KR101776906B1 - Mehtod for biotransformation using proteins left in enzymatic pretreated lignocellulosic biomass - Google Patents
Mehtod for biotransformation using proteins left in enzymatic pretreated lignocellulosic biomass Download PDFInfo
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Abstract
The present invention relates to a method for producing a biocomponent by applying a saccharide enzyme protein contained in a biosquare by using a strain that can be used at one time without requiring a separate filtration process. More specifically, the present invention relates to a biosugar- The remaining saccharogenic enzyme interferes with the growth of the fermenting microorganism and becomes a factor which comes out as a solid substance due to sedimentation or denaturation and interferes with the entire process. Therefore, by applying a strain having such a saccharifying enzyme as a nitrogen source, To a method for producing a biocomponent without a filtration step of increasing the growth of the strain and removing a saccharification enzyme.
Description
The present invention relates to wood biomass utilization technology. Specifically, the saccharification enzymes used for the pretreatment and saccharification processes of biomass remain after the saccharification of the enzyme and are usually separated through filtration. However, The present invention relates to a method for producing a biocomponent while removing the protein without using a separate filtration process by applying a strain that can utilize the protein contained in the biosurge.
The amount of fossil fuels used as primary energy sources is limited depending on the region and the crisis of exhaustion is approaching. In addition, the use of fossil fuels causes global warming, environmental crises, and energy crises due to high oil prices. In this situation, biomass can be an alternative energy source in the future.
In particular, woody biomass can be supplied in an unlimited and stable manner because there is an unlimited distribution of resources in the region. It contains a large amount of carbon source, which is advantageous for use as a microbial substrate for a bioproduct. In addition, the emission of nitrogen dioxide, which is the cause of environmental pollution, is relatively small, so it is widely used as a green energy source with little environmental pollution. Among them, lignocelluloses are the core. Although it is an abundant resource on the earth, humanity is not able to use most of it, and because it mainly contains cellulose, which is a substrate capable of hydrolyzing glucose, which is a monosaccharide used for microbial fermentation, have.
The woody biomass is a concept that includes plants biosynthesized through photosynthesis, waste wood, rice straw, etc. It is anatomically composed of 40 ~ 60% of cellulosic, 20 ~ 40% of hemicelluloses, 10 ~ 25 of lignin % ≪ / RTI > is a main component of lignocellulose. Cellulose refers to the polysaccharide, which is the main constituent of cellobiose, which is the two glucose residues connected by β-1,4 glucosidic bonds as the substance with the largest molecular weight among the polysaccharides. Due to the hydrogen bonds between the carbon residues of the glucose moiety, they form a stable chain of linear structure. These chains are gathered to form fine fibers, which are closely arranged and bonded to each other to form a crystalline region and a porous region. It is physically and chemically stable and restricts hydrolysis. In order to utilize such cellulose as a microbial substrate, an appropriate pretreatment process is required.
Pre-treatment with acid is mainly used, but by-products are generated in the process, which hinders the growth of microorganisms. On the other hand, the pretreatment method using hot water requires pretreatment to weaken the structure of the tightly bound cellulose and to saccharify the enzyme such as cellulase. In this process, the enzymes remaining after the saccharification process are required to be recovered, and the remaining enzymes are left in the medium to inhibit the growth of microorganisms. Therefore, they are separated by filtration and then recycled. However, the costs and time- (Korean Patent No. 10-1536132, Korean Patent No. 10-1447534, Korean Patent No. 10-1504197, and Korean Patent No. 10-1449552).
In the saccharification process, saccharogenic enzymes of about 0.5 to 2% of the total mass are used. This saccharifying enzyme is an element that interferes with the growth of microorganisms in the fermentation utilizing biosugar. Because of the precipitation and denaturation, Resulting in interference with the entire process. Therefore, in general, an additional filtration process is introduced and filtered to remove from the biosurge, and the obtained protein is mainly discarded. However, in most cases, the filtration process is costly and the economical efficiency of the whole process is lowered, and additional resources are required to be input.
Accordingly, the present inventors have made intensive efforts to develop a technique for effectively utilizing remaining enzymes after the saccharification process in a pretreatment process of making biosugars using biomass, and as a result, it has been found that a strain having a saccharification enzyme of a saccharification process as a nitrogen source (for example, Bacillus The present invention has been accomplished by developing a method for producing a biocompound at the same time that a protein is directly used as a substrate to increase the growth rate and eliminate a separate saccharification enzyme by using a subtilis.
It is an object of the present invention to provide a process for producing a biomolecule which inhibits the whole process of inhibiting the growth of microorganisms and producing a biomolecule by remaining saccharification enzymes which are essentially used for pretreatment and saccharification of biomass, The present invention provides a method for producing a biocomponent by applying a strain that can be used at one time without the necessity of a separate filtration process for the protein contained in the biosquare will be.
In order to achieve the above object, the present invention provides a method for culturing a strain of the genus Bacillus or Streptomyces using the saccharification enzyme remaining after the pretreatment and saccharification of biomass as a nitrogen source.
The present invention also relates to a method for removing saccharification enzymes remaining after saccharification by culturing a strain of the genus Bacillus or Streptomyces using the saccharification enzyme remaining after the pretreatment and saccharification of biomass as a nitrogen source to provide.
The present invention also relates to a method for producing a biosurfactant, which comprises culturing a genus of Bacillus or Streptomyces using a saccharifying enzyme remaining after pretreatment and saccharification of biomass as a nitrogen source, (Fermentation sugar) is fermented simultaneously with the saccharification process.
The present invention also relates to a method for producing a biocomponent from a biomass by culturing a strain of the genus Bacillus or Streptomyces using the saccharification enzyme remaining after the pretreatment and saccharification of the biomass as a nitrogen source, The present invention provides a method for efficiently producing a biocompound without separating and purifying the biocompound produced, without requiring a separate filtration step of the saccharifying enzyme.
The present invention can utilize the saccharifying enzyme remaining after the biosugar process as a substrate using a strain having proteolytic ability without a separate filtration process. This can be applied not only to the growth of the strain but also to the biocomponent production, thereby reducing the process and enabling effective fermentation. It can also solve environmental and energy problems through bio-products such as isobutanol as well as economic benefits.
FIG. 1 is a view showing a biosurge process from a conventional woody biomass and a biosurge process according to the present invention. FIG.
FIG. 2 is a graph showing the degree of growth of each strain when a biosugar containing saccharogenic enzyme obtained through saccharification is used as a nitrogen source.
FIG. 3 is a graph showing the degree of growth of Bacillus subtilis according to time when a biosugar containing saccharogenic enzyme obtained through saccharification is used as a nitrogen source.
FIG. 4 is a graph showing the results of production of isobutanol using a biosugar containing a glycated enzyme obtained from a recombinant Bacillus subtilis vector pHCMC05 recombinant strain through a glycation process as a nitrogen source.
Hereinafter, the present invention will be described in detail.
The present invention provides a method for culturing a strain of the genus Bacillus or Streptomyces with the saccharification enzyme remaining after the pretreatment and saccharification of the biomass as a nitrogen source.
The biomass is preferably a woody biomass, and both woody biomass and herbaceous biomass are usable.
The pretreatment is preferably a hydrothermal treatment, and the hydrothermal treatment is preferably performed with hot water at 100 to 250 ° C. for 1 to 60 minutes.
The saccharification process is a treatment of a saccharification enzyme, and the saccharification enzyme is preferably a complex enzyme including cellulase or cellulase.
The Bacillus subtilis is preferably Bacillus subtillis , Bacillus cereus or Bacillus thuringiensis , Bacillus subtillis is most preferable, and Bacillus subtilis is Bacillus subtilis . All Bacillus spp. Strains with resolution can be used.
The Streptomyces is Streptomyces into the nose Eli coke (Streptomyces coelicolor) or Streptomyces Arbor US subtilis (Streptomyces avermitilis ).
The protease produced in the strain of the genus Bacillus or Streptomyces can be used to digest the glycation enzyme protein remaining in the biosugar (fermentation sugar). The digested saccharogenic enzyme protein can be used as a nitrogen source, such as yeast extract, to increase the growth of the strain.
The present invention also provides a method for producing a biomass, comprising: 1) pretreating a biomass and saccharifying the saccharification enzyme; And 2) culturing a strain of the genus Bacillus or Streptomyces using the saccharification enzyme remaining after the saccharification as a nitrogen source. The present invention also provides a method for removing the remaining saccharification enzyme after saccharification.
Also, the present invention provides a method for producing a biomass comprising: 1) pretreating a biomass, And 2) culturing the strain of genus Bacillus or Streptomyces with the saccharification enzyme remaining after the saccharification process as a nitrogen source. Thereby providing a method of fermenting sugar.
In the present invention, the saccharifying enzymes which are essentially used for the pretreatment and saccharification of biomass are left after the saccharification of the enzyme to inhibit the growth of the microorganism and hinder the entire process of producing the biomolecule. The present invention is based on the discovery that the remaining saccharifying enzyme is removed by applying a strain capable of utilizing the remaining saccharification enzyme protein so as to eliminate the need for such a separate filtration step, To provide a method to do so.
As shown in one embodiment of the present invention, when a variety of microorganisms having a protein degrading ability were cultured using a saccharifying enzyme remaining after saccharification as a nitrogen source, most of the strains were inhibited from growth, whereas Bacillus subtilis was grown The present invention is to select an optimal strain capable of using a saccharifying enzyme as a nitrogen source, not all of the strains having a protein degrading ability.
Also, the present invention provides a method for producing a biomass comprising: 1) pretreating a biomass, 2) culturing a strain of the genus Bacillus or Streptomyces using the saccharification enzyme remaining after the saccharification process of the step 1) as a nitrogen source; And (3) separating or purifying the biocompound produced from the cultured medium of step (2). The present invention also provides a method for producing a biocomponent from a biomass without a separate saccharification enzyme filtration step.
In this method, the biocompound is composed of isobutanol, polyhydroxyl alkanoate (PHA), fatty acid, cadaverine, autinorhodin and avermectin. , And is preferably isobutanol.
In the above method, the strain of genus Bacillus or Streptomyces is selected from the group consisting of isobutanol, polyhydroxyl alkanoate (PHA), fatty acid and cadaverine. It is preferable to use a recombinant strain optimized to produce a biomolecule-producing metabolic pathway by genetic engineering.
As shown in one embodiment of the present invention, a Bacillus subtilis pHCMC05 vector recombinant strain producing isobutanol was used. This strain produced a recombinant strain using KivD, yqhD gene, pHCMC05 vector, which produces isobutanol in Bacillus subtilis. In the process, the KivD and yqhD genes derived from E. coli were amplified by PCR cloning, and the recombination vectors were constructed by ligating the vector and the gene into the ligase respectively. Recombinant pHCMC05 vector was transformed into competant cell by shock to the cell using ultrasonic wave. This recombinant strain was used for overexpression of genes that produce isobutanol. As a result of the production of isobutanol from a biosugar substrate containing the saccharogenic enzyme protein remaining after the saccharification process, the growth of the strain and the production of isobutanol are both remarkably excellent. Thus, But it has identified how to efficiently produce bio-products.
Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
< Example 1> Pretreatment of cellulose and Glycation After the process Glycation Identification of residues of enzymes
The lignocellulose of Empty Fruit Bunches (EFB) was subjected to a general hydrothermal treatment and saccharification process. Pressure reactor and then subjected to hydrothermal treatment at 160 to 230 ° C for 0.001 to 60 minutes depending on the temperature. Thereafter, only the liquid phase was separated using a solid-liquid separation method such as centrifugation or pressure filtration. Thereafter, cellulose was saccharified using a cellulase through a washing step to obtain a sugar including glucose (see Korean Patent No. 10-1636132). The bio-sucrose composition was analyzed and shown in Table 1 below. Using hydrothermal treatment, it was confirmed that the well known toxic substance furfural was hardly produced, and that the amount of protein due to the remaining enzyme was as high as 1.9%.
HPLC (PerkinElme, USA) was used for the analysis of saccharified liquid of bio-sugar. The measurement conditions were a flow rate of 0.6 ml / min and a column oven temperature of 60 ° C. The UV detector and the RI detector were simultaneously measured at 210 nm and 50 ° C., respectively. The column used was HPX-87H, and the solvent was measured using 0.008 N HSO. Protein was measured using a BSA assay (96-well microplate reader (TECAN, USA)).
< Example 2> Protein-based Bacillus Subtilers ( Bacillus subtilis )of Growth Confirmation
In the <Example 1>, the microorganism growth experiment was carried out with the biosugar containing the saccharifying enzyme obtained through the saccharification process. The culture medium used for the growth experiments was 8% by weight of biosuque based on M9 media 1x, and the experiment was carried out without adding another nitrogen source. Here, the synthetic medium used as a control was a medium prepared by equally mixing the components except for the protein in the biosurge. Growth conditions were grown overnight at 37 ° C, and OD values were determined by measuring at 595 nm with a 96-well microplate reader (TECAN, USA). The strains used were purchased from KCTC of Microbiology Resource Center.
As a result, the protein was found to be an obstacle to growth in most strains (R. eutropha, E. coli, C. glutamicum, Lactobacillus, and S. cerevisiae), whereas Bacillus subtilis 168 Wild type was used to increase growth. Each of the strains weighed 1% by weight of the medium, and a rapid increase in growth was observed in the growth curve using only Bacillus subtilis (FIGS. 2 and 3).
< Example 3> Protein-Based Bacillus Subtilers ( Bacillus subtilis )of this Confirmation of production of isobutanol
Using the protein in biosugar, we confirmed that it could be applied not only to the growth of microorganisms but also to the production of biochemicals. The experiment was carried out using Bacillus subtilis, a recombinant strain of pathway produced by isobutanol in Bacillus subtilis, to the pHCMC05 vector.
The production of isobutanol with the biosugar as a substrate was carried out using the Bacillus subtilis pHCMC05 vector recombinant strain. The culture medium was the same as that used in the growth experiment of Example 2. The culturing conditions were incubated at 37 ° C for 4 hours, and 3 mM of IPTG was added thereto to close the lid tightly to make micro aerobic conditions. After induction and incubation for 3 days, isobutanol and OD were measured. Isobutanol was separated from the chloroform layer by centrifugation for 1 minute with 1: 1 shaking with the cell supernatant using chloroform, filtered, and analyzed by gas chromatography gas chromatography, GC) (Young Lin, Seoul, Korea).
As a result, the growth of Bacillus subtilis was confirmed and it was confirmed that 40 mg / L of isobutanol was produced (FIG. 4).
Claims (13)
2) culturing Bacillus subtilis containing the remaining saccharifying enzyme after the saccharification process;
Wherein the strain is cultured without filtration of the remaining saccharification enzyme after the saccharification process.
2) culturing Bacillus subtilis containing the remaining saccharifying enzyme after the saccharification process;
Wherein the fermentation sugar is fermented without filtration of the remaining saccharification enzyme after the saccharification process.
2) culturing Bacillus subtilis containing the remaining saccharifying enzyme after the saccharification process; And
3) separating or purifying the biocompounds produced from the cultured medium of step 2);
≪ / RTI > wherein the biomass is a biomass.
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