KR102466543B1 - Method for preparing turanose - Google Patents

Abstract

The present invention relates to an enzyme for producing turanose and a method for efficiently producing turanose using the same.

Description

Method for preparing turanose {Method for preparing turanose}

The present invention relates to an enzyme for producing turanose and a method for efficiently producing turanose using the same.

Turanose is a reducing disaccharide naturally occurring in bees, which is an analog of sugar with a sweetness equivalent to about 50% of that of sugar, and has the chemical structure of 3-O-α-D-glucopyranosyl-D-fructose. Since turanose is not fermented by dental caries-causing microorganisms, it helps prevent tooth decay and can be used as a calorie-free sweetener, thus playing an important role in the food, cosmetic and pharmaceutical industries.

Korean Patent No. 10-11772184 includes a method for producing turanose using sugar and fructose as substrates using amylosucrase. However, existing enzymes fail to show or maintain activity at high temperatures, and lose activity before sucrose is completely consumed during the turanose conversion reaction at 50° C., which is the optimum temperature suitable for the process. As a result, during the conversion reaction of turanose by amylosucrase, sugar remains in the final conversion solution, which becomes a decisive obstacle to high-purity separation of turanose later. In addition, the production of turanose by conventional enzymes is not suitable for commercialization because the reaction rate is very slow. Therefore, it is very important to develop an amylosucrase having an improved reaction rate and consuming all sucrose during the turanose conversion reaction at a temperature of 50° C. or higher.

Amylosucrases having turanose-producing activity are known to be derived from microorganisms such as Neisseria polysaccharea, Deinococcus geothermalis, and Alteromonas macleodii.

When turanose is prepared using sucrose-containing saccharides as a substrate using amylosucrase having turanose-producing activity, the reaction product includes not only turanose but also various saccharides, and the reaction product contains turanose in high purity and high yield. In order to produce lanose, a catalyst composition containing an enzyme that produces turanose at a high conversion rate is required, but also minimizes the effect of the presence of saccharides other than turanose contained in the reaction product in the separation and purification steps and achieves high purity. You need to get Turanos.

An object of the present invention is to provide an amylosucrase enzyme protein derived from Bifidobacterium thermophilum or a variant thereof, which produces turanose at a high conversion rate and minimizes the production of other sugars.

Another object of the present invention relates to a method for producing turanose using the amylosucrase enzyme protein or a variant thereof.

A further object of the present invention is to produce high-purity turanose, including the step of producing turanose using the amylosucrase enzyme protein or a variant thereof and obtaining high-purity turanose using simulated homogeneous layer chromatography. It's about how to produce.

An example of the present invention is an amylosucrase enzyme protein or variant thereof derived from Bifidobacterium thermophilum, which produces turanose at a high conversion rate and minimizes the production of other saccharides, and the enzyme protein or variant thereof It relates to a method for producing turanose using

An example of the present invention is an enzyme protein or mutant enzyme protein derived from Bifidobacterium thermophilum and having turanose conversion activity, a recombinant microorganism expressing the same, a culture of the recombinant microorganism, or a lysate of the recombinant microorganism ( Or a supernatant of the lysate) provides a composition for producing turanose comprising at least one selected from the group consisting of.

As another example of the present invention, an enzyme protein derived from Bifidobacterium thermophilum, characterized in that it produces turanose, a recombinant microorganism expressing the same, a culture of the recombinant microorganism, or a lysate of the recombinant microorganism (or Supernatant of lysate) provides a method for producing turanose comprising reacting a composition for producing turanose containing at least one selected from the group consisting of a substrate with a substrate. Matters concerning the enzyme protein may be equally applied to the composition and manufacturing method for producing turanose.

In addition, an example of the present invention is a high-purity solution comprising the steps of producing turanose using the amylosucrase enzyme protein or a variant thereof and obtaining high-purity turanose using simulated homogeneous layer chromatography. It's about how to produce Lanos.

When turanose is prepared using sucrose-containing saccharides as a substrate, the amylosucrase having turanose-producing activity according to the present invention not only produces turanose at a high conversion rate, but also saccharides other than turanose in the reaction product. For example, since the content of trehalulose and oligosaccharides is low, it is possible to obtain turanose with high purity while minimizing the effect of the presence of saccharides other than turanose contained in the reaction product in the separation and purification steps.

Hereinafter, the present invention will be described in more detail.

An example of the present invention is an enzyme protein or mutant enzyme protein derived from Bifidobacterium thermophilum and having turanose conversion activity, a recombinant microorganism expressing the same, a culture of the recombinant microorganism, or a lysate of the recombinant microorganism ( Or a supernatant of the lysate) provides a composition for producing turanose comprising at least one selected from the group consisting of.

An example of the present invention is an enzyme protein or mutant enzyme protein derived from Bifidobacterium thermophilum and having turanose conversion activity, a recombinant microorganism expressing the same, a culture of the recombinant microorganism, or a lysate of the recombinant microorganism ( or a supernatant of a lysate) reacting with a composition for producing turanose containing at least one selected from the group consisting of sucrose or a substrate containing sucrose and fructose to produce turanose.

In addition, the present invention relates to a method for producing turanose comprising the step of producing turanose using an amylosucrase enzyme protein derived from Bifidobacterium thermophilum or a variant thereof, and also to produce turanose. In addition to the step of doing, a step of separating using simulated homogeneous layer chromatography is included. When turanose is prepared using sugar by a biological method, it is difficult to selectively separate only turanose on a separation column because of oligomers and trehalulose that are closely attached back and forth with respect to turanose in the reaction product. In particular, there is a problem in that oligomer, turanose, and trehalulose are continuously adjacent to each other to form peaks on the chromatogram, making separation difficult.

The composition for producing turanose applied in the present invention is an enzyme protein or mutant enzyme protein derived from Bifidobacterium thermophilum and having turanose conversion activity, a recombinant microorganism expressing the same, a culture of the recombinant microorganism, or the above It may include at least one selected from the group consisting of a lysate of a recombinant microorganism (or a supernatant of the lysate). The composition comprises the steps of centrifuging a culture of a recombinant microorganism expressing an enzyme protein derived from Bifidobacterium thermophilum; And crushing the precipitate, which may include the centrifuged concentrate; and obtaining a supernatant of the lysate.

The enzyme of the present invention may be an enzyme protein derived from Bifidobacterium thermophilum. The enzyme protein of the present invention is an amylosucrase that produces turanose, and can produce turanose from sucrose alone or a substrate solution containing sucrose and fructose.

The amylosucrase enzyme and its mutant enzyme protein having turanose production characteristics applicable to the turanose production method according to the present invention can be used as long as the production conditions are satisfied. The enzyme protein derived from Bifidobacterium thermophilum may be a protein having the amino acid sequence of SEQ ID NO: 1, but is not limited thereto as long as it has an activity capable of producing turanose, and 1 of the amino acid sequence of SEQ ID NO: 1 It may include all mutant proteins in which the above amino acids are substituted, inserted, or deleted. For example, the enzyme protein or its variant enzyme is 70% or more, preferably 80% or more, more preferably 90% or more, 93% or more or 95% or more, more preferably 90% or more, more preferably 90% or more of the amino acid sequence of SEQ ID NO: 1 Preferably, it may be an enzyme protein having an amino acid sequence with 98% or more, 99% or more, or 99.5% or more homology.

The composition for producing turanose applied in the present invention is derived from Bifidobacterium thermophilum, and an example of a mutant enzyme protein by substitution mutation having turanose conversion activity is the 200th amino acid sequence having SEQ ID NO: 1 , 202nd, 265th, 305th, 393rd, 414th, 420th and 542th position may be one or more amino acids selected from the substituted. For example, at least one amino acid selected from the group consisting of positions 200, 202, 265, 305, 393, 414, 420 and 542 of the amino acid sequence having SEQ ID NO: 1 is arginine, It may be substituted with at least one amino acid selected from the group consisting of histidine, aspartic acid, glutamic acid, isoleucine, phenylalanine, and threonine.

In a specific example, the example of the mutant enzyme protein by the substitution mutation is the group consisting of the 200th, 202nd, 265th, 305th, 393rd, 414th and 420th positions of the amino acid sequence having SEQ ID NO: 1 One or more amino acids selected from may be substituted with one amino acid selected from the group consisting of arginine, isoleucine, phenylalanine, and threonine. The amino acids of the amino acid sequence having SEQ ID NO: 1 may contain one or more of the following amino acid substitutions:

The 200th amino acid is substituted with arginine.

The 202nd amino acid is substituted with isoleucine,

The 265th amino acid is substituted with phenylalanine,

The 305th amino acid is substituted with isoleucine,

The 393rd amino acid is substituted with arginine,

The 414th amino acid is substituted with phenylalanine, and

The 420th amino acid is substituted with threonine.

For example, the variant enzyme protein may have any one of the amino acid sequences of SEQ ID NOs: 2 to 10 having one amino acid mutation, or the 200th, 202nd, Amino acid variation 2 in which amino acids at two or more positions selected from the group consisting of positions 265, 305, 393, 414, and 420 are substituted with at least one amino acid selected from the group consisting of arginine, isoleucine, phenylalanine, and threonine. It may be a mutant enzyme comprising 2 or more, for example, 2 selected from the group consisting of the 200th, 202nd, 265th, 305th, 393rd, 414th and 420th positions of the amino acid sequence having SEQ ID NO: 1 A mutant enzyme comprising two mutations in which an amino acid is substituted with at least one amino acid selected from the group consisting of arginine, isoleucine, phenylalanine, and threonine, specifically selected from the amino acid sequences of SEQ ID NOs: 11 to 13 can

Specifically, the enzyme protein of the present invention may be an enzyme protein comprising any one amino acid sequence selected from an amino acid sequence comprising the amino acid sequence of SEQ ID NO: 2 (mutant enzyme V542K) or SEQ ID NO: 3 (mutant enzyme V542D). The enzyme of SEQ ID NO: 1 is a wild-type enzyme, SEQ ID NO: 2 is an enzyme having a V542D mutation in the amino acid sequence of the wild-type enzyme of SEQ ID NO: 1, and SEQ ID NO: 3 is an enzyme having a V542K mutation in the amino acid sequence of the night-type enzyme of SEQ ID NO: 1. it means. Since the activity of the mutant enzyme protein having the V542D mutation or the V542K mutation is increased by more than two times, an effect of improving productivity problems directly related to cost reduction can be expected.

Examples of wild-type enzymes and mutant enzymes according to the present invention are shown in Table 1 below.

division sequence type sequence number wild type enzyme amino acid sequence One Mutase V542K amino acid sequence 2 Mutase V542D amino acid sequence 3 Mutase P200R amino acid sequence 4 Mutase V202I amino acid sequence 5 Mutase Y265F amino acid sequence 6 Mutase V305I amino acid sequence 7 Mutase K393R amino acid sequence 8 Mutase S420T amino acid sequence 9 Mutase Y414F amino acid sequence 10 Mutase Y414F*P200R amino acid sequence 11 Mutase Y414F*V202I amino acid sequence 12 Mutase Y414F*Y265F amino acid sequence 13 wild type enzyme nucleotide sequence 14 Mutase V542K nucleotide sequence 15 Mutase V542D nucleotide sequence 16 Mutase P200R nucleotide sequence 17 Mutase V202I nucleotide sequence 18 Mutase Y265F nucleotide sequence 19 Mutase V305I nucleotide sequence 20 Mutase K393R nucleotide sequence 21 Mutase S420T nucleotide sequence 22 Mutase Y414F nucleotide sequence 23 Mutase Y414F*P200R nucleotide sequence 24 Mutase Y414F*V202I nucleotide sequence 25 Mutase Y414F*Y265F nucleotide sequence 26

Conventionally known amylosucrases have an optimal activity temperature of around 35 ° C, and when the conversion reaction is performed at 50 ° C, all sucrose is consumed in the conversion solution due to low activity and lack of heat resistance, leaving only products containing turanose. Since the activity of the enzyme was not maintained until then, there was difficulty in industrialization. However, the enzyme protein of the present invention can be said to satisfy the conditions for industrialization because it is possible to maintain the reaction until all sucrose is decomposed at 50 ° C.

The enzyme protein of the present invention has an optimum temperature of 30 to 65°C, an optimum pH of 5.0 to 9.0, and an activity to generate turanose from a sugar-containing substrate, preferably (i) pH 6.0 and a substrate of 2M water at a temperature of 50°C. When cross is reacted, the turanose conversion rate calculated as the concentration of turanose produced with respect to 100% of the substrate concentration is 10 to 60% and/or (ii) per hour when the substrate 2M sucrose is reacted at pH 6.0 and a temperature of 50 ° C. The produced turanose may have a characteristic of producing 3 to 80 g/L/h.

The optimum temperature is the temperature at which the enzyme's turanose production activity is maximized, and is preferably 30 to 65°C, preferably 40 to 65°C, 41 to 65°C, 42 to 65°C, or 41 to 60°C, more preferably It may be 45 to 55 °C, more preferably 50 °C. Conventionally known amylosucrases have an optimal activity temperature of around 35 ℃, and when the conversion reaction is carried out at a temperature of 40 ℃ or more, for example, 45 ℃ or 50 ℃, due to low activity and lack of heat resistance, it can be in the conversion solution. There was difficulty in industrialization because the activity of the enzyme was not maintained until only the product including turanose remained without cross. However, the enzyme protein of the present invention is prepared at a temperature of 40 ° C or higher or higher than 40 ° C, for example, 41 ° C, 42 ° C?, 43 ° C?, 45 ° C? Alternatively, since the reaction is maintained at a temperature of 50° C. until all sucrose is decomposed, it has excellent heat resistance and is preferable for industrialization.

The optimal pH of the mutant enzyme P200R, Y265F, V305I, K393R, S420T, Y414F, Y414F*P200R, Y414F*V202I or Y414F*Y265F is a pH at which the enzyme's turanose production activity is maximized, and is pH 5.0 to 9.0 or pH 5.0 to 7.0, preferably pH 5.5 to 6.5, more preferably pH 6.0.

The turanose conversion rate of the enzyme protein of the present invention is 10 to 70%, preferably turanose conversion calculated by the concentration of turanose produced with respect to 100% of the substrate concentration when the substrate 2M sucrose is reacted at pH 6.0 and a temperature of 50 ° C. Preferably it may be 20 to 40% or 35 to 60%, more preferably 45% to 55%. When the enzyme protein of the present invention reacts with the substrate 2M sucrose at pH 6 and a temperature of 50 ° C, the amount of turanose produced per hour is 3 to 80 g / L / h, preferably 10 to 30 g / L / h or 20 to 60 g/L/h, more preferably 50 g/L/h to 60 g/L/h.

NpAS (Neisseria polysaccharea) is known as an enzyme with a high turanose production rate among amylosucrase enzymes that have been preceded by many studies. NpAS is known to have an optimum pH of 7.0, an optimum temperature of 35°C, and a turanose conversion rate of 50.9% based on 2 M sucrose as a substrate. The novel amylosucrase wild-type enzyme of the present invention is more stable due to its high heat resistance and is industrially useful due to its high conversion yield of turanose.

In the present specification, the turanose content in the sugar syrup composition produced from the substrate containing sugar by the enzyme protein is 1 to 48 hours, preferably 3 to 48 hours, more preferably 3 to 48 hours with the substrate at a temperature of 50 ° C. and pH 6.0. It is defined as the amount of turanose contained in the product at the time when sucrose does not remain in the reaction solution after 12 to 48 hours of reaction, that is, the weight% of turanose relatively converted from the substrate. For example, the turanose conversion rate of the enzyme protein having the amino acid sequence of SEQ ID NO: 1 may be 35 to 45% by weight, preferably 40 to 43% by weight, and the enzyme having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 The turanose conversion rate of the protein may be 40 to 50% by weight, preferably 43 to 49% by weight.

In addition, the enzyme protein of the present invention is expressed in a GRAS (Generally Recognized as Safe) strain to show activity, for example, a wild-type enzyme protein having the amino acid sequence of SEQ ID NO: 1, as well as a mutated enzyme protein such as SEQ ID NO: 2 Alternatively, the effect of increasing activity due to modification with the mutant enzyme protein having the amino acid sequence of SEQ ID NO: 3 is also reproduced in the GRAS strain. Therefore, the present invention can express turanose in a GRAS strain to provide it as a safe food raw material, and secure an enzyme having high reaction stability and activity through enzyme improvement, so it is suitable for industrial application.

Another example of the present invention provides a polynucleotide encoding the enzyme protein, a recombinant vector containing the polynucleotide encoding the protein, or a recombinant microorganism expressing the protein.

Matters concerning the enzyme protein may be equally applied to a polynucleotide encoding the enzyme protein, a recombinant vector containing the same, or a recombinant microorganism expressing the same.

The polynucleotide encoding the enzyme protein of the present invention may be a polynucleotide encoding any one amino acid sequence selected from the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 13, and preferably the polynucleotide encoding the amino acid sequence It may be any one of SEQ ID NO: 14 to SEQ ID NO: 26, but also includes a sequence showing substantial identity to the above nucleotide sequence. The above substantial identity is, when the nucleotide sequence of the present invention and any other sequence described above are arranged to correspond as much as possible, and the sequence is analyzed, 70% or more of the nucleotide sequence of SEQ ID NO: 14 to SEQ ID NO: 26, more preferably contains a nucleotide sequence with 80% or more, more preferably 90% or more, most preferably 98% or more homology.

For example, the polynucleotide encoding the enzyme protein of the present invention may be a polynucleotide encoding any one amino acid sequence selected from the amino acid sequences of SEQ ID NOs: 1 to 13, and the polynucleotides of SEQ ID NOs: 14 to 26 It may be one or more selected from the group consisting of nucleotide sequences.

The recombinant expression vector of the present invention may include, for example, a polynucleotide encoding any one amino acid sequence selected from among the amino acid sequences of SEQ ID NOs: 1 to 13, and includes the polynucleotide encoding the amino acid sequence of SEQ ID NO: 1. The recombinant expression vector to be may have a cleavage map of FIG. The recombinant microorganism of the present invention is, for example, a recombinant expression vector containing a polynucleotide encoding any one amino acid sequence selected from among the amino acid sequences of SEQ ID NOs: 1 to 13 and expressing an amylosucrase enzyme protein. It may be a strain.

In the turanose production method, for efficient production of turanose, sucrose alone or fructose may be additionally used as the substrate used for the sucrose.

In the method for producing turanose according to the present invention, when fructose is added to a substrate and used as a mixed substrate of sucrose and fructose, when the weight of fructose relative to sugar in the substrate is increased, the production of oligomers is reduced and the sugar ratio is relatively increased. As it decreases, the amount of turanose produced decreases. In a specific example of the present invention, trehalulose contained in the reaction product is not greatly affected by the fructose to sugar ratio and is kept at a relatively low level. Therefore, in the case of using a mixed substrate of sucrose and fructose in the method for producing turanose according to the present invention, the content of turanose, which is the target product, is increased among the total sugars included in the reaction product, and the undesirable by-product, trehalul The composition of the substrate can be appropriately adjusted to lower the loss content and the oligosaccharide content. Based on the total saccharide solid content of the reaction product obtained in the production of turanose according to the present invention, the preferred sugar composition is 20% by weight or less, 18% by weight or less, 17% by weight or less, 15% by weight or less, 13% by weight or less. or less, 11% by weight or less, or 10% by weight or less, and the condition that the trehalulose content is 7% by weight or less, 6% by weight or less, 5% by weight, 4.5% by weight or less, or 4.0% by weight or less is satisfied, and turanose The higher the content, the better, for example, 30% by weight or more, 35% by weight or more, 40% by weight or more, 41% by weight or more, 43% by weight or more, 45% by weight or more, 47% by weight or more, 49% by weight or more, or 50 may be greater than or equal to weight percent. Most of the oligosaccharides have a degree of polymerization higher than DP3 and DP4 in which glucose is linked by an alpha 1-4 linkage, and DP3 is the main component. About 80 to 85% by weight of the oligosaccharide can be decomposed into glucose by glucoamylase enzyme treatment.

In one example of the present invention, the composition of the substrate in which the oligosaccharide content is reduced relative to the amount of turanose produced in the reaction product is 5 to 250 parts by weight, 5 to 200 parts by weight, 5 to 175 parts by weight, 5 to 150 parts by weight of fructose based on 100 parts by weight of sugar 5 to 125 parts by weight, 5 to 100 parts by weight, 5 to 90 parts by weight, 5 to 80 parts by weight, 5 to 75 parts by weight, 5 to 70 parts by weight, 10 to 250 parts by weight, 10 to 200 parts by weight , 10 to 175 parts by weight, 10 to 150 parts by weight, 10 to 125 parts by weight, 10 to 100 parts by weight, 10 to 90 parts by weight, 10 to 80 parts by weight, 10 to 75 parts by weight, 10 to 70 parts by weight, 15 to 250 parts by weight, 15 to 200 parts by weight, 15 to 175 parts by weight, 15 to 150 parts by weight, 15 to 125 parts by weight, 15 to 100 parts by weight, 15 to 90 parts by weight, 15 to 80 parts by weight, 15 to 75 parts by weight 15 to 70 parts by weight, 20 to 250 parts by weight, 20 to 200 parts by weight, 20 to 175 parts by weight, 20 to 150 parts by weight, 20 to 125 parts by weight, 20 to 100 parts by weight, 20 to 90 parts by weight , 20 to 80 parts by weight, 20 to 75 parts by weight, 20 to 70 parts by weight, 30 to 250 parts by weight, 30 to 200 parts by weight, 30 to 175 parts by weight, 30 to 150 parts by weight, 30 to 125 parts by weight, 30 to 100 parts by weight, 30 to 90 parts by weight, 30 to 80 parts by weight, or 30 to 75 parts by weight, 30 to 70 parts by weight, 35 to 250 parts by weight, 35 to 200 parts by weight, 35 to 175 parts by weight, 35 to 150 parts by weight, 35 to 125 parts by weight, 35 to 100 parts by weight, 35 to 90 parts by weight, 35 to 80 parts by weight, 35 to 75 parts by weight, or 35 to 70 parts by weight, for example, 50 parts by weight to be.

The method for preparing turanose according to the present invention reduces the content of oligosaccharides of DP3 or higher relative to the amount of turanose produced in the reaction product of the reaction for preparing turanose, and is therefore suitable for SMB chromatography. If the content of fructose added to sugar is too high, the content of oligosaccharide in the reaction product is low, but the content of turanose is low, so the yield is low and separation and purification are not easy. Therefore, the sugar composition of the mixed matrix can be appropriately set so that the content of turanose contained in the reaction product is high and the content of one or more selected from the group consisting of oligosaccharide, trehalulose and fructose is low, and preferably the content of oligosaccharide is low. have.

For example, the concentration of sucrose (sugar) in the substrate reaction for preparing turanose of the present invention may include 0.1M to 2.5M, preferably 0.5M to 2.0M. In addition, when the substrate for preparing turanose of the present invention includes sucrose and fructose, the concentration of fructose in the substrate solution may further include 0.1 to 4M, or 0.75 to 3M.

The enzyme protein of the present invention has a sucrose concentration of 0.1 to 2.5M, 0.25 to 2.5M, 0.75 to 2.5M, 1.0 to 2.5M, 0.1 to 2.25M, 0.25 to 2.25M, 0.75 to 2.25M, 1.0 to 2.5M in the substrate reaction. 2.25M, 0.1 to 2.0M, 0.25 to 2.0M, 0.75 to 2.0M, 1.0 to 2.0M, 0.1 to 1.5M, 0.25 to 1.5M, 0.75 to 1.5M, 1.0 to 1.5M, 0.1 to 1.25M, 0.25 to 0.25M 1.25 M, 0.75 to 1.25 M, or 1.0 to 1.25 M.

The step of reacting the enzyme protein with the substrate may be performed by contacting the protein with the substrate. The step of contacting the enzyme protein with the substrate may be performed, for example, by mixing the enzyme protein with the substrate or contacting the substrate with a carrier on which the enzyme protein or the like is immobilized. In another example, the step of reacting the enzyme protein with the substrate may be performed by culturing the cells of the recombinant strain in a culture medium containing the substrate. As such, the enzyme protein can convert sucrose or fructose into turanose to produce turanose from sucrose or fructose.

The production method comprises culturing a recombinant microorganism expressing an enzyme protein having at least one amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 13; and reacting the recombinant microorganism or an enzyme protein isolated from the recombinant microorganism with a substrate.

The step of culturing the recombinant microorganism may be performed under a medium and culture conditions easily selected by a person skilled in the art according to the characteristics of the microorganism used, for example, a strain. For example, the medium includes any culture medium, solution, solid, semi-solid or rigid support capable of supporting or containing any host cells, including E. coli, and cell contents, preferably 2YT medium, LB medium, SOB medium or TB medium, etc. The culture may be continuous, semi-continuous, or batch culture.

The cells can be obtained by performing centrifugation, filtration, etc. on the culture of the microorganisms, and the supernatant obtained by homogenizing and centrifuging the obtained cells or fractionating the supernatant, or through chromatography, etc. Enzyme proteins can be obtained by separation and purification. For example, the recovered cells are suspended in 50 mM phosphate buffer, disrupted and centrifuged, and only the supernatant is adsorbed on a Ni-NTA column (Qiagen), and then the target protein can be recovered at a concentration of 20 mM or 200 mM imidazole.

In the turanose production method, for efficient production of turanose, the amount of enzyme protein used may be 50 U/ml to 1500 U/ml based on total reactants (including both substrate and protein). If the amount of enzyme used is lower than the above concentration, turanose conversion efficiency may be lowered, and if the concentration is higher than the above concentration, economic feasibility in the industry is lowered, so the above range is suitable.

In addition, the step of reacting with the substrate may be preferably performed under optimal activation conditions of the enzyme protein of the present invention. That is, the reaction may be performed at a temperature of 30 to 65°C, preferably 40 to 60°C, more preferably 45 to 55°C, and more preferably 50°C. The reacting with the substrate may be performed at pH 5.0 to 9.0, preferably pH 5.5 to 8.0, or pH 5.5 to 6.5.

Turanose obtained by the method of the present invention can be separated with high purity using simulated homogeneous layer chromatography. In the separation method. For example, one or more steps selected from the group consisting of centrifugation, filtration, ion purification, and combinations thereof may be further performed.

In the method for preparing turanose according to the present invention, a turanose-containing sugar composition is separated by simulated moving bed (SMB) chromatography to obtain a turanose fraction and a raffinate. It's about manufacturing methods.

The production process of turanose according to the present invention can be used both in a continuous process and in a batch process, and is preferably a continuous process.

In the present specification, the term "raffinate" is also referred to as raffinate, and the product obtained by passing the raw material introduced into the separation process through the separation process includes a target fraction including the target substance to be increased in content by the separation process and , It includes a residual liquid containing substances to be removed or reduced in content in the separation process, and the residual liquid is called raffinate. In one example of the present invention, the product obtained in the turanose conversion process is a mixture containing sucrose and/or fructose as a raw material substrate and turanose as a product. A lanose fraction and a residual liquid are obtained, and since the residual liquid contains a large amount of fructose, which is a substrate or product of the turanose conversion reaction, it may mean fructose raffinate.

In one example of the present invention, the high-purity separation process using SMB chromatography is a separation method that is easy to secure the stability of the material because there is no phase change during the separation process. Among these adsorption separation methods, a chromatographic separation method is widely used as a liquid phase adsorption separation method. Among them, the simulated moving bed adsorption separation method (simulated moving bed, SMB) is a separation technique proposed in US Patent No. 2,985,589 in 1961, which uses multiple columns to continuously separate the purity and productivity compared to conventional batch chromatography. It is excellent and has the advantage of being able to use a small amount of solvent. The simulated moving bed (SMB) adsorption separation process is a process in which the injection of the mixture to be separated and the production of raffinate and extract are continuously performed.

As an ion purification process after SMB separation, one or three separation towers filled with ion exchange resin are used, and the ion exchange resin can be carried out at 35 to 50 ° C using strong acid cation exchange resin and weakly basic anion exchange resin. have.

As another example of the present invention, a turanose-containing sugar composition produced by the turanose production method is provided. The enzyme protein and the method for producing turanose may be equally applied to the turanose-containing sugar composition.

A sugar composition containing turanose produced by reacting the enzyme protein with sucrose alone or with a substrate comprising sucrose and fructose, for example, turanose, sucrose, fructose, trehalulose, glucose and A sugar composition comprising at least one saccharide selected from the group consisting of oligosaccharides and rare saccharides (allulose, allose, etc.) may be prepared.

It is preferable that the reaction product contains a high content of turatose as a target product and a low content of oligosaccharide and trehalulose as impurities. For example, based on 100% by weight of the saccharide solid content contained in the reaction product, turanose is 30% by weight or more, 35% by weight or more, 40% by weight or more, 41% by weight or more, 43% by weight or more, 45% by weight or more. It may be more than 47% by weight, more than 49% by weight or more than 50% by weight. Based on the total saccharide solid content of the reaction product obtained in the production of turanose according to the present invention, the preferred sugar composition is 20% by weight or less, 18% by weight or less, 17% by weight or less, 15% by weight or less, 13% by weight or less. % or less, 11 wt% or less, or 10 wt% or less, and the trehalulose content is 7 wt% or less, 6 wt% or less, 5 wt%, 4.5 wt% or less, or 4.0 wt% or less.

In a specific example, the amylosucrase enzyme according to the present invention is reacted with a reaction substrate at a temperature of 50 ° C. and pH 6.0 for 24 hours, based on 100% by weight of the solid content of the reaction product, 30% by weight or more of turanose, oligosaccharide A reaction product containing 20% by weight or less and 7% by weight or less of trehalulose can be prepared.

The enzyme protein derived from Bifidobacterium thermophilum according to the present invention can produce turanose from sucrose in high yield and is useful for industrial use of turanose.

1 is a view showing an expression vector pRSet (A) containing recombinant DNA for expressing recombinant turanose 3-epimerase according to an embodiment of the present invention.
Figure 2 is an SDS-PAGE diagram showing the expression of Bifidobacterium thermophilum amylosucrase according to an example of the present invention.
Figure 3 is a result showing the relative activity according to the temperature of wild-type amylosucrase.
Figure 4 is a result showing the relative enzyme activity according to the pH of wild-type amylosucrase.
5 is a result showing the relative enzyme activity according to the temperature of wild-type amylosucrase.
6 is a result showing the turanose conversion rate according to the concentration of sucrose and fructose as substrates according to an example of the present invention.
7 is a view showing the measured amount of concentration of turanose produced per hour according to the enzyme concentration of the mutant V542D (SEQ ID NO: 6) of bifidobacterium thermophilum amylosucrase purified according to an example of the present invention. to be.
8 is a view showing the measured amount of concentration of turanose produced per hour according to the enzyme concentration of the mutant V542K (SEQ ID NO: 5) of bifidobacterium thermophilum amylosucrase purified according to an example of the present invention. to be.
9 is a result showing the conversion of turanose calculated after reacting with a substrate 2 M sucrose at 400 U/L and 1600 U/L at pH 6 and a temperature of 50° C. according to an example of the present invention.
10 is a result showing the consumption of the substrate sucrose calculated after reacting with the substrate 2 M sucrose at 400 U / L and 1600 U / L at pH 6 and a temperature of 50 ° C. according to an example of the present invention.

Hereinafter, the present invention will be described in more detail by specific examples. However, the present invention is not limited to the following examples.

Example 1. Production of wild-type amylosucrase

A polynucleotide (SEQ ID NO: 1) encoding amylosucrase (SEQ ID NO: 4) derived from Bifidobacterium thermophilum was requested from Bioneer.Co.Korea to be optimized for E. coli to be used as an expression strain. synthesized. The synthesized polynucleotide of SEQ ID NO: 1 was inserted into the same restriction enzyme site of the expression vector pRSet(A) (Invitrogen) using restriction enzymes NheI and XhoI (NEB) to recombinant vector pRSet(A)-amylosucrasase. (pRSet(A)-BtAS) was prepared (FIG. 1).

Thereafter, E. coli BL21 (DE3) (Invitrogen) was transformed with the prepared recombinant vector by the heat shock method (Sambrook and Russell: Molecular Cloning) to prepare a recombinant strain containing a polynucleotide encoding the amino acid sequence of SEQ ID NO: 4. .

The transformed recombinant strain was inoculated into 5ml LB-ampicillin medium (Difco) and cultured with shaking at 37°C and 200rpm until the absorbance (OD) at 600nm reached 1.5. Inoculation into ampicillin medium, shaking culture at a temperature of 37 ℃ and 200rpm. When the absorbance at 600 nm of the culture medium was 0.5, 0.1 mM IPTG was added to induce overexpression of the target enzyme. (At this time, it is preferable to maintain the culture conditions for 16 hours by switching to a temperature of 16 ° C and 150 rpm from the time of induction of overexpression, but it is irrelevant to experiment at a temperature of 30 ° C.

Thereafter, cells were recovered by centrifugation at 6000 rpm for 20 minutes in a centrifuge. The recovered cells were washed twice with 0.85% (w/v) NaCl, turbid in lysis buffer (PBS buffer, 10 mM imidazole), and disrupted by rotating the bead beater three times for 20 seconds. After collecting only the supernatant by centrifuging the lysate at 13000 rpm for 20 minutes, it was applied to a Ni-NTA column (Ni-NTA Superflow. Qiagen) equilibrated with lysis buffer in advance, and then a buffer solution containing 40 mM imidazole in PBS buffer was applied. flowed sequentially. The target protein was eluted by flowing 200 mM imidazole into PBS buffer, which was the last step. The eluted protein was converted into a buffer solution for measuring enzyme activity (50 mM Tris-HCl pH 7.0) and used in the next experiment.

Partially purified amylosucrase was obtained in this way, and it was confirmed through SDS-PAGE that the size of the monomer was about 68 kilodaltons (kDa) (FIG. 2).

Example 2. Preparation of mutant amylosucrase

2.1 Mutant Construction and Cloning by PCR

Amylosucrase mutants were constructed using Stratagene's Quikchange site-directed mutagenesis method. Specifically, instead of amplifying only the insert through PCR and subcloning it into plasmid DNA, a method of amplifying the entire plasmid DNA through PCR and digesting the template DNA with the DpnI enzyme was used. Quikchange site-directed mutagenesis has the advantage of being able to induce mutations more quickly.

Plasmid DNA cloned into the vector DNA of DNA pET21a encoding the wild-type amylosucrase enzyme was used as a template for replication. The expression vector containing the prepared recombinant DNA for expressing the mutant amylosucrase is a sequence corresponding to the insert in the vector shown in Figure 1, that is, pRSet (A) -BtAS V542D, pRSet in which one nucleotide is substituted. (A) -BtAS V542K.

To prepare the V542D mutant, PCR was set in a final volume of 50 ul containing 250 ng of template DNA, 125 ng of V542D forward primer, 125 ng of V542D reverse primer, 2.5 ul of pfu ultra high fidelity DNA polymerase, 5 ul of 10X reaction buffer, and 1 ul of 10 mM dNTP. The reaction was performed in the GeneAmp PCR system 9700, 1 cycle at 95 ° C (1 minute), 18 cycles of [95 ° C (50 seconds), 60 ° C (50 seconds), 68 ° C (6 minutes)], 68 ° C (7 minutes) minutes) in one cycle. 500 U (10 U/ul) of DpnI enzyme, which recognizes and degrades unmutated template DNA, was added to a reaction solution in a volume of 50 ul after PCR, and treated at 37° C. for 1 hour.

In addition, to prepare the V5425K mutant, PCR was set in a final volume of 50 ul containing 250 ng of template DNA, 125 ng of V5425K forward primer, 125 ng of V5425K reverse primer, 2.5 ul of pfu ultra high fidelity DNA polymerase, 5 ul of 10X reaction buffer, and 1 ul of 10 mM dNTP. did Primer base sequences required for the reaction are shown in Table 1. The reaction was performed in the GeneAmp PCR system 9700, 1 cycle at 95 ° C (1 minute), 18 cycles of [95 ° C (50 seconds), 60 ° C (50 seconds), 68 ° C (7 minutes)], 68 ° C (7 minutes) minutes) in one cycle. 500 U (10 U/ul) of DpnI enzyme, which recognizes and degrades unmutated template DNA, was added to a reaction solution in a volume of 50 ul after PCR, and treated at 37° C. for 1 hour.

The sequences of the primers used to construct the mutants are shown in the table below, and the PCR process is summarized in the table below.

designation order Tm V542D_FWD (SEQ ID NO: 27) 5′-CGCCCCTGACGCTTGGGACACGACGTGGGACGCGC-3′ 73℃ V542D_RVS (SEQ ID NO: 28) 5′-GCGCGTCCCACGTCGTGTCCCAAGCGTCAGGGGCG-3′ 72℃ V542K_FWD (SEQ ID NO: 29) 5′-CGCCCCTGACGCTTGGAAGACGACGTGGGACGCGC-3′ 73℃ V542K_RVS (SEQ ID NO: 30) 5′-GCGCGTCCCACGTCGTCTTCCAAGCGTCAGGGGCG-3′ 72℃

segment step cycles temperature(℃) time One initialization One 95 1min 2 denaturation 18 95 50 seconds annealing 118 60 50 seconds elongation 18 68 7min 3 final elongation One 68 ∞

2.2 Transformation and expression

E. coli DH10B was used as the initial host. DpnI was added to the PCR reaction described in Example 2.1, and the mixture was immediately transformed into DH10B. Pipette 5ul of the mixed solution of the PCR reaction solution and DpnI, and transform 50ul of DH10B, a competent cell prepared by the CaCl ₂ method, stored in a cryogenic freezer by the heat shock method. Plated on LB-ampicillin solid medium and incubated at 37 °C. Three colonies were selected from the colonies formed on the solid medium, cultured in 3ml LB-ampicillin liquid medium, and plasmid DNA was extracted using a miniprep kit (Quiagen). Sequencing was performed by an external institution (Macrogen, Korea), and as a result, mutations were induced in the DNA region encoding the amylosucrase enzyme for turanose production. Specifically, the amino acid sequence V542D mutation (SEQ ID NO: 2) and the amino acid sequence It was confirmed that the V542K mutation (SEQ ID NO: 3) was induced. The plasmid DNA for which mutagenesis was confirmed was transformed into 50ul of E.coli BL21 solu (DE3), a competent cell prepared by the CaCl ₂ method stored in a freezer, by the heat shock method. Plated on LB-ampicillin solid medium and incubated at 37°C.

Colonies of E.coli BL21 solu (DE3) containing the plasmid DNA encoding the amylosucrase mutation obtained by transformation were picked and inoculated into 3 ml LB-ampicillin liquid medium and incubated at 37 °C for 12 hours. 1 ml of the microorganisms in the 3 ml culture volume that had grown cloudy was pipetted and inoculated into 50 ml of LB-ampicillin liquid medium contained in a 250 ml Erlenmeyer flask and cultured at 37 °C. When the absorbance at 600 nm was 0.6, IPTG was inducted to 0.1 mM, followed by incubation at 16°C. All cultures were performed in a 200 rpm agitated incubator. After 12 hours of incubation at 16° C., microbial cells were recovered by centrifugation for 30 minutes in a 3500 gX centrifuge.

After turbidity of the recovered cells in lysis buffer (PBS buffer, 10 mM imidazole), the bead beater is disrupted by rotating the bead beater three times for 20 seconds. The lysate was centrifuged at 13000 rpm for 20 minutes to collect only the supernatant, applied to a Ni-NTA column (Ni-NTA Superflow. Qiagen) equilibrated with lysis buffer in advance, and then a buffer solution containing 40 mM imidazole in PBS buffer. flowed sequentially. The target protein was eluted by flowing 200 mM imidazole into PBS buffer, which was the last step. The eluted protein was converted into a buffer solution (50 mM Tris-HCl pH 7.0) for measuring enzyme activity and used in subsequent experiments.

Example 3. Preparation of mutant amylosucrase

To prepare a single point mutant enzyme, a recombinant vector pBT7-N-His/amylosucrasse was prepared by mutagenesis using the primers shown in the table below and a Quickchange site-directed mutagenesis kit.

gene direction sequence number order BtAS P200R Forward 31 5′-GCCGTCGTCCGCCAAGTCTTC-3′ Reverse 32 5′-GAAGACTTGGCGGACGACGGC-3′ BtAS V202I Forward 33 5′-GTCCCGCAAATCTTCCCGACC-3′ Reverse 34 5′-GGTCGGGAAGATTTGCGGGAC-3′ BtAS-Y265F Forward 35 5′-GACGCGGTGCCGTTCATCTGGAAGCAA-3′ Reverse 36 5'-TTGCTTCCAGATGAACGGCACCGCGTC-3' BtAS V305I Forward 37 5′-AAAGGTGAAGTCATCATGGCTCCCAAG-3′ Reverse 38 5′-CTTGGGAGCCATGATGACTTCACCTTT-3′ BtAS K393R Forward 39 5′-CCGCTCAAGCACAGGGAATTCCTCTAC-3′ Reverse 40 5′-GTAGAGGAATTCCCTGTGCTGAGCGG-3′ BtAS S420T Forward 41 5′-TATGATCCGGCGACGGGTGACGCGCGC-3′ Reverse 42 5′-GCGCGCGTCACCCGTCGCCGGATCATA-3′ BtAS-Y414F Forward 43 5′-ATGGGCGAGCTGTTCAACTATGATCCG-3′ Reverse 44 5'-CGGATCATAGTTGAACAGCTCGCCCAT-3'

In the induced point mutation, the 200th proline of the wild-type amylosucrase enzyme is arginine (SEQ ID NO: 4), the 202nd valine is isoleucine (SEQ ID NO: 5), and the 265th tyrosine is phenylalanine (SEQ ID NO: 6 ) Valine at position 305 is replaced with isoleucine (SEQ ID NO: 7), lysine at position 393 is replaced with arginine (SEQ ID NO: 8), tyrosine at position 414 is replaced with phenylalanine (SEQ ID NO: 9), and serine at position 420 is replaced with threonine (SEQ ID NO: 10) will be.

The plasmid was transformed into E. coli BL21 (DE3) by the transformation method, and the mutant amylosucrase enzyme was isolated in the same manner as in Example 2.2.

Example 4. Preparation of mutant amylosucrase enzymes

In order to prepare a mutant enzyme having a double point mutation, the Y414F mutant and the double point mutation (applying P200R to Y414F) of the mutant with a faster reaction completion time than the wild type enzyme were grafted.

A double point mutant amylosucrase enzyme, Y414F*P200R (SEQ ID NO: 9), was prepared using the P200R primer in the pBT7-N-His/amylosucrase Y414F mutant enzyme vector. The plasmid was transformed into Escherichia coli BL21 (DE3) by a transformation method, and the stock culture of the recombinant E.coli BL21 was inoculated into 5 mL of LB medium at a temperature of 37 ° C for 12 hours to mass-produce amylosucrase. After pre-incubation at 180 rpm for 15 minutes, the entire culture was inoculated into 1 L LB medium contained in a 2 L flask sterilized at 120 ° C for 15 minutes, and then in the presence of 0.01% (w / v) ampicillin, the culture temperature was 37 ° C. , The stirring speed was incubated at 180 rpm. When the absorbance at 600 nm reached 0.6, mass expression of the enzyme was induced by adding isopropyl-β-D-thiogalactopyranoside at a final concentration of 0.2 mM. After adding isopropyl-β-D-thiogalactopyranoside, the culture temperature was lowered to 16° C. and cultured for 20 hours. Cultures of the overexpressed amylosucrase enzyme were centrifuged at 3,000 x g for 10 min at 4 °C to harvest the precipitate. The precipitate was completely resuspended by vortexing in 45 mL of 50 mM Tris-HCl buffer solution (pH 7), and then the cell solution was disrupted using a sonicator (Sonic Dismembrator 550, Fisher Scientific Co.) Model D100. The cell lysate is centrifuged at 11,000 x g for 20 minutes at 4° C. to obtain only the supernatant excluding cell debris as a precipitate. The reaction product obtained in this process was named cell extract.

A small amount of the harvested cell extract is collected and stored to determine the yield of enzyme purification. After filtering the cell supernatant through a sterile 0.45 μm syringe filter, 45 mL of the filtrate was passed through a nickel-nitrilotriacetic acid (Ni-NTA) affinity column to obtain recombinant His-tagged amylase. Purified amylosucrase enzyme was obtained by passing through sucrase. The column was washed first with 40 mL Lysis buffer (50 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 7.0), then with 40 mL Elution buffer (50 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole). dazole, pH 7.0) to obtain the desired protein. The purified protein was concentrated at 4° C. using Amicon Centrifugal Filter Devices (Millipore Corporation, Billerica, USA) and isolated as an amylosucrase enzyme used in the production of turanose.

Comparative Example 1. Preparation of Neisseria subflava-derived amylosucrase

Polynucleotide (SEQ ID NO: 4) encoding amylosucrase (SEQ ID NO: 1) derived from Neisseria subflava was synthesized by commissioning Bioneer.Co.Korea to be optimized for E. coli to be used as an expression strain. .

The synthesized polynucleotide of SEQ ID NO: 4 was inserted into the same restriction enzyme site of the expression vector pRSet(A) (invitrogen) using restriction enzymes NheI and XhoI (NEB) to recombinant vector pRSet(A)-amylosucrase. (pRSet(A)-NsAS) was prepared. Thereafter, E. coli BL21 (DE3) (invitrogen) was transformed with the prepared recombinant vector by the heat shock method (Sambrook and Russell Molecular Cloning.) to prepare a recombinant strain containing a polynucleotide encoding the amino acid sequence of SEQ ID NO: 1.

The transformed recombinant strain was inoculated into 5ml LB-ampicilline medium (Difco), cultured with shaking at 37°C and 200rpm until the absorbance (OD) at 600nm reached 1.5, and then the culture medium was incubated in 500ml LB-ampicillin medium. Inoculated in ampicilline medium, and cultured with shaking at a temperature of 37 ° C and 200 rpm. When the absorbance at 600 nm of the culture medium was 0.5, 0.1 mM IPTG was added to induce overexpression of the target enzyme. (At this time, from the time of overexpression induction, the culture conditions were switched to a temperature of 16 ° C and 150 rpm and maintained for 16 hours. Then, the cells were recovered by centrifugation at 6000 rpm for 20 minutes. The recovered cells were 0.85% (w / v) After washing twice with NaCl, the cells were used for production of turanose and purification of enzymes.

Example 5. Activity assay of wild-type amylosucrase

5.1 Enzyme activity as a function of temperature

The effect of temperature on the enzymatic activity of the amylosucrase enzyme obtained in Example 1 was determined in the range of 25 to 65°C. The reaction mixture in a total volume of 0.5 mL containing 0.1 M sucrose, 50 mM Tris-HCl buffer (pH 7.0) and diluted enzyme was incubated at 25, 30, 35, 40, 45, 50, 55, 60 and 65 °C. was cultured in The temperature dependence of BtAS activity is a DNS method for determining the amount of reducing sugar released at various temperatures, that is, the enzyme activity at each temperature was obtained through a reducing equivalent measurement method using a DNS solution and expressed as a relative activity. The results are shown in FIG. 3 . Specifically, in the measurement of enzyme activity by the DNS method, 100 mM sucrose as a substrate is treated with 0.02 mg/ml of amylosucrase, reacted at the pH and temperature conditions for 30 minutes, and DNS reagent is added to stop the reaction. After heating at 100 ° C. for 5 minutes to induce a color reaction of reducing sugar in DNS, cooling on ice, ABS was measured at 575 nm, and then calculated by comparing with fructose standard.

As can be seen from the results of FIG. 3, the ability to hydrolyze sucrose, which is a substrate, was maximized at 50 °C. It shows 98% relative activity at 55℃ and about 85% residual activity at 60℃. It was confirmed that most of the activity decreased at 65 ° C. Therefore, wild-type amylosucrase BtAS showed maximum activity while being stable even at 50 ° C, so 50 ° C was confirmed as the optimum temperature.

5.2 Assay of activity according to pH

In order to confirm the optimal pH for the amylosucrase enzyme purified in Example 1, the optimal pH for enzyme activity was measured in the range of 4.0 to 10.0. Four buffer systems were used to analyze the effect of pH on enzyme activity: 50 mM sodium acetate buffer (pH 4.0 to 6.0); 50 mM sodium phosphate buffer (pH6.0 - 7.5); 50 mM Tris-HCl buffer (pH 7.0 - 9.0); A reaction mixture containing 50 mM glycine-sodium hydroxide (pH 8.0 - 10.0) 0.1 M sucrose as a substrate, 50 mM each buffer at different pH and diluted enzyme was carried out at 50°C for 30 minutes. Enzyme activity was measured using the DNS method presented in Example 5-1 and the results are shown in FIG. 4 .

As a result, the highest relative activity of the wild-type enzyme was observed in 50 mM sodium acetate buffer at pH 6.0. When the pH was moved from the optimum pH 6.0 to the acidic (pH 5.5-4.0) and alkaline (pH 7.0-9.0) ranges, their relative activity decreased significantly. The buffer system should not affect the enzyme activity, but in the present invention, the enzyme activity was significantly affected by the type of buffer component even at the same pH. Therefore, pH 6.0 in 50 mM sodium acetate buffer was used for further experiments.

At the optimal temperature and optimal pH of 6.0, amylosucrasase derived from Bifidobacterium thermophilum amylosucrase mutant strain V542D was 1.7 U/mg, and amylsucrase derived from bifidobacterium thermophilum amylosucrase mutant strain V542K Rosucrase has an optimal activity of 1.4 U/mg, and bifidobacterium thermophilum amylosucrase mutant strain Y414F*P200R-derived amylosucrasase has an optimal activity of 2.6 U/mg.

5.3. Evaluation of heat resistance and enzyme inactivation constant (Kd) of amylosucrase

In order to evaluate the heat resistance and enzyme deactivation constant of wild-type amylosucrase, the wild-type amylosucrase purified in Example 1 was heat-treated in a constant-temperature shaking water bath for up to 120 hours. After heat treatment at temperatures of 45, 50, 55, and 60 ° C, 10 μl of a dilution of the heat-treated enzyme was added to 490 μl of 50 mM buffer containing 0.1 M sucrose to measure the residual enzyme activity, and then at 35 ° C. After reacting for 30 minutes, 500 μl of DNS solution was added to the reaction solution, boiled at 100 ° C for 5 minutes, and then cooled in ice water for 5 minutes. By measuring the absorbance at Abs575 and measuring the reducing equivalent from the standard curve, the relative value of the enzyme activity according to the temperature was derived, and the results are shown in Figure 5 (A, 45 ℃; B, 50 ℃?; C, 55 ℃?; D, 60°C?). Based on this value, the relative activity according to temperature and time was expressed as a function, and converted into a natural logarithmic function to obtain kd and temperature half-life, which are shown in the table below.

Temperature (℃) Kd(h ^-1 ) t _1/2 (h) 45 0.00003 23104.9 50 0.0012 577.6 55 0.0098 70.7 60 0.762 0.9

As can be seen from the table above, the wild-type amylosucrase BtAS has a temperature half-life of about 23,104 hours at 45 ° C and a temperature half-life of about 577 hours at 50 ° C. seemed At 55 ° C, which is 5 ° C higher than this temperature, the temperature half-life tended to decrease rapidly to about 70 hours, but the overall stability to heat was excellent.

Example 6. Turanose Conversion Reaction of Amylosucrase

6.1 Bifidobacterium thermophilum-derived amylosucrase turanose conversion rate

In order to examine the turanose conversion rate of amylosucrase of Examples 1 to 4, a substrate was prepared by mixing sucrose and fructose at a weight ratio of 1:0.2 and dissolving them to a concentration of 63.5bx, and adjusting the pH to 6.0. did 700 U/L of the wild type of Bifidobacterium thermophilum-derived turanose converting enzyme (BtAS) of Example 1 was added, and the reaction was performed at 50° C. for 24 hours. Variants 1 (V542D) and 2 (V542K) of BtAS of Example 2, and variant 3 (Y414F*P200R) of Example 4 were reacted under the same conditions as the wild type. After the conversion reaction of each enzyme, the sugar composition was analyzed by HPLC under the conditions shown in the table below.

column Bio-rad Aminex HPX-87C injection volume 10 μl flow rate 0.6ml/min column temperature 80℃ mobile phase Water 100%

The sugar composition results after the conversion reaction according to the BtAS enzyme derived from Bifidobacterium thermophilum are shown in the table below. In the above table, the sugar composition analysis of the product obtained as a result of conversion shows the solid content (weight) of each constituent saccharide as a percentage based on the total saccharide solid content (weight) contained in the reaction product (the unit of the table below is % by weight to be). The oligosaccharide is mainly a glucose oligomer in which glucose is linked by an alpha 1-4 linkage, and most of the oligosaccharide is composed of DP3 and DP4 or higher saccharides.

division oligosaccharide sugar Turanos trehalulose fruit sugar BtAS wild type 16.1 0 46.7 3.3 33.9 BtAS V542D 18.5 0 46.9 3.4 31.2 BtAS V542K 18.1 0 46.1 4.2 31.6 BtAS Y414F*P200R 15.8 0 50.0 3.2 31.0

As confirmed in the table above, bifidobacterium-derived amylosucrases has a fast reaction rate as the reaction temperature is as high as 50°C, consumes all sugar substrates and produces maximum turanose with 700 U/L of enzyme within 24 hours. and the reaction could be terminated.

6.2 Neisseria subflava-derived amylosucrase turanose conversion rate

In order to examine the turanose conversion rate of the Neisseria subflava-derived amylosucrasase of Comparative Example 1, the analysis was performed under the same conditions and methods as in Example 6.1, but the Neisseria subflava-derived turanose converting enzyme (NsAS) 500, 700, 1000, 1400, and 1900 U/L were added, and the reaction was conducted at 35° C. for 48 hours, and the results are shown in the table below.

division oligomer sugar Turanos trehalulose fruit sugar NSAS 500U/L 1.9 41.8 25.2 8.8 22.3 NSAS 700U/L 3.5 29.7 35.3 8.8 22.7 NSAS 1000U/L 6.5 16.6 47.7 7.7 21.5 NSAS 1400U/L 13.0 0 55.9 7.7 23.4 NSAS 1900U/L 12.6 0 56.7 7.9 22.8

As confirmed in the table above, NsAS has a slow reaction rate as the reaction temperature is as low as 35°C, and it is difficult to complete the reaction within 48 hours with the input of 1000 U/L of enzyme. It was confirmed that many enzymes of 1400 U/L or more are required for maximum production. Looking at the results of turanose production using Neisseria subflava-derived amylosucrase, it was confirmed that the trehalulose content was high, which was not preferable for separation and purification of turanose.

Therefore, compared to the amylosucrase derived from Neisseria subflava, Bifidobacterium-derived amylosucrase rapidly consumes the substrate with a smaller amount of enzyme input and can quickly terminate the entire conversion reaction, thereby reducing the amount of enzyme input. And it was confirmed that the reaction rate was shortened.

Example 7. Conversion reaction of mutant amylosucrase

7.1 Wild-type amylosucrase turanose conversion reaction

Sucrose at concentrations of 1.0, 1.5, 2.0 and 2.5 M (1.0S, 1.5S, 2.0S and 2.5S, respectively) and fructose at 0.25, 0.5, 0.75 and 1M in 50 mM sodium acetate buffer (pH 6.0). Turanose was synthesized from a substrate containing at concentrations of (0.25F, 0.5F, 0.75F and 1F, respectively). Enzymatic reactions were performed for 48 hours with 400 U/L of wild-type enzyme. After incubation, the reaction mixture was heat-treated in boiling water for 10 min to inactivate the enzyme. The inactivated reaction solution was filtered through a 0.2 μm syringe filter and the soluble fraction was analyzed by HPAEC. The soluble fraction was analyzed by high-performance anion exchange chromatogram coupled with pulsed amperometric detection (HPAEC-PAD) on a CarboPacTM PA1 analytical column (4 x 250 mm, DIONEX, SUNNYVALE, CA, USA) to detect low amounts of glucose, fructose, sucrose and oxalate. Lanose was quantified. The supernatant of the reaction was appropriately diluted with distilled water and filtered through a 0.2 μm syringe filter. The injection volume was 50 μl and analyzed at a flow rate of 1.0 mL/min with 100 mM NaOH solution. The content composition of saccharides was determined by area comparison with standard materials.

The effect of the substrate composition on the turanose conversion rate was performed at a temperature of 50° C. with 400 U/L of BtAS and a 50 mM sodium acetate buffer at pH 6.0, and the analyzed results are shown in FIG. 6 . The turanose conversion rate means the production amount of turanose converted from a substrate (sucrose), and is expressed as a ratio (%) of the turanose concentration produced to the substrate concentration.

As can be seen from the experimental results, after the enzymatic reaction for 48 hours, the final turanose conversion significantly increased as the fructose concentration increased up to 1.0 M. This suggested that exogenous fructose as a co-substrate promotes turanose production. By adding 1.0 M fructose in the reaction mixture, the turanose conversion increased from 22.7% to 43.3% with 1.0 M sucrose and from 23.7% to 39.4% with 1.5 M sucrose. Therefore, it was confirmed that the yield of turanose was highly dependent on the initial fructose concentration of the BtAS biotransformation system.

That is, it seems that the fructose portion derived from sucrose is directly used as an acceptor for producing turanose without being released from the enzyme, and the exogenous addition of fructose further improves the yield of turanose production. Optimal substrate conditions were 1.0 M each of sucrose and fructose, and the turanose conversion rate was 43.3%.

7.2. Turanose productivity of the mutant enzyme of Example 2

In order to examine the turanose productivity of the two mutant amylosucrases of Example 2, 100 U / L to 500 U / L of amylosucrase in substrates of 2 M sugar and 0.75 M fructose (where Unit is hydrolysis As an activity, it means the amount of fructose produced per minute when 100 mM sugar is treated with 1 mg of amylosucrase and reacted at 50 ° C. for 30 minutes.) was added to start the turanose conversion reaction.

7 and 8 are graphs showing the correlation between the concentration of enzyme and the concentration of turanose produced per hour. In both the formulas of FIGS. 7 and 8, the x value means the concentration (U / L) of the enzyme added, and the y value represents the concentration of turanose produced per hour in g / L, and the unit is g / L / hr . The formula is y = 0.082x + 7.3862, and the turanose produced per hour was calculated when the mutant amylosucrase V542D was added to 2M sugar and 0.75M fructose. As a result, it was confirmed that the turanose productivity of amylosucrase V542D derived from Bifidobacterium thermophilum was 8.9 U/mg.

In the same way, 100 U/L to 500 U/L of amylosucrase V542K was added to 2M sugar and 0.75M fructose, and samples were taken and analyzed at each time point. As a result, the correlation between enzyme concentration and turanose production as shown in FIG. The relationship was y = 0.1879x + 8.0043. Through this, it was confirmed that the turanose productivity of the mutant amylosucrase V542K derived from Bifidobacterium thermophilum was 9.9 U/mg.

When reacted at 50°C with 2M sugar or 0.75M fructose as a substrate, the activity of mutant amylosucrase V542D was 8.9 U/mg and that of mutant amylosucrase V542K was 9.9 U/mg. It is a value that is more than twice that of sucrase 3.0U/mg, and it can be said that the enzyme activity has been significantly improved by DNA mutation method.

7.3 Activity and productivity of the mutant enzyme of Example 3

The enzyme activity of the single point mutation type amylosucrase enzyme of Example 3 was measured in the same manner as in Example 5.1, and the turanose conversion rate of the single point mutation type amylosucrase enzyme was measured at pH 6 and 50 ° C. At this temperature, the ratio (%) of the concentration of turanose produced to the concentration of the substrate after reacting with 2M sucrose as a substrate at 400 U/L was calculated. In addition, the concentration of turanose produced from the substrate was divided by the reaction completion time to calculate the turanose (concentration) produced per hour when turanose was converted from the substrate, and is shown in the table below.

When Y414F reacted at 400 U/L, sucrose was not completely consumed and the rate slowed down. reacted with The measured and calculated results are shown in the table below.

After purification amount of reaction enzyme
(unit/L) protein amount
(mg/10ml) enzyme inactivity
(unit/mg) Turanose Conversion Rate
(%) Reaction completion time (h) Turanose produced per hour (g/L/h) Wild-type enzyme (BtAS) 400 3 1.3±0.1 25±2.5 12 14.25 P200R 400 8 0.5±0.0 27±1.5 12 15.39 V202I 400 7 0.9±0.0 29±2.0 12 16.53 Y265F 400 6 0.6±0.2 23±1.0 6 26.22 V305I 400 3 1.6±0.1 21±1.0 24 6 K393R 400 4 1.0±0.0 22±0.0 12 12.54 S420T 400 4 1.5±0.3 20 24 5.7 Y414F 400 One 4.1±0.8 12.5±2.5 >24 - Y414F 1600 4 4.1±0.8 35±1.4 12 19.95

As shown in the above results, compared to the wild-type enzyme, the specific activity of the mutant in which the 414th tyrosine was substituted with phenylalanine was specifically 4-fold higher. In order to confirm the turanose conversion rate of all mutants, as a screening concept, the reaction was performed by adjusting the amount of enzyme to 400 U / L. As a result, P200R and V202I showed higher turanose conversion rates than wild-type enzymes, and Y265F confirmed that the reaction rate was faster. Could. In addition, as a result of reacting Y414F at 1600 U/L, sucrose was completely consumed and the reaction was terminated in 12 hours, and the conversion rate of turanose was about 35%. It was confirmed that the Lanose conversion rate was the highest.

7.4. Activity and productivity of double-point mutant enzymes

The double-point mutant amylosucrase enzyme of Example 4 was reacted with the substrate 2 M sucrose at 400 U/L and 1600 U/L at pH 6 and a temperature of 50° C. in the same manner as in the experiment of 7.3 above. After turanose conversion and sucrose consumption were calculated, they are shown in FIGS. 9 and 10 .

After purification amount of reaction enzyme
(unit/L) protein amount
(mg/10ml) enzyme inactivity
(unit/mg) Turanose Conversion Rate (%) Reaction completion time (h) Turanose produced per hour (g/L/h) Wild-type enzyme (BtAS) 400 3 1.3±0.1 25±2.5 12 14.25 Y414F*P200R 400 1.5 2.6±0.5 17±0.5 >24 - Y414F*V202I 400 One 4.0±0.2 14±2.5 >24 - Y414F*Y265F 400 One 4.2±0.1 13±2.0 >24 - Y414F*P200R 1600 6 2.6±0.5 50±4 6 57 Y414F*V202I 1600 4 4.0±0.2 41.5±4.9 6 47 Y414F*Y265F 1600 4 4.2±0.1 40.5±2.1 6 46

Like the Y414F single-point mutant mutant, the Y414F-based double-point mutant mutant did not completely consume sucrose when reacted at 400 U/L, so the maximum turanose conversion rate was not confirmed. However, as a result of the reaction at 1600 U / L, the turanose conversion rate was high, and among them, the Y414F * P200R double point mutation mutant grafted with the Y414F mutant and P200R showed the highest turanose conversion rate at 50%.

Example 8. Production of Turanose (Cell Extract) Containing Double Point Mutant Enzyme

After pre-heating 10 ml of 50 mM sodium acetate buffer (pH 6.0) containing 2 M sucrose and a mixed substrate of 2 M sucrose and 0.75 M fructose, respectively, for 10 minutes, Y414F*P200R mutant enzyme cell extract state of Example 4 16 units of the previously purified novel amylosucrase were added to the sucrose buffer solution to a concentration of 16 units/10ml. The precipitate harvested in Example 4 was completely resuspended by vortexing in 45 mL of 50 mM Tris-HCl buffer solution (pH 7), and then the cell solution was sonicated using a sonicator (Sonic Dismembrator 550, Fisher Scientific Co.) Model D100. destroyed. The cell lysate was centrifuged at 11,000 x g for 20 minutes at 4 to obtain a supernatant and cell extract except for cell debris as a precipitate.

Enzymatic reaction was performed at 50° C. for 12 hours, and after heating in boiling water for 10 minutes to terminate the enzymatic reaction, the obtained reaction product was diluted 10000 times and analyzed using an UltiMate 3000 HPLC system (Dionex, USA). At this time, the enzyme reaction was analyzed using glucose, fructose, sucrose, turanose, maltose (Sigma-Aldrich Chemical Co., St. Louis, MO), and trehalulose (Samyang, Seongnam, Korea) as standard materials. The conversion rate of turanose was calculated using a standard curve obtained by analyzing turanose standards at various concentrations.

Before analysis of turanose, the sample is analyzed after pre-treatment with Invertase to remove sucrose. The reason for removing sucrose is that the peaks of turanose and sucrose overlap and affect the quantification of turanose. As the turanose analysis conditions, Bio-rad Aminex HPX-87C was used for the HPLC column, the sample injection volume was 10 ul, the column oven temperature was 80 ° C, and the mobile phase solvent was 100% water.

The sugar analysis results of the samples 6 hours after the reaction using 2M sucrose as substrates and the sugar analysis results of the samples after 6 hours of reaction using 2M sucrose and 0.75M fructose as substrates are shown in the table below. Organized.

temperament oligomer
(weight%) Turanos
(weight%) Glucose + Trehalulose
(weight%) 2M sucrose 30.02 42.01 0.16 2M sucrose +0.75M fructose 17.05 51.12 0.95

As shown in the above results, when the substrate is reacted with only 2M sucrose, turanose is 42.01%, and when the substrate is reacted with 2M sucrose as well as 0.75M fructose, turanose is produced at a higher level of 51.1%. confirmed that.

Example 9. Amylosucrase Conversion Reaction According to Substrate Composition

In order to examine the reactivity according to the substrate composition in the conversion reaction of amylosucrase, the solid weight ratio of sugar and fructose used as substrate was 1:0, 1:0.2, 1:0.5, 1:0.75, 1:1, 1 : 1.5 and water was added to prepare a substrate by dissolving to a concentration of 63.5bx, and the pH was adjusted to 6. The sugar solid content corresponds to 2M, and the specific weight of sugar and fructose is shown in Table 12 below.

Sugar:fructose mixing ratio Sugar (g/L) Sugar molarity (M) Fructose (g/L) Fructose molarity (M) 1:0.0 685 2 0 0 1:0.2 685 2 135 0.75 1:0.5 685 2 342 1.9 1:0.7 685 2 513 2.8 1:1.0 685 2 685 3.8 1:1.5 685 2 1027 5.7

700 U/L of the BtAS (Y414F*P200R) enzyme of Example 4 was added to substrates having different compositions of sugar and fructose, and the reaction was performed at 50° C. for 24 hours. After enzymatic conversion of each substrate, analysis of sugar composition was performed under the same conditions as in Example 6 above.

The sugar composition results after the conversion reaction of the BtAS (Y414F*P200R) enzyme according to the substrate composition are shown in the table below. In Table 13 below, the contents of each saccharide contained in 100% by weight of the total saccharide solid content in the reaction product are expressed in weight%.

Sugar:fructose mixing ratio oligosaccharide Oligosaccharide/Turanose Ratio sugar Turanos trehalulose fruit sugar 1:0.0 32.4 82.2% 0 39.4 2.9 25.3 1:0.2 16.5 33.0% 0 50.0 3.5 30.0 1:0.5 8.2 16.5% 0 49.7 3.5 38.6 1:0.7 5.8 12.8% 0 45.2 3.3 45.7 1:1.0 4.0 9.7% 0 41.3 3.1 51.6 1:1.5 2.4 7.0% 0 34.4 2.5 60.7

As confirmed from the above results, it was confirmed that when the weight of fructose relative to sugar in the substrate is increased, the production of oligosaccharide decreases and the ratio of sugar decreases, so the amount of turanose produced decreases. Trehalulose was maintained at a level of 3 to 4% by weight without being significantly affected by the fructose ratio. The composition of the substrate, in which oligosaccharides significantly decreased compared to the amount of turanose production, started from sugar:fructose = 1:0.2 and greatly decreased at 1:0.5, and then gradually decreased as the fructose content increased. Looking at the mixing ratio of sugar and fructose, as the content of fructose mixed with the same sugar solid content increased, the oligosaccharide content compared to the amount of turanose produced decreased, and it was determined that the sugar composition was suitable for SMB chromatography. However, if the fructose content added to the sugar substrate is too high, the amount of turanose produced decreases, so a product with a low ratio of oligosaccharide content as an impurity to turanose content while securing the amount of turanose produced above a certain ?t amount It is desirable to appropriately select the mixing ratio of sugar and fructose to obtain.

Example 10. High-purity separation of turanose from amylosucrase conversion reaction composition

10.1 Bifidobacterium thermophilum-derived amylosucrase conversion reaction composition

Bifidobacterium thermophilum-derived amylosucrase conversion reaction composition of Example 9 by adding 700 U / L of the BtAS (Y414F * P200R) enzyme of Example 4 with the composition of the substrate as sugar: fructose = 1: 0.5 It was obtained by conversion reaction in the same way as in

The conversion reaction composition by amylosucrase contains 0.3% by weight, PH 4.5, pH 4.5, Oligosaccharide was decomposed into glucose by reacting at 60 °C. Thereafter, the turanose fraction was separated using a Na+ type cation exchange resin. The sugar composition of the composition before and after separation of the turanose fraction by SMB chromatography is shown in the table below.

division oligosaccharide sugar Turanos trehalulose glucose fruit sugar before separation 1.5 0 52.2 2.8 7.0 36.5 after separation 4.0 0 91.6 3.8 0.6 0

As a result of high-purity separation of turanose using SMB chromatography, a 91.6% content of turanose composition was obtained in a yield of 65%. SMB chromatography is designed to be suitable for two-phase separation, so oligosaccharides are decomposed into glucose by glucoamylase enzyme treatment, and two fractions on both sides are obtained based on the point between the turanose peak and the trehalulose peak on the SMB chromatogram. Separation was made between lanose and trehalulose, and as a result, since only turanose and some oligomers exist in the left fraction, the purity of turanose in the left fraction increases to obtain a turanose fraction, and the right fraction has almost no turanose Therefore, it became a raffinate, and eventually, a high-purity turanose fraction was obtained.

The BtAS enzyme conversion reaction composition had a low trehalulose content before SMB separation, so the purity and yield of turanose after separation by SMB chromatography could be increased. That is, the turanose peak and the trehalulose peak overlap slightly, so when both fractions are obtained from between the two, the two substances are not completely separated and each of the two fractions is accompanied little by little. Therefore, when the trehalulose content is small, the amount of trehalulose separated toward the turanose fraction decreases, so that turanose can be obtained with a higher content and purity from the turanose fraction.

10.2 Conversion reaction composition of Neisseria subflava-derived amylosucrase

A composition for the conversion reaction of Neisseria subflava-derived enzyme was obtained by inputting 1400 U/L of the NsAS enzyme of Comparative Example 1 and performing the conversion reaction in the same manner as in Example 10.1. In order to facilitate SBM chromatography, the conversion reaction composition reacts Glucoamlyase (Novozyme) at 0.3% by weight, pH 4.5, based on the solid content (weight) of the reaction product obtained after the turanose conversion reaction at 60 ° C to form oligomers. broken down into glucose.

Thereafter, a turanose fraction was separated using a Na+ type cation exchange resin. The sugar composition of the composition before and after separation of the turanose fraction by SMB chromatography is shown in the table below.

division oligosaccharide sugar Turanos trehalulose glucose fruit sugar before separation 1.6 0 60.6 8.0 7.4 22.4 after separation 4.8 0 87.2 7.3 0.7 0

In the case of the conversion reaction product of Neisseria subflava-derived amylosucrase, a turanose composition having an 87.2% content was obtained in a yield of 38% after high-purity separation using SMB chromatography. The purity of turanose separation did not reach 90% or more due to the high content of trehalulose, which is closely associated with turanose.

<110> SAMYANG CORPORATION INDUSTRY ACADEMY COOPERATION FOUNDATION OF SEJONG UNIVERSITY <120> Method of producing turanose <130> DPP20173326KR <160> 44 <170> KoPatentIn 3.0 <210> 1 <211> 603 <212> PRT <213> Artificial Sequence <220 > <223> amylosucrase BtAS wild type <400> 1 Met Glu Ala Thr Tyr Arg Asp Ser Val Phe Ala Glu Arg Leu Ala Pro 1 5 10 15 Arg Cys Ala Glu Leu Glu Gln Leu Phe Arg Ser Leu Tyr Gly Asp Ser 20 25 30 Pro Glu Phe Asp His Phe Glu Gln Val Met Ala Lys Ala His Ala Asp 35 40 45 Arg Pro Ala Asp Leu Lys Arg Leu Asp Ala Ala Arg Glu His Asp Pro 50 55 60 Gln Trp Tyr Arg Arg Gly Asp Met Phe Gly Met Thr Met Tyr Thr Asp 65 70 75 80 Leu Phe Ala Gly Lys Leu Thr Asp Leu Ala Lys His Ile Asp Tyr Leu 85 90 95 Lys Glu Gln His Leu Thr Tyr Leu His Leu Met Pro Leu Leu Thr Met 100 105 110 Pro His Pro Asp Asn Asp Gly Gly Tyr Ala Ile Glu Asp Phe Asp Thr 115 120 125 Val Asp Pro Thr Ile Gly Thr Asn Glu Asp Leu Ala Asp Leu Thr Ala 130 135 140 Lys Leu Arg Glu Ala Gly Ile Ser Leu Cys Leu Asp Phe Val Met Asn 145 150 155 160 His Thr Ala Ser Thr His Arg Trp Ala Lys Ala Ala Gln Ala Gly Asp 165 170 175 Pro Glu Tyr Gln Asp Tyr Tyr Phe Cys Tyr Asp Asp Arg Thr Ile Pro 180 185 190 Asp Gln Tyr Asp Ala Val Val Pro Gln Val Phe Pro Thr Ala Ala Pro 195 200 205 Gly Asn Phe Thr Trp Asn Glu Gln Met Gly Lys Trp Val Met Thr Gln 210 215 220 Phe Tyr Pro Phe Gln Trp Asp Leu Asn Tyr Arg Asn Pro Lys Val Phe 225 230 235 240 Val Val Met Met Ser Ser Leu Leu His Leu Ala Asn Leu Gly Val Glu 245 250 255 Val Phe Arg Ile Asp Ala Val Pro Tyr Ile Trp Lys Gln Leu Gly Thr 260 265 270 Asn Cys Arg Asn Leu Pro Gln Val His Thr Ile Val Arg Met Met Arg 275 280 285 Ile Met Ser Glu Ile Val Cys Pro Ala Val Val Phe Lys Gly Glu Val 290 295 300 Val Met Ala Pro Lys Glu Leu Ala Ala Tyr Phe Gly Thr Pro Glu Lys 305 310 315 320 Pro Glu Cys His Met Leu Tyr Asn Val Ser Val Met Val Asn Leu Trp 325 330 335 Ser Ala Leu Ala Asn Gly Asp Thr Arg Leu Leu Lys Thr Gln Ile Asp 340 345 350 Lys Leu Asp Ala Leu Pro Asp Asn Cys Trp Phe Val Asn Tyr Leu Arg 355 360 365 Cys His Asp Asp Ile Gly Trp Gly Leu Asp Glu Asp Val Glu Arg Gln 370 375 380 Leu Gly Ile Asp Pro Leu Lys His Lys Glu Phe Leu Tyr His Phe Tyr 385 390 395 400 Glu Gly Met Val Pro Gly Ser Trp Ala Met Gly Glu Leu Tyr Asn Tyr 405 410 415 Asp Pro Ala Ser Gly Asp Ala Arg Ser Cys Gly Thr Thr Ala Ser Leu 420 425 430 Cys Gly Ile Glu Arg Ala Leu Ile Thr His Asp Arg Pro Leu Tyr Glu 435 440 445 Arg Ser Ile Gln Arg Asp Leu Leu Met His His Ala Met Gly Phe Leu 450 455 460 Arg Gly Phe Pro Met Leu Asn Cys Gly Asp Glu Ile Gly Gln Leu Asn 465 470 475 480 Gly Trp Asp Tyr Lys Glu Asp Pro Asp Arg Val Ala Asp Ser Arg Asn 485 490 495 Leu His Arg Ser Lys Phe Asn Trp Lys Asn Ala Ala Lys Arg Asp Val 500 505 510 Pro Gly Thr Leu Pro Asn Arg Leu Trp Glu Gly Met Ala Asp Val Arg 515 520 525 Gln Met Arg Ser Asp Pro Cys Phe Ala Pro Asp Ala Trp Val Thr Thr Thr 530 535 540 Trp Asp Ala His Asp Asp Gly Ile Leu Ala Met Val Arg Gln Ser Gly 545 550 555 560 Gly Arg Thr Leu Leu Gly Val Phe Asn Phe Ala Asn Arg Asp Ala Thr 565 570 575 Ala Thr Leu Asp Ser Ile Glu Gly Val Ser Leu Pro Arg Thr Val Ala 580 585 590 Leu Lys Pro Tyr Glu Trp Lys Ile Glu Ala Cys 595 600 <210> 2 <211> 603 <212> PRT <213> Artificial Sequence <220> <223> amylosucrase BtAS V542K <400> 2 Met Glu Ala Thr Tyr Arg Asp Ser Val Phe Ala Glu Arg Leu Ala Pro 1 5 10 15 Arg Cys Ala Glu Leu Glu Gln Leu Phe Arg Ser Leu Tyr Gly Asp Ser 20 25 30 Pro Glu Phe Asp His Phe Glu Gln Val Met Ala Lys Ala His Ala Asp 35 40 45 Arg Pro Ala Asp Leu Lys Arg Leu Asp Ala Ala Arg Glu His Asp Pro 50 55 60 Gln Trp Tyr Arg Arg Gly Asp Met Phe Gly Met Thr Met Tyr Thr Asp 65 70 75 80 Leu Phe Ala Gly Lys Leu Thr Asp Leu Ala Lys His Ile Asp Tyr Leu 85 90 95 Lys Glu Gln His Leu Thr Tyr Leu His Leu Met Pro Leu Leu Thr Met 100 105 110 Pro His Pro Asp Asn Asp Gly Gly Tyr Ala Ile Glu Asp Phe Asp Thr 115 120 125 Val Asp Pro Thr Ile Gly Thr Asn Glu Asp Leu Ala Asp Leu Thr Ala 130 135 140 Lys Leu Arg Glu Ala Gly Ile Ser Leu Cys Leu Asp Phe Val Met Asn 145 150 155 160 His Thr Ala Ser Thr His Arg Trp Ala Lys Ala Ala Gln Ala Gly Asp 165 170 175 Pro Glu Tyr Gln Asp Tyr Tyr Phe Cys Tyr Asp Asp Arg Thr Ile Pro 180 185 190 Asp Gln Tyr Asp Ala Val Val Pro Gln Val Phe Pro Thr Ala Ala Pro 195 200 205 Gly Asn Phe Thr Trp Asn Glu Gln Met Gly Lys Trp Val Met Thr Gln 210 215 220 Phe Tyr Pro Phe Gln Trp Asp Leu Asn Tyr Arg Asn Pro Lys Val Phe 225 230 235 240 Val Val Met Met Ser Ser Leu Leu His Leu Ala Asn Leu Gly Val Glu 245 250 255 Val Phe Arg Ile Asp Ala Val Pro Tyr Ile Trp Lys Gln Leu Gly T hr 260 265 270 Asn Cys Arg Asn Leu Pro Gln Val His Thr Ile Val Arg Met Met Arg 275 280 285 Ile Met Ser Glu Ile Val Cys Pro Ala Val Val Phe Lys Gly Glu Val 290 295 300 Val Met Ala Pro Lys Glu Leu Ala Ala Tyr Phe Gly Thr Pro Glu Lys 305 310 315 320 Pro Glu Cys His Met Leu Tyr Asn Val Ser Val Met Val Asn Leu Trp 325 330 335 Ser Ala Leu Ala Asn Gly Asp Thr Arg Leu Leu Lys Thr Gln Ile Asp 340 345 350 Lys Leu Asp Ala Leu Pro Asp Asn Cys Trp Phe Val Asn Tyr Leu Arg 355 360 365 Cys His Asp Asp Ile Gly Trp Gly Leu Asp Glu Asp Val Glu Arg Gln 370 375 380 Leu Gly Ile Asp Pro Leu Lys His Lys Glu Phe Leu Tyr His Phe Tyr 385 390 395 400 Glu Gly Met Val Pro Gly Ser Trp Ala Met Gly Glu L eu Tyr Asn Tyr 405 410 415 Asp Pro Ala Ser Gly Asp Ala Arg Ser Cys Gly Thr Thr Ala Ser Leu 420 425 430 Cys Gly Ile Glu Arg Ala Leu Ile Thr His Asp Arg Pro Leu Tyr Glu 435 440 445 Arg Ser Ile Gln Arg Asp Leu Leu Met His His Ala Met Gly Phe Leu 450 455 460 Arg Gly Phe Pro Met Leu Asn Cys Gly Asp Glu Ile Gly Gln Leu Asn 465 470 475 480 Gly Trp Asp Tyr Lys Glu Asp Pro Asp Arg Val Ala Asp Ser Arg Asn 485 490 495 Leu His Arg Ser Lys Phe Asn Trp Lys Asn Ala Ala Lys Arg Asp Val 500 505 510 Pro Gly Thr Leu Pro Asn Arg Leu Trp Glu Gly Met Ala Asp Val Arg 515 520 525 Gln Met Arg Ser Asp Pro Cys Phe Ala Pro Asp Ala Trp Lys Thr Thr Thr 530 535 540 Trp Asp Ala His Asp Asp Gly Ile Leu Ala Met Val Arg Gln S er Gly 545 550 555 560 Gly Arg Thr Leu Leu Gly Val Phe Asn Phe Ala Asn Arg Asp Ala Thr 565 570 575 Ala Thr Leu Asp Ser Ile Glu Gly Val Ser Leu Pro Arg Thr Val Ala 580 585 590 Leu Lys Pro Tyr Glu Trp Lys Ile Glu Ala Cys 595 600 <210> 3 <211> 603 <212> PRT <213> Artificial Sequence <220> <223> amylosucrase BtAS V542D <400> 3 Met Glu Ala Thr Tyr Arg Asp Ser Val Phe Ala Glu Arg Leu Ala Pro 1 5 10 15 Arg Cys Ala Glu Leu Glu Gln Leu Phe Arg Ser Leu Tyr Gly Asp Ser 20 25 30 Pro Glu Phe Asp His Phe Glu Gln Val Met Ala Lys Ala His Ala Asp 35 40 45 Arg Pro Ala Asp Leu Lys Arg Leu Asp Ala Ala Arg Glu His Asp Pro 50 55 60 Gln Trp Tyr Arg Arg Gly Asp Met Phe Gly Met Thr Met Tyr Thr Asp 65 70 75 80 Leu Phe Ala Gly Lys Leu Thr Asp Leu Ala Lys His Ile Asp Tyr Leu 85 90 95 Lys Glu Gln His Leu Thr Tyr Leu His Leu Met Pro Leu Leu Thr Met 100 105 110 Pro His Pro Asp Asn Asp Gly Gly Tyr Ala Ile Glu Asp Phe Asp Thr 115 120 125 Val Asp Pro Thr Ile Gly Thr Asn Glu Asp Leu Ala Asp Leu Thr Ala 130 135 140 Lys Leu Arg Glu Ala Gly Ile Ser Leu Cys Leu Asp Phe Val Met Asn 145 150 155 160 His Thr Ala Ser Thr His Arg Trp Ala Lys Ala Ala Gln Ala Gly Asp 165 170 175 Pro Glu Tyr Gln Asp Tyr Tyr Phe Cys Tyr Asp Asp Arg Thr Ile Pro 180 185 190 Asp Gln Tyr Asp Ala Val Val Pro Gln Val Phe Pro Thr Ala Ala Pro 195 200 205 Gly Asn Phe Thr Trp Asn Glu Gln Met Gly Lys Trp Val Met Thr Gln 210 215 220 Phe Tyr Pro Phe Gln Trp Asp Leu Asn Tyr Arg Asn Pro Lys Val Phe 225 230 235 240 Val Val Met Met Ser Ser Leu Leu His Leu Ala Asn Leu Gly Val Glu 245 250 255 Val Phe Arg Ile Asp Ala Val Pro Tyr Ile Trp Lys Gln Leu Gly Thr 260 265 270 Asn Cys Arg Asn Leu Pro Gln Val His Thr Ile Val Arg Met Met Arg 275 280 285 Ile Met Ser Glu Ile Val Cys Pro Ala Val Val Phe Lys Gly Glu Val 290 295 300 Val Met Ala Pro Lys Glu Leu Ala Ala Tyr Phe Gly Thr Pro Glu Lys 305 310 315 320 Pro Glu Cys His Met Leu Tyr Asn Val Ser Val Met Val Asn Leu Trp 325 330 335 Ser Ala Leu Ala Asn Gly Asp Thr Arg Leu Leu Lys Thr Gln Ile Asp 340 345 350 Lys Leu Asp Ala Leu Pro Asp Asn Cys Trp Phe Val Asn Tyr Leu Arg 355 360 365 Cys His Asp Asp Ile Gly Trp Gly Leu Asp Glu Asp Val Glu Arg Gln 370 375 380 Leu Gly Ile Asp Pro Leu Lys His Lys Glu Phe Leu Tyr His Phe Tyr 385 390 395 400 Glu Gly Met Val Pro Gly Ser Trp Ala Met Gly Glu Leu Tyr Asn Tyr 405 410 415 Asp Pro Ala Ser Gly Asp Ala Arg Ser Cys Gly Thr Thr Ala Ser Leu 420 425 430 Cys Gly Ile Glu Arg Ala Leu Ile Thr His Asp Arg Pro Leu Tyr Glu 435 440 445 Arg Ser Ile Gln Arg Asp Leu Leu Met His His Ala Met Gly Phe Leu 450 455 460 Arg Gly Phe Pro Met Leu Asn Cys Gly Asp Glu Ile Gly Gln Leu Asn 465 470 475 480 Gly Trp Asp Tyr Lys Glu Asp Pro Asp Arg Val Ala Asp Ser Arg Asn 485 490 495 Leu His Arg Ser Lys Phe Asn Trp Lys Asn Ala Ala Lys Arg Asp Val 500 505 510 Pro Gly Thr Leu Pro Asn Arg Leu Trp Glu Gly Met Ala Asp Val Arg 515 520 525 Gln Met Arg Ser Asp Pro Cys Phe Ala Pro Asp Ala Trp Asp Thr Thr 530 535 540 Trp Asp Ala His Asp Asp Gly Ile Leu Ala Met Val Arg Gln Ser Gly 545 550 555 560 Gly Arg Thr Leu Leu Gly Val Phe Asn Phe Ala Asn Arg Asp Ala Thr 565 570 575 Ala Thr Leu Asp Ser Ile Glu Gly Val Ser Leu Pro Arg Thr Val Ala 580 585 590 Leu Lys Pro Tyr Glu Trp Lys Ile Glu Ala Cys 595 600 <210> 4 <211> 603 <212> PRT <213> Artificial Sequence <220> <223> BtAS P200R <400 > 4 Met Glu Ala Thr Tyr Arg Asp Ser Val Phe Ala Glu Arg Leu Ala Pro 1 5 10 15 Arg Cys Ala Glu Leu Glu Gln Leu Phe Arg Ser Leu Tyr Gly Asp Ser 20 25 30 Pro Glu Phe Asp His Phe Glu Gln Val Met Ala Lys Ala His Ala Asp 35 40 45 Arg Pro Ala Asp Leu Lys Arg Leu Asp Ala Ala Arg Glu His Asp Pro 50 55 60 Gln Trp Tyr Arg Arg Gly Asp Met Phe Gly Met Thr Met Tyr Thr Asp 65 70 75 80 Leu Phe Ala Gly Lys Leu Thr Asp Leu Ala Lys His Ile Asp Tyr Leu 85 90 95 Lys Glu Gln Hi s Leu Thr Tyr Leu His Leu Met Pro Leu Leu Thr Met 100 105 110 Pro His Pro Asp Asn Asp Gly Gly Tyr Ala Ile Glu Asp Phe Asp Thr 115 120 125 Val Asp Pro Thr Ile Gly Thr Asn Glu Asp Leu Ala Asp Leu Thr Ala 130 135 140 Lys Leu Arg Glu Ala Gly Ile Ser Leu Cys Leu Asp Phe Val Met Asn 145 150 155 160 His Thr Ala Ser Thr His Arg Trp Ala Lys Ala Ala Gln Ala Gly Asp 165 170 175 Pro Glu Tyr Gln Asp Tyr Tyr Phe Cys Tyr Asp Asp Arg Thr Ile Pro 180 185 190 Asp Gln Tyr Asp Ala Val Val Arg Gln Val Phe Pro Thr Ala Ala Pro 195 200 205 Gly Asn Phe Thr Trp Asn Glu Gln Met Gly Lys Trp Val Met Thr Gln 210 215 220 Phe Tyr Pro Phe Gln Trp Asp Leu Asn Tyr Arg Asn Pro Lys Val Phe 225 230 235 240 Va l Val Met Met Ser Ser Leu Leu His Leu Ala Asn Leu Gly Val Glu 245 250 255 Val Phe Arg Ile Asp Ala Val Pro Tyr Ile Trp Lys Gln Leu Gly Thr 260 265 270 Asn Cys Arg Asn Leu Pro Gln Val His Thr Ile Val Arg Met Met Arg 275 280 285 Ile Met Ser Glu Ile Val Cys Pro Ala Val Val Phe Lys Gly Glu Val 290 295 300 Val Met Ala Pro Lys Glu Leu Ala Ala Tyr Phe Gly Thr Pro Glu Lys 305 310 315 320 Pro Glu Cys His Met Leu Tyr Asn Val Ser Val Met Val Asn Leu Trp 325 330 335 Ser Ala Leu Ala Asn Gly Asp Thr Arg Leu Leu Lys Thr Gln Ile Asp 340 345 350 Lys Leu Asp Ala Leu Pro Asp Asn Cys Trp Phe Val Asn Tyr Leu Arg 355 360 365 Cys His Asp Asp Ile Gly Trp Gly Leu Asp Glu Asp Val Glu Arg Gln 370 375 380 Leu Gly Il e Asp Pro Leu Lys His Lys Glu Phe Leu Tyr His Phe Tyr 385 390 395 400 Glu Gly Met Val Pro Gly Ser Trp Ala Met Gly Glu Leu Tyr Asn Tyr 405 410 415 Asp Pro Ala Ser Gly Asp Ala Arg Ser Cys Gly Thr Thr Ala Ser Leu 420 425 430 Cys Gly Ile Glu Arg Ala Leu Ile Thr His Asp Arg Pro Leu Tyr Glu 435 440 445 Arg Ser Ile Gln Arg Asp Leu Leu Met His His Ala Met Gly Phe Leu 450 455 460 Arg Gly Phe Pro Met Leu Asn Cys Gly Asp Glu Ile Gly Gln Leu Asn 465 470 475 480 Gly Trp Asp Tyr Lys Glu Asp Pro Asp Arg Val Ala Asp Ser Arg Asn 485 490 495 Leu His Arg Ser Lys Phe Asn Trp Lys Asn Ala Ala Lys Arg Asp Val 500 505 510 Pro Gly Thr Leu Pro Asn Arg Leu Trp Glu Gly Met Ala Asp Val Arg 515 520 52 5 Gln Met Arg Ser Asp Pro Cys Phe Ala Pro Asp Ala Trp Val Thr Thr Thr 530 535 540 Trp Asp Ala His Asp Asp Gly Ile Leu Ala Met Val Arg Gln Ser Gly 545 550 555 560 Gly Arg Thr Leu Leu Gly Val Phe Asn Phe Ala Asn Arg Asp Ala Thr 565 570 575 Ala Thr Leu Asp Ser Ile Glu Gly Val Ser Leu Pro Arg Thr Val Ala 580 585 590 Leu Lys Pro Tyr Glu Trp Lys Ile Glu Ala Cys 595 600 <210> 5 <211> 603 <212> PRT <213> Artificial Sequence <220> <223> BtAS V202I <400> 5 Met Glu Ala Thr Tyr Arg Asp Ser Val Phe Ala Glu Arg Leu Ala Pro 1 5 10 15 Arg Cys Ala Glu Leu Glu Gln Leu Phe Arg Ser Leu Tyr Gly Asp Ser 20 25 30 Pro Glu Phe Asp His Phe Glu Gln Val Met Ala Lys Ala His Ala Asp 35 40 45 Arg Pro Ala Asp Leu Lys Arg Leu Asp Ala Ala Arg Glu His Asp Pro 50 55 60 Gln Trp Tyr Arg Arg Gly Asp Met Phe Gly Met Thr Met Tyr Thr Asp 65 70 75 80 Leu Phe Ala Gly Lys Leu Thr Asp Leu Ala Lys His Ile Asp Tyr Leu 85 90 95 Lys Glu Gln His Leu Thr Tyr Leu His Leu Met Pro Leu Leu Thr Met 100 105 110 Pro His Pro Asp Asn Asp Gly Gly Tyr Ala Ile Glu Asp Phe Asp Thr 115 120 125 Val Asp Pro Thr Ile Gly Thr Asn Glu Asp Leu Ala Asp Leu Thr Ala 130 135 140 Lys Leu Arg Glu Ala Gly Ile Ser Leu Cys Leu Asp Phe Val Met Asn 145 150 155 160 His Thr Ala Ser Thr His Arg Trp Ala Lys Ala Ala Gln Ala Gly Asp 165 170 175 Pro Glu Tyr Gln Asp Tyr Tyr Phe Cys Tyr Asp Asp Arg Thr Ile Pro 180 185 190 Asp Gln Tyr Asp Ala Val Val Pro Gln Ile Phe Pro Thr Ala Ala Pro 195 200 205 Gly Asn Phe Thr Trp Asn Glu Gln Met Gly Lys Trp Val Met Thr Gln 210 215 220 Phe Tyr Pro Phe Gln Trp Asp Leu Asn Tyr Arg Asn Pro Lys Val Phe 225 230 235 240 Val Val Met Met Ser Ser Leu Leu His Leu Ala Asn Leu Gly Val Glu 245 250 255 Val Phe Arg Ile Asp Ala Val Pro Tyr Ile Trp Lys Gln Leu Gly Thr 260 265 270 Asn Cys Arg Asn Leu Pro Gln Val His Thr Ile Val Arg Met Met Arg 275 280 285 Ile Met Ser Glu Ile Val Cys Pro Ala Val Val Phe Lys Gly Glu Val 290 295 300 Val Met Ala Pro Lys Glu Leu Ala Ala Tyr Phe Gly Thr Pro Glu Lys 305 310 315 320 Pro Glu Cys His Met Leu Tyr Asn Val Ser Val Met Val Asn Leu Trp 325 330 335 Ser Ala Leu Ala Asn Gly Asp Thr Arg Leu Leu Lys Thr Gln Ile Asp 340 345 350 Lys Leu Asp Ala Leu Pro Asp Asn Cys Trp Phe Val Asn Tyr Leu Arg 355 360 365 Cys His Asp Asp Ile Gly Trp Gly Leu Asp Glu Asp Val Glu Arg Gln 370 375 380 Leu Gly Ile Asp Pro Leu Lys His Lys Glu Phe Leu Tyr His Phe Tyr 385 390 395 400 Glu Gly Met Val Pro Gly Ser Trp Ala Met Gly Glu Leu Tyr Asn Tyr 405 410 415 Asp Pro Ala Ser Gly Asp Ala Arg Ser Cys Gly Thr Thr Ala Ser Leu 420 425 430 Cys Gly Ile Glu Arg Ala Leu Ile Thr His Asp Arg Pro Leu Tyr Glu 435 440 445 Arg Ser Ile Gln Arg Asp Leu Leu Met His His Ala Met Gly Phe Leu 450 455 460 Arg Gly Phe Pro Met Leu Asn Cys Gly Asp Glu Ile Gly Gln Leu Asn 465 470 475 480 Gly Trp Asp Tyr Lys Glu Asp Pro Asp Arg Val Ala Asp Ser Arg Asn 485 490 495 Leu His Arg Ser Lys Phe Asn Trp Lys Asn Ala Ala Lys Arg Asp Val 500 505 510 Pro Gly Thr Leu Pro Asn Arg Leu Trp Glu Gly Met Ala Asp Val Arg 515 520 525 Gln Met Arg Ser Asp Pro Cys Phe Ala Pro Asp Ala Trp Val Thr Thr Thr 530 535 540 Trp Asp Ala His Asp Asp Gly Ile Leu Ala Met Val Arg Gln Ser Gly 545 550 555 560 Gly Arg Thr Leu Leu Gly Val Phe Asn Phe Ala Asn Arg Asp Ala Thr 565 570 575 Ala Thr Leu Asp Ser Ile Glu Gly Val Ser Leu Pro Arg Thr Val Ala 580 585 590 Leu Lys Pro Tyr Glu Trp Lys Ile Glu Ala Cys 595 600 <210> 6 <211> 603 <212> PRT <213> Artificial Sequence <220> <223> BtAS Y265F <400> 6 Met Glu Ala Thr Tyr Arg Asp Ser Val Phe Ala Glu Arg Leu Ala Pro 1 5 10 15 Arg Cys Ala Glu Leu Glu Gln Leu Phe Arg Ser Leu Tyr Gly Asp Ser 20 25 30 Pro Glu Phe Asp His Phe Glu Gln Val Met Ala Lys Ala His Ala Asp 35 40 45 Arg Pro Ala Asp Leu Lys Arg Leu Asp Ala Ala Arg Glu His Asp Pro 50 55 60 Gln Trp Tyr Arg Arg Gly Asp Met Phe Gly Met Thr Met Tyr Thr Asp 65 70 75 80 Leu Phe Ala Gly Lys Leu Thr Asp Leu Ala Lys His Ile Asp Tyr Leu 85 90 95 Lys Glu Gln His Leu Thr Tyr Leu His Leu Met Pro Leu Leu Thr Met 100 105 110 Pro His Pro Asp Asn Asp Gly Gly Tyr Ala Ile Glu Asp Phe Asp Thr 115 120 125 Val Asp Pro Thr Ile Gly Thr Asn Glu Asp Leu Ala Asp Leu Thr Ala 130 135 140 Lys Leu Arg Glu Ala Gly Ile Ser Leu Cys Leu Asp Phe Val Met Asn 145 150 155 160 His Thr Ala Ser Thr His Arg Trp Ala Lys Ala Ala Gln Ala Gly Asp 165 170 175 Pro Glu Tyr Gln Asp Tyr Tyr Phe Cys Tyr Asp Asp Arg Thr Ile Pro 180 185 190 Asp Gln Tyr Asp Ala Val Val Pro Gln Val Phe Pro Thr Ala Ala Pro 195 200 205 Gly Asn Phe Thr Trp Asn Glu Gln Met Gly Lys Trp Val Met Thr Gln 210 215 220 Phe Tyr Pro Phe Gln Trp Asp Leu Asn Tyr Arg Asn Pro Lys Val Phe 225 230 235 240 Val Val Met Met Ser Ser Leu Leu His Leu Ala Asn Leu Gly Val Glu 245 25 0 255 Val Phe Arg Ile Asp Ala Val Pro Phe Ile Trp Lys Gln Leu Gly Thr 260 265 270 Asn Cys Arg Asn Leu Pro Gln Val His Thr Ile Val Arg Met Met Arg 275 280 285 Ile Met Ser Glu Ile Val Cys Pro Ala Val Val Phe Lys Gly Glu Val 290 295 300 Val Met Ala Pro Lys Glu Leu Ala Ala Tyr Phe Gly Thr Pro Glu Lys 305 310 315 320 Pro Glu Cys His Met Leu Tyr Asn Val Ser Val Met Val Asn Leu Trp 325 330 335 Ser Ala Leu Ala Asn Gly Asp Thr Arg Leu Leu Lys Thr Gln Ile Asp 340 345 350 Lys Leu Asp Ala Leu Pro Asp Asn Cys Trp Phe Val Asn Tyr Leu Arg 355 360 365 Cys His Asp Asp Ile Gly Trp Gly Leu Asp Glu Asp Val Glu Arg Gln 370 375 380 Leu Gly Ile Asp Pro Leu Lys His Lys Glu Phe Leu Tyr His Phe Tyr 385 390 395 400 Glu Gly Met Val Pro Gly Ser Trp Ala Met Gly Glu Leu Tyr Asn Tyr 405 410 415 Asp Pro Ala Ser Gly Asp Ala Arg Ser Cys Gly Thr Thr Ala Ser Leu 420 425 430 Cys Gly Ile Glu Arg Ala Leu Ile Thr His Asp Arg Pro Leu Tyr Glu 435 440 445 Arg Ser Ile Gln Arg Asp Leu Leu Met His His Ala Met Gly Phe Leu 450 455 460 Arg Gly Phe Pro Met Leu Asn Cys Gly Asp Glu Ile Gly Gln Leu Asn 465 470 475 480 Gly Trp Asp Tyr Lys Glu Asp Pro Asp Arg Val Ala Asp Ser Arg Asn 485 490 495 Leu His Arg Ser Lys Phe Asn Trp Lys Asn Ala Ala Lys Arg Asp Val 500 505 510 Pro Gly Thr Leu Pro Asn Arg Leu Trp Glu Gly Met Ala Asp Val Arg 515 520 525 Gln Met Arg Ser Asp Pro Cys Phe Ala Pro Asp Ala Trp Val Thr Thr 530 535 540 Trp Asp Ala His Asp Asp Gly Ile Leu Ala Met Val Arg Gln Ser Gly 545 550 555 560 Gly Arg Thr Leu Leu Gly Val Phe Asn Phe Ala Asn Arg Asp Ala Thr 565 570 575 Ala Thr Leu Asp Ser Ile Glu Gly Val Ser Leu Pro Arg Thr Val Ala 580 585 590 Leu Lys Pro Tyr Glu Trp Lys Ile Glu Ala Cys 595 600 <210> 7 <211> 603 <212> PRT <213> Artificial Sequence <220> <223> BtAS V305I <400 > 7 Met Glu Ala Thr Tyr Arg Asp Ser Val Phe Ala Glu Arg Leu Ala Pro 1 5 10 15 Arg Cys Ala Glu Leu Glu Gln Leu Phe Arg Ser Leu Tyr Gly Asp Ser 20 25 30 Pro Glu Phe Asp His Phe Glu Gln Val Met Ala Lys Ala His Ala Asp 35 40 45 Arg Pro Ala Asp Leu Lys Arg Leu Asp Ala Ala Arg Glu His Asp Pro 50 55 60 Gln Trp Tyr Arg Arg Gly Asp Met Phe Gly Met Thr Met Tyr Thr Asp 65 70 75 80 Leu Phe Ala Gly Lys Leu Thr Asp Leu Ala Lys His Ile Asp Tyr Leu 85 90 95 Lys Glu Gln His Leu T hr Tyr Leu His Leu Met Pro Leu Leu Thr Met 100 105 110 Pro His Pro Asp Asn Asp Gly Gly Tyr Ala Ile Glu Asp Phe Asp Thr 115 120 125 Val Asp Pro Thr Ile Gly Thr Asn Glu Asp Leu Ala Asp Leu Thr Ala 130 135 140 Lys Leu Arg Glu Ala Gly Ile Ser Leu Cys Leu Asp Phe Val Met Asn 145 150 155 160 His Thr Ala Ser Thr His Arg Trp Ala Lys Ala Ala Gln Ala Gly Asp 165 170 175 Pro Glu Tyr Gln Asp Tyr Tyr Phe Cys Tyr Asp Asp Arg Thr Ile Pro 180 185 190 Asp Gln Tyr Asp Ala Val Val Pro Gln Val Phe Pro Thr Ala Ala Pro 195 200 205 Gly Asn Phe Thr Trp Asn Glu Gln Met Gly Lys Trp Val Met Thr Gln 210 215 220 Phe Tyr Pro Phe Gln Trp Asp Leu Asn Tyr Arg Asn Pro Lys Val Phe 225 230 235 240 Val Val M et Met Ser Ser Leu Leu His Leu Ala Asn Leu Gly Val Glu 245 250 255 Val Phe Arg Ile Asp Ala Val Pro Tyr Ile Trp Lys Gln Leu Gly Thr 260 265 270 Asn Cys Arg Asn Leu Pro Gln Val His Thr Ile Val Arg Met Met Arg 275 280 285 Ile Met Ser Glu Ile Val Cys Pro Ala Val Val Phe Lys Gly Glu Val 290 295 300 Ile Met Ala Pro Lys Glu Leu Ala Ala Tyr Phe Gly Thr Pro Glu Lys 305 310 315 320 Pro Glu Cys His Met Leu Tyr Asn Val Ser Val Met Val Asn Leu Trp 325 330 335 Ser Ala Leu Ala Asn Gly Asp Thr Arg Leu Leu Lys Thr Gln Ile Asp 340 345 350 Lys Leu Asp Ala Leu Pro Asp Asn Cys Trp Phe Val Asn Tyr Leu Arg 355 360 365 Cys His Asp Asp Ile Gly Trp Gly Leu Asp Glu Asp Val Glu Arg Gln 370 375 380 Leu Gly Ile Asp P ro Leu Lys His Lys Glu Phe Leu Tyr His Phe Tyr 385 390 395 400 Glu Gly Met Val Pro Gly Ser Trp Ala Met Gly Glu Leu Tyr Asn Tyr 405 410 415 Asp Pro Ala Ser Gly Asp Ala Arg Ser Cys Gly Thr Thr Ala Ser Leu 420 425 430 Cys Gly Ile Glu Arg Ala Leu Ile Thr His Asp Arg Pro Leu Tyr Glu 435 440 445 Arg Ser Ile Gln Arg Asp Leu Leu Met His His Ala Met Gly Phe Leu 450 455 460 Arg Gly Phe Pro Met Leu Asn Cys Gly Asp Glu Ile Gly Gln Leu Asn 465 470 475 480 Gly Trp Asp Tyr Lys Glu Asp Pro Asp Arg Val Ala Asp Ser Arg Asn 485 490 495 Leu His Arg Ser Lys Phe Asn Trp Lys Asn Ala Ala Lys Arg Asp Val 500 505 510 Pro Gly Thr Leu Pro Asn Arg Leu Trp Glu Gly Met Ala Asp Val Arg 515 520 525 Gln M et Arg Ser Asp Pro Cys Phe Ala Pro Asp Ala Trp Val Thr Thr Thr 530 535 540 Trp Asp Ala His Asp Asp Gly Ile Leu Ala Met Val Arg Gln Ser Gly 545 550 555 560 Gly Arg Thr Leu Leu Gly Val Phe Asn Phe Ala Asn Arg Asp Ala Thr 565 570 575 Ala Thr Leu Asp Ser Ile Glu Gly Val Ser Leu Pro Arg Thr Val Ala 580 585 590 Leu Lys Pro Tyr Glu Trp Lys Ile Glu Ala Cys 595 600 <210> 8 <211> 603 <212> PRT <213> Artificial Sequence <220> <223> BtAS K393R <400> 8 Met Glu Ala Thr Tyr Arg Asp Ser Val Phe Ala Glu Arg Leu Ala Pro 1 5 10 15 Arg Cys Ala Glu Leu Glu Gln Leu Phe Arg Ser Leu Tyr Gly Asp Ser 20 25 30 Pro Glu Phe Asp His Phe Glu Gln Val Met Ala Lys Ala His Ala Asp 35 40 45 Arg Pro Ala Asp Leu Lys Arg Leu Asp Ala Ala Arg Glu His Asp Pro 50 55 60 Gln Trp Tyr Arg Arg Gly Asp Met Phe Gly Met Thr Met Tyr Thr Asp 65 70 75 80 Leu Phe Ala Gly L ys Leu Thr Asp Leu Ala Lys His Ile Asp Tyr Leu 85 90 95 Lys Glu Gln His Leu Thr Tyr Leu His Leu Met Pro Leu Leu Thr Met 100 105 110 Pro His Pro Asp Asn Asp Gly Gly Tyr Ala Ile Glu Asp Phe Asp Thr 115 120 125 Val Asp Pro Thr Ile Gly Thr Asn Glu Asp Leu Ala Asp Leu Thr Ala 130 135 140 Lys Leu Arg Glu Ala Gly Ile Ser Leu Cys Leu Asp Phe Val Met Asn 145 150 155 160 His Thr Ala Ser Thr His Arg Trp Ala Lys Ala Ala Gln Ala Gly Asp 165 170 175 Pro Glu Tyr Gln Asp Tyr Tyr Phe Cys Tyr Asp Asp Arg Thr Ile Pro 180 185 190 Asp Gln Tyr Asp Ala Val Val Pro Gln Val Phe Pro Thr Ala Ala Pro 195 200 205 Gly Asn Phe Thr Trp Asn Glu Gln Met Gly Lys Trp Val Met Thr Gln 210 215 220 Phe Tyr Pro Phe Gln Trp Asp Leu Asn Tyr Arg Asn Pro Lys Val Phe 225 230 235 240 Val Val Met Met Ser Ser Leu Leu His Leu Ala Asn Leu Gly Val Glu 245 250 255 Val Phe Arg Ile Asp Ala Val Pro Tyr Ile Trp Lys Gln Leu Gly Thr 260 265 270 Asn Cys Arg Asn Leu Pro Gln Val His Thr Ile Val Arg Met Met Arg 275 280 285 Ile Met Ser Glu Ile Val Cys Pro Ala Val Val Phe Lys Gly Glu Val 290 295 300 Val Met Ala Pro Lys Glu Leu Ala Ala Tyr Phe Gly Thr Pro Glu Lys 305 310 315 320 Pro Glu Cys His Met Leu Tyr Asn Val Ser Val Met Val Asn Leu Trp 325 330 335 Ser Ala Leu Ala Asn Gly Asp Thr Arg Leu Leu Lys Thr Gln Ile Asp 340 345 350 Lys Leu Asp Ala Leu Pro Asp Asn Cys Trp Phe Val Asn Tyr Leu Arg 355 360 365 Cys His Asp Asp Ile Gly Trp Gly Leu Asp Glu Asp Val Glu Arg Gln 370 375 380 Leu Gly Ile Asp Pro Leu Lys His Arg Glu Phe Leu Tyr His Phe Tyr 385 390 395 400 Glu Gly Met Val Pro Gly Ser Trp Ala Met Gly Glu Leu Tyr Asn Tyr 405 410 415 Asp Pro Ala Ser Gly Asp Ala Arg Ser Cys Gly Thr Thr Ala Ser Leu 420 425 430 Cys Gly Ile Glu Arg Ala Leu Ile Thr His Asp Arg Pro Leu Tyr Glu 435 440 445 Arg Ser Ile Gln Arg Asp Leu Leu Met His His Ala Met Gly Phe Leu 450 455 460 Arg Gly Phe Pro Met Leu Asn Cys Gly Asp Glu Ile Gly Gln Leu Asn 465 470 475 480 Gly Trp Asp Tyr Lys Glu Asp Pro Asp Arg Val Ala Asp Ser Arg Asn 485 490 495 Leu His Arg Ser Lys Phe Asn Trp Lys Asn Ala Ala Lys Arg Asp Val 500 505 510 Pro Gly Thr Leu Pro Asn Arg Leu Trp Glu Gly Met Ala Asp Val Arg 515 520 525 Gln Met Arg Ser Asp Pro Cys Phe Ala Pro Asp Ala Trp Val Thr Thr 530 535 540 Trp Asp Ala His Asp Asp Gly Ile Leu Ala Met Val Arg Gln Ser Gly 545 550 555 560 Gly Arg Thr Leu Leu Gly Val Phe Asn Phe Ala Asn Arg Asp Ala Thr 565 570 575 Ala Thr Leu Asp Ser Ile Glu Gly Val Ser Leu Pro Arg Thr Val Ala 580 585 590 Leu Lys Pro Tyr Glu Trp Lys Ile Glu Ala Cys 595 600 <210> 9 <211> 603 <212> PRT <213> Artificial Sequence <220> <223> BtAS S420T <400> 9 Met Glu Ala Thr Tyr Arg Asp Ser Val Phe Ala Glu Arg Leu Ala Pro 1 5 10 15 Arg Cys Ala Glu Leu Glu Gln Leu Phe Arg Ser Leu Tyr Gly Asp Ser 20 25 30 Pro Glu Phe Asp His Phe Glu Gln Val Met Ala Lys Ala His Ala Asp 35 40 45 Arg Pro Ala Asp Leu Lys Arg Leu Asp Ala Ala Arg Glu His Asp Pro 50 55 60 Gln Trp Tyr Arg Arg Gly Asp Met Phe Gly Met Thr Met Tyr Thr Asp 65 70 75 80 Leu Phe Ala Gly Lys Leu Thr Asp Leu Ala Lys His Ile Asp Tyr Leu 85 90 95 Lys Glu Gln His Leu Thr Tyr Leu His Leu Met Pro Leu Leu Thr Met 100 105 110 Pro His Pro Asp Asn Asp Gly Gly Tyr Ala Ile Glu Asp Phe Asp Thr 115 120 125 Val Asp Pro Thr Ile Gly Thr Asn Glu Asp Leu Ala Asp Leu Thr Ala 130 135 140 Lys Leu Arg Glu Ala Gly Ile Ser Leu Cys Leu Asp Phe Val Met Asn 145 150 155 160 His Thr Ala Ser Thr His Arg Trp Ala Lys Ala Ala Gln Ala Gly Asp 165 170 175 Pro Glu Tyr Gln Asp Tyr Tyr Phe Cys Tyr Asp Asp Arg Thr Ile Pro 180 185 190 Asp Gln Tyr Asp Ala Val Val Pro Gln Val Phe Pro Thr Ala Ala Pro 195 200 205 Gly Asn Phe Thr Trp Asn Glu Gln Met Gly Lys Trp Val Met Thr Gln 210 215 220 Phe Tyr Pro Phe Gln Trp Asp Leu Asn Tyr Arg Asn Pro Lys Val Phe 225 230 235 240 Val Val Met Met Ser Ser Leu Leu His Leu Ala Asn Leu Gly Val Glu 245 250 255 Val Phe Arg Ile Asp Ala Val Pro Tyr Ile Trp Lys Gln Leu Gly Thr 260 265 270 Asn Cys Arg Asn Leu Pro Gln Val His Thr Ile Val Arg Met Met Arg 275 280 285 Ile Met Ser Glu Ile Val Cys Pro Ala Val Val Phe Lys Gly Glu Val 290 295 300 Val Met Ala Pro Lys Glu Leu Ala Ala Tyr Phe Gly Thr Pro Glu Lys 305 310 315 320 Pro Glu Cys His Met Leu Tyr Asn Val Ser Val Met Val Asn Leu Trp 325 330 335 Ser Ala Leu Ala Asn Gly Asp Thr Arg Leu Leu Lys Thr Gln Ile Asp 340 345 350 Lys Leu Asp Ala Leu Pro Asp Asn Cys Trp Phe Val Asn Tyr Leu Arg 355 360 365 Cys His Asp Asp Ile Gly Trp Gly Leu Asp Glu Asp Val Glu Arg Gln 370 375 380 Leu Gly Ile Asp Pro Leu Lys His Lys Glu Phe Leu Tyr His Phe Tyr 385 390 395 400 Glu Gly Met Val Pro Gly Ser Trp Ala Met Gly Glu Leu Tyr Asn Tyr 405 410 415 Asp Pro Ala Thr Gly Asp Ala Arg Ser Cys Gly Thr Thr Ala Ser Leu 420 425 430 Cys Gly Ile Glu Arg Ala Leu Ile Thr His Asp Arg Pro Leu Tyr Glu 435 440 445 Arg Ser Ile Gln Arg Asp Leu Leu Met His His Ala Met Gly Phe Leu 450 455 460 Arg Gly Phe Pro Met Leu Asn Cys Gly Asp Glu Ile Gly Gln Leu Asn 465 470 475 480 Gly Trp Asp Tyr Lys Glu Asp Pro Asp Arg Val Ala Asp Ser Arg Asn 485 490 495 Leu His Arg Ser Lys Phe Asn Trp Lys Asn Ala Ala Lys Arg Asp Val 500 505 510 Pro Gly Thr Leu Pro Asn Arg Leu Trp Glu Gly Met Ala Asp Val Arg 515 520 525 Gln Met Arg Ser Asp Pro Cys Phe Ala Pro Asp Ala Trp Val Thr Thr 530 535 540 Trp Asp Ala His Asp Asp Gly Ile Leu Ala Met Val Arg Gln Ser Gly 545 550 555 560 Gly Arg Thr Leu Leu Gly Val Phe Asn Phe Ala Asn Arg Asp Ala Thr 565 570 575 Ala Thr Leu Asp Ser Ile Glu Gly Val Ser Leu Pro Arg Thr Val Ala 580 585 590 Leu Lys Pro Tyr Glu Trp Lys Ile Glu Ala Cys 595 600 <210> 10 <211> 603 <212> PRT <213> Artificial Sequence < 220> <223> BtAS Y414F <400> 10 Met Glu Ala Thr Tyr Arg Asp Ser Val Phe Ala Glu Arg Leu Ala Pro 1 5 10 15 Arg Cys Ala Glu Leu Glu Gln Leu Phe Arg Ser Leu Tyr Gly Asp Ser 20 25 30 Pro Glu Phe Asp His Phe Glu Gln Val Met Ala Lys Ala His Ala Asp 35 40 45 Arg Pro Ala Asp Leu Lys Arg Leu Asp Ala Ala Arg Glu His Asp Pro 50 55 60 Gln Trp Tyr Arg Arg Gly Asp Met Phe Gly Met Thr Met Tyr Thr Asp 65 70 75 80 Leu Phe Ala Gly Lys Leu Thr Asp Leu Ala L ys His Ile Asp Tyr Leu 85 90 95 Lys Glu Gln His Leu Thr Tyr Leu His Leu Met Pro Leu Leu Thr Met 100 105 110 Pro His Pro Asp Asn Asp Gly Gly Tyr Ala Ile Glu Asp Phe Asp Thr 115 120 125 Val Asp Pro Thr Ile Gly Thr Asn Glu Asp Leu Ala Asp Leu Thr Ala 130 135 140 Lys Leu Arg Glu Ala Gly Ile Ser Leu Cys Leu Asp Phe Val Met Asn 145 150 155 160 His Thr Ala Ser Thr His Arg Trp Ala Lys Ala Ala Gln Ala Gly Asp 165 170 175 Pro Glu Tyr Gln Asp Tyr Tyr Phe Cys Tyr Asp Asp Arg Thr Ile Pro 180 185 190 Asp Gln Tyr Asp Ala Val Val Pro Gln Val Phe Pro Thr Ala Ala Pro 195 200 205 Gly Asn Phe Thr Trp Asn Glu Gln Met Gly Lys Trp Val Met Thr Gln 210 215 220 Phe Tyr Pro Phe Gln Trp Asp Leu Asn Tyr Arg Asn Pro Lys Val Phe 225 230 235 240 Val Val Met Met Ser Ser Leu Leu His Leu Ala Asn Leu Gly Val Glu 245 250 255 Val Phe Arg Ile Asp Ala Val Pro Tyr Ile Trp Lys Gln Leu Gly Thr 260 265 270 Asn Cys Arg Asn Leu Pro Gln Val His Thr Ile Val Arg Met Met Arg 275 280 285 Ile Met Ser Glu Ile Val Cys Pro Ala Val Val Phe Lys Gly Glu Val 290 295 300 Val Met Ala Pro Lys Glu Leu Ala Ala Tyr Phe Gly Thr Pro Glu Lys 305 310 315 320 Pro Glu Cys His Met Leu Tyr Asn Val Ser Val Met Val Asn Leu Trp 325 330 335 Ser Ala Leu Ala Asn Gly Asp Thr Arg Leu Leu Lys Thr Gln Ile Asp 340 345 350 Lys Leu Asp Ala Leu Pro Asp Asn Cys Trp Phe Val Asn Tyr Leu Arg 355 360 365 Cys His Asp Asp Ile Gly Trp Gly Leu Asp Glu Asp Val Glu Arg Gln 370 375 380 Leu Gly Ile Asp Pro Leu Lys His Lys Glu Phe Leu Tyr His Phe Tyr 385 390 395 400 Glu Gly Met Val Pro Gly Ser Trp Ala Met Gly Glu Leu Phe Asn Tyr 405 410 415 Asp Pro Ala Ser Gly Asp Ala Arg Ser Cys Gly Thr Thr Ala Ser Leu 420 425 430 Cys Gly Ile Glu Arg Ala Leu Ile Thr His Asp Arg Pro Leu Tyr Glu 435 440 445 Arg Ser Ile Gln Arg Asp Leu Leu Met His His Ala Met Gly Phe Leu 450 455 460 Arg Gly Phe Pro Met Leu Asn Cys Gly Asp Glu Ile Gly Gln Leu Asn 465 470 475 480 Gly Trp Asp Tyr Lys Glu Asp Pro Asp Arg Val Ala Asp Ser Arg Asn 485 490 495 Leu His Arg Ser Lys Phe Asn Trp Lys Asn Ala Ala Lys Arg Asp Val 500 505 510 Pro Gly Thr Leu Pro Asn Arg Leu Trp Glu Gly Met Ala Asp Val Arg 515 520 525 Gln Met Arg Ser Asp Pro Cys Phe Ala Pro Asp Ala Trp Val Thr Thr Thr 530 535 540 Trp Asp Ala His Asp Asp Gly Ile Leu Ala Met Val Arg Gln Ser Gly 545 550 555 560 Gly Arg Thr Leu Leu Gly Val Phe Asn Phe Ala Asn Arg Asp Ala Thr 565 570 575 Ala Thr Leu Asp Ser Ile Glu Gly Val Ser Leu Pro Arg Thr Val Ala 580 585 590 Leu Lys Pro Tyr Glu Trp Lys Ile Glu Ala Cys 595 600 <210> 11 <211 > 603 <212> PRT <213> Artificial Sequence <220> <223> BtAS Y414F*P200R <400> 11 Met Glu Ala Thr Tyr Arg Asp Ser Val Phe Ala Glu Arg Leu Ala Pro 1 5 10 15 Arg Cys Ala Glu Leu Glu Gln Leu Phe Arg Ser Leu Tyr Gly Asp Ser 20 25 30 Pro Glu Phe Asp His Phe Glu Gln Val Met Ala Lys Ala His Ala Asp 35 40 45 Arg Pro Ala Asp Leu Lys Arg Leu Asp Ala Ala Arg Glu His Asp Pro 50 55 60 Gln Trp Tyr Arg Arg Gly Asp Met Phe Gly Met Thr Met Tyr Thr Asp 65 70 75 80 Leu Phe Ala Gly Lys Leu Thr Asp Leu Ala Lys His Ile Asp Tyr Leu 85 90 95 Lys Glu Gln His Leu Thr Tyr Leu His Leu Met Pro Leu Leu Thr Met 100 105 110 Pro His Pro Asp Asn Asp Gly Gly Tyr Ala Ile Glu Asp Phe Asp Thr 115 120 125 Val Asp Pro Thr Ile Gly Thr Asn Glu Asp Leu Ala Asp Leu Thr Ala 130 135 140 Lys Leu Arg Glu Ala Gly Ile Ser Leu Cys Leu Asp Phe Val Met Asn 145 150 155 160 His Thr Ala Ser Thr His Arg Trp Ala Lys Ala Ala Gln Ala Gly Asp 165 170 175 Pro Glu Tyr Gln Asp Tyr Tyr Phe Cys Tyr Asp Asp Arg Thr Ile Pro 180 185 190 Asp Gln Tyr Asp Ala Val Val Arg Gln Val Phe Pro Thr Ala Ala Pro 195 200 205 Gly Asn Phe Thr Trp Asn Glu Gln Met Gly Lys Trp Val Met Thr Gln 210 215 220 Phe Tyr Pro Phe Gln Trp Asp Leu Asn Tyr Arg Asn Pro Lys Val Phe 225 230 235 240 Val Val Met Met Ser Ser Leu Leu His Leu Ala Asn Leu Gly Val Glu 245 250 255 Val Phe Arg Ile Asp Ala Val Pro Tyr Ile Trp Lys Gln Leu Gly Thr 260 265 270 Asn Cys Arg Asn Leu Pro Gln Val His Thr Ile Val Arg Met Met Arg 275 280 285 Ile Met Ser Glu Ile Val Cys Pro Ala Val Val Phe Lys Gly Glu Val 290 295 300 Val Met Ala Pro Lys Glu Leu Ala Ala Tyr Phe Gly Thr Pro Glu Lys 305 310 315 320 Pro Glu Cys His Met Leu Tyr Asn Val Ser Val Met Val Asn Leu Trp 325 330 335 Ser Ala Leu Ala Asn Gly Asp Thr Arg Leu Leu Lys Thr Gln Ile Asp 340 345 350 Lys Leu Asp Ala Leu Pro Asp Asn Cys Trp Phe Val Asn Tyr Leu Arg 355 360 365 Cys His Asp Asp Ile Gly Trp Gly Leu Asp Glu Asp Val Glu Arg Gln 370 375 380 Leu Gly Ile Asp Pro Leu Lys His Lys Glu Phe Leu Tyr His Phe Tyr 385 390 395 400 Glu Gly Met Val Pro Gly Ser Trp Ala Met Gly Glu Leu Phe Asn Tyr 405 410 415 Asp Pro Ala Ser Gly Asp Ala Arg Ser Cys Gly Thr Thr Ala Ser Leu 420 425 430 Cys Gly Ile Glu Arg Ala Leu Ile Thr His Asp Arg Pro Leu Tyr Glu 435 440 445 Arg Ser Ile Gln Arg Asp Leu Leu Met His His Ala Met Gly Phe Leu 450 455 460 Arg Gly Phe Pro Met Leu Asn Cys Gly Asp Glu Ile Gly Gln Leu Asn 465 470 475 480 Gly Trp Asp Tyr Lys Glu Asp Pro Asp Arg Val Ala Asp Ser Arg Asn 485 490 495 Leu His Arg Ser Lys Phe Asn Trp Lys Asn Ala Ala Lys Arg Asp Val 500 505 510 Pro Gly Thr Leu Pro Asn Arg Leu Trp Glu Gly Met Ala Asp Val Arg 515 520 525 Gln Met Arg Ser Asp Pro Cys Phe Ala Pro Asp Ala Trp Val Thr Thr Thr 530 535 540 Trp Asp Ala His Asp Asp Gly Ile Leu Ala Met Val Arg Gln Ser Gly 545 550 555 560 Gly Arg Thr Leu Leu Gly Val Phe Asn Phe Ala Asn Arg Asp Ala Thr 565 570 575 Ala Thr Leu Asp Ser Ile Glu Gly Val Ser Leu Pro Arg Thr Val Ala 580 585 590 Leu Lys Pro Tyr Glu Trp Lys Ile Glu Ala Cys 595 600 <210> 12 <211> 603 <212> PRT <213> Artificial Sequence <220> <223> BtAS Y414F*V202I <400> 12 Met Glu Ala Thr Tyr Arg Asp Ser Val Phe Ala Glu Arg Leu Ala Pro 1 5 10 15 Arg Cys Ala Glu Leu Glu Gln Leu Phe Arg Ser Leu Tyr Gly Asp Ser 20 25 30 Pro Glu Phe Asp His Phe Glu Gln Val Met Ala Lys Ala His Ala Asp 35 40 45 Arg Pro Ala Asp Leu Lys Arg Leu Asp Ala Ala Arg Glu His Asp Pro 50 55 60 Gln Trp Tyr Arg Arg Gly Asp Met Phe Gly Met Thr Met Tyr Thr Asp 65 70 75 80 Leu Phe Ala Gly Lys Leu Thr Asp Leu Ala Lys His Ile Asp Tyr Leu 85 90 95 Lys Glu Gln His Leu Thr Tyr Leu His Leu Met Pro Leu Leu Thr Met 100 105 110 Pro His Pro Asp Asn Asp Gly Gly Tyr Ala Ile Glu Asp Phe Asp Thr 115 120 125 Val Asp Pro Thr Ile Gly Thr Asn Glu Asp Leu Ala Asp Leu Thr Ala 130 135 140 Lys Leu Arg Glu Ala Gly Ile Ser Leu Cys Leu Asp Phe Val Met Asn 145 150 155 160 His Thr Ala Ser Thr His Arg Trp Ala Lys Ala Ala Gln Ala Gly Asp 165 170 175 Pro Glu Tyr Gln Asp Tyr Tyr Phe Cys Tyr Asp Asp Arg Thr Ile Pro 180 185 190 Asp Gln Tyr Asp Ala Val Val Pro Gln Ile Phe Pro Thr Ala Ala Pro 195 200 205 Gly Asn Phe Thr Trp As n Glu Gln Met Gly Lys Trp Val Met Thr Gln 210 215 220 Phe Tyr Pro Phe Gln Trp Asp Leu Asn Tyr Arg Asn Pro Lys Val Phe 225 230 235 240 Val Val Met Met Ser Ser Leu Leu His Leu Ala Asn Leu Gly Val Glu 245 250 255 Val Phe Arg Ile Asp Ala Val Pro Tyr Ile Trp Lys Gln Leu Gly Thr 260 265 270 Asn Cys Arg Asn Leu Pro Gln Val His Thr Ile Val Arg Met Met Arg 275 280 285 Ile Met Ser Glu Ile Val Cys Pro Ala Val Val Phe Lys Gly Glu Val 290 295 300 Val Met Ala Pro Lys Glu Leu Ala Ala Tyr Phe Gly Thr Pro Glu Lys 305 310 315 320 Pro Glu Cys His Met Leu Tyr Asn Val Ser Val Met Val Asn Leu Trp 325 330 335 Ser Ala Leu Ala Asn Gly Asp Thr Arg Leu Leu Lys Thr Gln Ile Asp 340 345 350 Lys Leu As p Ala Leu Pro Asp Asp Asn Cys Trp Phe Val Asn Tyr Leu Arg 355 360 365 Cys His Asp Asp Ile Gly Trp Gly Leu Asp Glu Asp Val Glu Arg Gln 370 375 380 Leu Gly Ile Asp Pro Leu Lys His Lys Glu Phe Leu Tyr His Phe Tyr 385 390 395 400 Glu Gly Met Val Pro Gly Ser Trp Ala Met Gly Glu Leu Phe Asn Tyr 405 410 415 Asp Pro Ala Ser Gly Asp Ala Arg Ser Cys Gly Thr Thr Ala Ser Leu 420 425 430 Cys Gly Ile Glu Arg Ala Leu Ile Thr His Asp Arg Pro Leu Tyr Glu 435 440 445 Arg Ser Ile Gln Arg Asp Leu Leu Met His His Ala Met Gly Phe Leu 450 455 460 Arg Gly Phe Pro Met Leu Asn Cys Gly Asp Glu Ile Gly Gln Leu Asn 465 470 475 480 Gly Trp Asp Tyr Lys Glu Asp Pro Asp Arg Val Ala Asp Ser Arg Asn 485 490 49 5 Leu His Arg Ser Lys Phe Asn Trp Lys Asn Ala Ala Lys Arg Asp Val 500 505 510 Pro Gly Thr Leu Pro Asn Arg Leu Trp Glu Gly Met Ala Asp Val Arg 515 520 525 Gln Met Arg Ser Asp Pro Cys Phe Ala Pro Asp Ala Trp Val Thr Thr 530 535 540 Trp Asp Ala His Asp Asp Gly Ile Leu Ala Met Val Arg Gln Ser Gly 545 550 555 560 Gly Arg Thr Leu Leu Gly Val Phe Asn Phe Ala Asn Arg Asp Ala Thr 565 570 575 Ala Thr Leu Asp Ser Ile Glu Gly Val Ser Leu Pro Arg Thr Val Ala 580 585 590 Leu Lys Pro Tyr Glu Trp Lys Ile Glu Ala Cys 595 600 <210> 13 <211> 603 <212 > PRT <213> Artificial Sequence <220> <223> BtAS YY414F*Y265F <400> 13 Met Glu Ala Thr Tyr Arg Asp Ser Val Phe Ala Glu Arg Leu Ala Pro 1 5 10 15 Arg Cys Ala Glu Leu Glu Gln Leu Phe Arg Ser Leu Tyr Gly Asp Ser 20 25 30 Pro Glu Phe Asp His Phe Glu Gln Val Met Ala Lys Ala His Ala Asp 35 40 45 Arg Pro Ala Asp Leu Lys Arg Leu Asp Ala Ala Arg Glu His Asp Pro 50 55 60 Gln Trp Tyr Arg Arg Gly Asp Met Phe Gly Met Thr Met Tyr Thr Asp 65 70 75 80 Leu Phe Ala Gly Lys Leu Thr Asp Leu Ala Lys His Ile Asp Tyr Leu 85 90 95 Lys Glu Gln His Leu Thr Tyr Leu His Leu Met Pro Leu Leu Thr Met 100 105 110 Pro His Pro Asp Asn Asp Gly Gly Tyr Ala Ile Glu Asp Phe Asp Thr 115 120 125 Val Asp Pro Thr Ile Gly Thr Asn Glu Asp Leu Ala Asp Leu Thr Ala 130 135 140 Lys Leu Arg Glu Ala Gly Ile Ser Leu Cys Leu Asp Phe Val Met Asn 145 150 155 160 His Thr Ala Ser Thr His Arg Trp Ala Lys Ala Ala Gln Ala Gly Asp 165 170 175 Pro Glu Tyr Gln Asp Tyr Tyr Phe Cys Tyr Asp Asp Arg Thr Ile Pro 180 185 190 Asp Gln Tyr Asp Ala Val Val Pro Gln Val Phe Pro Thr Ala Ala Pro 195 200 205 Gly Asn Phe Thr Trp Asn Glu Gln Met Gly Lys Trp Val Met Thr Gln 210 215 220 Phe Tyr Pro Phe Gln Trp Asp Leu Asn Tyr Arg As n Pro Lys Val Phe 225 230 235 240 Val Val Met Met Ser Ser Leu Leu His Leu Ala Asn Leu Gly Val Glu 245 250 255 Val Phe Arg Ile Asp Ala Val Pro Phe Ile Trp Lys Gln Leu Gly Thr 260 265 270 Asn Cys Arg Asn Leu Pro Gln Val His Thr Ile Val Arg Met Met Arg 275 280 285 Ile Met Ser Glu Ile Val Cys Pro Ala Val Val Phe Lys Gly Glu Val 290 295 300 Val Met Ala Pro Lys Glu Leu Ala Ala Tyr Phe Gly Thr Pro Glu Lys 305 310 315 320 Pro Glu Cys His Met Leu Tyr Asn Val Ser Val Met Val Asn Leu Trp 325 330 335 Ser Ala Leu Ala Asn Gly Asp Thr Arg Leu Leu Lys Thr Gln Ile Asp 340 345 350 Lys Leu Asp Ala Leu Pro Asp Asn Cys Trp Phe Val Asn Tyr Leu Arg 355 360 365 Cys His Asp Asp Ile Gly Trp Gly Le u Asp Glu Asp Val Glu Arg Gln 370 375 380 Leu Gly Ile Asp Pro Leu Lys His Lys Glu Phe Leu Tyr His Phe Tyr 385 390 395 400 Glu Gly Met Val Pro Gly Ser Trp Ala Met Gly Glu Leu Phe Asn Tyr 405 410 415 Asp Pro Ala Ser Gly Asp Ala Arg Ser Cys Gly Thr Thr Ala Ser Leu 420 425 430 Cys Gly Ile Glu Arg Ala Leu Ile Thr His Asp Arg Pro Leu Tyr Glu 435 440 445 Arg Ser Ile Gln Arg Asp Leu Leu Met His His Ala Met Gly Phe Leu 450 455 460 Arg Gly Phe Pro Met Leu Asn Cys Gly Asp Glu Ile Gly Gln Leu Asn 465 470 475 480 Gly Trp Asp Tyr Lys Glu Asp Pro Asp Arg Val Ala Asp Ser Arg Asn 485 490 495 Leu His Arg Ser Lys Phe Asn Trp Lys Asn Ala Ala Lys Arg Asp Val 500 505 510 Pro Gly Thr Leu Pro As n Arg Leu Trp Glu Gly Met Ala Asp Val Arg 515 520 525 Gln Met Arg Ser Asp Pro Cys Phe Ala Pro Asp Ala Trp Val Thr Thr Thr 530 535 540 Trp Asp Ala His Asp Asp Gly Ile Leu Ala Met Val Arg Gln Ser Gly 545 550 555 560 Gly Arg Thr Leu Leu Gly Val Phe Asn Phe Ala Asn Arg Asp Ala Thr 565 570 575 Ala Thr Leu Asp Ser Ile Glu Gly Val Ser Leu Pro Arg Thr Val Ala 580 585 590 Leu Lys Pro Tyr Glu Trp Lys Ile Glu Ala Cys 595 600 <210> 14 <211> 1812 <212> DNA <213> Artificial Sequence <220> <223> amylosucrase BtAS wild type <400> 14 atggaagcca catatcgcga ttccgtattc gccgaacggc tcgccccgcg ctgcgcagaa 60 cttgaacaac ttttccgctc gttgtacggg gattcccctg aattcgacca cttcgaacag 120 gtcatggcca aggcccacgc cgaccggccca gccgacctca aacgcctcga cgccgcccgt 180 gaacacgatc cgcaatggta ccgtcgcggc gacatgttcg gcatgaccat gtacaccgac 240 ctgttcgccg gcaaactcac cgatctcgcc aagcatatcg actatctcaa agagcagcat 300 ctgacctacc tgcacctcat gccgctgctg accatgcccc accccgacaa cgacggcggc 360 tacgccatcg aggatttcga caccgtcgac ccgactatcg gcaccaatga ggacctcgcc 420 gacctcaccg cgaaactgcg cgaagccggc atcagcctgt gccttgattt cgtcatgaac 480 cacaccgcat ccacccaccg gtgggcgaaa gccgcacaag ccggcgaccc cgaataccag 540 gactactact tctgctatga cgaccgcacc atccccgacc aatatgacgc cgtcgtcccg 600 caagtcttcc cgaccgccgc ccccggcaac ttcacatgga atgagcagat gggcaaatgg 660 gtcatgaccc agttctaccc gttccaatgg gacctcaact accgcaatcc caaggtcttc 720 gtcgtcatga tgtccagcct gctgcacctg gccaacctcg gcgtcgaagt cttccgcatc 780 gacgcggtgc cgtacatctg gaagcaactc ggcaccaact gccgcaacct gccgcaagtc 840 cacaccatcg tgcgcatgat gcgcatcatg tccgaaatcg tctgcccggc cgtcgtgttc 900 aaaggtgaag tcgtcatggc tcccaaggag ctcgccgcct acttcggcac ccccgagaag 960 cccgaatgcc acatgctgta caacgtgtcc gtcatggtca acttgtggag cgcgctcgcc 1020 aacggcgaca cccgcctgct taaaacccag atcgacaagc tcgacgccct gcccgacaac 1080 tgctggttcg tcaactatct gcgctg ccat gacgatatcg gctggggtct ggacgaggat 1140 gtcgaacgcc agttgggcat cgacccgctc aagcacaagg aattcctcta ccacttctac 1200 gagggcatgg tgcccggcag ctgggcgatg ggcgagctgt acaactatga tccggcgtcc 1260 ggtgacgcgc gcagctgcgg caccacggcg agcttgtgcg gtattgagcg tgcgctgatc 1320 acgcatgacc ggccgctgta tgagcgttcc atccagcgtg atctgctcat gcaccacgct 1380 atgggcttcc tgcgtgggtt cccgatgctc aactgcggcg acgagatcgg ccagctcaac 1440 ggctgggatt ataaggaaga cccggaccgt gtcgctgaca gccgcaatct gcaccgcagc 1500 aagttcaact ggaagaacgc cgcgaagcgc gatgtccccg gaaccttgcc aaaccggctg 1560 tgggaaggca tggcggatgt gcggcagatg cgctcggacc catgcttcgc ccctgacgct 1620 tgggtgacga cgtgggacgc gcatgatgac ggtattctcg cgatggtccg gcagtcaggt 1680 gggcgcacac tgctcggcgt gttcaatttc gcgaaccgtg acgccacggc gacgcttgac 1740 agcatcgagg gcgtgagcct gccgcgtacg gtggcgctca agccatacga gtggaagatc 1800 gaggcctgct ga 1812 <210> 15 <211> 1812 <212> DNA <213> Artificial Sequence <220> <223> amylosucrase BtAS V542K <400> 15 atggaagcca catatcgcga ttccgtattc gccgaacggc tcgccccgcg c tgcgcagaa 60 cttgaacaac ttttccgctc gttgtacggg gattcccctg aattcgacca cttcgaacag 120 gtcatggcca aggcccacgc cgaccggcca gccgacctca aacgcctcga cgccgcccgt 180 gaacacgatc cgcaatggta ccgtcgcggc gacatgttcg gcatgaccat gtacaccgac 240 ctgttcgccg gcaaactcac cgatctcgcc aagcatatcg actatctcaa agagcagcat 300 ctgacctacc tgcacctcat gccgctgctg accatgcccc accccgacaa cgacggcggc 360 tacgccatcg aggatttcga caccgtcgac ccgactatcg gcaccaatga ggacctcgcc 420 gacctcaccg cgaaactgcg cgaagccggc atcagcctgt gccttgattt cgtcatgaac 480 cacaccgcat ccacccaccg gtgggcgaaa gccgcacaag ccggcgaccc cgaataccag 540 gactactact tctgctatga cgaccgcacc atccccgacc aatatgacgc cgtcgtcccg 600 caagtcttcc cgaccgccgc ccccggcaac ttcacatgga atgagcagat gggcaaatgg 660 gtcatgaccc agttctaccc gttccaatgg gacctcaact accgcaatcc caaggtcttc 720 gtcgtcatga tgtccagcct gctgcacctg gccaacctcg gcgtcgaagt cttccgcatc 780 gacgcggtgc cgtacatctg gaagcaactc ggcaccaact gccgcaacct gccgcaagtc 840 cacaccatcg tgcgcatgat gcgcatcatg tccgaaatcg tctgcccggc cgtcgtgttc 900 aaaggtg aag tcgtcatggc tcccaaggag ctcgccgcct acttcggcac ccccgagaag 960 cccgaatgcc acatgctgta caacgtgtcc gtcatggtca acttgtggag cgcgctcgcc 1020 aacggcgaca cccgcctgct taaaacccag atcgacaagc tcgacgccct gcccgacaac 1080 tgctggttcg tcaactatct gcgctgccat gacgatatcg gctggggtct ggacgaggat 1140 gtcgaacgcc agttgggcat cgacccgctc aagcacaagg aattcctcta ccacttctac 1200 gagggcatgg tgcccggcag ctgggcgatg ggcgagctgt acaactatga tccggcgtcc 1260 ggtgacgcgc gcagctgcgg caccacggcg agcttgtgcg gtattgagcg tgcgctgatc 1320 acgcatgacc ggccgctgta tgagcgttcc atccagcgtg atctgctcat gcaccacgct 1380 atgggcttcc tgcgtgggtt cccgatgctc aactgcggcg acgagatcgg ccagctcaac 1440 ggctgggatt ataaggaaga cccggaccgt gtcgctgaca gccgcaatct gcaccgcagc 1500 aagttcaact ggaagaacgc cgcgaagcgc gatgtccccg gaaccttgcc aaaccggctg 1560 tgggaaggca tggcggatgt gcggcagatg cgctcggacc catgcttcgc ccctgacgct 1620 tggaagacga cgtgggacgc gcatgatgac ggtattctcg cgatggtccg gcagtcaggt 1680 gggcgcacac tgctcggcgt gttcaatttc gcgaaccgtg acgccacggc gacgcttgac 1740 agcatcgagg gcg tgagcct gccgcgtacg gtggcgctca agccatacga gtggaagatc 1800 gaggcctgct ga 1812 <210> 16 <211> 1812 <212> DNA <213> Artificial Sequence <220> <223> amylosucrase BtAS V542D <400> 16 atggaagcca catatcgcga ttccgtattc gccgaacggc tcgccccgcg ctgcgcagaa 60 cttgaacaac ttttccgctc gttgtacggg gattcccctg aattcgacca cttcgaacag 120 gtcatggcca aggcccacgc cgaccggcca gccgacctca aacgcctcga cgccgcccgt 180 gaacacgatc cgcaatggta ccgtcgcggc gacatgttcg gcatgaccat gtacaccgac 240 ctgttcgccg gcaaactcac cgatctcgcc aagcatatcg actatctcaa agagcagcat 300 ctgacctacc tgcacctcat gccgctgctg accatgcccc accccgacaa cgacggcggc 360 tacgccatcg aggatttcga caccgtcgac ccgactatcg gcaccaatga ggacctcgcc 420 gacctcaccg cgaaactgcg cgaagccggc atcagcctgt gccttgattt cgtcatgaac 480 cacaccgcat ccacccaccg gtgggcgaaa gccgcacaag ccggcgaccc cgaataccag 540 gactactact tctgctatga cgaccgcacc atccccgacc aatatgacgc cgtcgtcccg 600 caagtcttcc cgaccgccgc ccccggcaac ttcacatgga atgagcagat gggcaaatgg 660 gtcatgaccc agttctaccc gttccaatgg gacctcaact accgcaatcc caaggtcttc 720 gtcgtcatga tgtccagcct gctgcacctg gccaacctcg gcgtcgaagt cttccgcatc 780 gacgcggtgc cgtacatctg gaagcaactc ggcaccaact gccgcaacct gccgcaagtc 840 cacaccatcg tgcgcatgat gcgcatcatg tccgaaatcg tctgcccggc cgtcgtgttc 900 aaaggtgaag tcgtcatggc tcccaaggag ctcgccgcct acttcggcac ccccgagaag 960 cccgaatgcc acatgctgta caacgtgtcc gtcatggtca acttgtggag cgcgctcgcc 1020 aacggcgaca cccgcctgct taaaacccag atcgacaagc tcgacgccct gcccgacaac 1080 tgctggttcg tcaactatct gcgctgccat gacgatatcg gctggggtct ggacgaggat 1140 gtcgaacgcc agttgggcat cgacccgctc aagcacaagg aattcctcta ccacttctac 1200 gagggcatgg tgcccggcag ctgggcgatg ggcgagctgt acaactatga tccggcgtcc 1260 ggtgacgcgc gcagctgcgg caccacggcg agcttgtgcg gtattgagcg tgcgctgatc 1320 acgcatgacc ggccgctgta tgagcgttcc atccagcgtg atctgctcat gcaccacgct 1380 atgggcttcc tgcgtgggtt cccgatgctc aactgcggcg acgagatcgg ccagctcaac 1440 ggctgggatt ataaggaaga cccggaccgt gtcgctgaca gccgcaatct gcaccgcagc 1500 aagttcaact ggaagaacgc cgcgaagcgc gatgtccccg gaaccttgcc aaaccggctg 1560 tgggaaggca tggcggatgt gcggcagatg cgctcggacc catgcttcgc ccctgacgct 1620 tgggacacga cgtgggacgc gcatgatgac ggtattctcg cgatggtccg gcagtcaggt 1680 gggcgcacac tgctcggcgt gttcaatttc gcgaaccgtg acgccacggc gacgcttgac 1740 agcatcgagg gcgtgagcct gccgcgtacg gtggcgctca agccatacga gtggaagatc 1800 gaggcctgct ga 1812 <210> 17 <211> 1812 <212> DNA <213> Artificial Sequence < 220> <223> BtAS P200R <400> 17 atggaagcca catatcgcga ttccgtattc gccgaacggc tcgccccgcg ctgcgcagaa 60 cttgaacaac ttttccgctc gttgtacggg gattcccctg aattcgacca cttcgaacag 120 gtcatggcca aggcccacgc cgaccggcca gccgacctca aacgcctcga cgccgcccgt 180 gaacacgatc cgcaatggta ccgtcgcggc gacatgttcg gcatgaccat gtacaccgac 240 ctgttcgccg gcaaactcac cgatctcgcc aagcatatcg actatctcaa agagcagcat 300 ctgacctacc tgcacctcat gccgctgctg accatgcccc accccgacaa cgacggcggc 360 tacgccatcg aggatttcga caccgtcgac ccgactatcg gcaccaatga ggacctcgcc 420 gacctcaccg cgaaactgcg cgaagccggc atcagcctgt gccttgattt cgtcatgaac 480 cacaccgcat ccacccaccg gtgggcgaaa gccgcacaag ccggcgaccc cgaataccag 540 gactactact tctgctatga cgaccgcacc atccccgacc aatatgacgc cgtcgtccgc 600 caagtcttcc cgaccgccgc ccccggcaac ttcacatgga atgagcagat gggcaaatgg 660 gtcatgaccc agttctaccc gttccaatgg gacctcaact accgcaatcc caaggtcttc 720 gtcgtcatga tgtccagcct gctgcacctg gccaacctcg gcgtcgaagt cttccgcatc 780 gacgcggtgc cgtacatctg gaagcaactc ggcaccaact gccgcaacct gccgcaagtc 840 cacaccatcg tgcgcatgat gcgcatcatg tccgaaatcg tctgcccggc cgtcgtgttc 900 aaaggtgaag tcgtcatggc tcccaaggag ctcgccgcct acttcggcac ccccgagaag 960 cccgaatgcc acatgctgta caacgtgtcc gtcatggtca acttgtggag cgcgctcgcc 1020 aacggcgaca cccgcctgct taaaacccag atcgacaagc tcgacgccct gcccgacaac 1080 tgctggttcg tcaactatct gcgctgccat gacgatatcg gctggggtct ggacgaggat 1140 gtcgaacgcc agttgggcat cgacccgctc aagcacaagg aattcctcta ccacttctac 1200 gagggcatgg tgcccggcag ctgggcgatg ggcgagctgt acaactatga tccggcgtcc 1260 ggtgacgcgc gcagctgcgg caccacggcg agcttgtgcg gtattgagcg tgcgctgatc 1320 acgcatgacc ggccgctgta tgagcgttcc atccagcgtg atctgctcat gca ccacgct 1380 atgggcttcc tgcgtgggtt cccgatgctc aactgcggcg acgagatcgg ccagctcaac 1440 ggctgggatt ataaggaaga cccggaccgt gtcgctgaca gccgcaatct gcaccgcagc 1500 aagttcaact ggaagaacgc cgcgaagcgc gatgtccccg gaaccttgcc aaaccggctg 1560 tgggaaggca tggcggatgt gcggcagatg cgctcggacc catgcttcgc ccctgacgct 1620 tgggtgacga cgtgggacgc gcatgatgac ggtattctcg cgatggtccg gcagtcaggt 1680 gggcgcacac tgctcggcgt gttcaatttc gcgaaccgtg acgccacggc gacgcttgac 1740 agcatcgagg gcgtgagcct gccgcgtacg gtggcgctca agccatacga gtggaagatc 1800 gaggcctgct ga 1812 <210> 18 <211> 1812 <212> DNA <213> Artificial Sequence <220> <223> BtAS V202I <400> 18 atggaagcca catatcgcga ttccgtattc gccgaacggc tcgccccgcg ctgcgcagaa 60 cttgaacaac ttttccgctc gttgtacggg gattcccctg aattcgacca cttcgaacag 120 gtcatggcca aggcccacgc cgaccggcca gccgacctca aacgcctcga cgccgcccgt 180 gaacacgatc cgcaatggta ccgtcgcggc gacatgttcg gcatgaccat gtacaccgac 240 ctgttcgccg gcaaactcac cgatctcgcc aagcatatcg actatctcaa agagcagcat 300 ctgacctacc tgcacctcatg gccgctg acctg ccatgcccc accccgacaa cgacggcggc 360 tacgccatcg aggatttcga caccgtcgac ccgactatcg gcaccaatga ggacctcgcc 420 gacctcaccg cgaaactgcg cgaagccggc atcagcctgt gccttgattt cgtcatgaac 480 cacaccgcat ccacccaccg gtgggcgaaa gccgcacaag ccggcgaccc cgaataccag 540 gactactact tctgctatga cgaccgcacc atccccgacc aatatgacgc cgtcgtcccg 600 caaatcttcc cgaccgccgc ccccggcaac ttcacatgga atgagcagat gggcaaatgg 660 gtcatgaccc agttctaccc gttccaatgg gacctcaact accgcaatcc caaggtcttc 720 gtcgtcatga tgtccagcct gctgcacctg gccaacctcg gcgtcgaagt cttccgcatc 780 gacgcggtgc cgtacatctg gaagcaactc ggcaccaact gccgcaacct gccgcaagtc 840 cacaccatcg tgcgcatgat gcgcatcatg tccgaaatcg tctgcccggc cgtcgtgttc 900 aaaggtgaag tcgtcatggc tcccaaggag ctcgccgcct acttcggcac ccccgagaag 960 cccgaatgcc acatgctgta caacgtgtcc gtcatggtca acttgtggag cgcgctcgcc 1020 aacggcgaca cccgcctgct taaaacccag atcgacaagc tcgacgccct gcccgacaac 1080 tgctggttcg tcaactatct gcgctgccat gacgatatcg gctggggtct ggacgaggat 1140 gtcgaacgcc agttgggcat cgacccgctc aagcacaagg aattcct cta ccacttctac 1200 gagggcatgg tgcccggcag ctgggcgatg ggcgagctgt acaactatga tccggcgtcc 1260 ggtgacgcgc gcagctgcgg caccacggcg agcttgtgcg gtattgagcg tgcgctgatc 1320 acgcatgacc ggccgctgta tgagcgttcc atccagcgtg atctgctcat gcaccacgct 1380 atgggcttcc tgcgtgggtt cccgatgctc aactgcggcg acgagatcgg ccagctcaac 1440 ggctgggatt ataaggaaga cccggaccgt gtcgctgaca gccgcaatct gcaccgcagc 1500 aagttcaact ggaagaacgc cgcgaagcgc gatgtccccg gaaccttgcc aaaccggctg 1560 tgggaaggca tggcggatgt gcggcagatg cgctcggacc catgcttcgc ccctgacgct 1620 tgggtgacga cgtgggacgc gcatgatgac ggtattctcg cgatggtccg gcagtcaggt 1680 gggcgcacac tgctcggcgt gttcaatttc gcgaaccgtg acgccacggc gacgcttgac 1740 agcatcgagg gcgtgagcct gccgcgtacg gtggcgctca agccatacga gtggaagatc 1800 gaggcctgct ga 1812 <210> 19 <211> 1812 <212> DNA <213> Artificial Sequence <220> <223> BtAS Y265F <400> 19 atggaagcca catatcgcga ttccgtattc gccgaacggc tcgccccgcg ctgcgcagaa 60 cttgaacaac ttttccgctc gttgtacggg gattcccctg aattcgacca cttcgaacag 120 gtcatggcca aggcccacgc cg accggcca gccgacctca aacgcctcga cgccgcccgt 180 gaacacgatc cgcaatggta ccgtcgcggc gacatgttcg gcatgaccat gtacaccgac 240 ctgttcgccg gcaaactcac cgatctcgcc aagcatatcg actatctcaa agagcagcat 300 ctgacctacc tgcacctcat gccgctgctg accatgcccc accccgacaa cgacggcggc 360 tacgccatcg aggatttcga caccgtcgac ccgactatcg gcaccaatga ggacctcgcc 420 gacctcaccg cgaaactgcg cgaagccggc atcagcctgt gccttgattt cgtcatgaac 480 cacaccgcat ccacccaccg gtgggcgaaa gccgcacaag ccggcgaccc cgaataccag 540 gactactact tctgctatga cgaccgcacc atccccgacc aatatgacgc cgtcgtcccg 600 caagtcttcc cgaccgccgc ccccggcaac ttcacatgga atgagcagat gggcaaatgg 660 gtcatgaccc agttctaccc gttccaatgg gacctcaact accgcaatcc caaggtcttc 720 gtcgtcatga tgtccagcct gctgcacctg gccaacctcg gcgtcgaagt cttccgcatc 780 gacgcggtgc cgttcatctg gaagcaactc ggcaccaact gccgcaacct gccgcaagtc 840 cacaccatcg tgcgcatgat gcgcatcatg tccgaaatcg tctgcccggc cgtcgtgttc 900 aaaggtgaag tcgtcatggc tcccaaggag ctcgccgcct acttcggcac ccccgagaag 960 cccgaatgcc acatgctgta caacgtgtcc gtcatggtca acttgtggag cgcgctcgcc 1020 aacggcgaca cccgcctgct taaaacccag atcgacaagc tcgacgccct gcccgacaac 1080 tgctggttcg tcaactatct gcgctgccat gacgatatcg gctggggtct ggacgaggat 1140 gtcgaacgcc agttgggcat cgacccgctc aagcacaagg aattcctcta ccacttctac 1200 gagggcatgg tgccc ggcag ctgggcgatg ggcgagctgt acaactatga tccggcgtcc 1260 ggtgacgcgc gcagctgcgg caccacggcg agcttgtgcg gtattgagcg tgcgctgatc 1320 acgcatgacc ggccgctgta tgagcgttcc atccagcgtg atctgctcat gcaccacgct 1380 atgggcttcc tgcgtgggtt cccgatgctc aactgcggcg acgagatcgg ccagctcaac 1440 ggctgggatt ataaggaaga cccggaccgt gtcgctgaca gccgcaatct gcaccgcagc 1500 aagttcaact ggaagaacgc cgcgaagcgc gatgtccccg gaaccttgcc aaaccggctg 1560 tgggaaggca tggcggatgt gcggcagatg cgctcggacc catgcttcgc ccctgacgct 1620 tgggtgacga cgtgggacgc gcatgatgac ggtattctcg cgatggtccg gcagtcaggt 1680 gggcgcacac tgctcggcgt gttcaatttc gcgaaccgtg acgccacggc gacgcttgac 1740 agcatcgagg gcgtgagcct gccgcgtacg gtggcgctca agccatacga gtggaagatc 1800 gaggcctgct ga 1812 <210> 20 <211> 1812 <212> DNA <213> Artificial Sequence <220> <223> BtAS V305I <400> 20 atggaagcca catatcgcga ttccgtattc gccgaacggc tcgccccgcg ctgcgcagaa 60 cttgaacaac ttttccgctc gttgtacggg gattcccctg aattcgacca cttcgaacag 120 gtcatggcca aggcccacgc cgaccggcca gccgacctca aacgccgcga cgccgcga cccgt 180 gaacacgatc cgcaatggta ccgtcgcggc gacatgttcg gcatgaccat gtacaccgac 240 ctgttcgccg gcaaactcac cgatctcgcc aagcatatcg actatctcaa agagcagcat 300 ctgacctacc tgcacctcat gccgctgctg accatgcccc accccgacaa cgacggcggc 360 tacgccatcg aggatttcga caccgtcgac ccgactatcg gcaccaatga ggacctcgcc 420 gacctcaccg cgaaactgcg cgaagccggc atcagcctgt gccttgattt cgtcatgaac 480 cacaccgcat ccacccaccg gtgggcgaaa gccgcacaag ccggcgaccc cgaataccag 540 gactactact tctgctatga cgaccgcacc atccccgacc aatatgacgc cgtcgtcccg 600 caagtcttcc cgaccgccgc ccccggcaac ttcacatgga atgagcagat gggcaaatgg 660 gtcatgaccc agttctaccc gttccaatgg gacctcaact accgcaatcc caaggtcttc 720 gtcgtcatga tgtccagcct gctgcacctg gccaacctcg gcgtcgaagt cttccgcatc 780 gacgcggtgc cgtacatctg gaagcaactc ggcaccaact gccgcaacct gccgcaagtc 840 cacaccatcg tgcgcatgat gcgcatcatg tccgaaatcg tctgcccggc cgtcgtgttc 900 aaaggtgaag tcatcatggc tcccaaggag ctcgccgcct acttcggcac ccccgagaag 960 cccgaatgcc acatgctgta caacgtgtcc gtcatggtca acttgtggag cgcgctcgcc 1020 aacggcgac a cccgcctgct taaaacccag atcgacaagc tcgacgccct gcccgacaac 1080 tgctggttcg tcaactatct gcgctgccat gacgatatcg gctggggtct ggacgaggat 1140 gtcgaacgcc agttgggcat cgacccgctc aagcacaagg aattcctcta ccacttctac 1200 gagggcatgg tgcccggcag ctgggcgatg ggcgagctgt acaactatga tccggcgtcc 1260 ggtgacgcgc gcagctgcgg caccacggcg agcttgtgcg gtattgagcg tgcgctgatc 1320 acgcatgacc ggccgctgta tgagcgttcc atccagcgtg atctgctcat gcaccacgct 1380 atgggcttcc tgcgtgggtt cccgatgctc aactgcggcg acgagatcgg ccagctcaac 1440 ggctgggatt ataaggaaga cccggaccgt gtcgctgaca gccgcaatct gcaccgcagc 1500 aagttcaact ggaagaacgc cgcgaagcgc gatgtccccg gaaccttgcc aaaccggctg 1560 tgggaaggca tggcggatgt gcggcagatg cgctcggacc catgcttcgc ccctgacgct 1620 tgggtgacga cgtgggacgc gcatgatgac ggtattctcg cgatggtccg gcagtcaggt 1680 gggcgcacac tgctcggcgt gttcaatttc gcgaaccgtg acgccacggc gacgcttgac 1740 agcatcgagg gcgtgagcct gccgcgtacg gtggcgctca agccatacga gtggaagatc 1800 gaggcctgct ga 1812 <210> 21 <211 > 1812 <212> DNA <213> Artificial Sequence <220> <223> BtAS K393R <400> 21 atggaagcca catatcgcga ttccgtattc gccgaacggc tcgccccgcg ctgcgcagaa 60 cttgaacaac ttttccgctc gttgtacggg gattcccctg aattcgacca cttcgaacag 120 gtcatggcca aggcccacgc cgaccggcca gccgacctca aacgcctcga cgccgcccgt 180 gaacacgatc cgcaatggta ccgtcgcggc gacatgttcg gcatgaccat gtacaccgac 240 ctgttcgccg gcaaactcac cgatctcgcc aagcatatcg actatctcaa agagcagcat 300 ctgacctacc tgcacctcat gccgctgctg accatgcccc accccgacaa cgacggcggc 360 tacgccatcg aggatttcga caccgtcgac ccgactatcg gcaccaatga ggacctcgcc 420 gacctcaccg cgaaactgcg cgaagccggc atcagcctgt gccttgattt cgtcatgaac 480 cacaccgcat ccacccaccg gtgggcgaaa gccgcacaag ccggcgaccc cgaataccag 540 gactactact tctgctatga cgaccgcacc atccccgacc aatatgacgc cgtcgtcccg 600 caagtcttcc cgaccgccgc ccccggcaac ttcacatgga atgagcagat gggcaaatgg 660 gtcatgaccc agttctaccc gttccaatgg gacctcaact accgcaatcc caaggtcttc 720 gtcgtcatga tgtccagcct gctgcacctg gccaacctcg gcgtcgaagt cttccgcatc 780 gacgcggtgc cgtacatctg gaagcaactc gccaccaact gccgcaacct gccgcaagtc 840 cacaccatcg tgcgcatgat gcgcatcatg tccgaaatcg tctgcccggc cgtcgtgttc 900 aaaggtgaag tcgtcatggc tcccaaggag ctcgccgcct acttcggcac ccccgagaag 960 cccgaatgcc acatgctgta caacgtgtcc gtcatggtca acttgtggag cgcgctcgcc 1020 aacggcgaca cccgcctgct taaaacccag atcgacaagc tcgacgccct gcccgacaac 1080 tgctggttcg tcaactatct gcgctgccat gacgatatcg gctggggtct ggacgaggat 1140 gtcgaacgcc agttgggcat cgacccgctc aagcacaggg aattcctcta ccacttctac 1200 gagggcatgg tgcccggcag ctgggcgatg ggcgagctgt acaactatga tccggcgtcc 1260 ggtgacgcgc gcagctgcgg caccacggcg agcttgtgcg gtattgagcg tgcgctgatc 1320 acgcatgacc ggccgctgta tgagcgttcc atccagcgtg atctgctcat gcaccacgct 1380 atgggcttcc tgcgtgggtt cccgatgctc aactgcggcg acgagatcgg ccagctcaac 1440 ggctgggatt ataaggaaga cccggaccgt gtcgctgaca gccgcaatct gcaccgcagc 1500 aagttcaact ggaagaacgc cgcgaagcgc gatgtccccg gaaccttgcc aaaccggctg 1560 tgggaaggca tggcggatgt gcggcagatg cgctcggacc catgcttcgc ccctgacgct 1620 tgggtgacga cgtgggacgc gcatgatgac ggtattctcg cgatggtccg gcagtcaggt 1680 gggcgcac ac tgctcggcgt gttcaatttc gcgaaccgtg acgccacggc gacgcttgac 1740 agcatcgagg gcgtgagcct gccgcgtacg gtggcgctca agccatacga gtggaagatc 1800 gaggcctgct ga 1812 <210> 22 <211> 1812 <212> DNA <213> Artificial Sequence <220> <223> BtAS S420T <400> 22 atggaagcca catatcgcga ttccgtattc gccgaacggc tcgccccgcg ctgcgcagaa 60 cttgaacaac ttttccgctc gttgtacggg gattcccctg aattcgacca cttcgaacag 120 gtcatggcca aggcccacgc cgaccggcca gccgacctca aacgcctcga cgccgcccgt 180 gaacacgatc cgcaatggta ccgtcgcggc gacatgttcg gcatgaccat gtacaccgac 240 ctgttcgccg gcaaactcac cgatctcgcc aagcatatcg actatctcaa agagcagcat 300 ctgacctacc tgcacctcat gccgctgctg accatgcccc accccgacaa cgacggcggc 360 tacgccatcg aggatttcga caccgtcgac ccgactatcg gcaccaatga ggacctcgcc 420 gacctcaccg cgaaactgcg cgaagccggc atcagcctgt gccttgattt cgtcatgaac 480 cacaccgcat ccacccaccg gtgggcgaaa gccgcacaag ccggcgaccc cgaataccag 540 gactactact tctgctatga cgaccgcacc atccccgacc aatatgacgc cgtcgtcccg 600 caagtcttcc cgaccgccgc ccccggcaac ttcacatgga atgagcagat gggca aatgg 660 gtcatgaccc agttctaccc gttccaatgg gacctcaact accgcaatcc caaggtcttc 720 gtcgtcatga tgtccagcct gctgcacctg gccaacctcg gcgtcgaagt cttccgcatc 780 gacgcggtgc cgtacatctg gaagcaactc ggcaccaact gccgcaacct gccgcaagtc 840 cacaccatcg tgcgcatgat gcgcatcatg tccgaaatcg tctgcccggc cgtcgtgttc 900 aaaggtgaag tcgtcatggc tcccaaggag ctcgccgcct acttcggcac ccccgagaag 960 cccgaatgcc acatgctgta caacgtgtcc gtcatggtca acttgtggag cgcgctcgcc 1020 aacggcgaca cccgcctgct taaaacccag atcgacaagc tcgacgccct gcccgacaac 1080 tgctggttcg tcaactatct gcgctgccat gacgatatcg gctggggtct ggacgaggat 1140 gtcgaacgcc agttgggcat cgacccgctc aagcacaagg aattcctcta ccacttctac 1200 gagggcatgg tgcccggcag ctgggcgatg ggcgagctgt acaactatga tccggcgacg 1260 ggtgacgcgc gcagctgcgg caccacggcg agcttgtgcg gtattgagcg tgcgctgatc 1320 acgcatgacc ggccgctgta tgagcgttcc atccagcgtg atctgctcat gcaccacgct 1380 atgggcttcc tgcgtgggtt cccgatgctc aactgcggcg acgagatcgg ccagctcaac 1440 ggctgggatt ataaggaaga cccggaccgt gtcgctgaca gccgcaatct gcaccgcagc 1500 a agttcaact ggaagaacgc cgcgaagcgc gatgtccccg gaaccttgcc aaaccggctg 1560 tgggaaggca tggcggatgt gcggcagatg cgctcggacc catgcttcgc ccctgacgct 1620 tgggtgacga cgtgggacgc gcatgatgac ggtattctcg cgatggtccg gcagtcaggt 1680 gggcgcacac tgctcggcgt gttcaatttc gcgaaccgtg acgccacggc gacgcttgac 1740 agcatcgagg gcgtgagcct gccgcgtacg gtggcgctca agccatacga gtggaagatc 1800 gaggcctgct ga 1812 <210> 23 <211> 1812 <212> DNA <213> Artificial Sequence <220> <223> BtAS Y414F <400> 23 atggaagcca catatcgcga ttccgtattc gccgaacggc tcgccccgcg ctgcgcagaa 60 cttgaacaac ttttccgctc gttgtacggg gattcccctg aattcgacca cttcgaacag 120 gtcatggcca aggcccacgc cgaccggcca gccgacctca aacgcctcga cgccgcccgt 180 gaacacgatc cgcaatggta ccgtcgcggc gacatgttcg gcatgaccat gtacaccgac 240 ctgttcgccg gcaaactcac cgatctcgcc aagcatatcg actatctcaa agagcagcat 300 ctgacctacc tgcacctcat gccgctgctg accatgcccc accccgacaa cgacggcggc 360 tacgccatcg aggatttcga caccgtcgac ccgactatcg gcaccaatga ggacctcgcc 420 gacctcaccg cgaaactgcg cgaagccggc atcagcctgt gccttg attt cgtcatgaac 480 cacaccgcat ccacccaccg gtgggcgaaa gccgcacaag ccggcgaccc cgaataccag 540 gactactact tctgctatga cgaccgcacc atccccgacc aatatgacgc cgtcgtcccg 600 caagtcttcc cgaccgccgc ccccggcaac ttcacatgga atgagcagat gggcaaatgg 660 gtcatgaccc agttctaccc gttccaatgg gacctcaact accgcaatcc caaggtcttc 720 gtcgtcatga tgtccagcct gctgcacctg gccaacctcg gcgtcgaagt cttccgcatc 780 gacgcggtgc cgtacatctg gaagcaactc ggcaccaact gccgcaacct gccgcaagtc 840 cacaccatcg tgcgcatgat gcgcatcatg tccgaaatcg tctgcccggc cgtcgtgttc 900 aaaggtgaag tcgtcatggc tcccaaggag ctcgccgcct acttcggcac ccccgagaag 960 cccgaatgcc acatgctgta caacgtgtcc gtcatggtca acttgtggag cgcgctcgcc 1020 aacggcgaca cccgcctgct taaaacccag atcgacaagc tcgacgccct gcccgacaac 1080 tgctggttcg tcaactatct gcgctgccat gacgatatcg gctggggtct ggacgaggat 1140 gtcgaacgcc agttgggcat cgacccgctc aagcacaagg aattcctcta ccacttctac 1200 gagggcatgg tgcccggcag ctgggcgatg ggcgagctgt tcaactatga tccggcgtcc 1260 ggtgacgcgc gcagctgcgg caccacggcg agcttgtgcg gtattgagcg tgcgctgatc 1320 acgcatgacc ggccgctgta tgagcgttcc atccagcgtg atctgctcat gcaccacgct 1380 atgggcttcc tgcgtgggtt cccgatgctc aactgcggcg acgagatcgg ccagctcaac 1440 ggctgggatt ataaggaaga cccggaccgt gtcgctgaca gccgcaatct gcaccgcagc 1500 aagttcaact ggaagaacgc cgcgaagcgc gatgtccccg gaaccttgcc aaaccggctg 1560 tgggaaggca tggcggatgt gcggcagatg cgctcggacc catgcttcgc ccctgacgct 1620 tgggtgacga cgtgggacgc gcatgatgac ggtattctcg cgatggtccg gcagtcaggt 1680 gggcgcacac tgctcggcgt gttcaatttc gcgaaccgtg acgccacggc gacgcttgac 1740 agcatcgagg gcgtgagcct gccgcgtacg gtggcgctca agccatacga gtggaagatc 1800 gaggcctgct ga 1812 <210> 24 <211> 1812 <212> DNA <213> Artificial Sequence <220> <223> BtAS Y414F*P200R <400> 24 atggaagcca catatcgcga ttccgtattc gccgaacggc tcgccccgcg ctgcgcagaa 60 cttgaacaac ttttccgctc gttgtacggg gattcccctg aattcgacca cttcgaacag 120 gtcatggcca aggcccacgc cgaccggcca gccgacctca aacgcctcga cgccgcccgt 180 gaacacgatc cgcaatggta ccgtcgcggc gacatgttcg gcatgaccat gtacaccgac 240 ctgttcgccg gcatactcgccg cgactactcgccc agcatatcg actatctcaa agagcagcat 300 ctgacctacc tgcacctcat gccgctgctg accatgcccc accccgacaa cgacggcggc 360 tacgccatcg aggatttcga caccgtcgac ccgactatcg gcaccaatga ggacctcgcc 420 gacctcaccg cgaaactgcg cgaagccggc atcagcctgt gccttgattt cgtcatgaac 480 cacaccgcat ccacccaccg gtgggcgaaa gccgcacaag ccggcgaccc cgaataccag 540 gactactact tctgctatga cgaccgcacc atccccgacc aatatgacgc cgtcgtccgc 600 caagtcttcc cgaccgccgc ccccggcaac ttcacatgga atgagcagat gggcaaatgg 660 gtcatgaccc agttctaccc gttccaatgg gacctcaact accgcaatcc caaggtcttc 720 gtcgtcatga tgtccagcct gctgcacctg gccaacctcg gcgtcgaagt cttccgcatc 780 gacgcggtgc cgtacatctg gaagcaactc ggcaccaact gccgcaacct gccgcaagtc 840 cacaccatcg tgcgcatgat gcgcatcatg tccgaaatcg tctgcccggc cgtcgtgttc 900 aaaggtgaag tcgtcatggc tcccaaggag ctcgccgcct acttcggcac ccccgagaag 960 cccgaatgcc acatgctgta caacgtgtcc gtcatggtca acttgtggag cgcgctcgcc 1020 aacggcgaca cccgcctgct taaaacccag atcgacaagc tcgacgccct gcccgacaac 1080 tgctggttcg tcaactatct gcgctgccat gacgatatcg gctggggt ct ggacgaggat 1140 gtcgaacgcc agttgggcat cgacccgctc aagcacaagg aattcctcta ccacttctac 1200 gagggcatgg tgcccggcag ctgggcgatg ggcgagctgt tcaactatga tccggcgtcc 1260 ggtgacgcgc gcagctgcgg caccacggcg agcttgtgcg gtattgagcg tgcgctgatc 1320 acgcatgacc ggccgctgta tgagcgttcc atccagcgtg atctgctcat gcaccacgct 1380 atgggcttcc tgcgtgggtt cccgatgctc aactgcggcg acgagatcgg ccagctcaac 1440 ggctgggatt ataaggaaga cccggaccgt gtcgctgaca gccgcaatct gcaccgcagc 1500 aagttcaact ggaagaacgc cgcgaagcgc gatgtccccg gaaccttgcc aaaccggctg 1560 tgggaaggca tggcggatgt gcggcagatg cgctcggacc catgcttcgc ccctgacgct 1620 tgggtgacga cgtgggacgc gcatgatgac ggtattctcg cgatggtccg gcagtcaggt 1680 gggcgcacac tgctcggcgt gttcaatttc gcgaaccgtg acgccacggc gacgcttgac 1740 agcatcgagg gcgtgagcct gccgcgtacg gtggcgctca agccatacga gtggaagatc 1800 gaggcctgct ga 1812 <210> 25 <211> 1812 <212> DNA <213> Artificial Sequence <220> <223> BtAS Y414F*V202I <400> 25 atggaagcca catatcgcga ttccgtattc gccgaacggc tcgccccgcg ctgcgcagaa 60 cttgaacaac ttttccg ctc gttgtacggg gattcccctg aattcgacca cttcgaacag 120 gtcatggcca aggcccacgc cgaccggcca gccgacctca aacgcctcga cgccgcccgt 180 gaacacgatc cgcaatggta ccgtcgcggc gacatgttcg gcatgaccat gtacaccgac 240 ctgttcgccg gcaaactcac cgatctcgcc aagcatatcg actatctcaa agagcagcat 300 ctgacctacc tgcacctcat gccgctgctg accatgcccc accccgacaa cgacggcggc 360 tacgccatcg aggatttcga caccgtcgac ccgactatcg gcaccaatga ggacctcgcc 420 gacctcaccg cgaaactgcg cgaagccggc atcagcctgt gccttgattt cgtcatgaac 480 cacaccgcat ccacccaccg gtgggcgaaa gccgcacaag ccggcgaccc cgaataccag 540 gactactact tctgctatga cgaccgcacc atccccgacc aatatgacgc cgtcgtcccg 600 caaatcttcc cgaccgccgc ccccggcaac ttcacatgga atgagcagat gggcaaatgg 660 gtcatgaccc agttctaccc gttccaatgg gacctcaact accgcaatcc caaggtcttc 720 gtcgtcatga tgtccagcct gctgcacctg gccaacctcg gcgtcgaagt cttccgcatc 780 gacgcggtgc cgtacatctg gaagcaactc ggcaccaact gccgcaacct gccgcaagtc 840 cacaccatcg tgcgcatgat gcgcatcatg tccgaaatcg tctgcccggc cgtcgtgttc 900 aaaggtgaag tcgtcatggc tcccaaggag ctcgc cgcct acttcggcac ccccgagaag 960 cccgaatgcc acatgctgta caacgtgtcc gtcatggtca acttgtggag cgcgctcgcc 1020 aacggcgaca cccgcctgct taaaacccag atcgacaagc tcgacgccct gcccgacaac 1080 tgctggttcg tcaactatct gcgctgccat gacgatatcg gctggggtct ggacgaggat 1140 gtcgaacgcc agttgggcat cgacccgctc aagcacaagg aattcctcta ccacttctac 1200 gagggcatgg tgcccggcag ctgggcgatg ggcgagctgt tcaactatga tccggcgtcc 1260 ggtgacgcgc gcagctgcgg caccacggcg agcttgtgcg gtattgagcg tgcgctgatc 1320 acgcatgacc ggccgctgta tgagcgttcc atccagcgtg atctgctcat gcaccacgct 1380 atgggcttcc tgcgtgggtt cccgatgctc aactgcggcg acgagatcgg ccagctcaac 1440 ggctgggatt ataaggaaga cccggaccgt gtcgctgaca gccgcaatct gcaccgcagc 1500 aagttcaact ggaagaacgc cgcgaagcgc gatgtccccg gaaccttgcc aaaccggctg 1560 tgggaaggca tggcggatgt gcggcagatg cgctcggacc catgcttcgc ccctgacgct 1620 tgggtgacga cgtgggacgc gcatgatgac ggtattctcg cgatggtccg gcagtcaggt 1680 gggcgcacac tgctcggcgt gttcaatttc gcgaaccgtg acgccacggc gacgcttgac 1740 agcatcgagg gcgtgagcct gccgcgtacg gtggcgctca a gccatacga gtggaagatc 1800 gaggcctgct ga 1812 <210> 26 <211> 1812 <212> DNA <213> Artificial Sequence <220> <223> BtAS Y414F*Y265F <400> 26 atggaagcca catatcgcga ttccgtattc gccgaacggc tcgccccgcg ctgcgcagaa 60 cttgaacaac ttttccgctc gttgtacggg gattcccctg aattcgacca cttcgaacag 120 gtcatggcca aggcccacgc cgaccggcca gccgacctca aacgcctcga cgccgcccgt 180 gaacacgatc cgcaatggta ccgtcgcggc gacatgttcg gcatgaccat gtacaccgac 240 ctgttcgccg gcaaactcac cgatctcgcc aagcatatcg actatctcaa agagcagcat 300 ctgacctacc tgcacctcat gccgctgctg accatgcccc accccgacaa cgacggcggc 360 tacgccatcg aggatttcga caccgtcgac ccgactatcg gcaccaatga ggacctcgcc 420 gacctcaccg cgaaactgcg cgaagccggc atcagcctgt gccttgattt cgtcatgaac 480 cacaccgcat ccacccaccg gtgggcgaaa gccgcacaag ccggcgaccc cgaataccag 540 gactactact tctgctatga cgaccgcacc atccccgacc aatatgacgc cgtcgtcccg 600 caagtcttcc cgaccgccgc ccccggcaac ttcacatgga atgagcagat gggcaaatgg 660 gtcatgaccc agttctaccc gttccaatgg gacctcaact accgcaatcc caaggtcttc 720 gtccagccgat gctgcacctg gccaacctcg gcgtcgaagt cttccgcatc 780 gacgcggtgc cgttcatctg gaagcaactc ggcaccaact gccgcaacct gccgcaagtc 840 cacaccatcg tgcgcatgat gcgcatcatg tccgaaatcg tctgcccggc cgtcgtgttc 900 aaaggtgaag tcgtcatggc tcccaaggag ctcgccgcct acttcggcac ccccgagaag 960 cccgaatgcc acatgctgta caacgtgtcc gtcatggtca acttgtggag cgcgctcgcc 1020 aacggcgaca cccgcctgct taaaacccag atcgacaagc tcgacgccct gcccgacaac 1080 tgctggttcg tcaactatct gcgctgccat gacgatatcg gctggggtct ggacgaggat 1140 gtcgaacgcc agttgggcat cgacccgctc aagcacaagg aattcctcta ccacttctac 1200 gagggcatgg tgcccggcag ctgggcgatg ggcgagctgt tcaactatga tccggcgtcc 1260 ggtgacgcgc gcagctgcgg caccacggcg agcttgtgcg gtattgagcg tgcgctgatc 1320 acgcatgacc ggccgctgta tgagcgttcc atccagcgtg atctgctcat gcaccacgct 1380 atgggcttcc tgcgtgggtt cccgatgctc aactgcggcg acgagatcgg ccagctcaac 1440 ggctgggatt ataaggaaga cccggaccgt gtcgctgaca gccgcaatct gcaccgcagc 1500 aagttcaact ggaagaacgc cgcgaagcgc gatgtccccg gaaccttgcc aaaccggctg 1560 tgggaaggca tggcggatgt gcggcagatg cgctcggacc catgcttcgc ccctgacgct 1620 tgggtgacga cgtgggacgc gcatgatgac ggtattctcg cgatggtccg gcagtcaggt 1680 gggcgcacac tgctcggcgt gttcaatttc gcgaaccgtg acgccacggc gacgcttgac 1740 agcatcgagg gcgtgagcct gccgcgtacg gtggcgctca agccatacga gtggaagatc 1800 gaggcc tgct ga 1812 <210> 27 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> BtAS V542D forward primer <400> 27 cgcccctgac gcttggggaca cgacgtggga cgcgc 35 <210> 28 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> BtAS V542D reverse primer <400> 28 gcgcgtccca cgtcgtgtcc caagcgtcag gggcg 35 <210> 29 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> BtAS V542K forward primer <400> 29 cgcccctgac gcttggaaga cgacgtggga cgcgc 35 <210> 30 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> BtAS V542K reverse primer <400> 30 gcgcgtccca cgtcgtcttc caagcgtcag gggcg 35 <210> 31 <211> 212> DNA <213> Artificial Sequence <220> <223> BtAS P200R forward primer <400> 31 gccgtcgtcc gccaagtctt c 21 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> BtAS P200R reverse primer <400> 32 gaagacttgg cggacgacgg c 21 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> BtAS V202I forward primer <400> 33 gtcccgcaaa tcttcccgac c 21 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> BtAS V202I reverse primer <400> 34 ggtcgggaag atttgcggga c 21 <210> 35 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> BtAS Y265F forward primer <400> 35 gacgcggtgc cgttcatctg gaagcaa 27 <210> 36 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> BtAS Y265F reverse primer <400> 36 ttgcttccag atgaacggca ccgcgtc 27 <210> 37 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> BtAS Y305I forward primer <400> 37 aaaggtgaag tcatcatggc tcccaag 27 <210> 38 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> BtAS Y305I reverse primer <400> 38 cttgggagcc atgatgactt caccttt 27 <210> 39 <211 > 27 <212> DNA <213> Artificial Sequence <220> <223> BtAS K393R forward primer <400> 39 ccgctcaagc acagggaatt cctctac 27 <210> 40 <211> 27 <212> DNA <213> Artificial Sequence <220> < 223> BtAS K393R reverse primer <400> 40 gtagaggaat tccctgtgct tgagcgg 27 <210> 41 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> BtAS S420T forward primer <400> 41 tatgatccgg cgacgggtga cgcgcgc 27 < 210> 42 <211> 27 <212> DNA <213> A rtificial Sequence <220> <223> BtAS S420T reverse primer <400> 42 gcgcgcgtca cccgtcgccg gatcata 27 <210> 43 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> BtAS Y414F forward primer <400> 43 atgggcgagc tgttcaacta tgatccg 27 <210> 44 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> BtAS Y414F reverse primer<400> 44 cggatcatag ttgaacagct cgcccat 27

Claims (15)

preparing a reaction product containing turanose by reacting a catalyst composition containing an amylosucrase enzyme with at least one reaction substrate selected from the group consisting of sugar and fructose; and
Separating the reaction product into a turanose fraction and a raffinate using simulated moving bed (SMB) chromatography;
The amylosucrase enzyme is an enzyme consisting of one or more sequences selected from the group consisting of amino acid sequences of SEQ ID NOs: 2, 3, 10, 11, 12 and 13,
Method for producing turanos. The method of claim 1, further comprising reacting the reaction product with a glucoamylase enzyme. delete The method according to claim 1, wherein the reaction product has a trehalulose content of 7.0% by weight or less based on 100% by weight of the total saccharide solid content. The method according to claim 1, wherein the turanose fraction has a trehalulose content of 7.0% by weight or less based on 100% by weight of the saccharide solid content. The method according to claim 1, wherein the turanose fraction is 70% by weight or more based on 100% by weight of the saccharide solid content in the turanose fraction. The method of claim 1, wherein the reaction substrate contains 5 to 250 parts by weight of fructose solids based on 100 parts by weight of sugar solids. The method according to claim 1, wherein the reaction product contains 20% by weight of oligosaccharides of DP3 or higher and 30% by weight or more of turanose, based on 100% by weight of the solid content of the reaction product. The method of claim 1, wherein the catalyst composition comprises at least one selected from the group consisting of an amylosucrase enzyme, a recombinant microorganism producing the amylosucrase enzyme, a culture of the recombinant microorganism, and a lysate of the recombinant microorganism. Including, the manufacturing method. The method according to claim 1, further comprising at least one step selected from the group consisting of desalting, decoloring, concentration, crystallization, centrifugation and filtration. The method of claim 1, wherein the amylosucrase enzyme has one or more properties selected from the group consisting of the following properties:
(1) the molecular weight of the monomer is 65 to 75 kDa
(2) The optimum temperature is 30 to 65 ° C.
(3) an optimum pH of 6.0 to 9.0. The method of claim 1, wherein the amylosucrase enzyme is reacted with the reaction substrate at a temperature of 50 ° C. and pH 6.0 for 24 hours, based on 100% by weight of solid content of the reaction product, 30% by weight or more of turanose, oligosaccharide A method for producing a reaction product containing 20% by weight or less and 7% by weight or less of trehalulose. delete delete delete

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