an economical and environmentally friendly manner, comprising the step of adding a small amount of a secondary biomass substrate containing cumaric acid to the lacase to supplement the vehicle.

Also preferably, the secondary biomass substrate may be selected from the group consisting of wheat bran, rice husks and populus nigra.

Also, the amount of the secondary biomass substrate may be such that 1.5 mM of p-cumaric acid can be supplied.

The present invention also provides a method for producing a biomass comprising: a first step of mixing a biomass, a secondary biomass substrate different from the biomass and containing p-coumaric acid, and pulverizing the ground biomass, and immersing the pulverized biomass; And a second step of preparing a saccharide by simultaneously pretreating and saccharifying the biomass mixture immersed in an enzyme mixture and a laccase produced by inoculating a fungal consortium producing a lignocellulase in a culture medium, And a simultaneous pretreatment and saccharification method of the biomass.

Preferably, the amount of the secondary biomass substrate to be mixed can be 1.5 mM of p-cumaric acid.

Preferably, the fungal consortium is selected from the group consisting of Pholiota It may be a mixture of adiposa and Armillaria gemina .

Preferably, the saccharification can be carried out for 36 hours with stirring at a pH of 5.0 and 35 ° C at a rate of 90 to 110 RPM.

Further, preferably, in the second step, a surfactant may be further added as a protective agent.

Also, preferably, the surfactant may be polyoxyethylene sorbitan monolaurate (Tween-20).

The invention also Pholiota to the culture medium for producing sugar from the biomass storage adiposa and Armillaria the present invention provides a simultaneous pretreatment and composition for glycation of biomass, which comprises as an active ingredient a secondary biomass substrate comprising a gemina fungal consortium, lacase , and p-coumaric acid.

Preferably, the composition for simultaneous pretreatment and saccharification of the biomass may further comprise a surfactant of polyoxyethylene sorbitan monolaurate (Tween-20).

According to the present invention, at the simultaneous pretreatment and glycosylation of woody biomass, by adding a small amount of a low-cost secondary biomass substrate different from the biomass, a biomass is obtained by a natural mediator contained in the secondary substrate, The detoxification activity of the lacase which removes toxic substances which may be generated in the decomposition of the mass is promoted.

In addition, since the secondary substrate used is low in environmental toxicity, it does not require an additional cleaning step and is used as it is without purification, so it is economical and environmentally friendly because it is inexpensive and has less environmental toxicity.

Therefore, the detoxification method according to the present invention can be applied to the simultaneous pretreatment and saccharification (SPS) processes, so that the saccharide can be produced with high saccharification yield from biomass quickly, economically and environmentally.

1 is a schematic diagram of a simultaneous pretreatment and saccharification method (SPS) of the present invention and a sequential pretreatment and saccharification method (SqPS) of the prior art.
Fig. 2 is a graph showing the change in pH of a phenol compound derived from immersed rice straw (CRS) when treating rice straw in a medium containing or not containing a lacase (10 U / g-substrate) PH 4.0 (?); PH 4.5 (?); PH 5.0 (?); And pH 5.5 (?)).
3 is a graph showing the degree of solubilization of a phenol compound derived from immersed rice straw at pH 4.5 according to the dose of lacase when a rice straw is treated in a medium containing a lacase in an experimental example of the present invention (?); 15 U / g-substrate (?); 20 U / g-substrate (?).
4 is a graph showing the degree of solubilization of a phenol compound derived from an immersed rice straw according to the addition of a mediator when treating rice straw in a medium containing a lacase in an experimental example of the present invention (?); HBT (?); AS (?); PCA (?);
FIG. 5 is a photograph (FIG. 5) showing a transmission electron microscope image of the rice straw before and after the saccharification, and FIG. 5 (c) After simultaneous pretreatment and saccharification (SPS) reactions including detoxification process)

Hereinafter, the present invention will be described in detail.

The method of enhancing the detoxification activity of a lactic acid according to the present invention is different from the target biomass in the detoxification process of reducing toxic substances produced in the biomass to lacase during the pretreatment or saccharification process and is a natural mediator, And adding a small amount of a secondary biomass substrate containing p-cumaric acid as a carrier to supplement the intermediates to the lactase.

Lignin oxidase is well known for their broad substrate specificity and lower redox potentials. The use of an organic mediator improves the redox potential of the lignin oxidase, thus enhancing the rate of catalysis. However, not all commercial vehicles are highly environmentally toxic and thus unsuitable for eco-friendly reactions, and natural intermediates also elevated the detoxification potential of TcLac (see FIG. 4), but the application of commercially available, Lt; RTI ID = 0.0 > a < / RTI > removal step to remove these residual concentrates. When the residual natural intermediates were not removed, the overall saccharification yield was sharply reduced (see Table 1), which was due to free radicals generated during the action of the intermediates. The total concentration of these free radicals was very high, and the radicals were highly reactive species, which could then easily denature the enzymes used in the hydrolysis step, thus requiring a washing step.

The need for extensive cleaning with distilled water increases the overall cost of the process, creates a large number of environmental toxic effluents, and this wash step alone has not completely eliminated the residual mediator.

Thus, the inventors have considered providing the intermediary from a primary biomass substrate and a different secondary biomass substrate in order to solve all the problems associated with the application of commercial natural intermediates. Plant and agricultural residues are known to contain a variety of phenolic compounds as their constituent structural materials [B. Donohoe, P.N. Ciesielski, T.B. Vinzant Methods Mol Biol., 2012, 908, 31-47], since almost all available intermediates are also phenolic in nature, a variety of agricultural-residue materials can be utilized for intermediary diffusion.

It is preferable that the secondary biomass substrate contains p-coumaric acid as a natural mediator. It is preferable that the secondary biomass substrate includes p-cumaric acid as a natural mediator, In the detoxification potential test of TcLac, the p-cumaric acid increased the detoxification potential of TcLac after 1-HBT (see FIG. 4). Since it was derived from a plant, Because it has been shown to be effective in improving TcLac activity without inducing significant inhibition (see Table 3). In the case of 1-HBT, its environmental toxicity was so large that it was excluded from the viewpoint of environment friendliness.

The secondary biomass substrate containing p-coumaric acid may be bran, rice hull, Populus nigra or the like, but bran is preferably used.

At this time, it is preferable that the amount of the secondary biomass substrate is such that 1.5 mM of p-cumaric acid can be supplied. If the amount of p-coumaric acid supplied is less than the above range, the detoxifying potential of TcLac is not observed If it exceeds the above range, it is difficult to remove the residual intermediates, which results in a problem that the saccharification yield is rapidly lowered.

The present invention aims at maximizing economic and environmentally friendly release of toxic substances or non-toxic substances as a biomass process technology. Accordingly, according to the present invention, it is possible to provide a biomass- This eliminates the need for an additional cleaning step and thus eliminates the need for processing costs for potential wastewater treatment and the cost of synthesized organic chemicals as a result of the cleaning step, thereby reducing the overall process cost and being environmentally friendly. The natural intermediates released from tea biomass increase the detoxification activity of lacase, thus reducing the hydrolytic modification and consequently improving the saccharification yield.

In addition, pretreatment and saccharification in the same vessel can facilitate the process, reduce energy consumption, and facilitate the treatment of residual biomass, so that the detoxification process using the secondary biomass substrate can be performed simultaneously, Can be included in the saccharification (SPS) method.

The SPS method according to the present invention is a simultaneous pretreatment and saccharification method of biomass capable of reducing the generation of toxic by-products and loss of biomass and improving the yield of saccharification. The biomass is different from the biomass, and p- A first step of mixing and pulverizing a secondary biomass substrate containing the pulverized biomass, and immersing the pulverized biomass; And a second step of preparing a saccharide by simultaneously pretreating and saccharifying the biomass mixture immersed in an enzyme mixture and a laccase produced by inoculating a fungal consortium producing a lignocellulase in a culture medium, .

In the first step according to the present invention, the biomass is derived from a plant. Preferably, the biomass may be lignocellulosic biomass, such as willow, rice straw or the like, It is not limited. In the embodiment of the present invention, rice straw of a species of Oryza sativa L. was used as a biomass material.

In the first step, the biomass may be pulverized by a method used in the art, and in the case of rice straw (Oryza sativa L.), it may be cut to a length of about 5 cm and pulverized to a size of about 4 mm. Secondary biomass substrates include bran, rice husk, Populus containing p-coumaric acid nigra and the like may be used without limitation. Preferably, bran can be used. In this case, the amount of the secondary biomass substrate may be sufficient to contain only a small amount of 1.5 mM of p-coumaric acid.

The pulverized solid biomass can then be subjected to a physical treatment by mixing with a buffer solution at a ratio of 1:15 (w / v) and soaking. At this time, the buffer may preferably be a sodium acetate buffer having a pH of 5.0 to optimize an enzyme produced in a fungal consortium with a sodium acetate buffer having a concentration of 0.1 molar (M).

The second step according to the present invention is a simultaneous pretreatment and saccharification (SPS) process in which the cellulose in the biomass section is subjected to ethanol fermentation through a single combined step of substrate modification and hydrolysis via enzymatic means Such as glucose, into disaccharides and disaccharides.

As shown in FIG. 1, since the pretreatment, the detoxification and the hydrolysis are simultaneously performed by the enzyme, the SPS process reduces the generation of toxic by-products and the loss of biomass, , It is very important to increase enzyme activity for hydrolysis and enzyme activity for detoxification.

First, in order to increase the enzyme activity for the hydrolysis of woody biomass, it is required to produce a highly active enzyme which decomposes lignin and lignocellulose, which are complex matrices, which is biologically a fungal consortium and fungal consortiums in the form of a combination of strains can be achieved.

The fungal consortium includes Pholiota Adiposa (PA) and Armillaria gemina ( AG ) may be used.

The AG endoglucanase (EG; 65 kDa) belongs to the glycoside hydrolase (GH) family 61, which is the most abundant of the reported catalytic efficiencies for carboxymethylcellulose (CMC) Glucopyranoside (pNPG) as a substrate and? -Glucosidase (BGL) as a substrate exhibited a high value (k _cat / K _m , 3590 mg ml ^-1 s ^-1 ) The combination of the two fungal cultures can provide a synergistic effect on the enzymatic saccharification of the lignocellulosic biomass since it represents the highest value of the V _max reported for both bios.

The results showed that the synergistic interactions of the PA and AG cultures were 2.1-3.0 times higher (2.57 FPU / ml) than those produced by the individual AG (1.72 FPU / ml) and PA (1.25 FPU / ml) .

Optimized enzyme production from a fungal consortium is described in [S. S. Jagtap, S. S. Dhiman, T.-S. Kim, I.-W. Kim and J.-K. Lee, Appl. Microbiol. Biotechnol., 2014, 98, 661-669 .; And S. S. Jagtap, S. S. Dhiman, M. Jeya, Y. C. Kang, J.-H. Choi and J.-K. Lee, Biores. May be obtained from a 7 liters jar fermenter using peptone as the nitrogen source at 25 ° C, pH 5.0. have.

The crude extract from the fungal consortium under conditions for producing the above optimized enzymes contained 1,870 ± 250 U / ml xylanase, 176 ± 23 U / ml endoglucanase (EG), cellobiose hydrolase , 44 ± 5 U / ml of cellobiohydrolase (CBH), 39 ± 5 U / ml of β-glucosidase (BGL), and 2.6 ± 0.5 of filter paper unit (FPU).

In the SPS process according to the present invention, when using rice straw as the biomass, the optimum dose of the enzyme produced from the fungal consortium was 20 FPU / g-substrate. Lower doses produced a significant amount of reducing sugar , And higher doses do not significantly affect the increase in the final saccharification yield (see Table 5).

The detoxification according to the present invention is a reaction for reducing or eliminating the toxic substance of the phenolic compound as described above produced in the simultaneous pretreatment and saccharification steps by a biological method, and in one embodiment, producing laccase Tyromyces chioneus was inoculated into the medium and incubated at 30 ° C with a revolution speed of 150 RPM for 4 hours. Biomass of 1 g dry weight was immersed in 25 ml of 50 mM buffer. TcLac The 'TcLac' refers to a strain producing laccase), and the lacase produced in this strain can be detoxified to contain 15 U / mL.

In general, Laccase has low substrate specificity and is widely used in phenols such as polyphenol, methoxy-substituted phenol, and diamine. Can be oxidized. Lacase produced by fungi is also known to be involved in degradation of lignin.

Therefore, TcLac can be added together with the fungal consortium as it can be used for pretreatment of biomass as well as detoxification to oxidize and remove phenolic compounds derived from biomass through production of lacase.

At this time, since the secondary biomass substrate is added together, the natural intermediates released from the secondary biomass increase the detoxification activity of the lactase, so that the modification of the hydrolytic enzyme is reduced, have. The secondary biomass substrate is less environmentally toxic, is inexpensive and can be used in an unprocessed state, thereby being eco-friendly and economical. The secondary biomass substrate can increase the detoxification activity of the lactase even if only a small amount of p-cumaric acid can be supplied at 1.5 mM.

In the present invention, the saccharification step can be carried out for 36 hours with stirring at a pH of 5.0 and 35 ° C at a rate of 90 to 110 RPM.

Specifically, at pH, the glycosylation process is preferably performed at pH 5.0, since it is the optimum pH for lignocellulosic enzymes and TcLac secreted by PA and AG.

At reaction temperature, the fungal consortium enzyme maintained a relative activity (FPU unit) of 74% for 36 hours at 35 ° C. for immersed rice straw (CRS), which is sufficiently stable to perform the hydrolysis of the substrate. Also, the use of this temperature range is advantageous because a simultaneous fermentation process for biofuel production can also be integrated.

For the stirring speed, it is preferable to use an intermediate stirring of 100 10 RPM to save energy. This suggests that RPM is not an important factor affecting the final saccharification yield (%) (%) for the substrate as the saccharification yield increases only 0.8% or 1.3% as the RPM increases to 150 or 200 RPM .

The reaction time is preferably 36 hours, but longer incubation times can lead to inactivation of the hydrolytic enzyme.

In the second step, a surfactant may be further added. The surfactants serve to protect against thermal shock and to inhibit woody materials, facilitate desorption of cellulase enzymes from the lignin surface [S. S. Jagtap, S. S. Dhiman, T.-S. Kim, I.-W. Kim and J.-K. Lee, Appl. Microbiol. Biotechnol., 2014, 98, 661-669.], And is also expected to help prevent the inhibition caused by highly reactive free radicals.

The non-ionic surfactant may be used as the surfactant. For example, polyoxyethylene sorbitan monolaurate (Tween-20) may be used.

The invention also Pholiota to the culture medium for producing sugar from the biomass storage adiposa and Armillaria the present invention provides a simultaneous pretreatment and composition for glycation of biomass, which comprises as an active ingredient a secondary biomass substrate comprising a gemina fungal consortium, lacase , and p-coumaric acid.

The composition for simultaneous pretreatment and saccharification of the biomass may further comprise a surfactant of polyoxyethylene sorbitan monolaurate (Tween-20).

The composition according to the present invention is a composition used in the simultaneous pretreatment and saccharification methods of the above-mentioned biomass. In order to avoid the excessive complexity of the specification according to the repetitive description, the common description of both inventions is omitted.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not limited to these embodiments.

< Example  1> Secondary Biomass  Simultaneous pretreatment of substrate with rice straw Glycation ( SPS ) fair

(One) Biomass Immersion

Biomass samples were obtained from Hwaseong Co. (Daejeon, Korea). Rice straw (Oryza sativa L.) was used as the primary biomass substrate for hydrolysis and stored at 4 ° C until the next step. For activation, the rice straw was mixed with the buffer solution at a ratio of 1: 45 (w / v) and stored at a temperature of 75 DEG C for 65 days.

(2) from the fungal consortium Lignocellulase  Preparation of enzyme mixture

Pholiota Adiposa (PA) and Armillaria A crude extract of the concentrated culture of the fungal consortium of gemina (AG) was used as the lignocellulase enzyme mixture.

Optimized enzyme production from the fungal consortium was obtained at 25 ° C and pH 5.0. In the optimized enzyme production conditions, the crude extract from the fungal consortium also contained xylanase: 1,870 ± 250 U / ml, endoglucanase (EG): 176 ± 23 U / ml, celobiohydrolase ): 44 ± 5 U / ml, β-glucosidase (BGL): 39 ± 5 U / ml, and FPU (filter paper activity unit): 2.6 ± 0.5.

(3) Lacase  Produce

Tyracytes (TcLac) were prepared by inoculating Tyromyces chioneus into the medium and incubating at 30 ° C with shaking at 150 RPM for 4 hours.

(4) Lacase  Secondary as a natural intermediary supplier Biomass  Substrate preparation

A wheat bran (WB) containing natural p-coumaric acid as a secondary biomass substrate was immersed in the buffer solution in the same manner as the above-described primary biomass substrate.

The immersed bran was then used in the SPS process as a natural mediator source.

(5) Simultaneous pretreatment and Glycation ( SPS ) Through the process Glycoside  Produce

To provide the optimum concentration (1.5 mM) of p-cumaric acid (pCA) as a natural mediator from wheat bran, 9.8 mg of immersed secondary substrate (wheat bran) was mixed with 10 g of immersed primary substrate (rice straw) Subsequently, 20 FPU / g-substrate of lignocellulase mixture prepared from a fungal consortium and 15 U / ml of lacase were added to the reaction tank, and the mixture was stirred for 36 hours at 35 ° C and pH 5.0 at 100 RPM, Respectively.

The yield of glycation (SY) (%) was 67.3 ± 7.4%.

In the SPS process, some sugar contents were filtered during the immersion of the primary and secondary biomass, but were easily recovered using the phase extraction method because there were no chemicals or contaminants during the immersion step.

Therefore, the amount of sugar present in the residue of rice straw (48 mg / g-substrate) and the amount of sugar present in the residue of bran (29 mg / g-substrate) , The final yield of saccharification (%) obtained from the immersed rice straw was 84.3%.

< Comparative Example > Sequential pretreatment of rice straw Glycation  ( SqPS ) Through the process Glycoside  Produce

(One) Alkaline  Preprocessing step

10 g of the primary substrate (rice straw) was mixed with the buffer at a 1:45 ratio (w / v) and immersed in the buffer, and then stored at a temperature of 75 ° C for 65 days. Thereafter, the immersed substrate was pretreated with alkali (NaOH; 0.2% w / v).

After pretreatment, the substrate was washed with water to remove some of the generated toxicities.

The wastewater was generated by the washing step.

(2) Glycation  step

Pretreatment of rice straw was added to the reaction tank, and saccharification was carried out in the same manner as in Example except that the secondary biomass was added. Specifically, a lignocellulase mixture 20 FPU / g-substrate and 15 U / ml lactase prepared from a fungal consortium were added, and the resulting mixture was stirred at 35 ° C and pH 5.0 at 100 RPM for 36 hours.

The yield of glycation (SY) (%) was 71.4 ± 7.6%.

However, wastewater was generated twice or more as compared with the examples.

<Analysis>

In the conventional method in which pretreatment and saccharification are separated and proceeded sequentially, a cleaning process is required every time the process proceeds to the next step in the case of 1 kg of substrate. In this case, about 50 L of clean water is required for the washing process. However, the present invention does not require such a washing process, can proceed at the same time, and can produce biofuels from biomass quickly, economically, and environmentally friendly as compared with saccharification yield or conventional methods.

< Experimental Example  1> Lacase  Optimization of detoxification activity

In the SPS process according to the present invention, saccharification yield can be increased due to the detoxification process of lactase (TcLac). Therefore, in order to optimize the detoxification activity of the lactase, the latachemical detoxification potential was measured as follows.

Specifically, the methodology proposed by Lee et al., (2012) [K.-M. Lee, D. Kalyani, M. K. Tiwari, T.-S. Kim, S.S. Dhiman, J.-K. Lee and I.-W. Kim, Biores. Preliminary analysis was carried out at 30 ° C for 4 hours with or without a vehicle on a rotary vibrator (150 rpm) using 5 U / ml of Laccase according to the method described in US Pat. Samples were taken periodically and analyzed by the Folin-Ciocalteau method (V. L. Singleton and J. A. Ross, Am. J. Enol. Viticult., 1965, 16, 144-158]. Results are expressed as grams of catechol equivalents (CE) per liter of liquid phase.

(1) pH dependent Lacase  Effect of detoxification activity

The detoxification potential of lacarge at a fixed dose of 10 U / ml was measured at different pHs of 4.0-5.5. Also, the solubilization of phenol without TcLac was checked and considered as a control. The measurement results are shown in Fig.

2 is a graph showing the degree of solubilization of phenol compounds derived from immersed rice straw (CRS) when treating rice straw in a medium containing or not containing racaaca (10 U / ml) PH 4.5 (?), PH 5.0 (?), And pH 5.5 (?)). The higher the degree of solubilization of the phenolic compound, the higher the latachematous detoxification potential.

As shown in FIG. 2, the solubility of the phenol compound increased from the reaction mixture as the pH increased from 4.0 to 4.5, but no significant adverse effect was observed at pH 5.0 and 5.5. As a result, pH 4.5 was selected as the optimum pH for the biomass detoxification.

The maximum admiral level obtained for CRS immersed with 10 U / ml TcLac was 49.7%.

(2) Dose (ED) Lacase  Effect of detoxification activity

At an optimized pH of 4.5, the latachemical detoxification potential was determined according to the dose (5-20 U / ml). The measurement results are shown in Fig.

Figure 3 is a graph showing the solubilization of phenolic compounds derived from immersed rice straw at pH 4.5 according to the dose of lacase (10 U / ml (∘); 15 U / ml ▼); 20 U / mL (DELTA)).

As shown in FIG. 3, the solubility of the phenol compound from the reaction mixture increased with increasing the dose of lacase from 5 to 15 U / ml, but no remarkable adverse effect was observed at 20 U / ml. Therefore, 15 U / ml was chosen as the optimal dose for the biocide admission.

Therefore, in the detoxification process using lacase, the detoxification activity of lactase was optimized at pH 4.5 to 15 U / ml.

When the enzymatic hydrolysis was carried out using a fungal consortium of 20 FPU / g-substrate at the optimum conditions, the yield of saccharification was 54.6%, which was not detoxified Which is 55% higher than that of untreated rice straw.

(3) Depending on the type of intermediary Lacase  Effect of detoxification activity

TcLac (15 U / g-substrate) was reacted with 1-hydroxybenzatriazole (HBT) (O), acetosyringone (AS) Para-coumaric acid (pCA) (Δ), and syringaldehyde (SA) (■). The control reaction (●) was carried out with 15 U / ml of TcLac without any mediator. The detoxification potential of lacase was measured, and the measurement results are shown in Fig.

As shown in FIG. 4, the intermediates significantly increase the latacious potential of the lacase and increase the detoxification potential of TcLac in the order of 1-HBT, pCA, AS, and SA. However, since 1-HBT is highly toxic to the environment (see Table 4), p-coumaric acid (pCA) and acetosyringone (AS), which are natural intermediates from plants and have a synergistic effect on the detoxification potential of TcLac, Respectively.

As shown in FIG. 5, the additional adverse effect of biomass morphology was observed using a transmission electron microscope, and compared with the pure structure (a) of the raw material as shown in FIG. 5, (C) was completely deformed after the saccharification, and the constituent materials and sugar were released from the biomass.

A vehicle with a higher concentration (> 1.5 mM) was not tested because it was difficult to remove the residual vehicle after an independent wash step.

< Experimental Example  2> Selection of intermediaries

(1) Depending on the type of intermediary Biomass Glycation  Yield measurement

To determine the suitability of the selected mediator, saccharification was performed with a conjugate enzyme of fixed dose (20 FPU / g-substrate) after adding p-coumaric acid (pCA) or acetosyringone (AS) And the results are shown in Table 1.

TcLac
(U / g-substrate) Mediator
(mM) Enzyme dose
(FPU / g-substrate) Reducing sugar
(mg / g-substrate) Glycosylation yield (%) Perform intermittent washing steps (performed after detoxification, pH 5.0 at 35 ° C, 36 hours at RPM 200) 15 none 30 69 ± 8 15.3 ± 2.1 ^# 15 none 30 156 ± 18 34.6 ± 4.1 15 Acetoxylin (0.5) 30 192 ± 19 42.6 ± 4.6 15 p-coumaric acid (0.5) 30 169 ± 18 37.5 ± 3.9 15 Acetoxylin (1.0) 30 203 ± 21 45.1 ± 5.1 15 p-coumaric acid (1.0) 30 183 ± 20 40.6 ± 4.2 15 Acetoxylin (1.5) 30 238 ± 22 52.9 ± 5.2 15 p-coumaric acid (1.5) 30 213 ± 22 47.3 ± 4.9 No cleaning steps 15 Acetoxylin (1.5) 30 112 ± 14 24.8 ± 2.8 15 p-coumaric acid (1.5) 30 139 ± 16 30.8 ± 3.2 # Unconditioned biomass was used.

However, as shown in Table 1, even though natural intermediates increase the latent potential of the lacase, they are unsuitable for application to the SPS process.

As shown in Table 1, natural brokers require an independent washing step to remove residual concentrate after the detoxification reaction, and when the detoxification reaction is not performed, the total saccharification yield is rapidly . This inhibition of glycation yield (%) was due to the free radicals generated during the action of the intermediates. The total concentration of these free radicals was very high, and the radicals are highly reactive species, which can then easily denature the enzyme used in the hydrolysis step.

The need for extensive cleaning with distilled water increases the overall cost of the process and also creates many environmental toxic effluents. Also, this cleaning step alone does not completely remove the residual vehicle.

(2) Assessment of secondary substrate as raw material of natural intermediates

In order to overcome the problem of the necessity of the cleaning process related to the application of the commercial vehicle, an evaluation of the utilization of the secondary biomass substrate for the supply of intermediaries was carried out.

Specifically, seven different secondary substrates based on their availability and cost-effectiveness through the production year, namely wheat bran, rice husk and populus nigra, salix koreensis, Pinus rediata, rice bran, and palm kernel were selected.

High performance liquid chromatography (HPLC) was used to identify and quantify mediator-like compounds in the secondary matrix. Specifically, after immersing the secondary substrate, the immersed secondary substrate was extensively treated with TcLac (20 U / g-substrate) to remove excess concentrations of unwanted phenolic compounds (PC). Dionex HPLC was performed on a C ₁₈ TSKgel CDS-80TS column (4.6 mm ㅧ 25 cm) at 35 째 C with a flow rate of 0.4 ml / min as the mobile phase: acetonitrile (85:15) . &Lt; / RTI &gt; The concentration of each predictable mediator was determined based on the peaks of the standard compound and the extracted compound.

The inhibition constant (K _i ) for all observed phenolic compounds derived from the secondary substrate was then determined for TcLac (Table 2) and for fungal consortium enzymes (Table 3).

Specifically, K _i values were measured using 20 U / ml TcLac and 1.5 mM concentrations of each standard broth, and samples were incubated at 35 ° C for 4 hours. Residual TcLac activity was measured using 2,6-DMP as a substrate.

Likewise, K _i values for each catalytic subunit of fungal complexes were calculated using standard procedures after 1.5 mM of each standard broth and 20 FPU / ml of enzyme were incubated at 35 ° C for 36 hours.

compound Initial concentration
(mM) Inhibition constant
(K _i , mM) Residual concentration
(mM) Gallic acid 7 4 2 Para-cumaric acid
(para-Coumaric acid) 9 11 4 Ferulic acid 10 6 Not detected Caffeic acid 6 2 Not detected 4-hydroxybenzoic acid 12 9 One Vanillic acid 11 8 Not detected Syringic acid 8 6 2 Syringaldehyde 12 9 Not detected

compound Xylanase EG CBH BGL FPU Gallic acid 2 4 4 6 4 Para-cumaric acid
(para-Coumaric acid) 10 16 8 10 11 Ferulic acid 8 8 6 6 8 Caffeic acid 6 12 8 6 6 4-hydroxybenzoic acid 6 10 10 8 9 Vanillic acid 6 8 6 8 8 Syringic acid 4 10 8 6 6 Syringaldehyde 8 10 10 10 9

The inhibitory response of each vehicle towards the fungal consortium enzyme is more important than the inhibition of the lacase activity by the same vehicle because the fungal consortium enzyme is responsible for the final saccharification yield of the biomass. Based on the K _i values for the fungal consortium enzyme (Table 3), pCA and SA were selected as possible NMs that could be supplied by a secondary substrate for SPS. However, inhibitory potential results have been found to be more effective in enhancing TcLac activity without inducing significant inhibition of pCA.

Among all seven tested secondary substrates (pCA in μg / g substrate), bran (2), chaff (0.8) and populus nigra (1.2), willow (Salix koreensis) Pine (Pinus rediata), rice bran and palm kernel, and bran (WB) was chosen as the secondary substrate for glycation experiments because it was shown to contain the highest concentration of pCA. The absence of pCA in the rice straw was confirmed by previous experiments [K.-M. Lee, D. Kalyani, M. K. Tiwari, T.-S. Kim, S.S. Dhiman, J.-K. Lee and I.-W. Kim, Biores. Technol., 2012, 123, 636-645].

(3) Environmental toxicity analysis

In the saccharification process according to the present invention, the following toxicity test was conducted in order to examine the environmental toxicity of natural intermediates.

Specifically, the Microtox test results of immersion, alkali pretreatment, and effluent wastewater after the medicinal addition process with intermediates were entered into the program worksheet and corrected to alleviate the color differences of the different samples. In addition, acute toxicity units (TOU) were calculated for each sample after 5 and 15 min incubation. The sensitivity of each lyophilized bacterial sample was checked periodically in the laboratory using zinc sulfate (ZnSO ₄ .7H ₂ O) as a reference material.

The results are shown in Table 4.

sample EC ₅₀ (%) TOU (%) 5 minutes 15 minutes Wastewater from immersion 69 56 141 Wastewater obtained by alkali treatment 144 132 284 1-hydroxybenzotriazole 186 163 312 p-cumaric acid 117 99 226 Sealing Aldehyde 162 133 279 Acetoxylin 144 127 255 P-cumaric acid extracted from wheat bran
(p-CA _WB ) 103 86 202

As shown in Table 4, since the SPS process according to the present invention does not perform chemical pretreatment, the wastewater obtained from the immersion in the environmental toxicity While the conventional SqPS process using chemical pretreatment showed 284% TOU. Therefore, it can be seen that the SPS process according to the present invention shows environmental toxicity twice as much as the wastewater generated from the conventional SqPS process, and thus it is an environmentally friendly process.

In addition, as a result of the environmental toxicity analysis of the mediator, the environmental toxicity of p-cumaric acid extracted from bran, which is a secondary substrate, was lowest as shown in Table 4.

Accordingly, the present invention can increase the saccharification yield in the SPS process by using the secondary biomass substrate having low environmental toxicity, thereby increasing the detoxification activity of the lactase in a low cost and environmentally friendly manner.

< Experimental Example  3> Optimization of Fungal Consortium Enzyme Doses

In the SPS process according to the present invention, the optimal concentration of immobilized TcLac (15 U / ml) and 10 to 40 FPU / g-substrate to determine the optimal dose of the enzyme produced from the fungal consortium, The saccharification yield was measured after the saccharification process of the rice straw was performed by changing the dosage, and the results are shown in Table 5 below.

Enzyme dose Reduction sugar (mg / g) Glycosylation yield (%) 10 88 ± 7.7 19.6 ± 2.2 15 119 ± 20 26.4 ± 3.2 20 156 ± 18 34.6 ± 4.1 25 178 ± 19 39.1 ± 4.1 30 228 ± 22 50.6 ± 4.1 35 236 ± 23 52.4 ± 4.2 40 241 ± 22 53.1 ± 4.4

As a result, the optimal dose of the enzyme produced from the fungal consortium was 20 FPU / g-substrate, lower doses (10-15 FPU) did not produce significant amounts of reducing sugars, and higher doses (Greater than 30 FPU / g-substrate) did not significantly affect the increase in the final saccharification yield.

Enzymatic cocktails of lower EDs failed to attack the loosened cellulose and hemicellulosic contents, resulting in lower saccharification yields (%).

However, ED above the 30 FPU / g-substrate for precipitated rice straw did not give any significant improvement in final saccharification yield (%) even with a linear increase in ED. It is likely that substrate saturation is probably the reason for the slight improvement in final SY (%) with a linear increase in ED. The optimal dose (ED) of 20 FPU / g-substrate for precipitated rice straw as the primary substrate resulted in the release of 228 ± 22 mg of reducing sugar (RS) per gram of CRS after 36 h of culture Respectively.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (10)

In the detoxification process for reducing the toxic substances produced in the biomass to lacase during the pretreatment or glycation process,
Adding a secondary biomass substrate which is different from the biomass and contains p-coumaric acid as a natural mediator to supplement the intermediates to the lacase,
Wherein the secondary biomass substrate is wheat bran, and p-cumaric acid is added so as to be supplied at 1.5 mM. The method of claim 1, wherein the secondary biomass substrate is included in an amount of 0.098% or less based on the weight of the biomass substrate. A first step of mixing the biomass with a secondary biomass substrate which is different from the biomass and containing p-coumaric acid, pulverizing the same, and immersing the pulverized biomass; And
And a second step of preparing a saccharide by simultaneously pretreating and saccharifying the biomass mixture immersed in an enzyme mixture and a laccase produced by inoculating a fungal consortium producing a lignocellulase in a culture medium In addition,
Wherein the secondary biomass substrate is wheat bran and p-cumaric acid is added so as to provide 1.5 mM. 4. The method of claim 3, wherein the secondary biomass substrate comprises less than 0.098% by weight of the biomass substrate. 4. The method of claim 3, wherein the fungal consortium is selected from the group consisting of Pholiota A method for simultaneous pretreatment and saccharification of biomass characterized by being a mixture of adiposa and Armillaria gemina 4. The method of simultaneous pretreatment and saccharification of biomass according to claim 3, wherein the saccharification is performed at a pH of 5.0 at 35 DEG C with stirring at a speed of 90 to 110 RPM for 36 hours. 4. The method of claim 3, wherein the second step further comprises adding a surfactant as a protective agent. The method of claim 7, wherein the surfactant is polyoxyethylene sorbitan monolaurate (Tween-20). A secondary biomass substrate comprising Pholiota adiposa and Armillaria gemina fungal consortia, lacase, and p-coumaric acid as active ingredients, wherein the secondary biomass substrate is wheat bran and p-cumaric acid is supplied in a concentration of 1.5 mM 0.098%, based on the weight of the primary biomass substrate, of the biomass. 10. The composition for simultaneous pretreatment and saccharification of biomass of claim 9, wherein the composition further comprises a surfactant that is polyoxyethylene sorbitan monolaurate (Tween-20).

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