KR20100091289A - NEW OAT β-GLUCOSIDASE MUTANTS WITH IMPROVED SUBSTRATE SPECIFICITY BY A MODIFICATION OF AGLYCONE BINDING SITE AND A METHOD OF MANUFACTURING THEM - Google Patents

Abstract

PURPOSE: An oat beta-glucosidase mutant containing mutated AsGlu1 is provided to improve specificity to flavonoid-beta-glucoside substrate. CONSTITUTION: A mutated AsGlu1 of AsGlu1S186V, AsGlu1H197F, or AsGlu1F371W has improved specificity to flavonoid substrate by mutation of aglycon binding site. A mutated beta-glucosidase includes the mutated AsGlu1 of AsGlu1S186V, AsGlu1H197F, or AsGlu1F371W. The flavonoid substrate is isoflavon, flanonone, flavonol, or flavone. The isoflavon is genistin, daidzin, or glycitin. The flanonone is naringenin-7Glc. The flavonol is kaempferol-7-Glc or quercetin-3-Glc.

Description

Novel oat β-glucosidase mutants with improved substrate specificity by a modification of aglycone binding site and a method of manufacturing them }

The present invention, oat β with improved substrate specificity (substrate specificity) to the deformation of the aglycone-binding site-relates to-glucosidase variant-containing oat β-glucosidase of AsGlu1 subunit variants and this variant. The present invention also relates to a method of obtaining these variants.

Polyphenolic compounds (polyphenolic compounds) are one of the most abundant substances in plants and are generally produced by the secondary metabolism of plants and are mainly attached to sugar (glycoside), but in some cases aglycone (Karakaya, 2004). The reason why polyphenol compounds are attracting attention in recent years is that they have anti-oxidation function, that is, anti-mutation, anti-cancer and anti-inflammatory activity in vivo, and are expected to contribute to maintaining health and preventing diseases ( Grimmer et al. , 1992; Hollman and Katan, 1997; Parr and Bolwell, 2000). In addition, polyphenolic compounds act to lower blood cholesterol levels by preventing cholesterol from being absorbed into the digestive tract (Bose et al. , 2008).

Types of Flavonoids

Flavonoids are the largest group of polyphenol compounds and are pale yellow or yellow pigment compounds in fruits and vegetables. Flavonoids are also called 'vitamin P' and are widely distributed in fruits (apples, lemons, oranges), vegetables (tomatoes, cabbage, potatoes, onions), beverages (tea, wine), shells of plant stems and roots, and pollen. It is a water soluble chemical that has an important effect on the color and flavor of food (Bravo, 1998). They have diphenyl propane (C ₆ -C ₃ -C ₆ ), in which two aromatic rings are bonded through three carbon chains, as base skeletons, and glucoside (also known as glucoside; And glycosides) (Karakaya, 2004). The A and B rings of flavonoids are produced by different synthetic pathways, respectively, and the A ring is structurally located at position 5 and 7, and the B ring is located at position 4 ', 3' 4 'or 3' 4 '5'. OH) (Croft, 1998).

Basic structure of flavonoids

Flavonoids are flavones, flavonols, flavanones, flavanols, anthocyanidins, isoflavones according to the heterocyclic C-ring. It can be divided into six (Fig. 1), and more than 5,000 compounds belonging to this flavonoid subclass has been identified (Harbourne, 1993). Flavonoids are relatively small in animals and present in many plant leaves, flowers, roots, fruits, and stems. Especially, dried green tea leaves contain about 30% of the weight of green tea leaves (Wang et al., 2000). . The formation of flavonoids in plants is influenced not only by species diversity but also by various factors, including light conditions, germination maturation, storage, and so on. Cherry tomatoes, for example, contain six times more quercetin than regular tomatoes, due to the properties of flavonols that are synthesized in tomato skin and have a small size of hard skin ( Duthie and Crozier, 2000). In addition, there is a marked difference in procyanidin content in apples depending on the species (Hammerstone et al., 2000). Flavonoids exist in the form of glycosides, most of which are combined with sugars in plants, and are estimated to vary from about 23 to 1000 mg per person per day (Kuhnan, 1976; Hertog et al. , 1993; Leth and Justesen, 1998).

Flavonoid Function

In nature, there are various β -glucosides and glucose-free active aglycones have various drug activities (Table 1). Among them, flavonoids are known to have a wide range of antioxidant capacity. Vitamin E, widely known as a fat-soluble antioxidant, removes free radicals only from the fat-soluble part of the cell (cell membrane), while vitamin C, a representative component of water-soluble vitamins, removes free radicals from the water-soluble part of the cell (inside and outside of the cell). (Janisch et al. , 2005). Flavonoids, on the other hand, have many advantages, such as water solubility, fat solubility, or intermediate levels, which can exert antioxidant functions in various parts of cells (Birt et al. , 2001).

In general, the term 'antioxidative action' refers to inhibiting or incapacitating the action of representative components of oxidizing substances that cause aging or disease in the human body or skin. Oxidizing materials include oxygen radicals in which stable oxygen molecules are destabilized, lipid peroxides in oxidation forms of lipid components, and hydroxyl radicals. There are various free groups such as antioxidants. (Miller, 1994). Flavonoids such as quercetin and rutin, which can act as antioxidants, act as metal chelates that block many harmful heavy metals. Especially, 4-keto groups on the pyrone rings and flavonoids with B rings are effective antioxidants. Act as an agent (Thompson et al. , 1976; Park et al. , 1998).

Recently, flavonoids have been reported to act as antiviral, antihistamine, antioxidant, inhibit capillary permeation, lower blood pressure, insecticide, hepatocyte protection, lower blood sugar, etc. in the body (Hollman and Katan, 1997; Parr) and Bolwell, 2000). In addition, there is a growing interest in the development and utilization of flavonoid materials as it is known that it reduces the risk of cancer and inhibits the oxidation in vivo that causes all diseases. Many studies have shown that flavonoids have various effects on humans, but only a few representative flavonoids have been studied (Larkin et al. , 2008). Genistein in beans (Sakai and Kogiso, 2008), quercetin (Slimestad et al., 2007) found in apples and onions, red wine and grape seed extract, and proanthocyanidin, abundant in pine bark (Sovak) , 2001), catechins found in green tea (Babu and Liu, 2008), rutin and hesperidine found in citrus (Benavente-Garcia and Castillo, 2008) are representative flavonoids.

Recent studies have reported a significant decrease in blood glucose levels after taking cacao liquor proanthocyanidins in obese and diabetic rats (Tomaru et al., 2007). Proanthocyanidins are an antioxidant flavonoid that contains about 72% polyphenols. These results suggested that flavonoids have an effect on reducing blood sugar levels. In addition, flavonoids in green tea and black tea have been reported to prevent lung cancer (Cui et al., 2008). People who eat fruits and vegetables daily or drink black tea and green tea are at lower risk for lung cancer because of the increased intake of flavonoids from food. In addition, the most prophylactic flavonoids were found to be catechins (strawberry, green tea, black tea), camphorol (apple), quercetin (soybean, onion, apple) and the like.

It is known that the incidence of several diseases, including osteoporosis and cancer, is low in Asians who consume soy-based traditional Jang food (Hirayama et al. , 1982). Since then, it has been reported that soy isoflavones play an important role in the inhibition of osteoporosis, breast cancer and prostate cancer (Kennedy, 1995; Xu et al. , 1996). Soybeans contain 0.1-0.3% of isoflavonoids such as Genistein, Daidzein, Glycitein, and most of them (more than 80%) are present in glycosides ( β- D-glucoside). Among them, genistein (4,5,7-trihydroxy isoflavone) and its glycoside, genistin (genistein-7- β -D-glucopyranoside), account for 55% or more (Murphy et al., 1982). Genistein is called phytoestrogen because it is similar in structure to the female hormone estrogen.It suppresses menopausal syndrome, which is common in women in their 50s and 60s, and inhibits calcium resorption. Inhibiting secretion has the effect of inhibiting the development of osteoporosis and leukemia (Blair, et al. , 1996). In other words, it works competitively with estrogens to inhibit breast and prostate cancers caused by excess estrogens.

β Glucosidase Functions and Characteristics

β-glucosidase (β -D-glucoside glucohydrolases; EC 3.2.1.21) is produced in E. coli, fungi, mammals, aryl from all living things such as plants -, alkyl-β-glucoside, cells, various substrates such as agarobiose (cellobiose) As an isoenzyme that hydrolyzes (Woodward and Wiseman, 1982). Their functions are so diverse that they play an important role in biomass conversion, such as degradation of cellulose or cellobiose in fungi and E. coli (Wood and Bhat, 1988), and lysosomes found in humans (Wood and Bhat, 1988). lysosomal β -glucosidase is associated with Gaucher's disease (Beutler, 1992). In particular, plants hydrolyze β -glucosides such as auxin, gibberellin, and cytokinin to activate phytohormones (Estruch et al., 1991), or cyanogenic. Hydrolysis of glucosides to generate HCN (Poulton, 1988). Plant β -glucosidase is a protective mechanism against pests (Bell, 1981; Poluton, 1990), plant hormone activation (Brzobohaty et al., 1993), lignination (Dharmawardhana, 1995), and cell wall catabolism (Leah et al). , 1995).

Catalytic reaction of β -glucosidase occurs through a double displacement mechanism in which the anomeric configuration of the reducing product, the reaction product, is maintained before and after the reaction (Burmeister et al. , 1997). At the active site of β -glucosidase, two carboxylates (glutamate / aspartate) constitute a catalytic site, one acting as an acid / base reaction [ ¹⁸⁰ TFNEP ¹⁸⁴ ] One acts as a [ ³⁹⁷ Y (I / V) TENG ⁴⁰¹ ] nucleophile. The mechanism proceeds in two stages with a typical acid / base catalysis. In the first step, protonation is carried out to the substrate by an acid catalyst, which is attacked by the carboxylate group of the enzyme on the opposite side of the sugar ring, thus removing the aglycone portion. And a covalent glycosyl-enzyme intermediate is formed, with the anomeric batch reversed. In the second step, the glycosyl-enzyme is hydrolyzed by the water molecule to fall off into the glucose form with the substrate medium.

Therefore, enzyme specificity for various substrates is determined by the enzyme binding site (aglycone binding site), and exists in various forms of β-glucosidase depending on the type of aglycone.

In addition to the human small intestine, there are abundant cytosolic β -glucosidase (CBG) of a wide range of specificity in the liver, kidney and the like of mammals. Let human cytosolic β- gluconate dehydratase (hCBG) are phytoestrogens (phytoestrogen), simple phenolic compounds, cyanogen (cyanogen) of β - glucoside diet containing xenobiotic (dietary xenobiotic) β - glucoside and various aryl - β -Glucosides were hydrolyzed (Daniels et al., 1981). They showed high specificity for 7-chain flavonoid glucoside substrates (isoflavones, flavonols, flavones, flavonones) (Berrin et al., 2002). Due to these characteristics, hCBG was thought to be involved in xenobiotic metabolism, but its in vivo function is not yet known. Plant endophytic bacteria ( Pseudomonas ZD-8) have been identified for isoflavone conjugated hydrolysis β -glucosidase (ICHG) that is specific for these 7-chain isoflavones and flavone glucosides. (Yang et al., 2004). In addition, lactase phlorizin hydrolase (LPH) (EC 3.2.1.23 and 62) present in the human small intestine hydrolyzed flavonol and isoflavone glucosides (Day et al., 2000),

Plant β -glucosidase belongs to line 1 of the 69 glycosyl hydrolase (EC 3.2.xx) lines classified according to amino acid sequence. Line 1 glucosidase forms a ( β / α ) ₈ barrel form of protein tertiary structure, in which different secondary structures are inserted between the β / α units and the C-terminus of the strand. The length of the loop connecting the N-terminus of the helix and the helix is generally longer than the length of the loop connecting the C-terminus of the helix and the N-terminus of the strand. The lengths of the loops connecting the 6 strands and the 6 helix vary, which is known to play an important role in binding to the substrate (Sanz-Aparicio et al ., 1998).

These β shown in a plant-glucosidase are used to form a variety of oligomers (oligomer), look at the crystal structure thereof 1PBG, Lactococcus lactis (Lactococcus lactis) phospho-β-galactosidase (β -galactosidase phospho- ) Forms monomers (Wiesmann et al ., 1995), 1E1E, maize ( Zea mays ) β -glucosidase; 1 CBG, white clover ( Trifolium repens ) lanamar race linamarase; 1MYR, white mustard ( Sinapis alba ) myrosinase forms dimers (Czjzek et al ., 2001; Barrett et al ., 1995; Burmeister et al ., 1997), 1 GOW Sulfolobus solfataricus β -glucosidase; 1QVB, Thermosphaera aggregans β -glucosidase forms tetramers (Aguilar et al ., 1997; Chi et al ., 1999), 1BGA, Bacillus polymyxa β -glucosidase forms an octamer (Sanz-Aparicio et al ., 1998). In particular, β -glucosidase (1GOW) of Sulfolobus solfataricus ( Sulfolobus solfataricus ) is involved in the dimerization of loops between barrels 1, 3 and 4, and loops between barrels 7 and 7. The C-terminus has ionic and hydrogen bonds, and the C-terminus plays an important role in the structural stability of the entire protein. In addition, several oligomers exist in an overlapped form, and linarace of para rubber tree ( Heviea brasiliens ) and cassava ( Manihot esculenta ) are composed of monomer subunits. (Selmar et al ., 1987; Sermsuvityawong et al ., 1995). 1QOX, Bacillus circulans sp. Alkalophilus β -glucosidase, when octamer subunits overlap, forms long chains during crystal formation (Hakulinen et al ., 2000 The structure shows that the ionic bonds and hydrogen bonds between the loops and helixes 6 and 7 between the barrels 5 and 5 are involved in the dimerization, and these four dimers are the ionic bonds between the loops between the barrels 7 and 8. And octamers between the barrel 1 and the helix by hydrophobic interaction at the loop, C-terminus and N-terminus. In the crystalline state, ionic bonds between the 2 and 3 helices form a long chain.

oat β Structure and Function of Glucosidase

Oat β -glucosidase has been identified in the form of a stromacentre, in which cellulose extends in all directions (Gunning et al ., 1965). This structure has a unique fibrous protein-like long fibrillar multimer structure with a molecular weight of tens of millions of completely different from the aforementioned plant β -glucosidase (Kim et al ., 1998, 2000). It breaks down the steroid saponin-based avenacoside and converts it to 26-desglucoavenacoside, which inhibits mold growth (Nisius, 1988). I.e. the presence of oats for the chlorophyll β - glucosidase is not applied to the substrate which is located in the vacuole is considered as the reaction takes place when the plant tissue destroyed by the invasion of pathogens, oat β - glucosidase in vivo function of plants It is known to be involved in the defense mechanism of the.

Hydrolysis of Avenacoside B to 26-Desglucovenacoside B by Oat β -Glucosidase.

This long fibrillar multimer of oat β -glucosidase consists of two isozymes of type I and type II. Previous studies using cryo-electron tomography revealed the structure of small-fiber multimers of oat β -glucosidase and hexamers, the basic units of hexamers. Is a donut-shaped ring structure consisting of six monomers, in which the two trimers overlap to form a three-fold symmetry (FIG. 2). Each monomer is a 60 kDa protein consisting of AsGlu1 and AsGlu2 subunits, the basic unit of type I consists of homo-hexamer (AsGlu1), and type II is hetero-hexamer. (AsGlu1 and AsGlu2) (Kim et al ., 1996). And these structures overlap to form giant multimers, representing a unique stromacentre structure in chlorophyll (Nisius and Ruppel, 1987). These proteins exist in a very stable form, but type I is more stable than type II, type I is expressed in herbaceous leaves and type II is organ specific only in cotyledon (Kim et al ., 1996, 1998). However, when AsGlu1 and AsGlu2 genes were expressed in Escherichia coli, AsGlu1 formed a type I multimer with enzymatic activity, whereas AsGlu2 formed only a dimer. This means that AsGlu1 plays an important role in forming multimers (Kim et. al ., 2000).

Plant isoflavonoids, triterpenoid saponins, and steroid saponins are often present in the inactive form of β -glucoside. When glucose is removed through hydrolysis of certain β -glucosidase, the active form Aglycone is produced. For example, since inactive form of glucoside is inactive and does not absorb well in the body, it is essential to remove glucoside and convert it into active genistein. Is β -glucosidase with substrate specificity. Recently, studies have shown that β -glucosidase in the intestine converts genistin to genistein (Day et al ., 1998; Izumi et al ., 2000). There was this. Therefore, effective to hydrolyze the β that the genistin - it can be said that the glucosidase development is imperative in-β-glucosidase.

Oat β -glucosidase maintains stability by forming small-fiber multimer structures with molecular weights of tens of millions (Nisius, 1988). It is a functional material that maintains enzyme activity. Oat β-glucosidase is ρ-nitrophenyl-β -D- glucopyranoside and ρ-nitrophenyl-decomposing β -D- galacto pyrano seed singer but ρ-nitrophenyl-α -D- gluconic nose It has a typical β -glucosidase activity that does not degrade lanoxide. In particular, Genistin has a substrate similarity to the in vivo substrate avenacoside, although the enzyme activity of oat β -glucosidase on the two substrates differs in about 25-fold K _m values (Kim et al ., 2005), which is a significantly higher enzymatic activity compared to other β -glucosidases (Table 2).

For this reason, there is a need for the development of novel β -glucosidase variants capable of more effectively hydrolyzing the various types of glycoside flavonoid- β -glucosides present in large quantities in plants by changing the substrate specificity of oat β -glucosidase. do.

In order to solve the above problems, the present inventors analyze and confirm the aglycon binding site in the AsGlu1 subunit of wild type oat β -glucosidase, and site specific mutagenesis. -directed mutagenesis was used to modify the aglycone binding site to change the specificity for flavonoid substrates and to isolate AsGlu1 mutants with enhanced specificity for these substrates.

Through this work, the inventors have confirmed that the following AsGlu1S186V, AsGlu1H197F and AsGlu1F371W variants exhibit improved specificity for flavonoid substrates, and as a result, the hydrolysis efficiency for the flavonoid substrates is improved:

AsGlu1S186V: Substituted Ser ¹⁸⁶ → Val in the AsGlu1 subunit.

AsGlu1H197F: His ¹⁹⁷ → in AsGlu1 subunit Substituted with Phe.

AsGlu1F371W: Substituted by Phe ³⁷¹ → Trp in the AsGlu1 subunit.

Based on these results, the present invention devises AsGlu1 variants selected from AsGlu1S186V, AsGlu1H197F, or AsGlu1F371W, in which the aglycon binding site is modified to improve specificity for flavonoid substrates.

In addition, the present invention provides an oat β -glucosidase variant with improved specificity for the flavonoid substrate, including AsGlu1 variant selected from AsGlu1S186V, AsGlu1H197F, or AsGlu1F371W.

In addition, the present invention contemplates a method for obtaining AsGlu1 variants with improved specificity for flavonoid substrates by modification of the aglycone binding site, the method comprising the following steps:

a). Identifying an aglycone binding site in the AsGlu1 subunit of wild type oat β -glucosidase;

b). replacing at least one amino acid residue with a site-directed mutagenesis in the aglycone binding site identified in step a);

c). identifying substrate specificity in the variant substituted in step b);

d). Isolating variants exhibiting improved specificity for the flavonoid substrate, wherein in step b) the amino acid residue is selected from the polar residues Ser186, Phe371, or His197.

In the present invention, flavonoid substrates include, but are not limited to, isoflavon, flavonone, flavonol, or flavone. In the present invention, isoflavones include, but are not limited to, genistin, didzin, or glycidin. Flavonones include, but are not limited to naringenin-7-Glc. Flavonols include, but are not limited to, kaempferol-7-Glc or quercetin-3-Glc. Flavones include, but are not limited to, apigenin-7-Glc.

AsGlu1 variants according to the present invention and oat β -glucosidase variants comprising these AsGlu1 variants have improved specificity for various types of glycoside flavonoid- β -glucoside substrates present in large quantities in plants, which more effectively hydrolyze the flavonoid substrates. Can be disassembled.

Experiment method

1. Material

1.1 Reagent

Pfu DNA polymerase, an enzyme required for genetic engineering, is used in Stratagene (La Jolla, Calif., USA), while Eco Ri, Hind III, and T4 DNA ligase are used in conjunction with Fermentas Inc. (Hanover, MD, USA). In Promega (Madison, WI, USA), the MUGlc required for the enzyme activity assay was purchased from Sigma (St. Louis, MO, USA).

TLC plates for TLC (20 × 20 cm, silica gel 60, F254, 0.25 mm), methanol, chloroform, acetone, acetic acid were obtained from Merck Co. (Darmastade, Germany), and ρ -anisaldehyde was obtained from Fluka Co. (St. Louis, MO, USA), sulfuric acid was purchased from Fisher Scientific Co. (Fair Lawn, New Jersy, USA) respectively.

HA used for HA column chromatography for separation of recombinant proteins was prepared in the laboratory. Tris-base used for separation and ammonium sulfate used for protein precipitation were purchased from USB Corporation (Cleveland, OH, USA). All other reagents used for buffer and electrophoresis were It was purchased from Sigma and Bio-Rad Biochemical (Richmond, Califonia, USA).

The hexokinase and glucose-6-phosphate dehydrogenases used in the enzyme activity assay were extracted from Saccharomyces cerevisiae and adenosine 5 'from MP Biomedicals (Solon, OH 44139, USA). Triphosphate, NADP, MgCl ₂ , PGO enzymes were purchased from Sigma (St. Louis, MO, USA).

1.2 strains and plasmids

Transformant and the host for the plasmid propagation is E. coli (Escherichia coli) DH5α [ψ80 dlac ΔM15, rec A, end A, gyr A, thi -1, hsd R (r k -, m k +), sup E, rel A , deo R, Δ ( lac ZAY- arg F) U169] were used. As a host for recombinant protein production, BL21 star (DE3) [ F - omp - hsd SB gal dcm rne131 (DE3) ) purchased from Invitrogen (Carlsbad, California, USA) was used. And pET24a (Novagen) was used as a vector for expressing the protein in E. coli.

1.3 Cultivation of Oats

Oat seeds (Avaena sativa L.cv. Gary) used those purchased from New York Seed Improvement Cooperative, Inc. (Ithaca, NY) in a previous study. First, the oat seeds were sufficiently washed, soaked for 1 hour in a rock at 28 ° C., and then sprinkled uniformly on an aluminum plate (40 × 55 cm) covered with wet vermiculite, and covered with vermiculite so that the seeds were not visible. Maintaining good humidity in the rock at 27 ~ 28 ℃, sprayed a certain amount of water every day to grow for 4 ~ 5 days. When the seeds germinated and grew about 5 cm, the top 2-3 cm (coleoptile) of the seedlings were harvested and used as a sample.

1.4 Substrates Used for Enzymatic Properties

Avenacoside, an in vivo substrate used to characterize the enzymatic properties of the mutant protein, was directly isolated from oat seedlings, and ρ NPG, an in vitro substrate, was purchased from Sigma (St. Louis, MO, USA).

Flavonoid glucosides are isoflavones, genistein-7-Glc, dydzein-7-Glc, glycidin-7-Glc, flavonone naringenin-7-Glc, flavonol camphorol-3-Glc · quercetin 3-Glc, the flavone Apigenin-7-Glc was used. Isoflavones were purchased directly from LC Laboratories (Woburn, MA, USA). Naringenin-7-Glc, Camperol-3-Glc, and Apigenin-7-Glc were purchased from Prof. Ahn Joong-hoon (Department of Molecular Biotechnology, Konkuk University). Used.

2. Oats β Modeling of glucosidase

Modeling of AsGlu1 was performed using SWISS-MODEL program in ExPASY Proteomics server () with a template of maize β -glucosidase (PDB 1E1E) whose tertiary structure was identified. At this time, the structure is shown using Deep View program.

3. Mutated β Expression of glucosidase

3.1 Primer Construction and Domain Swapping and Site-Specific Mutagenesis

As for domain swapping, the C-terminal sites were replaced with AsGlu1 at amino acid positions 103, 193, 221, 425, 458, 477, and 496th based on AsGlu2. For this purpose, PCR was performed by preparing a primer so that amino acids were not changed in cGNAs of AsGlu1 and AsGlu2 (Table 3). That is, the N-terminal portion of the chimeric cDNA is a AsGlu2 template, and a sense primer (Glu2-SP) and a swapping antisense primer (SW-AP), and the C-terminal portion is a AsGlu1 template, and a swapping sense primer (SW-SP) Two fragments were made using antisense primers (Glu1-AP). The fragments thus prepared were made of full-length chimeric cDNA using sense (Glu2-SP) and antisense primer (Glu1-AP) as templates.

Mutated β -glucosidase according to the present invention was prepared through specific site mutagenesis using overlap extension PCR. The amino acid to be substituted was an aglycone binding site which is important for determining the specificity of substrate binding. Therefore, amino acids corresponding to β ₄ / α ₄ (Ser ¹⁸⁶ , His ¹⁹⁷ ) and β ₆ / α ₆ (Phe ³⁷¹ ) of AsGlu1 ( β / α ) _8- barrel structure were substituted as follows.

Wild type sequence of AsGlu1:

Ser ^{186 from} AsGlu1 → Val: S186V

His ¹⁹⁷ → Phe: H197F

Phe ³⁷¹ → Trp: F371W

N-terminal and C-terminal sites were made using mutagenesis primers (E495K, E183D, S186V, H197F, F371W-SP, -AP; Table 3) around the sites where amino acids were to be substituted. Later, the two fragments were overlapped to produce full length mutated cDNA. Genetic identification of the mutant cDNA was confirmed by sequencing at Genotech.

^* Underlined residues indicate mutation sites.

3.2 Expression Vector Preparation and Transformation into Expression Host BL21 star (DE3)

The prepared cDNA was ligated to the pET 24a vector using restriction enzymes of Eco Ri and Hind III. After transfection of the ligation reaction solution in DH5 α, it was confirmed the size of the gene inserted into the plasmid in a way to remove the soluble alkaline (alkaline lysis). This was transformed into BL21 star (DE3), a protein expression host.

3.3 Expression and Extraction of Mutant Proteins in Escherichia Coli

E. coli colonies were inoculated in 3 ml LB medium containing 50 μg / ml kanamycin and incubated at 37 ° C. until A ₆₀₀ became 0.4-0.6, then IPTG was added to a final concentration of 1 mM and 25 Expression was induced at 12 ° C. for 12 hours. After centrifugation, the strains were allowed to settle, and then suspended in a lysis buffer (50 mM Tris-HCl, 2 mM EDTA, 4 mM β -mercaptoethanol, 5% glycerol, pH 8.0) and an ultrasonic device (ultrasonicator). Ultrasonic grinding and centrifugation for 10 minutes at 10,000 x g, 4 ℃. Total cell extract (T), soluble cell lysate (S) and insoluble cell lysate (I) thus obtained were used as electrophoretic samples.

3.4 Expression of Mutant Proteins via SDS-PAGE

Extracts previously isolated were identified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE was added to protein samples in SDS-sample buffer [0.5 M Tris-Cl, pH 6.8, 10% (w / v) SDS, 50% (v / v) glycerol, 14.4 mM β -mercaptoethanol, 0.05% ( w / v) bromophenol blue] was added and boiled for 3 minutes, followed by a 10% gel according to the method of Laemmli (1970). After electrophoresis buffer [0.025 M Tris-Cl, pH 8.3, 0.195 M glycine, 0.1% (w / v) SDS] for 3 hours at 15 mA, staining solution [0.1% (w / v) Coomassie brilliant blue R-250, 40% (v / v) methanol, 10% (v / v) glacial acetic acid] for 1 hour, then bleach solution [30% (v / v) methanol, 10 Expression of the mutant protein was confirmed by decolorization with% (v / v) acetic acid, 1% (v / v) glycerol].

3.5 Enzyme Activity Assay through Native-PAGE and Zymogram

Native-PAGE was performed using SDS and β -mercaptoethanol according to the method of Laemmli (1970) using a 6% gel. Electrophoresis was carried out in a cold room for about 6 hours at a constant current of 5 mA. As in SDS-PAGE, Coomassie staining confirmed the presence of multimer formation of the mutant proteins. In addition, in order to measure the enzymatic activity of the mutant protein, the electrophoresis gel was replaced with citrate buffer (pH 5.5) three times at 10 minute intervals, and then reacted at room temperature by adding MUGlc so that the final concentration of the substrate was 2 mM. . Long wavelength UV light was confirmed by irradiation.

4. Mutated β -Purification of Glucosidase

4.1 Expression and Partial Purification of Mutant Proteins

E. coli colonies are inoculated in 3 ml LB medium containing 50 μg / ml kanamycin and incubated at 37 ° C. until A ₆₀₀ is 0.6-1.0. The cultured strains are inoculated in 800 ml LB medium containing the same antibiotics with an absorbance of 0.02. After incubation until A ₆₀₀ is 0.4-0.6, IPTG is added to a final concentration of 1 mM and expression is induced at 25 ° C. for 12 hours. The cells were allowed to settle by centrifugation at 5,000 × g, 4 ° C. for 10 minutes, and then in 80 ml of lysis buffer (50 mM Tris-HCl, 2 mM EDTA, 4 mM β -mercaptoethanol, 5% glycerol, pH 8.0). Suspension was added to lysozyme (25 ㎍ / ㎖) to react for 3 hours while stirring slowly at 4 ℃. Ultrasonic grinding was performed by an ultrasonic device for 5 minutes, and centrifuged at 10,000 × g for 20 minutes, 25 g of ammonium sulfate per 100 ml was slowly dissolved and stirred for 30 minutes. After the reaction, the precipitate obtained by centrifugation at 10,000 x g for 20 minutes was dissolved in the lysis buffer again. The precipitate suspension was dialyzed for 2 hours using Tris buffer (50 mM Tris-HCl, 5 mM EDTA, 14 mM β -mercaptoethanol, pH 7.8) and used for HA column chromatography.

4.2 HA column chromatography

The supernatant prepared prior to the HA column (2.1 x 5.8 cm) equilibrated with distilled water was adsorbed protein at a flow rate of 15 ml per hour. After washing with Tris buffer corresponding to 5 times the column volume, concentration gradient was performed using 5 mM KPB (5 mM potassium phosphate buffer, 5 mM EDTA, 14 mM β -mercaptoethanol, pH 7.8) and 20 mM KPB. Protein was eluted at a flow rate of 15 ml per hour by the method.

4.3 Activity Assay for Modified β -Glucosidase

In order to assay the activity of the isolated β -glucosidase, ρ NPG was used as a substrate and the reaction was performed in citrate buffer (pH 5.5) according to the method of Nisius and Ruppel (1987). The enzyme concentration was 0.01 μg / ml and the substrate concentration was 10 mM, followed by reaction at 30 ° C. for 15 minutes. To stop the reaction, 1 M Na ₂ CO ₃ was added in the same amount, and the activity was assayed by measuring the amount of the reaction product ρ -nitrophenol at 405 nm (ε = 18,500 M ⁻¹ cm ⁻¹ ). The activity of the enzyme on the gel was carried out in the same manner as in 3.5 above.

5. In vivo  Isolation of the avenacoside

In order to isolate the in vivo substrate of β -glucosidase, the top of oat seedlings grown on cancer for 4 days was cut into 3 cm in length and used as a sample.

To separate avenacosides, three times the volume of methanol was added, triturated in a Warning blender for 3 minutes, and stirred slowly for 30 minutes (Nisius, 1988). After centrifugation at 5,000 × g for 15 minutes, the supernatant was filtered through two layers of Miracloth and evaporated. The evaporated supernatant was again dissolved in TLC solvent CHCl ₃ : CH ₃ OH: H ₂ O = 70: 35: 5.5 (by vol).

In order to separate 26-desglucobenacoside into a glucose-free form from avenacoside, 1-fold volume of water was added, triturated in a Warning blender for 3 minutes, and then stirred slowly for 1 hour. Thereafter, three times the volume of methanol was added, and the mixture was ground again for 3 minutes in a Warning blender and stirred for 30 minutes. After centrifugation at 5,000 × g for 15 minutes, the supernatant was filtered through two layers of Miracloth and evaporated. The evaporated supernatant was dissolved in the same TLC solvent as in avenacoside.

The extracts thus separated were subjected to TLC on a silica gel 60 plate. After spraying ρ -anisaldehyde / sulfuric acid / acetic acid (1/1/48), which is only stained with steroids, and allowed to stand at 130 ° C. for 5 minutes, a band of avenacoside extract was confirmed. The locations of avenacosides A and B were identified by extracting each of the bands from the avenacoside extract and comparing the locations of the bands produced by reaction with β -glucosidase with those of the 26-desglucovenacoside extract. . The exact location of the Abenacosides A and B was extracted again from the silica gel, and the amount of glucose isolated was quantified by enzymatic method.

6. Mutated β -Stable Assay of Glucosidase

6.1 Stability Over Temperature

Ρ NPG was used as the substrate and was performed according to the method given in 4.3 above. That is, after reacting for 15 minutes at 30 ° C. in citrate buffer (pH 5.5), an equal volume of 1 M Na ₂ CO ₃ was added to stop the reaction, and the amount of ρ -nitrophenol as a reaction product was measured at 405 nm.

The optimal temperature of the mutant protein was confirmed by measuring enzyme activity in the range of 20-60 ° C. The stability to temperature was confirmed by first leaving the β -glucosidase enzyme at different temperatures (20-60 ° C.) for 30 minutes and measuring the enzyme activity according to the method given above after substrate addition.

6.2 Stability to pH

The optimal pH of the mutant protein was confirmed by measuring enzyme activity in the range of 3.0-11.0. At this time, citrate buffer in the range 3.0-5.5, phosphate buffer in the range 6.0-8.0, glycine-NaOH buffer in the range 8.5-11.0 was used as the buffer for each pH. The activity against pH was assayed under the same conditions except buffer. Stability to pH was first confirmed by leaving the β -glucosidase enzyme in different buffers (pH 3.0-11.0) for 2 hours at 30 ° C. and measuring activity by the method given above after substrate addition.

6.3 Changes in activity for various chemicals

Changes in activity for various chemicals of variant proteins were identified. Materials used were MgCl ₂ , CuSO ₄ , ZnSO ₄ , MnCl ₂ , AgNO ₃ , IAA, β -mercaptoethanol, dithiothreitol. As the substrate, ρ NPG was used, and these substances were added at a final concentration of 1 mM to confirm changes in enzyme activity.

7. Mutated β -Enzymatic properties of glucosidase

7.1 Properties for In vivo / in vitro Substrates

Avenacoside, an in vivo substrate, was used to measure the kinetics of modified β -glucosidase and binding using hexokinase and glucose-6-phosphate dehydrogenase to continuously measure the amount of glucose released through hydrolysis. A coupled assay method was used (Duerksen and Halvorson, 1958). Abenacosides A and B were mixed at a ratio of 1: 2 and, according to Nisius (1988), 0.7 mg NADP, 1.5 mg ATP, 1 mg MgSO ₄ , 2U hexokinase, in KPB buffer (pH 6.8), 1 U glucose-6-phosphate dehydrogenase was added and the reaction was performed. The enzyme concentration was 0.1 μg / ml and the substrate concentration was 5, 10, 15, 20, 30, 40, 50 μM, and after reaction at 30 ° C., A ₃₄₀ (extinction coefficient of NADPH: 6,200 M ⁻¹ · cm ⁻¹ ) and the activity change was measured continuously for 3 minutes. From the results were determined the K _m and V _max values of the enzyme using the Lineweaver-Bulk double reciprocal shown (double reciprocal plot) (Lineweaver and Burk, 1934).

In the case of ρ NPG, which is an in vitro substrate, enzyme concentration of 1 ㎍ / ml was used in KPB buffer (pH 6.8), and the concentration of substrate was 0.1, 0.3, 0.5, 0.7, 1, 3, 5, 7 mM. after reaction at ℃, a ₄₀₀ -: to measure the change in activity for 3 minutes continuously (ρ-nitrophenol extinction coefficient, pH 6.8 of 9,600 M ^-1 · ㎝ ^-1). As a result, the K _m and V _max values of the enzyme using the Lineweaver-Bulk shown were determined from double reciprocal.

7.2 Properties for Flavonoid Glucosides

Flavonoid glucosides are genistein-7-glucoside, isoflavone, diadin-7-glucoside, glycidin-7-glucoside, naringenin-7-glucoside, flavonone, camperol-3- Glucoside Quercetin-3-glucoside and flavone apigenin-7-glucoside were used. Camperol-3-glucoside was dissolved in methanol, and all other glucosides were dissolved in DMSO.

Even in this case, a coupled assay method was used to continuously measure the amount of glucose hydrolyzed and released from the substrate. 0.7 mg NADP, 1.5 mg ATP, 1 mg MgSO ₄ , 2U hexokinase, 1U glucose-6-phosphate dehydrogenase were added to KPB buffer (pH 6.8). Enzyme concentration was 1 μg / ml and substrate concentrations of isoflavone and quercetin-3-glucoside were 10, 30, 50, 70, 100, 150, 200 μM, and naringenin-7-glucoside, cAMP. For perol-3-glucoside, apigenin-7-glucoside, 1, 3, 5, 7, 10, 15, 20 μM was used. After reacting at 30 ° C., the activity change was measured continuously for 3 minutes at A ₃₄₀ (quenching coefficient of NADPH: 6,200 M ⁻¹ · cm ⁻¹ ), and from the result, the K of the enzyme was determined using the Lineweaver-Bulk double inverse plot. _m and V _max values were measured.

8. Protein Quantitation

The amount of protein was quantified by Coomassie-blue protein assay (Bradford, 1976) using Coomassie brilliant blue G-250, and BSA was used as a standard protein.

Experiment result

Identification of sites involved in multimer formation through domain swapping and site-specific mutagenesis

The subunits of oat β -glucosidase, AsGlu1 and AsGlu2, consist of 510 amino acids and these sequences show 90% identity and are very similar (FIG. 3). The secondary structure of the protein analyzed from these amino acid sequences forms a ( β / α ) ₈ barrel typical of the glucoside hydrolase strain 1. However, when each of these genes is expressed in E. coli, AsGlu1 forms a homomultimer of type I, whereas AsGlu2 forms a homodimer. And only when AsGlu1 and AsGlu2 are expressed at the same time, a type II heteromultimer is formed (Kim et. al ., 2000). In particular, unlike type I homomultimers, type II heteromultimers mostly exist as heterohexamers, which form overlapping multimers but not high molecular weight multimers. These results indicate that AsGlu1 is an important site for multimer formation.

Although AsGlu1 and AsGlu2 have high homogeneity in the amino acid sequence, structural differences exist so that sites substituted with different amino acids may be involved in multimer formation. However, in the comparison of the amino acid sequences of AsGlu1 and AsGlu2, the site of change is not limited to a part but is distributed throughout the amino acid (FIG. 3). Therefore, we tried to identify amino acids involved in multimer formation by substituting AsGlu1 for different domains using AsGlu2 forming homodimer only.

Domain swapping was performed by constructing primers such that restriction enzyme sites were added without changing the amino acids in the cDNAs of AsGlu1 and AsGlu2. AsGlu2 was substituted at 103, 193, 221, 425, 458, 477, and 496th amino acid positions, respectively. The substituted cDNA was cloned into pET24a vector and expressed as recombinant protein using BL21 star (DE3), an expression host.

The substituted β -glucosidases were all expressed as water soluble proteins with enzymatic activity. Among them, a multimer was formed from SW-1 substituted with AsGlu1 at amino acid position 103 to SW-6 substituted at 477, but SW-7 substituted at 496 formed a dimer. The site involved in the multimer formation of AsGlu2 was found to be between 477 and 495 amino acids based on AsGlu2. Comparing the amino acid sequences of AsGlu1 and AsGlu2 in this region, it is the 495th amino acid based on AsGlu2. This amino acid is thought to be an amino acid that plays an important role in multimer formation because it has different charges from AsGlu2 to glutamate (Glu, E) and AsGlu1 to lysine (Lys, K).

Accordingly, the AsGlu2 E495K variant was prepared by replacing glutamate (E), amino acid 495 of AsGlu2 with lysine (K), through specific site mutagenesis. The expressed variants formed multimers similar to AsGlu1. This suggests that the formation ability of hexamers and multimers is altered by differences in amino acid 495 relative to AsGlu2.

2. Oats β Enzymatic Properties of Glucosidase Multimers

Separation of avenacosides A and B was performed by extracting oat seedlings with methanol and then performing TLC using ρ -anisaldehyde / sulfuric acid / acetic acid (1: 1: 48) (v: v: v), which only stains steroids. The locations of avenacosides A and B were confirmed, which were extracted from silica gel and purified. Avenacosides A and B thus separated were used as substrates in vivo .

Thin layer chromatography results of hydrolysis of avenacoside to 26-desglucovenacoside by oat β -glucosidase.

The activity of β -glucosidase multimer was measured using Avenacocside A and B extracted previously. In the activity measurement using avenacoside, it was difficult to measure the amount of 26-desglucovenacoside, which is a direct product, and thus a coupled assay method was used to measure continuous changes (Duerksen and Halvorson, 1958). . That is, the released glucose was used as a substrate for hexokinase and glucose-6-phosphate dehydrogenase, and the amount of NADPH produced as a result was measured to indirectly measure the amount of glucose released by β -glucosidase. Continuous changes were measured at pH 6.8 to determine kinetic parameters [ K _m (substrate affinity), k _cat (metabolism), k _cat / K _m (catalyst efficiency)].

AsGlu1 to form a multi-Merced is K _m is 0.017 mM, the K _m AsGlu2 to form the dimer was higher about 3.3 times than AsGlu1 (Table 4). However, AsGlu2 E495K variant multimer was found to decrease K _m by multimer formation to 0.037 mM. For k _cat , AsGlu1, AsGlu2 and AsGlu2 E495K were almost similar, so no significant change between multimer and dimer was observed. In other words, k _cat / K _m, that is, the catalytic efficiency (Calytic efficiency) AsGlu1 is about 3 times higher than AsGlu2, AsGlu2 E495K was about 1.6 times higher than AsGlu2. These results show that the more the β -glucosidase forms a multimer, the higher the affinity for the substrate, so that the substrate can be more efficiently hydrolyzed.

In addition, the activity of ρ NPG was measured to confirm whether the properties of β -glucosidase multimers using in vivo substrates showed the same characteristics in in vitro substrates. The K _m of AsGlu1, AsGlu2, and AsGlu2 E495K was 4.1, 10.3, and 5.2 mM, respectively, and the K _m of the dimer was high, and K _m was decreased by the multimer formation. For k _cat is AsGlu2 2.0 (s ^-1) or, AsGlu2 E495K is the k _cat was reduced slightly to 1.05 (s ^-1) In analogy to AsGlu1. AsGlu1 was 1.6 times higher than AsGlu2, and AsGlu2 E495K was about 1.1 times higher than AsGlu2. That is, it can be seen that the substrate affinity of the multimer is higher than that of the dimer in the properties of β -glucosidase in vivo and in vitro substrate.

3. Oats β Identification of Aglycone Binding Sites of Glucosidase

The structure of plant β -glucosidase is well documented through X-ray crystallography in maize (Czjzek et al ., 2001). The model structure of oat β -glucosidase (AsGlu1) was predicted using SWISS-MODEL program using the crystal structure of maize β -glucosidase (ZmGlu1) as a template. As can be seen from the amino acid sequence above, a typical ( β / α ) ₈ barrel structure (FIG. 3), the C- and N-terminus are located close to each other, the β -strands are distributed only inside, and α- The helix is exposed to the outside, surrounding the β -strand. The two glutamate salts (Glu ¹⁸³ and Glu ³⁹⁹ ), which are catalytic sites of the enzyme active site, were located in β -strands 4 and 7 distributed therein.

Tertiary structure of the AsGlu1 monomer, wherein E183 and E399 indicate the catalytic glutamate in close proximity.

Recently, studies have been actively conducted on sites involved in substrate specificity of β -glucosidase (Verdoucq et al ., 2003, 2004). Among them, the sites involved in substrate specificity were identified through co-crystal structures (PDB 1E1E, 1E1F) bound to the substrate in ZmGlu1, and in particular, the glucose binding site and the aglycone binding site for the substrate were respectively identified. It is known to exist in isolation. Therefore, the AsGlu1 model structure was compared with the ZmGlu1 crystal structure to identify sites involved in substrate specificity of AsGlu1. First, among the enzyme active sites, the catalytic sites were the same in ZmGlu1 and AsGlu1 (Glu ¹⁹¹ · Glu ⁴⁰⁶ in ZmGlu1, Glu ¹⁸³ · Glu ³⁹⁹ in AsGlu1) (FIG. 4). Glucose binding sites to substrate were also the same in ZmGlu1 and AsGlu1 (Gln ^{38 in} ZmGlu1, His ¹⁴² , Asn ¹⁹⁰ , Trp ⁴⁵⁷ , Glu ⁴⁶⁴ , Trp ⁴⁶⁵ , Oat ³³ , His ¹³⁷ , Asn ¹⁸² , Trp ⁴⁵⁰ Glu Trp ⁴⁵⁷ · ⁴⁵⁸⁾ (Fig. 4A, B).

However, the aglycone binding sites were different in ZmGlu1 and AsGlu1. ZmGlu1 forms a hydrophobic pocket such as a slot, one of which is Phe ¹⁹⁸ · Phe ²⁰⁵ · Phe ⁴⁶⁶ and the other is Trp ³⁷⁸ and the corresponding AsGlu1 The amino acids are Leu ^190. His ^197. Ser ⁴⁵⁹ and Phe ³⁷¹ (FIG. 4A, B). That is, the aglycone binding site in AsGlu1 is more open than ZmGlu1, and this structure is consistent with the fact that avenacoside is larger than DIMBOA-glucoside, which is the substrate of ZmGlu1. When these structures were observed from the side, AsGlu1 was exposed to the outside of the aglycone binding site relatively compared with ZmGlu1. From these results, it was confirmed that the substrate specificity of β -glucosidase was determined by the aglycone properties of the substrate.

In other words, the possibility of increasing substrate affinity for various glucosides by changing the hydrophobicity of the aglycone binding site and the space of the aglycone binding site in oat β -glucosidase. Corn β -glucosidase (ZmGlu1) and sorghum β -glucosidase (ShDhr1) have a similar amino acid sequence of at least 70% (FIG. 5), although ShDhr1 has a narrow substrate specificity and ZmGlu1 has a broad substrate specificity. And cannot hydrolyze the durin, the substrate of ShDhr1 (Table 1). However, substituting the C-terminus of ZmGlu1 with a portion of the C-terminus (F466S, A467S, Y473F), which is the substrate binding site of ShDhr1, could change the substrate specificity to hydrolyze Durin. In addition, mutagenesis of the aglycone binding sites of the two enzymes resulted in a β ₄ / α ₄ loop (ZmGlu1 Phe ¹⁹⁸ , Phe ²⁰⁵ ) and β ₅ sheets (ShDhr Asn) in the ( β / α ) ₈ barrel structure. ²⁵⁹ , Phe ²⁶¹ ), a loop connecting the β ₆ / α ₆ helix (Trp ^{378 of} ZmGlu1) and β ₈ / α _{8 at} the C-terminal site (Phe ^{466 of} ZmGlu1) appeared to be important sites for substrate binding (FIG. 5).

Human cytoplasmic β -glucosidase (hCBG) is not yet known in vivo. The enzyme phytoestrogens, simple phenolic compounds, β of the cyanogen-expression xenobiotic comprising the glucoside (dietary xenobiotic) β-glucoside and aryl-β-glucoside (β -D- Foucault seed, β -D- Glucoside, β -D-galactosid, β- L-xyloxide) and weak enzymatic activity. When Val ¹⁶⁸ of the aglycone binding site was replaced with Try in hCBG, the enzymatic activity of the isoflavonoid β -glucoside was reduced by more than five times, suggesting that modification of the aglycone binding site can improve substrate specificity. Suggest.

Based on these results, the aglycone binding site present in oat β -glucosidase is Leu ¹⁹⁰ · Ser ¹⁸⁶ · His ¹⁹⁷ , β ₅ in the loop between β ₄ / α ₄ of ( β / α ) ₈ barrel structure. Asp ²⁵⁴ · Met ²⁵⁶ on the sheet, Phe ³⁷¹ in the loop between β ₆ / α ₆ and Ser ⁴⁵⁹ between the C-terminal β ₈ / α ₈ were expected (FIG. 4C, D).

4. Modification of Aglycone Binding Sites Through Specific Site Mutagenesis

Three significant sites were selected from the predicted aglycone binding site of the AsGlu1 model structure to determine changes in substrate specificity for various flavonoid glucosides. In other words, in order to increase the hydrophobicity of the aglycone binding site, Ser ¹⁸⁶ in the loop between β ₄ / α ₄ was substituted with Val. Phe ³⁷¹ in the loop between β ₆ / α ₆ was substituted to narrow the binding site. Trp and His ¹⁹⁷ in the loop between β ₄ / α _4, which can change both of these properties, was substituted with Phe ²⁰⁵ (FIG. 6).

These mutant genes were constructed by mutagenesis of specific sites using overlapping extension methods. That is, asGlu1 is used as a template, the N-terminal part is PCR using a primer (SP) of the template and the amino acid to be modified (M-AP), and the C-terminal part is M-SP and a primer (AP) of the template. Was performed. These two fragments were subjected to PCR once more using the primers of the template (SP, AP) to obtain a 1.5 kb mutant β -glucosidase cDNA (FIG. 7). Negative control produced E183D by substituting Asp for Glu ¹⁸³ , one of the catalytic sites of the enzyme active site.

5. Expression and Purification of Mutant Protein Expressed in Escherichia Coli

These mutant genes were cloned into pET24a, an expression vector, and then transformed into BL21 star (DE3), a protein expression strain, to induce protein expression at 1 mM IPTG. The expressed protein was divided into total cell extracts, soluble cell lysates and insoluble cell lysates, and SDS-PAGE was performed. As a result, all of them were expressed as water-soluble proteins (FIG. 8A). In addition, all of them formed structurally stable multimers (FIG. 8B). However, the activity of MUGlc, which is an in vitro substrate, was different. The negative control E183D had no enzymatic activity, but among the other mutant proteins, the S186V mutant protein was higher than the wild type AsGlu1, and H197F and F371W Similar activity was shown (FIG. 8C).

After confirming that all of the mutant proteins were expressed as water-soluble proteins and having a multimer having enzymatic activity, the mutant proteins were separated and purified by HA column chromatography to determine enzymatic properties. Since oat type I β -glucosidase elutes at 5 mM KPB, eluting with a slowly increasing concentration of potassium phosphate buffer (KPB) from 5 mM to 20 mM under similar conditions (FIG. 9). Mutant proteins expressed in E. coli were also mostly eluted in 5 mM KPB.

6. Modified according to the present invention β Activity Assay of Glucosidase

In order to assay the activity of the isolated and purified mutant proteins, we examined the activity of MUGlc, an in vitro substrate, and showed the same results as the assayed activity in the cell extract before separation and purification. In other words, E183D modified to inhibit the enzyme activity had no enzyme activity. Among other mutant proteins, S186V mutant protein was higher than wild type AsGlu1, and H197F and F371W mutant proteins showed similar activity. 10).

7. Modified according to the present invention β -Stability of Glucosidase

First, the optimum temperature of the separated and purified mutant proteins was confirmed between 20 ~ 60 ℃, temperature stability was reacted for 30 minutes at different temperatures (20 ~ 60 ℃), and then the activity was measured by the addition of a substrate. The optimum temperature of all enzymes was found to be 35 ° C., the activity at the optimum temperature was highest in S186V mutant protein, similar in H197F mutant protein and lowest in F371W mutant protein compared to wild type (FIG. 11). These enzymes showed stability only at about 30 ° C., showed an activity of about 80% at 35 ° C. or higher, but rapidly decreased at 40 ° C. or higher, and completely disappeared at higher temperatures. In addition, the difference in activity in the temperature stability of these mutant proteins was identical to the difference in activity at the optimum temperature.

Second, the optimum pH was confirmed between 3.0 and 11.0, the pH stability was reacted for 2 hours in a buffer having a different pH (3.0 ~ 11.0), and then the activity was measured by the addition of a substrate. The activity profile (activity profile) of the pH of these enzymes showed a bell shape, the optimum pH was found to be 5.5, it was confirmed that the most stable at pH 5.5 ~ 6.5 (Fig. 12). The activity difference of the mutant proteins according to pH was similar to that of the temperature-dependent activity, and showed the difference of activity in the order of S186V> H197F> wild type> F371W mutant protein.

Third, changes in activity for various chemicals were identified. Wild type (AsGlu1) showed no significant change in activity for these chemicals (Table 5), indicating that this is a structurally very stable enzyme. In the case of the mutant protein, S186V showed higher overall activity than the wild type, which is believed to increase the activity of these chemicals by structurally increasing the enzyme activity in the same manner as in the above activity assay. The H197F mutant protein increased the activity of these chemicals compared to the wild type, but showed no specific activity against metal chelators and reducing agents. And the F371W variant protein showed somewhat lower activity against these chemicals.

The reactant concentration used was 1 mM.

^a Activity in the absence of ^a reactant was considered 100% (specific activity = 1312 U / mg)

8. Modified according to the present invention β -Enzymatic properties of glucosidase

The substrate specificity of the mutated β -glucosidase was enzymatically analyzed using Avenacocsid B, p NPG and various flavonoid glucosides. The combined assay method described above was used to measure continuous changes, and the specific activity and kinetic parameters of these enzymes were measured.

1) Specific activity of the modified β -glucosidase according to the present invention

First, the enzymatic activity of various substrates in multimer formation was as follows. The specific activity of the multimer AsGlu1 was highest at 428.87 × 10 ³ unit · mg ⁻¹ for the in vivo substrate Avenacoside B. The activity of the in vitro substrate ρ NPG was 325 higher than that of Avenacoside B. It was found to be reduced fold (Table 6). In the case of flavonoid glucoside, the activity of genistein-7-Glc (genistin) and apigenin-7-Glc (apigetrin) was 5.8 times and 9.4 times lower than avenacoside, respectively, but compared to ρ NPG and other flavonoid glucosides. Its activity was high. In addition, the activities of Dadezin-7-Glc (daidzin), Glycithin-7-Glc (glycitin), and Camperol-3-Glc (astragalin) are almost similar, and quercetin-3-Glc in flavonoid glucoside (isoquercitrin) showed the lowest activity and no enzymatic activity against naringenin-7-Glc (Prunin).

Similar results were shown for AsGlu2 and AsGlu2 E495K mutant proteins (Table 6). That is, activity against avenacoside B was high and activity against ρ NPG was lower than that of avenacoside B. While the activity of isoflavone, zenithine, is similar to that of AsGlu1, the activity of diadzin and glycidin is about 2-3 times higher than that of AsGlu1 multimer and AsGlu2 E495K mutant multimer. High. The activity against camperol-3-Glc and apigenin-7-Glc was observed lower in AsGlu2 than in other substrates, but increased 4-6 fold in AsGlu2 E495K mutant multimers. In addition, the enzyme activities for quercetin-3-Glc and naringenin-7-Glc were the same as in AsGlu1. These results demonstrated that AsGlu1, which has high activity against Avenacoside B, has low activity against flavonoid glucoside, and increases its activity by forming multimers.

As a result of comparing the activity of the three mutated β -glucosidases with that of AsGlu1, the activity of A186-Abenacoside B and ρ NPG was increased by about 2 times while that of H197F and F371W was about 1-2. Decreased by a factor of 2. In the case of S186V, the activity of isoflavones, such as zenithine and dydazine, was increased by about 1.6 times, and the activity of glycidine was increased by 18 times, showing much higher activity. In the case of H197F, the activity on genistin was similar, but the activity on dydazine was increased by about 10 times and the activity on glycidine by 3.4 times. F371W showed a 1.6-fold decrease in activity against genistin and no activity for dydzin and glycidine. These mutated β -glucosidases had decreased activity against camperol-3-Glc and slightly increased activity against quercetin-3-Glc, which was the lowest as compared to AsGlu1. However, S186V did not show activity against Apigenin-7-Glc with AsGlu1 similar activity to that of Genistin, and H197F and F371W decreased significantly. Interestingly, AsGlu1 and other mutant proteins showed no activity for the flavonone naringenin-7-Glc, while the activity of 7.92 × 10 ³ unit · mg ⁻¹ was observed only in S186V. Although it is about 112 times lower than the activity against avenacoside B (886.33 × 10 ³ units · mg ⁻¹ ), it is a notable result considering the lack of activity on the substrate in AsGlu1. Substitution of Ser ¹⁸⁶ with Val among these mutated β -glucosidase is thought to induce high enzymatic activity against various substrates as well as in vivo substrates.

2) Enzymatic Characterization of In vivo / in vitro Substrates (Avenacoside B / ρ NPG)

The modified β -glucosidases according to the present invention exhibited various enzymatic activities against different substrates. We investigated enzymatic properties of in vivo / in vitro substrates. The properties of AsGlu1 multimers, AsGlu2 dimers, and AsGlu2 E495K mutant multimers for avenacoside B and ρ NPG were provided above (Table 4).

in vivo substrate of Abbe Tamara H197F and F371W of the seed B is K _m is however the affinity for each of the substrates to 0.023 and 0.030 mM similar AsGlu1, S186V has a substrate affinity than AsGlu1 or other mutant proteins in 0.109 mM Low It could be confirmed (Table 7). In H197F and F371W, k _cat was decreased compared to AsGlu1, and as a result, k _cat / K _m was decreased than AsGlu1. Also, S186V also increased k _cat to 832 (s ^-1 ) by about 2 times, but decreased k _cat / K _m because K _m was higher than AsGlu1.

In the in vitro substrate, ρ NPG, K _m had AsGlu1 of 4.1 mM, whereas S186V and H197F decreased to 1.44 and 3.4 mM, respectively, and F371W was 8.6 mM. In other words, these two mutant proteins increased substrate affinity for ρ NPG but decreased F371W. k _cat was the highest at 2.9 (s ^-1 ) at S186V, almost similar at AsGlu1 and H197F, and lowest at F371W. As a result, S186V showed lower K _m and higher k _cat than AsGlu1, resulting in a 6 times increase in k _cat / K _m (relative efficiency, 667%). For H197F to AsGlu1 than the K _m k _cat is a little low, but similar increase was about 1.2 times. On the other hand, in the case of F371W, K _m increased and k _cat decreased, so that the relative efficiency for k _cat / K _m decreased to 23%. From these results, in the case of Abenacoside B, which has a large size of aglycone, the affinity and k _cat of the mutated β -glucosidase due to the change in the characteristics or the size of the substrate recognition site are reduced, thereby hydrolyzing the substrate. It was confirmed that the efficiency was also reduced. On the other hand, ρ NPG is shown to have lower enzymatic activity than avenacoside B, but due to the small size of aglycone, the affinity for the substrate and k _cat are considered to increase the hydrolysis efficiency.

3) Enzymatic Properties of Flavonoid Glucoside

3-1) Isoflavones (Geninistin, Dydazine, Glycithin)

It was determined that the characteristics were changed along with the structural change of the substrate recognition site, and the enzymatic properties of the modified β -glucosidase according to the present invention for various flavonoid glucosides were investigated.

First, in the case of isoflavones, the K _m of the AsGlu1 multimer and AsGlu2 E495K mutant multimer was about 4 times and 2.7 times higher than that of AsGlu2 dimer in Genistin (Table 8). However, there was no significant change in dydazine and the K _m of AsGlu1 and AsGlu2 E495K was about 1.6 and 2.0 times lower than AsGlu2 in glycine. In kinistin, k _cat was not changed much, but AsGlu2 showed a decreased substrate affinity, resulting in high k _cat / K _m . Didazine and glycidine slightly increased the substrate affinity with the formation of multimers, but as for AsGlu2, k _cat decreased and k _cat / K _m was the same as that of Genistin. That is, as the multimer was formed, the substrate affinity for genistin decreased and increased for glycidine, but the multimer had higher catalytic efficiency for isoflavones than the dimer due to the difference in k _cat .

Among the mutated β -glucosidase, S186V had a K _m of 0.05, 0.062, 0.12 mM for geninistin, dydzin, and glycidine, which was lower than K _m (0.017 mM) for Abenacoside B of AsGlu1 (Table 7). ), It showed higher substrate affinity than other mutated β -glucosidase. In addition, k _cat was increased in all, and relative efficiency for k _cat / K _m was increased by 28 times, 62 times, and 40 times, respectively, compared to AsGlu1. In addition, H197F decreased K _{m in} genitine and k _cat increased in dydzin and glycidine, resulting in 4.6, 4.2, and 2.2 times increase in the relative efficiency of k _cat / K _m compared to AsGlu1. F371W, on the other hand, had no change in catalytic efficiency for genistin and no activity for dydzin and glycidine. These results demonstrated a 28-62 fold increase in hydrolysis efficiency for S186V, suggesting an improved substrate specificity for the isoflavone of the novel mutated β -glucosidase S186V.

3-2) Flavonone (Naringenin-7-Glc)

Second, flavonones had no activity against naringenin-7-Glc in other mutant proteins except S186V, including AsGlu1 (Table 9). S186V showed K _m of 0.022 mM, similar to that of Avenacoside B of AsGlu1, and k _cat decreased by about 57-fold to 7.4 (s ^-1 ). Thus, k _cat / K _m of the S186V 340 (s ^-1 · mM ^-1) to AsGlu1 biological substrate ^{[23519 (s -1 · mM -1} )] of about 70-fold natatjiman, activity for the substrate in comparison to AsGlu1 Considering the absence of this, the results are quite noteworthy. In addition, due to the high substrate affinity, the catalytic efficiency was higher than in other flavonoid glucosides except isoflavones. These results suggest that a new substrate specificity for naringenin-7-Glc was formed in the S186V variant protein.

3-3) flavonols (camphorol-7-Glc, quercetin-3-Glc)

Third, it was in the case of the flavonols quercetin and camp Ferrol -3-Glc-Glc at -3 AsGlu1 and AsGlu2 E495K K _m and k _cat is higher than in AsGlu2 (Table 10). In other words, it was confirmed that the affinity for these substrates decreased as the multimer was formed. Therefore, AsGlu2 was about 2 times higher than AsGlu1, and AsGlu2 E495K, which forms a multimer, was similar to AsGlu1. This suggests that the catalytic efficiency of the dimer to flavonol is higher.

The properties of the mutated β -glucosidases for flavonols were in contrast to the previously mentioned isoflavones, flavonones. First, S186V, which showed high hydrolysis efficiency for other substrates, decreased both K _m and k _cat in camperol-3-Glc and increased both K _m and k _cat in quercetin-3-Glc (Table 10). Although the same type of substrates showed opposite results, the catalyst efficiencies were similar to those of AsGlu1 for these substrates. And H197F than S186V efficiency is low, but showed a high substrate specificity for isoflavones were not an activity to camp Ferrol -3-Glc, the K _m and k _cat are 1.4 and 2.5 times respectively at the quercetin -3-Glc As a result, the catalyst efficiency is about two times higher than AsGlu1. On the other hand, F371W, which has no activity against isoflavones and flavonones, showed the highest activity against flavonol. In camperol-3-Glc and quercetin-3-Glc, both K _m decreased to 0.019 and 0.115 mM, respectively, compared to AsGlu1, which was similar to the substrate affinity for biological substrates or isoflavones. And k _cat / K _m for the camp Ferrol -3-Glc and quercetin -3-Glc was 90 and ^{10.0 (s -1 · mM -1)} [ Relative efficiency of 306% and 313%, respectively; compared to about 3 to AsGlu1 Fold increased. Although F371W's catalytic efficiency for this substrate is lower than for other substrates, F371W is considered to have substrate specificity for flavonol, given its lack of activity for isoflavones and flavonones.

3-4) Flavones (Apigenin-7-Glc)

Finally, in the flavone Apigenin-7-Glc, AsGlu1 and AsGlu2 E495K had higher K _m than AsGlu2, which reduced the affinity for the substrate but increased k _cat (Table 11). As a result, AsGlu2 was about 2 times higher than AsGlu1, and AsGlu2 E495K, which forms a multimer, was similar to AsGlu1. These results were very similar to those found in flavonols.

The properties of the mutated β -glucosidase on the substrate were also different from those on other substrates. S186V, which showed high efficiency for isoflavones and flavonones, showed no activity at apigenin-7-Glc. H197F by k _cat is reduced but even though K _m is reduced by about 32-fold compared to AsGlu1 to 0.013 mM, resulting in k _cat / K _m is ^{100 (sec -1 · mM -1)} AsGlu1 as Relative efficiency is 330%; It is about three times higher than. On the other hand, in F371W, K _m and k _cat decreased in similar proportions, so there was no significant change in catalyst efficiency. This showed a significant difference from the substrate specificity of F371W for flavonol, described above.

conclusion

As previously identified, the modified β -glucosidases according to the present invention have improved or at least equivalent substrate specificity for flavonoid glucosides compared to wild type, thus improving hydrolysis efficiency and unique for each substrate. Characteristics.

In other words, the S186V mutant protein that changed the polarity of the aglycone binding site showed overall high substrate specificity compared to the wild type, especially high isoflavones and flavonones. The F371W and H197F variant proteins showed higher substrate specificities for flavonols and flavones compared to wild type.

Equivalent

Those skilled in the art will be able to recognize or identify many equivalents to the compounds described herein and methods for their preparation using routine experimentation. These equivalents are considered to be within the scope of this invention and are encompassed by the following claims. In this specification, all references and publications mentioned above are incorporated by reference in their entirety.

1 shows the chemical structure of the plant flavonoid aglycone.

2 shows the reconstruction of the hexamer A and the multimer form B from a tomographic tilt series.

3 shows the sequence alignment of AsGlu1 and AsGlu2 subunits of oat β -glucosidase. These two protein sequences were aligned with the ClustalW program and represented as ESPRIPT. Spirals and arrows above the sequence indicate the secondary structural elements of the ( β / α ) ₈ barrel structure. Homologous sequence regions are shown in the background. The number on the right indicates the position of the amino acid sequence. Two catalytic glutamate salts are marked with asterisks.

4 shows the active sites of ZmGlu1 β -glucosidase and AsGlu1 β -glucosidase analyzed by constructing a model with the SWISS-MODEL program and the Deep View program. A: active site (borrowed from PDB 1E1E) in ZmGlu1 β -glucosidase. B: Suggested model of active site in AsGlu1 β-glucosidase, derived from the known crystal structure of ZmGlu1. Suggested substrate binding sites of wild type (C) and variant (D) AsGlu1 β -glucosidase. Glucose binding residues (Q33, H137, N182, W450, E457, W458) and aglycone binding residues (S186, L190, H197, D254, M256, F371, S459) are shown in C. Amino acid substitutions of the aglycone binding site (S186V, L190F, H197F, D254F, M256F, F371W, S459F) are located in the vicinity of the aglycone binding site (D).

Figure 5 shows the amino acid sequence comparison and point mutation sites of corn (ZmGlu1), sugar cane (SbDhr1), oats (AsGlu1), cassava (Lina Marais) and human cytoplasm (hCBG) β -glucosidase do. The portion of the α -helix and β -strand of the ( β / α ) ₈ structure of β -glucosidase is shown above the sequence alignment, and the same sequence region is shown with a red background. The point mutation site of the aglycone binding site is shown below the sequence alignment. Two catalytic glutamate salts are indicated by arrows.

Figure 6 shows the analysis results of the active site of the wild type and mutated AsGlu1 model consisting of SWISS-MODEL program and Deep View program. A: hypothetical aglycone binding site of wild type AsGlu1, B-D: modeling of aglycone binding sites of AsGlu1 variant, S186V (B), H197F (C) and F371W (D).

Figure 7 illustrates agarose gel electrophoresis of mutagenesis of oat β -glucosidase. Specific site mutagenesis of oat β -glucosidase cDNA was performed by redundant kidney PCR techniques using two overlapping complementary primers. S186V, H197F and F371W: aglycone binding site variants; E183D: catalytic site variants.

FIG. 8 shows electrophoresis of variant oat β -glucosidase and activity staining (C) of native gels in SDS-gel (A) and native gels (B). A, pET24a: plasmid pET24a; pET-Glu1: wild type AsGlu1; pET-S186V: variant AsGlu1 S186V; T: total cell extract; S: soluble cell lysate; I: Insoluble cell lysate. B, pET-S186V, -F371W: aglycone binding site variant; pET-E183D: catalytic site variant. C, native gel (B) was stained with 2 mM MUGlc.

Figure 9 shows the elution profile (A) and electrophoresis on SDS-gel (B) and native gel (C) of oat β -glucosidase (AsGlu1) purified by HA column chromatography. Protein solution was applied to an HA column (2.1 × 5.8 cm). Proteins absorbed in the column were eluted with 5 mM and 20 mM potassium phosphate buffer. The upper digits in (B) and (C) indicate the fraction number of the eluant in (A).

10 shows in situ active staining of mutant oat β -glucosidase. Native gels were stained with 2 mM MUGlc. pET-Glu1: wild type AsGlu1; pET-S186V and -F371W: aglycone binding site variants; pET-E183D: catalytic site variant.

11 shows the effect of temperature on activity (A) and stability (B) of wild type and mutated oat β -glucosidase. The optimum temperature and thermal stability of the enzyme were analyzed by measuring enzyme activity at various temperatures (20-60 ° C.) in citrate buffer (pH 5.5). The thermal stability of these enzymes was determined by pre-incubating them for 30 minutes at various temperatures (20-60 ° C.).

12 shows the effect of pH on activity (A) and stability (B) of wild type and mutated oat β -glucosidase. The optimum pH of the enzyme was analyzed by measuring enzyme activity at various pHs (5.5-8.5). The pH stability of these enzymes was determined from residual activity after incubating the enzymes in a buffer of pH 5.5-8.5 for 2 hours at 30 ° C.

Claims (7)

AsGlu1 variant selected from AsGlu1S186V, AsGlu1H197F, or AsGlu1F371W, wherein the modification of the aglycon binding site improves specificity for the flavonoid substrate. An oat β -glucosidase variant with improved specificity for flavonoid substrates, including AsGlu1 variants selected from AsGlu1S186V, AsGlu1H197F, or AsGlu1F371W. The flavonoid substrate of claim 2, wherein the flavonoid substrate is selected from isoflavon, flavonone, flavonol, or flavone, with improved specificity for the flavonoid substrate. Oat β -glucosidase variant with improved hydrolysis efficiency. The hydrolysis efficiency of the substrate according to claim 3, wherein the isoflavone is selected from genistin, didzin, or glycidin. Enhanced oat β -glucosidase variant. The oat β -glucosidase variant of claim 3, wherein the flavonone is naringenin-7-Glc with improved specificity for the flavonoid substrate. The improved specificity for the flavonoid substrate, characterized in that the flavonol is selected from kaempferol-7-Glc or quercetin-3-Glc to improve the hydrolysis efficiency of the substrate. Oat β -glucosidase variant. The oat β -glucosidase variant of claim 3, wherein the flavone is apigenin-7-Glc with improved specificity for the flavonoid substrate.

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