KR20230051777A - Producing new mannanase enzyme and method for biomass saccharification using thereof - Google Patents

Abstract

The present invention relates to a biomass decomposition technology, and more specifically, to a novel mannanase, polynucleotide encoding the same, a vector containing the polynucleotide, a transformant in which the polynucleotide or the vector is inserted into a host cell, a composition containing the mannanase, a method for manufacturing the mannanase, and application products thereof. As a result of numerous studies, the present inventors have completed the present invention by developing the mannanase having a novel amino acid sequence derived from Bacillus lichenifomis (B. lichenifomis).

Description

Novel mannanase and biomass saccharification method using the same {Producing new mannanase enzyme and method for biomass saccharification using its}

The present invention relates to biomass decomposition technology, and more specifically, to a novel metase, a polynucleotide encoding the same, a vector containing the polynucleotide, a transformant in which the polynucleotide or the vector is inserted into a host cell , It relates to a composition containing the met kinase, the manufacturing method of the met kinase, and its applied products.

As the world's population continues to grow, resources become scarce and the amount of waste produced each year increases. Therefore, waste is considered as a resource, and research is being conducted on what types of renewable waste are and how to utilize them.

Among these wastes, especially coffee wastes that can be used as biomass, the amount of coffee grounds discarded is also increasing as the consumption and production of coffee increases. Coffee grounds left after coffee extraction contain various polysaccharides, and among them, the content of mannan is very high compared to other plants.

Mannan is a mannose-containing polysaccharide found in a variety of plants. Mannan is poorly soluble in an aqueous environment and, due to its physicochemical properties, produces viscous dispersions. In addition, mannan has a high water binding capacity. All of these properties cause problems in several industries including brewing, baking, animal nutrition, and laundry and cleaning applications.

For example, in plant-based diets, various β-mannans are present and, depending on their amount and nature, can impair nutrient digestion, microbial colonization and growth performance. Since enzymatic degradation of mannan reduces the viscosity of the digestate of highly water-soluble mannan and results in the production of manno-oligosaccharides from water-insoluble linear mannan present in leguminoseae, mannanase is used in all monogastric animals. increases average daily gain, feed efficiency, body weight uniformity and survival in Therefore, in the field of animal feed, such as feed for unit animals including grain diet, mannan is a factor contributing to the viscosity of intestinal contents, and thus adversely affects feed digestibility and animal growth rate. For ruminants, mannan represents a substantial component of the fiber intake, and more complete digestion of mannan can promote higher feed conversion efficiency.

In addition, mannose constituting mannan is known to have effects such as acute cystitis, urinary tract infection prevention, weight loss, diabetes prevention, and immunity promotion. However, coffee grounds contain not only mannan but also other carbohydrates including cellulose, making it difficult to produce only mannose.

In order to solve this problem, it is required to develop a technology for an enzyme system that selectively degrades only mannan in order to improve the purity of mannose.

Domestic Patent Registration No. 10-0762410

As a result of numerous studies, the present inventors Bacillus lichenifomis ( B. lichenifomis ) The present invention was completed by developing a metase having a derived novel amino acid sequence.

Therefore, an object of the present invention is a mannanase having a novel amino acid sequence derived from a B. lichenifomis strain, a polynucleotide that is a gene sequence encoding the same, a vector containing the polynucleotide, a transformant containing the vector, It is to provide a biomass saccharification method using a mannanase manufacturing method and a mannanase using a transformant.

The object of the present invention is not limited to the above-mentioned object, and even if not explicitly mentioned, the object of the invention that can be recognized by those skilled in the art from the description of the detailed description of the invention to be described later may also be included. .

In order to achieve the above object of the present invention, the present invention provides a metase consisting of the amino acid sequence of SEQ ID NO: 1.

In a preferred embodiment, the metase is expressed in the Bacillus lichenifomis strain (Accession No. KCTC 1918).

In a preferred embodiment, the met kinase has a molecular weight of 39.0 ~ 41.0 kDa.

In a preferred embodiment, the met kinase exhibits maximum activity at pH 5.5 to 7.5.

In a preferred embodiment, the met kinase exhibits maximum activity at 40 to 55 °C.

In a preferred embodiment, the met kinase has endo-beta (beta) -1,4-D- met kinase activity.

The present invention also provides a polynucleotide encoding the above-mentioned met kinase.

In a preferred embodiment, the polynucleotide consists of the nucleotide sequence of SEQ ID NO: 2.

In addition, the present invention provides a vector comprising the polynucleotide described above.

In a preferred embodiment, a transformant in which the above-described polynucleotide or the above-described vector is inserted into a host cell is provided.

In addition, the present invention comprises the step of expressing the enzyme met by culturing the above-mentioned transformant; It provides a method for preparing met kinase comprising; and recovering the expressed kinase.

In a preferred embodiment, the step of culturing and expressing E. coli Rosetta with isopropyl-β-D-thiogalactopyranoside (IPTG); and recovering the expressed metase from the cell disruption supernatant.

In addition, the present invention provides an enzyme composition comprising the above-mentioned met kinase.

In addition, the present invention provides a biomass saccharification method in which biomass, the main component of which is mannan, is decomposed into sugars by adding the above-described mannanase or the above-described enzyme composition.

In a preferred embodiment, the saccharide degraded in the biomass is one or more of mannooligosaccharides and mannose.

In a preferred embodiment, the biomass whose main component is mannan is coffee grounds.

In a preferred embodiment, the first pre-treatment step of treating the coffee grounds with an alkali metal salt solution; and a second pre-treatment step of treating the first pre-treated coffee grounds with hydrogen peroxide.

In addition, the present invention provides a feed additive for mannan saccharification comprising the above-described enzyme composition or the above-described enzyme composition.

In a preferred embodiment, the feed additive is added to the non-ruminant feed.

In addition, the present invention provides a vegetable feed with increased glycation degree of mannan containing the feed additive described above.

The above-described mannanase of the present invention has a novel amino acid sequence derived from a B. lichenifomis strain, and converts coffee grounds into one or more of mannooligosaccharides and mannose, such as converting mannan-based biomass into saccharides efficiently. Since it can be decomposed, it can be effectively used in various industries that use biomass, such as food additives and feed additives.

These technical effects of the present invention are not limited to the above-mentioned scope, and even if not explicitly mentioned, the effects of the invention that can be recognized by those skilled in the art from the description of specific contents for the practice of the invention described later included of course

Figure 1 shows the DNA sequence and amino acid sequence of the novel metase, beta (beta) -1,4-D- metase derived from B. licheniformis .
Figure 2 is a diagram showing the production process and cleavage map of a recombinant expression vector (pCold I manB) for producing beta-1,4-D-mannanase enzyme.
Figure 3 is an SDS-PAGE image of beta (beta) -1,4-D- metase derived from B. licheniformis expressed in E. coli.
4a and 4b are B. licheniformis- derived beta (beta) -1,4-D- the optimal temperature and pH result image of the mannanase.
5 is an image of a coffee waste pretreatment process.
Figure 6 is a comparison graph of saccharification of in-housing cellulase and B. licheniformis -derived beta (beta) -1,4-D-mannanase for pretreated coffee waste.
Figure 7 is a graph of comparison results of saccharification at 50 ° C and 35 ° C of B. licheniformis -derived beta -1,4-D-mannanase for pretreated coffee waste.
8 is a graph of comparison results of glycosylation by concentration of B. licheniformis -derived beta (beta) -1,4-D-mannanase for pretreated coffee waste.
9 is a schematic diagram of mannose production from coffee waste.

Terms used in the present invention are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, terms such as "comprise" or "having" are intended to indicate that there is a feature, number, step, operation, component, part, or combination thereof described in the description of the invention, but one or more other It should be understood that it does not preclude the possibility of addition or existence of features, numbers, steps, operations, components, parts, or combinations thereof.

Terms such as first and second may be used to describe various components, but the components should not be limited by the terms. These terms are only used for the purpose of distinguishing one component from another. For example, a first element may be termed a second element, and similarly, a second element may be termed a first element, without departing from the scope of the present invention.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in the present invention, they should not be interpreted in an ideal or excessively formal meaning. don't

In interpreting the components, even if there is no separate explicit description, it is interpreted as including the error range. In particular, when the terms "about", "substantially", etc. of degree are used, they may be construed as being used in a sense at or close to that number when manufacturing and material tolerances inherent in the stated meaning are given. .

In the case of a description of a temporal relationship, for example, when a temporal precedence relationship is described as 'after', 'continue to', 'after ~', 'before', etc., 'immediately' or 'directly' Including non-consecutive cases unless ' is used.

Hereinafter, the technical configuration of the present invention will be described in detail with reference to the accompanying drawings and preferred embodiments.

However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Like reference numbers used to describe the invention throughout the specification indicate like elements.

The inventors of the present invention obtained a novel mannanase from the Bacillus lichenifomis strain (Accession No. KCTC 1918), and the novel mannanase exhibits thermal stability at room temperature and at the same time shows a high reaction rate to coffee grounds, efficiently hydrolysates. It was confirmed that it could be obtained and the present invention was completed.

Thus, the metase of the present invention consists of the amino acid sequence of SEQ ID NO: 1. In particular, the mannanase of the present invention can efficiently obtain pure mannooligosaccharide as well as mannose from mannan-containing biomass in a shorter time compared to conventionally known mannanase, as can be seen in the experimental examples described later. The met kinase of the present invention has a molecular weight of 39.0 to 41.0 kDa, has endo-beta (beta) -1,4-D- met kinase activity, and has high reactivity at pH 5.5 to 7.5 and 40 to 55 ° C. characteristics were shown.

The term "mannanase" used in the present invention is defined according to what is known in the art as an enzyme having mannan endo-1,4-beta-mannanase activity, and as an alternative name, beta-mannanase and endo-1,4 - Also called mannanase, it means a mannanase enzyme that catalyzes the hydrolysis of 1,4-beta-D-mannosidic linkage in mannan, galactomannan, glucomannan and galactoglucomannan.

Mannanase according to the present invention may also include synthetic metase, as described in the specific examples of the present invention, as well as naturally inducible metase, provided that it has homology to the amino acid sequence of SEQ ID NO: 1. More specifically, the naturally inducible metase may be obtained from a Bacillus lichenifomis strain (Accession No. KCTC 1918), which may be obtained by separation and purification according to a conventional method known in the art from the strain. For example, an extracellular fraction may be obtained by centrifuging the strain culture solution to recover the supernatant, or the supernatant may be recovered and the remaining pellet may be washed, resuspended, and centrifuged to obtain an extracellular fraction, but is not limited thereto. The term "strain" includes live bacteria, dead bacteria, dried bacteria, or strain cultures. In addition, the term "culture medium" refers to the culture medium itself obtained by culturing the strain in a suitable liquid medium, a filtrate obtained by filtering or centrifuging the culture medium to remove the strain (filtrate or centrifuged supernatant), a concentrate obtained by concentrating the filtrate, and a cell disruption solution obtained by treating the culture medium with ultrasonic waves or treating the culture medium with lysozyme.

In the present invention, "mannanase" includes functional equivalents thereof. The "functional equivalent" is at least 85% or more, preferably 90% or more, more preferably 95% or more sequence homology with the amino acid sequence of SEQ ID NO: 1, and is substantially homologous to the metase according to the present invention refers to a protein that exhibits the activity of

In the present invention, the term "homology" is intended to indicate the degree of similarity with the amino acid sequence of mannanase or the nucleotide sequence of the polynucleotide encoding it, and is 85% or more, preferably the amino acid sequence or nucleotide sequence of the present invention It includes sequences having at least 90% identical sequences, more preferably at least 95% identical sequences. Comparison of such homology can be performed with the naked eye or using a comparison program that is easy to purchase. Commercially available computer programs can calculate homology between two or more sequences as a percentage (%), and the homology (%) can be calculated for adjacent sequences.

"Substantially homogeneous activity" has the same functional properties as met kinase, and in particular means having the functional properties of enzymes. In addition, the scope of the functional equivalent includes protein derivatives in which some of the chemical structures of the protein are modified while maintaining the basic skeleton and activity of the mannanase. For example, structural modifications to change the stability, storage stability, volatility or solubility of the protein of the present invention, and fusion proteins made by fusion with other proteins such as GFP while maintaining physiological activity are included therein. In addition, as fragments of proteins, polypeptides having substantially equivalent activity to mannanase form another preferred group of functional equivalents of mannanase. The "fragment" refers to an amino acid sequence corresponding to a part of a protein, which has a common element of origin, structure and mechanism of action within the scope of the present invention, and is present in mannanase or cleaved using proteases. It includes both raw and chemically cleaved.

The met kinase of the present invention may include a foreign amino acid at its N- or C-terminus, and the "foreign amino acid" is about 1 or more, about 3 amino acids that do not exist in natural (naturally occurring) met kinase. A stretch of at least about 5, at least about 6, or at least about 7 contiguous amino acids. Preferred extensions of foreign amino acids may include, but are not limited to, “tags” that facilitate purification of recombinantly produced mannanase.

Polypeptides and polynucleotides of the present invention, including mannanase, may be provided in isolated form or may be provided homogeneously.

In the present invention, the term "isolated" means that a substance is removed from its native environment (eg, its natural environment if it occurs naturally). For example, naturally occurring polynucleotides or polypeptides present in living microorganisms are not isolated, but are the same polynucleotides or polypeptides isolated from some or all coexisting materials in a natural system. Such polynucleotides may be part of a vector and/or part of a composition, and the vector or composition may be isolated so that it is not part of its natural environment. The isolated polypeptide is at least 80% pure, preferably at least 90% pure, more preferably at least 95% pure, and most preferably at least 99% pure. Purity can be determined according to methods known in the art, such as SDS-PAGE and subsequent protein staining, and protein bands can be quantified by densitometry, but are not limited thereto.

In addition, the polynucleotide of the present invention encodes a mannanase consisting of the amino acid sequence of SEQ ID NO: 1, and the polynucleotide may be isolated and / or purified.

In the present invention, the term "polynucleotide" means any single or double-stranded polynucleotide of cDNA, genomic DNA, synthetic DNA or RNA, PNAS or LNA origin, or mixtures thereof. The "polynucleotide" includes a) a nucleic acid sequence encoding the metase according to the present invention and a functional equivalent thereof, or b) hybridization with these sequences under high stringent conditions as described in known literature. All sequences are included. In addition, a base sequence having a sequence homology of at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% with the nucleic acid base sequences of a) and b) may be included. . In addition, fragments or complements of any of the polynucleotides of a) and b) above may be included.

Therefore, the polynucleotide encoding the mannanase of the present invention may have a base sequence encoding the mannanase represented by the amino acid sequence of SEQ ID NO: 1 and a base sequence homologous thereto, preferably the base sequence of SEQ ID NO: 2 or It may have a nucleotide sequence homologous to this.

The polynucleotide encoding the metase of the present invention can be obtained by a nucleic acid molecule construction method known in the art from the sequence information disclosed in the present invention. For example, a polynucleotide as a starting material can be isolated from a strain of Bacillus lichenifomis and/or prepared synthetically based on the nucleotide sequence disclosed in the present invention.

The polynucleotide encoding the mannanase of the present invention may be an isolated polynucleotide, that is, a polynucleotide that is substantially free of other base sequences such as DNA and RNA, but is not limited thereto. Polynucleotide purification methods known to those skilled in the art can be used to obtain isolated polynucleotides. Alternatively, the polynucleotide of the present invention relates to a functional polynucleotide to which a promoter, optionally a ribosome-binding site and a terminator are operably linked in the case of bacterial cells.

In addition, the vector of the present invention includes the polynucleotide according to the present invention described above.

In the present invention, the terms "plasmid", "vector" or "expression vector" are used interchangeably in the present invention and refer to a construct for in vivo or in vitro expression. . These constructs can be used to insert a polynucleotide encoding a mannanase into a host cell. In these constructs, the mannanase-encoding polynucleotide is operably linked to suitable regulatory sequences so that it can be expressed in a host cell, and when inserted into a host cell, the vector replicates and functions independently of the host genome. or, in some cases, integrated into the host genome itself. Plasmid vectors usually contain hundreds of plasmid vectors per host cell, a replication origin for efficient replication, an antibiotic resistance gene for selection of host cells inserted into the plasmid vector, and a foreign polynucleotide into which a foreign polynucleotide is to be inserted. It has a structure that includes a restriction enzyme cleavage site that allows Even if an appropriate restriction enzyme cleavage site does not exist, the vector and the foreign polynucleotide can be easily ligated by using a synthetic oligonucleotide adapter or linker according to a conventional method.

As used herein, “operably linked” means linked in such a way as to enable gene expression when appropriate polynucleotides are linked to regulatory sequences. "Recombinant" refers to a cell that replicates, expresses a heterologous polynucleotide, or expresses a peptide, a protein encoded by a heterologous peptide or a heterologous polynucleotide. Recombinant cells can express genes or gene segments not found in the wild-type form of the cell, either in sense or antisense form. In addition, recombinant cells can express genes found in wild-type cells, but the genes have been reintroduced into cells by artificial means as modified ones. A "vector" delivers a polynucleotide into a cell. A “vector” may further include any operator sequence for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences regulating termination of transcription and translation.

Vectors that can be used for expression in host cells are known in the art, and in particular suitable vectors that can be used for expression in E. coli are also known in the art. According to a specific embodiment of the present invention, pGEM T easy-manB was used as a vector, and the manufacturing process and cleavage map of the recombinant expression vector (pCold I manB) were specifically disclosed in FIG. 2, but are not limited thereto.

In addition, the transformant of the present invention is a recombinant microorganism in which the above-described polynucleotide or recombinant vector according to the present invention is inserted into a host cell.

In the present invention, the term "host cell" includes any cell containing the above-described polynucleotide or vector, and can be used for recombinant production of mannanase. As the host cell, a host cell having high polynucleotide introduction efficiency and high expression efficiency of the introduced polynucleotide is usually used. Host cells include prokaryotic or eukaryotic cells, and as recombinant microorganisms including such host cells, for example, bacteria, enzymes, fungi, etc. may be used. In the embodiment of the present invention, E. coli was used, Without being limited thereto, any kind of microorganisms may be used as long as the metase according to the present invention can be sufficiently expressed.

In the present invention, commonly known manipulation methods can be used to insert polynucleotides into host cells. For example, microprojectile bombardment, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, PEG-mediated fusion method (PEG- mediated fusion), microinjection, liposome-mediated method, in-planta transformation, vacuum infiltration method, floral meristem dipping method ), Agrobacteria spraying method can be used.

In addition, the met kinase production method of the present invention comprises the steps of culturing the above-described transformant to express the met kinase; and recovering the expressed metase. More specifically, culturing and expressing E. coli Rosetta with isopropyl-β-D-thiogalactopyranoside (IPTG); And recovering the expressed metase from the cell disruption supernatant; may include.

For (large-scale) cultivation of recombinant microorganisms, various culture methods can be applied, for example, large-scale production of expressed or overexpressed gene products from recombinant microorganisms can be achieved by batch or continuous culture methods. Batch and fed-batch culture methods are conventional and known in the art. Methods for controlling nutrients and growth factors for continuous culture processes, as well as techniques for maximizing product formation rates, are known in the microbial industry. In addition, as the culture medium, a medium composed of a carbon source, a nitrogen source, vitamins, and mitrals may be used, and may preferably include mannan as a carbon source, but is not limited thereto and may be composed according to known in the art.

In addition, the enzyme composition of the present invention includes the above-described metase according to the present invention.

The enzyme composition may be liquid or solid. The composition may include met kinase alone, may also include other proteins or enzymes together, may include additional additives to supplement the stability and / or activity of the met kinase, other proteins, or enzymes in the composition. Examples include, but are not limited to, glycerol, sorbitol, propylene glycol, salts, sugars, pH-buffers, preservatives, and carbohydrates. Liquid compositions are typically water or oil based slurries, suspensions or solvents. The solid composition can be prepared by spray-drying, lyophilisation, down-draught evaporation, thin-layer evaporation, centrifugal evaporation, conveyor drying, or from a liquid composition by a combination thereof. The solid composition can be granulated to a size suitable for application to food or feed.

The enzyme composition can be used in various industrial fields based on the characteristics of the mannanase according to the present invention, mannan decomposition, alcohol fermentation or production, food or feed processing, coffee extraction or coffee waste processing, food or feed supplements, detergents, It can be used in various fields such as processing of renewable resources or grain processing intended for biological fuel production, biofilm removal, biomass resource processing, etc., but is not limited thereto.

Various components may be added to the enzyme composition according to the present invention, for example, enzymes may be added according to the use of the composition, and the enzymes include beta-mannosidase, alpha-galactosidase, and cellobiohydrolase. , phytases, glucoamylases, alpha amylases, proteases, cellulases, and mixtures thereof.

In addition, the present invention provides a method for industrially utilizing the above-described metase according to the present invention, and in particular, it is possible to provide a method for converting biomass into sugar by adding the metase according to the present invention to biomass. there is. More specifically, in the present invention, the method of converting biomass into sugar, that is, the biomass saccharification method, can be performed by decomposing biomass containing mannan as a main component by adding the mannanase and / or enzyme composition according to the present invention. Here, the saccharides decomposed from biomass according to the biomass saccharification method of the present invention may be at least one of manno-oligosaccharides and mannose.

In the present invention, the term "biomass" means a target object containing hemicellulose, especially mannan, such as plant seeds, grains, tubers, corn (including cobs, fodder, fibers, etc.), grass (Indian grass , switchgrass, etc.), sorghum, sugarcane bagasse, etc. may be included, and plant wastes, by-products of food processing or industrial processing may also be included, but are not limited thereto.

In particular, when the biomass containing mannan as a main component is coffee grounds, as in the examples described below, a first pretreatment step of treating the coffee grounds with an alkali metal salt solution as shown in FIG. 10; And a second pre-treatment step of treating the first pre-treated coffee grounds with hydrogen peroxide; it may be obtained in the form shown in FIG.

In this way, when the coffee grounds are subjected to the first and second pretreatments, the mannanase of the present invention can efficiently obtain more pure manno-oligosaccharides as well as mannose in a shorter time.

In addition, the feed additive of the present invention may be useful for saccharification of mannan, including the above-described mannanase and / or enzyme composition.

Therefore, the feed additive of the present invention is added to the feed of non-ruminant animals such as pigs and chickens, where the efficiency of using starch or protein of grains contained in the cell wall is very low because there is no enzyme capable of decomposing the cell wall of the plant. The value of feed can be improved by saccharifying mannan, a major component of

Feed additives of the present invention may be included in an amount of 0.01 to 10 parts by weight, preferably 0.05 to 5 parts by weight, more preferably 0.12 parts by weight, based on the feed to which the metase and / or enzyme composition is added. In addition, the feed additive may additionally contain a carrier acceptable for unit animals. In the present invention, feed additives may be added as they are or known carriers and stabilizers may be added. If necessary, various nutrients such as vitamins, amino acids, and minerals, antioxidants, and other additives may be added. It may be in a suitable state such as granules, pellets, and suspensions. In the case of supplying the feed additive of the present invention, it can be supplied alone or mixed with feed for unit animals.

The feed additive of the present invention is added to the feed of a unit animal (non-ruminant), but is not limited thereto.

In this way, since the feed additive of the present invention is suitable as a feed additive for the purpose of enhancing mannan decomposition of vegetable feed, the vegetable feed of the present invention is usefully used as a vegetable feed having increased mannan glycosylation by including the above-described feed additive. It can be.

Example 1

Mannanase [beta-1,4-D- mannanase (manB)] from the B. lichenifomis strain was cloned as follows and expressed in E. coli.

1. Isolation of the metase gene

B. lichenifomis KCTC 1918 was purchased from Korea Center for Biological Resources. B. lichenifomis was cultured and genomic DNA was extracted and used as template DNA.

2. PCR and Mannanase clonal and genetic analysis

Genes were amplified by PCR using TAKARA Ex taq DNA polymerase. Forward primer (5'-gaattccacaccgtttctccggtaaacc-3') and reverse primer (5'-TCTAGATTCCACAACAGGCGTCAAAGAAC-3') were used. After insertion into Promega's pGEM T-easy vector, E. coli Top10 was transformed. E. coli transformed with the manB gene-inserted vector (pGEM T easy-manB) was selected and cultured to extract pGEM T easy-manB. By requesting DNA sequence analysis to Genotech, the mannanase (manB) gene was analyzed and the entire gene sequence analyzed is shown in FIG.

As described below, the metase gene consisting of the analyzed SEQ ID NO: 1 confirms that the metase of the present invention is a novel enzyme by confirming that it expresses an enzyme having a molecular structure of endo-beta-1,4-D-metase. did As shown in Figure 1, the metase gene (manB gene) of the present invention has a sequence of 1,080 bp in total, and was found to encode a 361 amino acid sequence.

3. Beta-1,4-D-mannanase expression and purification

The pGEM T easy-manB vector was digested with restriction enzymes EcoR I and Xba I, and then inserted into the pCold I vector digested with the same restriction enzymes to transform E. coli Top10. (FIG. 3) pColdI-manB transformed E. coli was selected, and pColdI-manB was extracted and transformed into E. coli Rosetta. Expression of the gene was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) and culturing at 17° C. for 24 hours. The cells were obtained by centrifugation at 6,000 g for 20 minutes at 4° C. and then resuspended in 20 mM phosphate buffer (pH 6.0) supplemented with 500 mM sodium chloride. The cells were disrupted using ultrasonic waves, and the supernatant obtained by centrifugation at 17,000 rpm for 20 minutes at 4 ° C was passed through a column with Ni-NTA agarose, and then 20 mM phosphate buffer containing 500 mM sodium chloride added with 20 mM imidazole. After washing with a solution (pH 6.0), beta-1,4-D-mannanase was eluted with the buffer solution to which 250 mM imidazole was added to obtain purified beta-1,4-D-mannanase.

Experimental Example 1. Analysis of metase enzyme

SDS-PAGE (sulfate-polyacrylamide gel electrophoresis) was performed under a 12.0% gel condition, and proteins were identified by Coomassie brilliant blue R-250 staining. As such, the purity and size were confirmed by SDS-PAGE, and the results are shown in FIG. 3 .

As shown in Figure 3, it was confirmed that the molecular weight of the metase of the present invention is an enzyme having a molecular weight of about 40kDa. Compared to β-1,4-metanases of microorganisms that have already been reported, the molecular weight of recombinant metanases is ManK (35.0 kDa) of strain Cellulosimicrobium sp.HY-13. and S. lividans (S. lividans) 66 of β- metase (36.0 kDa). In addition, the molecular weight of the recombinant gene was smaller than that of non-functional GH-5β-mannanase (>60.0 kDa), which is known to have a relatively large molecular weight.

Experimental Example 2. Optimum temperature and pH measurement of met kinase

The optimal reaction temperature of the metase was confirmed by measuring the activity at each temperature at 30, 40, 50, 60, 70 and 80 ℃,

Optimum reaction pH is in the range of 3.0 to 10.0 with citrate buffer (pH 3.0 to 5.5), phosphate buffer (pH 5.5 to 7.5), Tris-HCl buffer (pH 7.5 to 9.0), and glycine-NaOH buffer (pH 9.0). ~ 10.0) at a concentration of 50 mM, respectively, at 50° C. for 15 minutes to confirm the enzymatic reaction, and the results are shown in FIGS. 4a and 4b, respectively.

As a result, when reacting with coffee grounds at a temperature of 50 ° C. at pH 6.0, the degradation activity of metase for the substrate showed the maximum value (Figs. 4a and 4b).

Example 2

In order to produce mannose from coffee waste, the following procedure shown in FIG. 9 was performed.

1. Coffee grounds pre-processing step

After adding 4L of 0.3M NaOH solution to the prepared 1Kg coffee grounds, react at 100 degrees for 1 hour and wash with warm water to remove the remaining NaOH solution. Pretreatment by adding 4L of 20% H ₂ O ₂ solution to 1Kg of NaOH pre-treated coffee grounds, reacting at 100 degrees for 0.5 hour, washing at least 3 times with warm water to remove the remaining H ₂ O ₂ solution, and drying at 70 degrees. was performed. The coffee grounds subjected to the pretreatment in this way have the same shape as the photograph shown in FIG. 5 .

2. Protein quantification of cellulase and mannanase for hydrolysis

In-housing cellulase ( Trichoderma reesei 26921; self-produced cellulase) and B. lichenifomis beta-1,4-D-mannanase expressed in Escherichia coli were quantified by Bradford protein quantification method.

3. Comparative analysis of pretreated coffee grounds saccharification of in-housing cellulase and B. lichenifomis beta-1,4-D-mannanase

10% substrate (pretreated coffee grounds), in-housing cellulase and B. lichenifomis beta-1,4-D-mannanase (50mg/g substrate) were added to 0.1 M citrate buffer (pH 5.0) and incubated at 50℃ for 48 days. Time reaction was performed, and the results are shown in FIG. 6 .

From FIG. 6, as a result of saccharification of coffee grounds pretreated with in-housing cellulase and B. lichenifomis beta-1,4-D-mannanase, in-housing cellulase has excellent saccharification efficiency, but the production of glucose is high, so it is an additional step to remove glucose. It is expected that this will be necessary. It can be confirmed that B. lichenifomis beta-1,4-D-mannanase was more advantageous for the production of mannose.

4. Comparison of mannose production according to hydrolysis temperature

B. lichenifomis beta-1,4-D-mannanase was reacted with the substrate under the same conditions as above, but only at temperatures of 50 ° C and 35 ° C, and the results are shown in FIG. 7 .

As shown in FIG. 7, as a result of saccharification of coffee grounds pretreated with B. lichenifomis beta-1,4-D-mannanase at temperatures of 50 ° C and 35 ° C, the production of mannose showed little change, but the production of glucose was reduced to 1/4. Therefore, it can be seen that saccharification at 35 ° C is more advantageous for mannose production.

5. Comparison of hydrolysis by concentration of B. lichenifomis beta-1,4-D-mannanase

Under the same conditions as described above, B. licheniformis met kinase was treated at each concentration (25-200 mg / g substrate), and the glycosylation efficiency of each concentration of B. licheniformis met kinase was tested, and the results are shown in FIG. 8.

As shown in FIG. 8, it can be seen that the highest efficiency was obtained at 50 mg/g (substrate).

Although the present invention has been shown and described with preferred embodiments as described above, it is not limited to the above embodiments, and to those skilled in the art within the scope of not departing from the spirit of the present invention Various changes and modifications will be possible.

<110> Industry Foundation of Chonnam National University <120> Producing new mannanase enzyme and method for biomass saccharification using its <130> DPP-2021-0078-KR <160> 2 <170> KopatentIn 2.0 <210> 1 <211> 360 <212> PRT <213> Bacillus licheniformis <400> 1 Met Lys Lys Ser Ile Val Cys Ser Ile Phe Ala Leu Leu Leu Ala Phe 1 5 10 15 Ala Val Ser Gln Pro Ser Tyr Ala His Thr Val Ser Pro Val Asn Pro 20 25 30 Asn Ala Gln Pro Thr Thr Lys Ala Val Met Asn Trp Leu Ala His Leu 35 40 45 Pro Asn Arg Thr Glu Asn Arg Val Met Ser Gly Ala Phe Gly Gly Tyr 50 55 60 Ser Leu Asp Thr Phe Ser Leu Ala Glu Ala Asp Arg Ile Lys Gln Ala 65 70 75 80 Thr Gly Gln Leu Pro Ala Val Tyr Gly Cys Asp Tyr Ala Arg Gly Trp 85 90 95 Leu Glu Pro Glu Glu Ile Ala Asp Thr Ile Asp Tyr Ser Cys Asn Ser 100 105 110 Asp Leu Ile Ala Tyr Trp Lys Ser Gly Gly Ile Pro Gln Ile Ser Leu 115 120 125 His Leu Ala Asn Pro Ala Phe Thr Ser Gly His Tyr Lys Thr Gln Ile 130 135 140 Ser Asn Ser Gln Tyr Glu Arg Ile Leu Asp Ser Ser Thr Pro Glu Gly 145 150 155 160 Lys Arg Leu Glu Ala Met Leu Ser Lys Ile Ala Asp Gly Leu Gln Glu 165 170 175 Leu Glu Asn Glu Gly Val Pro Val Leu Phe Arg Pro Leu His Glu Met 180 185 190 Asn Gly Glu Trp Phe Trp Trp Gly Leu Thr Gln Tyr Asn Gln Lys Asp 195 200 205 Ser Ala Arg Ile Ser Leu Tyr Lys Arg Leu Tyr Val Lys Ile Tyr Asp 210 215 220 Tyr Met Thr Lys Thr Arg Gly Leu Asp His Leu Leu Trp Val Tyr Ala 225 230 235 240 Pro Asp Ala Asn Arg Asp Phe Lys Thr Asp Phe Tyr Pro Gly Ala Ser 245 250 255 Tyr Val Asp Ile Val Gly Leu Asp Ala Tyr Phe Asp Asp Pro His Ala 260 265 270 Ile Asp Gly Tyr Asp Gln Leu Thr Ser Leu Asn Lys Pro Phe Ala Phe 275 280 285 Thr Glu Val Gly Pro Gln Thr Thr Asn Gly Gly Leu Asp Tyr Ala Arg 290 295 300 Phe Ile His Ala Ile Lys Glu Lys Tyr Pro Asn Thr Thr Tyr Phe Leu 305 310 315 320 Ala Trp Asn Asp Gly Trp Ser Pro Ala Val Asn Lys Gly Ala Asp Thr 325 330 335 Leu Tyr Leu His Pro Trp Met Leu Asn Lys Gly Asp Ile Trp Asp Gly 340 345 350 Gly Ser Leu Thr Pro Val Val Glu 355 360 <210> 2 <211> 1083 <212> DNA <213> Bacillus licheniformis <400> 2 gtgaaaaaaa gcatcgtttg ctccatcttc gcgctgctcc ttgcttttgc cgtttcgcaa 60 ccgagctacg cacacaccgt ttctccggta aacccgaatg cccagccgac gacgaaagcg 120 gtgatgaact ggcttgccca cctgcccaat cggacggaaa atcgggtaat gtccggggca 180 ttcggaggat acagccttga cacattctcg ctggctgaag ccgaccggat caaacaggca 240 acaggacagc tgccagccgt atacggctgc gattatgcaa gaggatggct ggagccggag 300 gagatcgccg atacgattga ctacagctgc aacagcgatt tgatcgcata ctggaaaagc 360 ggaggcatac cgcaaatcag cctgcacctc gcaaaccccg cgtttacttc gggtcattat 420 aaaactcaga tttcgaacag ccagtatgag agaattttag attcttccac acccgaagga 480 aaacggcttg aggcgatgct gagcaaaatc gccgatggcc ttcaggagct tgaaaatgaa 540 ggtgtgcccg ttctgttcag accccttcac gaaatgaacg gcgaatggtt ctggtgggga 600 ctgacgcaat ataatcaaaa agacagcgcg agaatctcct tgtacaaacg gctctatgtg 660 aaaatctatg actatatgac aaagacaaga ggcttggatc atctgttgtg ggtgtatgcg 720 cctgatgcca acagagactt taaaacagac ttttatccgg gcgcatcata tgttgacatc 780 gtcgggcttg acgcttattt tgatgacccg cacgccattg atggctacga tcagctcaca 840 tctctgaaca agccgtttgc ctttacagag gtcgggccac agacgacaaa cggcgggctg 900 gattacgcgc ggtttatcca tgcaatcaaa gagaaatacc cgaatacgac gtacttcctg 960 gcgtggaacg atgggtggag ccctgctgg aataagggag cggacaccct ctatcttcat 1020 ccatggatgc tgaataaggg agacatctgg gacggcggtt ctttgacgcc tgttgtgggaa 1080 taa 1083

Claims (20)

Met kinase consisting of the amino acid sequence of SEQ ID NO: 1.
According to claim 1,
The met kinase is met kinase, characterized in that expressed in the Bacillus lichenifomis strain (Accession No. KCTC 1918).
According to claim 1,
The met kinase is characterized in that it has a molecular weight of 39.0 ~ 41.0 kDa.
According to claim 1,
The met kinase is met kinase, characterized in that exhibits the maximum activity at pH 5.5 ~ 7.5.
According to claim 1,
The met kinase is met kinase, characterized in that exhibits the maximum activity at 40 ~ 55 ℃.
According to claim 1,
The met kinase is endo-beta (beta) -1,4-D- met kinase, characterized in that having met kinase activity.
A polynucleotide encoding the kinase of claim 1.
According to claim 7,
The polynucleotide is characterized in that the polynucleotide consists of the nucleotide sequence of SEQ ID NO: 2.
A vector comprising the polynucleotide of claim 7.
A transformant in which the polynucleotide of claim 7 or the vector of claim 9 is inserted into a host cell.
Culturing the transformant of claim 10 to express the met kinase; and
Recovering the expressed kinase; met kinase production method comprising a.
According to claim 11,
culturing and expressing E. coli Rosetta with isopropyl-β-D-thiogalactopyranoside (IPTG); And the step of recovering the expressed metase from cell disruption supernatant; metase manufacturing method characterized in that it comprises a.
Enzyme composition containing the kinase of claim 1.
A biomass saccharification method in which biomass, the main component of which is mannan, is decomposed into sugars by adding the mannanase of claim 1 or the enzyme composition of claim 13. 15. The method of claim 14,
The saccharide decomposed from the biomass is a biomass saccharification method, characterized in that at least one of manno-oligosaccharides and mannose.
15. The method of claim 14,
Biomass saccharification method, characterized in that the biomass, the main component of which is mannan, is coffee grounds.
17. The method of claim 16,
A first pretreatment step of treating the coffee grounds with an alkali metal salt solution; and a second pre-treatment step of treating the first pre-treated coffee grounds with hydrogen peroxide.
A feed additive for mannan saccharification comprising the enzyme composition of claim 1 or the enzyme composition of claim 13.
According to claim 18,
The feed additive is a feed additive, characterized in that added to the feed of the unit animal (non-ruminant).
Vegetable feed with increased glycation degree of mannan containing the feed additive of claim 18.

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