Method for producing alpha-cyclodextrin

Abstract

Provided herein are methods of enzymatic production of alpha-cyclodextrin from sucrose. In some cases, the methods involve contacting sucrose with one or more enzymes to convert sucrose to amylose, and then contacting amylose with one or more enzymes to convert amylose to alpha-cyclodextrin. In some cases, the methods produce a higher yield of alpha-cyclodextrin relative to beta-cyclodextrin, gamma-cyclodextrin, or both.

Description

Background Art

[0001] Cyclodextrin is a class of cyclic oligosaccharides consisting of glucose cyclic oligomers.Cyclodextrin has a lipophilic central core and a hydrophilic outer surface, which makes them useful in pharmaceutical and other various industries.Natural cyclodextrins (i.e., α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin) are designated as generally recognized as safe (GRAS) by the U.S. Food and Drug Administration (FDA), and are widely used in food and pharmaceutical industries, etc.The standard method for producing cyclodextrins generally involves the enzymatic conversion of starch.However, there are various shortcomings in the standard production method, including supply chain shortage, scalability, quality changes, and purification and commodity costs.Therefore, it is necessary to solve these problems to produce improved methods of cyclodextrins. Summary of the invention

[0002] There is an unmet need for methods of producing cyclodextrins that do not involve starch conversion as a starting material. The present disclosure meets this unmet need by providing a method for producing cyclodextrins by biosynthesis without using starch as a starting material. Without being limited to any of the following, the advantages of the present disclosure provided herein over other methods (e.g., starch-based methods) include a higher total yield of cyclodextrin products, a more desirable purity, less by-product waste, and the by-products themselves can be used for other purposes.

[0003] In one aspect, a method for producing a composition comprising cyclodextrin is provided, the method comprising: (a) contacting sucrose with an enzyme or enzyme mixture capable of converting sucrose into amylose under conditions that allow the conversion of sucrose into amylose, thereby producing amylose; (b) contacting the amylose produced in (a) with an enzyme capable of converting amylose into cyclodextrin under conditions that allow the conversion of amylose into cyclodextrin, thereby producing a composition comprising cyclodextrin, wherein the enzyme capable of converting amylose into cyclodextrin in (b) is a variant enzyme that is capable of producing a greater amount and / or concentration of α-cyclodextrin than β-cyclodextrin, γ-cyclodextrin, or both relative to a wild-type enzyme capable of converting amylose into cyclodextrin, wherein the composition comprising cyclodextrin comprises α-cyclodextrin, and may optionally further comprise β-cyclodextrin, γ-cyclodextrin, or any combination thereof, and the amount and / or concentration of α-cyclodextrin contained in the composition comprising cyclodextrin is greater than that of β-cyclodextrin, γ-cyclodextrin, or both. In some cases, the enzyme of (a) is an amylosucrase, or the enzyme mixture of (a) comprises an amylosucrase. In some cases, the amylosucrase is a variant amylosucrase, which comprises at least one amino acid variant relative to a wild-type amylosucrase. In some cases, relative to a wild-type amylosucrase, the variant amylosucrase can produce a larger amount and / or concentration of straight-chain starch from sucrose. In some cases, the wild-type amylosucrase is a carbon source Cellulomonas T26 (Cellulomonas carboniz T26) amylosucrase. In some cases, the wild-type amylosucrase comprises or consists of the amino acid sequence of SEQ IDNO:1. In some cases, the wild-type amylosucrase is a polysaccharide Neisseria (Neisseria polysaccharea) amylosucrase. In some cases, the wild-type amylosucrase comprises or consists of the amino acid sequence of SEQ IDNO:2. In some cases, the variant amylosucrase comprises or consists of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity, to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the at least one amino acid variant comprises at least one amino acid substitution relative to the wild-type amylosucrase. In some cases, at least one amino acid substitution comprises an amino acid substitution at amino acid position 234 relative to the wild-type amylosucrase having the amino acid sequence of SEQ ID NO: 2. In some cases, the amino acid substitution at position 234 is selected from the group consisting of R234Q, R234G, R234A, R234S, R234M, R234C, R234K, R234I, R234D, R234Y, R234W, R234E, R234L and R234H. In some cases, the enzyme mixture of (a) comprises at least two enzymes, which together or in combination can convert sucrose into amylose.In some cases, the enzyme mixture comprises sucrose phosphorylase. In some cases, sucrose phosphorylase can convert sucrose into glucose-1-phosphate. In some cases, (a) contact also includes contacting sucrose with sucrose phosphorylase under conditions that allow sucrose to be converted into glucose-1-phosphate. In some cases, sucrose phosphorylase is selected from the group consisting of the following: Bifidobacterium longum sucrose phosphorylase, Leuconostoc mesenteroides sucrose phosphorylase and Streptococcus mutans sucrose phosphorylase. In some cases, sucrose phosphorylase comprises an amino acid sequence of any one of SEQ ID NO:17-20 or an amino acid sequence having at least about 70% sequence identity with an amino acid sequence of any one of SEQ ID NO:17-20 or consisting of it. In some cases, the enzyme mixture comprises alpha-glucan phosphorylase. In some cases, alpha-glucan phosphorylase can convert glucose-1-phosphate into amylose. In some cases, the contact of (a) further comprises contacting glucose-1-phosphate with α-glucan phosphorylase under conditions that allow glucose-1-phosphate to be converted into amylose. In some cases, α-glucan phosphorylase is selected from the group consisting of: potato (Solanum tuberosum) α-glucan phosphorylase, S. tokodaii strain 7 (S. tokodaii strain7) α-glucan phosphorylase and Corynebacterium callunae DSM 20145 (C. callunae DSM 20145) α-glucan phosphorylase. In some cases, α-glucan phosphorylase comprises an amino acid sequence of any one of SEQ ID NO: 21-24 or an amino acid sequence having at least about 70% sequence identity with an amino acid sequence of any one of SEQ ID NO: 21-24 or consisting of it. In some cases, the enzyme capable of converting amylose into cyclodextrin in (b) comprises a variant cyclodextrin glucanotransferase. In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to the wild-type cyclodextrin glucanotransferase. In some cases, the wild-type cyclodextrin glucanotransferase is Paenibacillus macerans cyclodextrin glucanotransferase. In some cases, the wild-type cyclodextrin glucanotransferase comprises or consists of an amino acid sequence of any one of SEQ ID NO:25-28. In some cases, the variant cyclodextrin glucanotransferase comprises or consists of an amino acid sequence having at least about 70% sequence identity with an amino acid sequence of any one of SEQ ID NO:25-28. In some cases, the at least one amino acid variant comprises at least one amino acid substitution relative to the wild-type cyclodextrin glucanotransferase.In some cases, at least one amino acid substitution comprises an amino acid substitution at amino acid position 146 relative to a wild-type cyclodextrin glucanotransferase having an amino acid sequence of SEQ ID NO: 28. In some cases, the amino acid substitution at position 146 is selected from the group consisting of: R146A and R146P. In some cases, at least one amino acid substitution comprises an amino acid substitution at amino acid position 147 relative to a wild-type cyclodextrin glucanotransferase having an amino acid sequence of SEQ ID NO: 28. In some cases, the amino acid substitution at position 147 is selected from the group consisting of: D147P and D147A. In some cases, at least one amino acid substitution comprises an amino acid substitution at amino acid position 146 relative to a wild-type cyclodextrin glucanotransferase having an amino acid sequence of SEQ ID NO: 28; and an amino acid substitution at amino acid position 147 relative to a wild-type cyclodextrin glucanotransferase having an amino acid sequence of SEQ ID NO: 28. In some cases, the amino acid substitution at position 146 is selected from the group consisting of: R146A and R146P. In some cases, the amino acid substitution at position 147 is selected from the group consisting of: D147P and D147A. In some cases, at least one amino acid substitution includes an amino acid substitution at amino acid position 372 relative to a wild-type cyclodextrin glucanotransferase having an amino acid sequence of SEQ ID NO: 26 or 28. In some cases, the amino acid substitution at position 372 is D372K. In some cases, at least one amino acid substitution includes an amino acid substitution at amino acid position 89 relative to a wild-type cyclodextrin glucanotransferase having an amino acid sequence of SEQ ID NO: 26 or 28. In some cases, the amino acid substitution at position 89 is Y89R. In some cases, at least one amino acid substitution includes an amino acid substitution at position 372 relative to a wild-type cyclodextrin glucanotransferase having an amino acid sequence of SEQ ID NO: 26 or 28; and an amino acid substitution at position 89 relative to a wild-type cyclodextrin glucanotransferase having an amino acid sequence of SEQ ID NO: 26 or 28. In some cases, the amino acid substitution at position 372 is D372K. In some cases, the amino acid substitution at position 89 is Y89R. In some cases, the contacting of (a), the contacting of (b), or both further comprises adding at least one additive during (a), (b), or both, the additive increasing the yield of alpha-cyclodextrin relative to beta-cyclodextrin, gamma-cyclodextrin, or both. In some cases, the at least one additive is CaCl. 2 In some cases, CaCl 2Added at a concentration of about 1mM to about 100mM. In some cases, at least one additive is ethanol. In some cases, ethanol is added at a concentration of about 1% v / v to about 10% v / v. In some cases, the contact of (a) and the contact of (b) occur sequentially. In some cases, the contact of (a) and the contact of (b) occur simultaneously or substantially simultaneously. In some cases, the straight-chain starch produced in (a) is not purified or separated before the contact of (b). In some cases, the contact of (a), the contact of (b) or both are carried out in vitro. In some cases, the contact of (a), the contact of (b) or both are carried out in a container, a vial, a jar, a test tube, a well, a plate or a package. In some cases, at least one enzyme in the enzyme or enzyme mixture of (a), the variant enzyme of (b) or both are purified enzymes, isolated enzymes or both. In some cases, at least one enzyme in the enzyme or enzyme mixture of (a), the variant enzyme of (b) or both are recombinantly produced enzymes. In some cases, the contact of (a), the contact of (b) or both are carried out in vivo. In some cases, the contact of (a), the contact of (b) or both are carried out in a recombinant host cell. In some cases, the recombinant host cell comprises a heterologous nucleic acid encoding at least one enzyme in the enzyme or enzyme mixture of (a), a variant enzyme of (b) or both. In some cases, the recombinant host cell is a microbial cell. In some cases, the microbial cell is a bacterial cell. In some cases, the bacterial cell is Escherichia coli. In some cases, at least one enzyme in the enzyme or enzyme mixture of (a), a variant enzyme of (b) or both are produced in a Pichia cell, such as a Pichia pastoris cell. In some cases, the ratio of alpha-cyclodextrin to beta-cyclodextrin in the composition comprising cyclodextrin is at least 2: 1. In some cases, the ratio of alpha-cyclodextrin to gamma-cyclodextrin in the composition comprising cyclodextrin is at least 2: 1.

[0004] Incorporated by Reference

[0005] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] The novel features of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description and accompanying drawings which set forth illustrative embodiments in which the principles of the present invention are utilized, wherein:

[0007] Figure 1A-Figure 1CThe structures of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin are depicted respectively.

[0008] Figure 2A Depicted is a non-limiting example of a single enzyme reaction for converting sucrose to amylose according to embodiments of the present disclosure.

[0009] Figure 2B Depicted is a non-limiting example of a dual enzyme reaction for converting sucrose to amylose according to embodiments of the present disclosure.

[0010] Figure 3 Depicted is a non-limiting example of an enzymatic reaction for converting amylose to α-cyclodextrin according to embodiments of the present disclosure.

[0011] Figure 4 Depicted are non-limiting examples of data demonstrating that variant cyclodextrin glucanotransferases are able to increase the ratio of α-cyclodextrin relative to β-cyclodextrin or γ-cyclodextrin produced from amylose.

[0012] Figure 5 Depicted is a non-limiting example of a one-pot enzymatic synthesis of sucrose to α-cyclodextrin using a variant amylosucrase and a variant cyclodextrin glucanotransferase.

[0013] Figure 6 A non-limiting example of the use of additives to enhance the production of α-cyclodextrin from sucrose in a one-pot enzymatic synthesis reaction is depicted.

[0014] 7 depicts a graph of the retained enzyme activity (%) of freeze-dried whole cell lysate or whole cell slurry of amylosucrase (with or without stabilizing compounds) as discussed in Example 4. The % retained enzyme activity was measured by comparing the enzyme activity to whole cell lysate or whole cell slurry that had not been freeze-dried. DETAILED DESCRIPTION

[0015] Current methods of producing cyclodextrins are plagued by supply chain shortages and issues of scalability, quality variation, purification, and commodity costs. In addition, there are several key issues with current cyclodextrin production for food and pharmaceutical use, such as, but not limited to, FDA certification of starch after each growing season, and the ability to scale up using standard agricultural techniques. The disclosed method overcomes these issues by providing a method for the facile enzymatic synthesis of a composition comprising cyclodextrins using sucrose as a starting material, preferably a one-pot enzymatic synthesis method.

[0016] Provided herein is a method for producing a composition comprising cyclodextrin. Also provided herein is an enzymatic synthesis method for α-cyclodextrin. Typically, the method provided herein does not involve the use of starch as a starting material. Preferably, the method provided herein involves the use of sucrose as a starting material; however, in some embodiments, other monosaccharides or disaccharides may be used. Also provided herein is a method for enzymatically converting sucrose into α-cyclodextrin using various enzymes. The method typically involves converting sucrose into amylose as the first step (step (a)) of the synthesis pathway. In one embodiment, the method involves converting sucrose into amylose using a single enzyme (e.g., amylosucrase). In another embodiment, the method involves converting sucrose into amylose using two enzymes (e.g., sucrose phosphorylase and α-glucan phosphorylase). The method typically also involves enzymatically converting amylose into α-cyclodextrin (e.g., using cyclodextrin glucanotransferase) in the second step (step (b)) of the synthesis pathway. In some embodiments, one or more enzymatic steps occur in vivo (e.g., within a microbial host cell). In some embodiments, one or more enzymatic steps occur in vitro (e.g., in a container, vial, jar, test tube, well, plate, package, e.g., using purified and / or isolated (e.g., recombinant) enzymes).

[0017] Cyclodextrins are formed by the conjugation of glucose pyranose units through α1,4 glycosidic bonds in a ring shape. Generally, cyclodextrins have three different forms: α-cyclodextrin ( Figure 1A ), β-cyclodextrin ( Figure 1B ) and γ-cyclodextrin ( Figure 1C ), depending on the number of glucose monomers that make up the ring arrangement. The number of glucose monomers that make up α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin are 6, 7, and 8, respectively. Cyclodextrins are widely used in the food, pharmaceutical, and chemical industries due to their low toxicity, low immunogenicity, and ability to form non-covalent complexes with guest molecules. For example, cyclodextrins are widely used as carriers to increase the water solubility of lipophilic vitamins and hormones. In Western countries, the intake of natural cyclodextrins is regulated by the Joint WHO / FAO Expert Committee on Food Additives (JECFA), while pharmaceutical applications fall under the jurisdiction of the European Medicines Agency (EMA) in Europe and the Food and Drug Administration (FDA) in the United States. Natural cyclodextrins (CDs) can be ingested without being absorbed in significant amounts and are therefore considered "generally recognized as safe" by the FDA and are often referred to as molecules with "GRAS status."

[0018] α-cyclodextrin is widely used in the pharmaceutical industry. In order to improve the oral bioavailability and solubility of cyclodextrin, different α-cyclodextrin derivatives have been prepared. For example, modifying the hydroxyl group of cyclodextrin with alkyl hydroxyl groups can significantly improve the solubility of cyclodextrin. Some potential derivatives include randomly methylated α-cyclodextrin and branched α-cyclodextrin.

[0019] In one aspect of the present disclosure, a method for producing a composition comprising cyclodextrin is provided. In some cases, the method includes (a) contacting sucrose with an enzyme or enzyme mixture capable of converting sucrose into amylose under conditions that allow sucrose to be converted into amylose, thereby producing amylose. In some cases, the method also includes (b) contacting amylose with an enzyme capable of converting amylose into cyclodextrin under conditions that allow amylose to be converted into cyclodextrin, thereby producing a composition comprising cyclodextrin. In some cases, the enzyme capable of converting amylose into cyclodextrin is a variant enzyme, relative to the wild-type enzyme capable of converting amylose into cyclodextrin, the variant enzyme is capable of producing alpha-cyclodextrin in greater amounts and / or concentrations (e.g., wt%, mol% or w / v) than beta-cyclodextrin, gamma-cyclodextrin or both. In some cases, the composition comprising cyclodextrin comprises alpha-cyclodextrin, and may optionally also comprise beta-cyclodextrin, gamma-cyclodextrin, or any combination thereof. In some cases, the composition comprising cyclodextrin comprises an amount and / or concentration (e.g., wt%, mol%, or w / v) of α-cyclodextrin greater than β-cyclodextrin, γ-cyclodextrin, or both. In some cases, the amount and / or concentration of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin is measured by high performance liquid chromatography (HPLC).

[0020] Method step (a): Enzymatic conversion of sucrose into amylose

[0021] The method provided herein relates to the enzymatic conversion of sucrose to amylose. In some cases, amylose is α-amylose. In some embodiments, the method relates to contacting sucrose with an enzyme or enzyme mixture capable of converting sucrose into amylose under conditions that allow sucrose to be converted into amylose, thereby producing amylose. In one aspect, the method relates to using a single enzyme to convert sucrose into amylose. In alternative aspects, the method relates to using an enzyme mixture (e.g., two enzymes), which convert sucrose into amylose together or in combination. In some cases, sucrose is deuterated sucrose (e.g., one or more hydrogens have been replaced by deuterium). In some cases, any one or more reagents used in sucrose and / or synthesis reactions are deuterated.

[0022] A single enzyme method for producing amylose from sucrose

[0023] In some aspects, the method of converting sucrose to amylose involves a single enzyme. In some cases, the enzyme is amylosucrase. Figure 2ADescribe the schematic diagram of the single enzyme method of producing amylose by sucrose. In this example, sucrose is contacted with amylosucrase, and amylosucrase converts sucrose into amylose. In some cases, amylosucrase is a wild-type amylosucrase. For example, a wild-type amylosucrase can be a carbon source cellulomonas T26 amylosucrase (e.g., NCBI accession number N868_11335). In some cases, a wild-type carbon source cellulomonas T26 amylosucrase may include or consist of an amino acid sequence according to SEQ ID NO:1. In some cases, a wild-type amylosucrase may be a polysaccharide Neisseria amylosucrase (e.g., NCBI accession number AJ011781). In some cases, a wild-type polysaccharide Neisseria amylosucrase may include or consist of an amino acid sequence according to SEQ ID NO:2. Table 1 below describes non-limiting examples of wild-type amylosucrase (and its amino acid sequence) that can be used according to the method provided herein.

[0024] Table 1. Non-limiting examples of wild-type amylosucrases

[0025]

[0026]

[0027] In some embodiments, amylosucrase is a variant amylosucrase comprising at least one amino acid variant relative to wild-type amylosucrase. Relative to wild-type amylosucrase, the variant amylosucrase may comprise one or more amino acid substitutions, deletions, insertions and / or modifications. In some cases, relative to wild-type amylosucrase, the variant amylosucrase is capable of producing a greater amount and / or concentration of amylose from sucrose.

[0028] In some cases, the variant amylosucrase comprises at least one amino acid variant relative to the wild-type carbon source Cellulomonas T26 amylosucrase. In some cases, the variant amylosucrase comprises at least one amino acid variant relative to SEQ ID NO: 1. In some cases, the variant amylosucrase comprises at least one amino acid variant relative to the wild-type polysaccharide Neisseria amylosucrase. In some cases, the variant amylosucrase comprises at least one amino acid variant relative to SEQ ID NO: 2. In some cases, the variant amylosucrase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more), preferably at least about 90% sequence identity relative to the wild-type carbon source Cellulomonas T26 amylosucrase. In some cases, the variant amylosucrase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more), preferably at least about 90% sequence identity, relative to the amino acid sequence of SEQ ID NO: 1. In some cases, the variant amylosucrase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity relative to wild-type Neisseria polysaccharida amylosucrase. In some cases, the variant amylosucrase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) relative to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity.

[0029] In some cases, the at least one amino acid variant comprises at least one amino acid replacement relative to wild-type amylosucrase. In some cases, the at least one amino acid variant comprises at least one amino acid replacement relative to wild-type carbon source Cellulomonas T26 amylosucrase. In some cases, the at least one amino acid variant comprises at least one amino acid replacement relative to wild-type polysaccharide Neisseria amylosucrase. In some cases, at least one amino acid replacement includes relative to SEQ ID NO:2 amino acid sequence, amino acid replacement at amino acid position 234. In some cases, relative to SEQ ID NO:2 amino acid sequence, amino acid replacement at amino acid position 234 is selected from the group consisting of the following: R234Q, R234G, R234A, R234S, R234M, R234C, R234K, R234I, R234D, R234Y, R234W, R234E, R234L and R234H. In a preferred embodiment, relative to the amino acid sequence of SEQ ID NO:2, the amino acid substitution at amino acid position 234 is selected from the group consisting of R234Q, R234G, R234A, R234S, R234M, R234C and R234K. In this regard, it should be understood that R234Q means that relative to the amino acid sequence of SEQ ID NO:2, the arginine (R) at amino acid position 234 is replaced by glutamine (Q), etc. In some cases, relative to the amino acid sequence of SEQ ID NO:2, the amino acid substitution at amino acid position 234 is R234Q (e.g., SEQ ID NO:3 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO:2, the amino acid substitution at amino acid position 234 is R234G (e.g., SEQ ID NO:4 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO:2, the amino acid substitution at amino acid position 234 is R234A (e.g., SEQ ID NO:5 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO: 2, the amino acid substitution at amino acid position 234 is R234S (e.g., SEQ ID NO: 6 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO: 2, the amino acid substitution at amino acid position 234 is R234M (e.g., SEQ ID NO: 7 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO: 2, the amino acid substitution at amino acid position 234 is R234C (e.g., SEQ ID NO: 8 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO: 2, the amino acid substitution at amino acid position 234 is R234K (e.g., SEQ ID NO: 9 in Table 2).In some cases, relative to the amino acid sequence of SEQ ID NO: 2, the amino acid substitution at amino acid position 234 is R234I (e.g., SEQ ID NO: 10 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO: 2, the amino acid substitution at amino acid position 234 is R234D (e.g., SEQ ID NO: 11 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO: 2, the amino acid substitution at amino acid position 234 is R234Y (e.g., SEQ ID NO: 12 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO: 2, the amino acid substitution at amino acid position 234 is R234W (e.g., SEQ ID NO: 13 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO: 2, the amino acid substitution at amino acid position 234 is R234E (e.g., SEQ ID NO: 14 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO: 2, the amino acid substitution at amino acid position 234 is R234L (e.g., SEQ ID NO: 15 in Table 2). In some cases, relative to the amino acid sequence of SEQ ID NO: 2, the amino acid substitution at amino acid position 234 is R234H (eg, SEQ ID NO: 16 in Table 2).

[0030] In some aspects, the variant amylosucrase comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 3-16 as depicted in Table 2. In preferred embodiments, the variant amylosucrase comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 3-9 as depicted in Table 2. In some cases, the variant amylosucrase comprises an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more), preferably at least about 90% sequence identity compared to the amino acid sequence of any one of SEQ ID NOs: 3-16 or 42 as depicted in Table 2, or an amino acid sequence having at least about 90% sequence identity compared to the amino acid sequence of any one of SEQ ID NOs: 3-16 or 42 as depicted in Table 2, or The amino acid sequence of any one of NO:3-16 or 42 has, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more). In preferred embodiments, the variant amylosucrase comprises or consists of an amino acid sequence having at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) sequence identity, preferably at least about 90% sequence identity, compared to the amino acid sequence of any one of SEQ ID NOs: 3-9 depicted in Table 2.

[0031] Table 2. Non-limiting examples of variant amylosucrases.

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039] In some aspects, the variant amylosucrase comprises an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution at amino acid position 234 relative to SEQ ID NO: 2, or consists of. In this regard, and as used throughout the disclosure, the sequence identity includes amino acid substitutions (i.e., the sequence identity is calculated based on the entire amino acid sequence of the variant enzyme, including the amino acid substitutions). In some cases, the variant amylosucrase comprises an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 3. NO:2 An amino acid substitution at amino acid position 234 selected from the group consisting of R234Q, R234G, R234A, R234S, R234M, R234C, R234K, R234I, R234D, R234Y, R234W, R234E, R234L and R234H, or consisting thereof. In preferred embodiments, the variant amylosucrase comprises an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution at amino acid position 234 relative to SEQ ID NO: 2 selected from the group consisting of R234Q, R234G, R234A, R234S, R234M, R234C and R234K, or consisting thereof.In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution R234Q relative to SEQ ID NO: 2. In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution R234G relative to SEQ ID NO: 2. In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity, and an amino acid substitution R234A relative to SEQ ID NO: 2. In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution R234S relative to SEQ ID NO: 2.In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution R234M relative to SEQ ID NO: 2. In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution R234C relative to SEQ ID NO: 2. In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution R234K relative to SEQ ID NO: 2. In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution R234I relative to SEQ ID NO: 2.In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity, and an amino acid substitution R234D relative to SEQ ID NO: 2. In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution R234Y relative to SEQ ID NO: 2. In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution R234W relative to SEQ ID NO: 2. In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution R234E relative to SEQ ID NO: 2.In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and the amino acid substitution R234L relative to SEQ ID NO: 2. In some cases, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity and an amino acid substitution R234H relative to SEQ ID NO: 2.

[0040] In some embodiments, amylosucrase is derived from microbial cells. In some cases, amylosucrase is separated and / or purified from microbial cells. In some cases, microbial cells are bacterial cells. In some cases, bacterial cells are Escherichia coli. In some embodiments, amylosucrase is derived from polysaccharide Neisseria. In some embodiments, amylosucrase is derived from carbon source Cellulomonas T26. In some embodiments, amylosucrase can be produced in microbial cells. In some embodiments, amylosucrase is expressed in recombinant host cells (for example, from recombinant polynucleotides). In some cases, amylosucrase is recombinantly produced. In some cases, amylosucrase is produced in yeast cells (for example, recombinantly produced). In some cases, yeast cells are Pichia cells, such as Pichia pastoris cells.

[0041] A dual-enzyme method for producing amylose from sucrose

[0042] In some aspects, the method involves contacting sucrose with an enzyme mixture capable of converting sucrose into amylose under conditions that allow sucrose to be converted into amylose, thereby producing amylose. In some cases, the method involves contacting sucrose with an enzyme mixture containing at least two enzymes, which together or in combination can convert sucrose into amylose. For example, the enzyme mixture can contain at least sucrose phosphorylase and alpha-glucan phosphorylase. The method can involve contacting sucrose with the at least two enzymes simultaneously or substantially simultaneously. Alternatively, the method can involve contacting sucrose with the at least two enzymes in sequence. Figure 2B Describe the schematic diagram of the double enzyme method of producing straight-chain starch by sucrose.In this example, sucrose is contacted with sucrose phosphorylase, and sucrose is converted into glucose-1-phosphate.Then glucose-1-phosphate is contacted with alpha-glucan phosphorylase, and glucose-1-phosphate is converted into straight-chain starch.In some cases, sucrose phosphorylase and alpha-glucan phosphorylase are contacted with sucrose simultaneously or substantially simultaneously.In other cases, sucrose phosphorylase and alpha-glucan phosphorylase are added successively (for example, sucrose phosphorylase is first contacted with sucrose to generate glucose-1-phosphate, and then alpha-glucan phosphorylase is added to generate straight-chain starch).In some cases, before glucose-1-phosphate is contacted with alpha-glucan phosphorylase, glucose-1-phosphate generated by the reaction of self-utilizing sucrose phosphorylase is separated and / or purified.In other cases, before glucose-1-phosphate is contacted with alpha-glucan phosphorylase, glucose-1-phosphate generated by the reaction of self-utilizing sucrose phosphorylase is not separated and / or purified. When used in the context of adding two or more components to a reaction mixture as described herein, the term "substantially simultaneously" means that the two or more components are added to the reaction mixture within 10 seconds or less of each other.

[0043] In some cases, the sucrose phosphorylase is a wild-type sucrose phosphorylase. For example, the wild-type sucrose phosphorylase can be a Bifidobacterium longum sucrose phosphorylase (e.g., NCBI accession number AAO84039). In some cases, the wild-type Bifidobacterium longum sucrose phosphorylase can have an amino acid sequence according to SEQ ID NO: 17. In some cases, the wild-type sucrose phosphorylase can be a mesenteroides-like leuconostoc sucrose phosphorylase (e.g., NCBI accession number D90314.1). In some cases, the wild-type mesenteroides-like leuconostoc sucrose phosphorylase can have an amino acid sequence according to SEQ ID NO: 18. In some cases, the wild-type sucrose phosphorylase can be a mutans streptococcus sucrose phosphorylase (e.g., NCBI accession number NZ_CP013237.1). In some cases, the wild-type mutans streptococcus sucrose phosphorylase can have an amino acid sequence according to SEQ ID NO: 19 (e.g., NCBI accession number P10249). In some cases, the sucrose phosphorylase is a variant sucrose phosphorylase. In some cases, the variant sucrose phosphorylase has one or more amino acid substitutions relative to the wild-type sucrose phosphorylase. In some cases, the variant sucrose phosphorylase has an amino acid substitution relative to SEQ ID NO: 19 at one or more or all of amino acid residues T47, S62, Y77, V128, K140, Q144, N155 and D249. In some cases, relative to SEQ ID NO: 19, the amino acid substitution at amino acid position 47 is T47S. In some cases, relative to SEQ ID NO: 19, the amino acid substitution at amino acid position 62 is S62P. In some cases, relative to SEQ ID NO: 19, the amino acid substitution at amino acid position 77 is Y77H. In some cases, relative to SEQ ID NO: 19, the amino acid substitution at amino acid position 128 is V128L. In some cases, relative to SEQ ID NO: 19, the amino acid substitution at amino acid position 140 is K140M. In some cases, the amino acid substitution at amino acid position 144 is Q144R relative to SEQ ID NO: 19. In some cases, the amino acid substitution at amino acid position 155 is N155S relative to SEQ ID NO: 19. In some cases, the amino acid substitution at amino acid position 249 is D249G relative to SEQ ID NO: 19. In some cases, the variant sucrose phosphorylase has amino acid substitutions T47S, S62P, Y77H, V128L, K140M, Q144R, N155S and D249G relative to SEQ ID NO: 19. In some cases, the variant sucrose phosphorylase comprises or consists of an amino acid sequence according to SEQ ID NO: 20.Table 3 below depicts non-limiting examples of sucrose phosphorylases (and their amino acid sequences) that can be used according to the methods provided herein.

[0044] Table 3. Non-limiting examples of sucrose phosphorylases

[0045]

[0046]

[0047] In some cases, the sucrose phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to a wild-type Bifidobacterium longum sucrose phosphorylase. In some cases, the sucrose phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 17. In some cases, the sucrose phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to a wild-type Leuconostoc mesenteroides sucrose phosphorylase. In some cases, the sucrose phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 18. In some cases, the sucrose phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to a wild-type mutans Streptococcus sucrose phosphorylase.In some cases, the sucrose phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 19. In some cases, the sucrose phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:20, and comprises amino acid substitutions T47S, S62P, Y77H, V128L, K140M, Q144R, N155S, and D249G relative to SEQ ID NO:19.

[0048] In some embodiments, sucrose phosphorylase is derived from microbial cells. In some cases, sucrose phosphorylase is isolated and / or purified from microbial cells. In some cases, microbial cells are bacterial cells. In some cases, bacterial cells are Escherichia coli. In some embodiments, sucrose phosphorylase is derived from Bifidobacterium longum. In some embodiments, sucrose phosphorylase is derived from Leuconostoc mesenteroides. In some embodiments, sucrose phosphorylase is derived from Streptococcus mutans. In some embodiments, sucrose phosphorylase can be produced in microbial cells. In some embodiments, sucrose phosphorylase is expressed in recombinant host cells (e.g., from recombinant polynucleotides). In some cases, sucrose phosphorylase is recombinantly produced. In some cases, sucrose phosphorylase is produced in yeast cells (e.g., recombinantly produced). In some cases, yeast cells are Pichia cells, such as Pichia pastoris cells.

[0049] In some aspects, the α-glucan phosphorylase is a wild-type α-glucan phosphorylase. In some cases, the wild-type α-glucan phosphorylase can be a potato α-glucan phosphorylase (e.g., NCBI accession number D00520.1). In some cases, the wild-type potato α-glucan phosphorylase can have an amino acid sequence according to SEQ ID NO:21. In some cases, the wild-type α-glucan phosphorylase can be a Sulfolobus fusogenus strain 7 α-glucan phosphorylase (e.g., NCBI accession number NC_003106.2). In some cases, the wild-type Sulfolobus fusogenus strain 7 α-glucan phosphorylase can have an amino acid sequence according to SEQ ID NO:22. In some cases, the wild-type α-glucan phosphorylase can be Corynebacterium heatherum DSM 20145 α-glucan phosphorylase (e.g., NCBI accession number AY102616.1). In some cases, the wild-type Corynebacterium heatherum DSM 20145 α-glucan phosphorylase can have an amino acid sequence according to SEQ ID NO:23. In some cases, the α-glucan phosphorylase is a variant α-glucan phosphorylase. In some cases, the variant α-glucan phosphorylase has one or more amino acid substitutions relative to the wild-type α-glucan phosphorylase. In some cases, the variant α-glucan phosphorylase has an amino acid substitution at one or more or all of amino acid residues F39, N135 and T706 relative to SEQ ID NO:21. In some cases, relative to SEQ ID NO:21, the amino acid substitution at amino acid position 39 is F39L. In some cases, relative to SEQ ID NO:21, the amino acid substitution at amino acid position 135 is N135S. In some cases, relative to SEQ ID NO:21, the amino acid substitution at amino acid position 706 is T706I. In some cases, the variant α-glucan phosphorylase has amino acid substitutions F39L, N135S, and T706I relative to SEQ ID NO: 21. In some cases, the variant α-glucan phosphorylase has an amino acid sequence according to SEQ ID NO: 24. Table 4 below depicts non-limiting examples of α-glucan phosphorylases (and amino acid sequences thereof) that can be used according to the methods provided herein.

[0050] Table 4. Non-limiting examples of α-glucan phosphorylases

[0051]

[0052]

[0053]

[0054] In some cases, the α-glucan phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to a wild-type potato α-glucan phosphorylase. In some cases, the α-glucan phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:21. In some cases, the α-glucan phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to a wild-type Sulfolobus solanum strain 7 α-glucan phosphorylase. In some cases, the α-glucan phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:22.In some cases, the α-glucan phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to wild-type Corynebacterium heatherum DSM 20145 α-glucan phosphorylase. In some cases, the α-glucan phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:23. In some cases, the sucrose phosphorylase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) to the amino acid sequence of SEQ ID NO:21, preferably at least about 90% sequence identity, and comprises amino acid substitutions F39L, N135S, and T706I relative to SEQ ID NO:21.

[0055] In some embodiments, α-glucan phosphorylase is derived from microbial cells. In some cases, α-glucan phosphorylase is isolated and / or purified from microbial cells. In some cases, microbial cells are bacterial cells. In some cases, bacterial cells are Escherichia coli. In some embodiments, α-glucan phosphorylase is derived from potato. In some embodiments, α-glucan phosphorylase is derived from Sulfolobus solanum strain 7. In some embodiments, α-glucan phosphorylase is derived from Corynebacterium heatherum DSM 20145. In some embodiments, α-glucan phosphorylase can be produced in microbial cells. In some embodiments, α-glucan phosphorylase is expressed in recombinant host cells (e.g., from recombinant polynucleotides). In some cases, α-glucan phosphorylase is recombinantly produced. In some cases, α-glucan phosphorylase is produced in yeast cells (e.g., recombinantly produced). In some cases, yeast cells are Pichia cells, such as Pichia pastoris cells.

[0056] Method step (b): Enzymatic conversion of amylose into α-cyclodextrin

[0057] In various aspects, the method further comprises enzymatically converting amylose (e.g., produced by the methods provided herein (e.g., method step (a))) into a cyclodextrin, preferably α-cyclodextrin. In some cases, the method comprises contacting amylose with an enzyme or enzyme mixture (e.g., two or more enzymes) capable of converting amylose into cyclodextrin under conditions that allow the conversion of amylose into cyclodextrin. In some cases, the enzyme capable of converting amylose into cyclodextrin is a variant enzyme that is capable of producing a greater amount and / or concentration of α-cyclodextrin than β-cyclodextrin, γ-cyclodextrin, or both, relative to a wild-type enzyme capable of converting amylose into cyclodextrin.

[0058] In some aspects, the enzyme capable of converting amylose to cyclodextrin comprises a variant cyclodextrin glucanotransferase. In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to a wild-type cyclodextrin glucanotransferase. Figure 3 Depicted is the enzymatic conversion of amylose to alpha-cyclodextrin using a cyclodextrin glucanotransferase. Preferably, the cyclodextrin glucanotransferase produces alpha-cyclodextrin from amylose in an amount and / or concentration greater than that of beta-cyclodextrin and / or gamma-cyclodextrin.

[0059] In some embodiments, cyclodextrin glucanotransferase is a variant cyclodextrin glucanotransferase, which comprises at least one amino acid variant relative to wild-type cyclodextrin glucanotransferase. Relative to wild-type cyclodextrin glucanotransferase, variant cyclodextrin glucanotransferase may comprise one or more amino acid substitutions, deletions, insertions and / or modifications. In some cases, relative to wild-type cyclodextrin glucanotransferase, variant cyclodextrin glucanotransferase can produce more alpha-cyclodextrin (relative to beta-cyclodextrin and / or gamma-cyclodextrin) in greater amounts and / or concentrations by amylose.

[0060] In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to a wild-type Paenibacillus macerans cyclodextrin glucanotransferase (e.g., NCBI Accession No. AAA22298.1 or X59045.1; e.g., SEQ ID NOs: 25-28). In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to any one of SEQ ID NOs: 25-28. In some cases, the variant cyclodextrin glucanotransferase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of wild-type Paenibacillus macerans cyclodextrin glucanotransferase. In some cases, the variant cyclodextrin glucanotransferase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more), preferably at least about 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 25-28.

[0061] In some cases, the at least one amino acid variant comprises at least one amino acid substitution relative to the wild-type cyclodextrin glucanotransferase. In some cases, at least one amino acid substitution includes an amino acid substitution at amino acid position 146 relative to the amino acid sequence of SEQ ID NO:28. In some cases, relative to the amino acid sequence of SEQ ID NO:28, the amino acid substitution at amino acid position 146 is R146A (e.g., SEQ ID NO:29 in Table 5). In some cases, relative to the amino acid sequence of SEQ ID NO:28, the amino acid substitution at amino acid position 146 is R146P (e.g., SEQ ID NO:30 in Table 5). In some cases, at least one amino acid substitution includes an amino acid substitution at amino acid position 147 relative to the amino acid sequence of SEQ ID NO:28. In some cases, relative to the amino acid sequence of SEQ ID NO:28, the amino acid substitution at amino acid position 147 is D147A (e.g., SEQ ID NO:31 in Table 5). In some cases, relative to the amino acid sequence of SEQ ID NO:28, the amino acid substitution at amino acid position 147 is D147P (e.g., SEQ ID NO:32 in Table 5). In some cases, at least one amino acid substitution includes amino acid substitutions at amino acid positions 146 and 147 relative to the amino acid sequence of SEQ ID NO: 28. In some cases, the amino acid substitution at amino acid position 146 relative to the amino acid sequence of SEQ ID NO: 28 is R146A, and the amino acid substitution at amino acid position 147 relative to the amino acid sequence of SEQ ID NO: 28 is D147P (e.g., SEQ ID NO: 33 in Table 5). In some cases, the amino acid substitution at amino acid position 146 relative to the amino acid sequence of SEQ ID NO: 28 is R146P, and the amino acid substitution at amino acid position 147 relative to the amino acid sequence of SEQ ID NO: 28 is D147A (e.g., SEQ ID NO: 34 in Table 5). In some cases, the amino acid substitution at amino acid position 146 relative to the amino acid sequence of SEQ ID NO: 28 is R146P, and the amino acid substitution at amino acid position 147 relative to the amino acid sequence of SEQ ID NO: 28 is D147P (e.g., SEQ ID NO: 35 in Table 5).

[0062] In some cases, at least one amino acid substitution comprises an amino acid substitution at amino acid position 372 relative to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 28. In some cases, the amino acid substitution at amino acid position 372 relative to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 28 is D372K (e.g., SEQ ID NO: 36 (relative to SEQ ID NO: 26) and SEQ ID NO: 39 (relative to SEQ ID NO: 28) in Table 5). In some cases, at least one amino acid substitution comprises an amino acid substitution at amino acid position 89 relative to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 28. In some cases, the amino acid substitution at amino acid position 89 relative to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 28 is Y89R (e.g., SEQ ID NO: 37 (relative to SEQ ID NO: 26) and SEQ ID NO: 40 (relative to SEQ ID NO: 28) in Table 5). In some cases, the at least one amino acid substitution includes an amino acid substitution at amino acid position 372 relative to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 28, and an amino acid substitution at amino acid position 89 relative to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 28. In some cases, the amino acid substitution at amino acid position 372 relative to the amino acid sequence of SEQ ID NO: 26 or 28 is D372K, and the amino acid substitution at amino acid position 89 relative to the amino acid sequence of SEQ ID NO: 26 or 28 is Y89R (e.g., SEQ ID NO: 38 (relative to SEQ ID NO: 26) and SEQ ID NO: 41 (relative to SEQ ID NO: 28) in Table 5).

[0063] In some aspects, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 25-41 as depicted in Table 5. In some aspects, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence having at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) sequence identity, preferably at least about 90% sequence identity, to the amino acid sequence of any one of SEQ ID NOs: 25-41 as depicted in Table 5.

[0064] In specific aspects, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence according to SEQ ID NO: 28, or comprises or consists of an amino acid sequence having at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) sequence identity, preferably at least about 90% sequence identity, to the amino acid sequence according to SEQ ID NO: 28.

[0065] In another specific aspect, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence according to SEQ ID NO: 33, or comprises or consists of an amino acid sequence having at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) sequence identity, preferably at least about 90% sequence identity, to the amino acid sequence according to SEQ ID NO: 33.

[0066] In another specific aspect, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence according to SEQ ID NO: 34, or comprises or consists of an amino acid sequence having at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) sequence identity, preferably at least about 90% sequence identity, to the amino acid sequence according to SEQ ID NO: 34.

[0067] In another specific aspect, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence according to SEQ ID NO: 35, or comprises or consists of an amino acid sequence having at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) sequence identity, preferably at least about 90% sequence identity, to the amino acid sequence according to SEQ ID NO: 35.

[0068] In another specific aspect, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence according to SEQ ID NO: 41, or comprises or consists of an amino acid sequence having at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) sequence identity, preferably at least about 90% sequence identity, to the amino acid sequence according to SEQ ID NO: 41.

[0069] Table 5. Non-limiting examples of cyclodextrin glucanotransferases

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079] In some aspects, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 28, preferably at least about 90% sequence identity and an amino acid substitution at amino acid position 146 relative to SEQ ID NO: 28. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) to the amino acid sequence of SEQ ID NO: 28, preferably at least about 90% sequence identity, and the amino acid substitution R146A relative to SEQ ID NO: 28. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) to the amino acid sequence of SEQ ID NO: 28, preferably at least about 90% sequence identity, and the amino acid substitution R146P relative to SEQ ID NO: 28.

[0080] In some aspects, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 28, preferably at least about 90% sequence identity and an amino acid substitution at amino acid position 147 relative to SEQ ID NO: 28. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) to the amino acid sequence of SEQ ID NO: 28, preferably at least about 90% sequence identity, and amino acid substitution D147P relative to SEQ ID NO: 28. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) to the amino acid sequence of SEQ ID NO: 28, preferably at least about 90% sequence identity, and amino acid substitution D147A relative to SEQ ID NO: 28.

[0081] In some aspects, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 28, preferably at least about 90% sequence identity, an amino acid substitution at amino acid position 146 relative to SEQ ID NO: 28, and an amino acid substitution at amino acid position 147 relative to SEQ ID NO: 28. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) to the amino acid sequence of SEQ ID NO: 28, preferably at least about 90% sequence identity, amino acid substitution R146A relative to SEQ ID NO: 28, and amino acid substitution D147P relative to SEQ ID NO: 28. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) to the amino acid sequence of SEQ ID NO: 28, preferably at least about 90% sequence identity, an amino acid substitution R146P relative to SEQ ID NO: 28, and an amino acid substitution D147A relative to SEQ ID NO: 28.In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) to the amino acid sequence of SEQ ID NO: 28, preferably at least about 90% sequence identity, an amino acid substitution R146P relative to SEQ ID NO: 28, and an amino acid substitution D147P relative to SEQ ID NO: 28.

[0082] In some aspects, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 26 or 28, preferably at least about 90% sequence identity and an amino acid substitution at amino acid position 372 relative to SEQ ID NO: 26 or 28. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 26 or 28, preferably at least about 90% sequence identity and an amino acid substitution D372K relative to SEQ ID NO: 26 or 28.

[0083] In some aspects, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 26 or 28, preferably at least about 90% sequence identity and an amino acid substitution at amino acid position 89 relative to SEQ ID NO: 26 or 28. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 26 or 28, preferably at least about 90% sequence identity and an amino acid substitution Y89R relative to SEQ ID NO: 26 or 28.

[0084] In some aspects, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more) to the amino acid sequence of SEQ ID NO: 26 or 28, preferably at least about 90% sequence identity, an amino acid substitution at amino acid position 372 relative to SEQ ID NO: 26 or 28, and an amino acid substitution at amino acid position 89 relative to SEQ ID NO: 26 or 28. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) to the amino acid sequence of SEQ ID NO: 26 or 28, preferably at least about 90% sequence identity, an amino acid substitution D372K relative to SEQ ID NO: 26 or 28, and an amino acid substitution Y89R relative to SEQ ID NO: 26 or 28.

[0085] In some embodiments, cyclodextrin glucanotransferase is derived from microbial cells. In some cases, cyclodextrin glucanotransferase is separated and / or purified from microbial cells. In some cases, microbial cells are bacterial cells. In some cases, bacterial cells are Escherichia coli. In some embodiments, cyclodextrin glucanotransferase is derived from Paenibacillus macerans. In some embodiments, cyclodextrin glucanotransferase can be produced in microbial cells. In some embodiments, cyclodextrin glucanotransferase is expressed in recombinant host cells (for example, from recombinant polynucleotides). In some cases, cyclodextrin glucanotransferase is recombinantly produced. In some cases, cyclodextrin glucanotransferase is produced in yeast cells (for example, recombinantly produced). In some cases, yeast cells are pichia cells, such as pichia pastoris cells.

[0086] In various aspects, one or more additives can be added to the reaction mixture. It should be understood that the reaction mixture refers to the reaction mixture present in method step (a), or in method step (b), or in method steps (a) and (b). If more than one additive is used, the additive can be introduced simultaneously or sequentially. In some cases, relative to the same reaction without using one or more additives, the one or more additives can increase the amount and / or concentration of the α-cyclodextrin produced. In some cases, relative to the same reaction without using one or more additives, the one or more additives can increase the ratio of α-cyclodextrin to β-cyclodextrin, γ-cyclodextrin or both. In some cases, one or more additives include calcium chloride (CaCl 2 ). In some cases, the one or more additives include ethanol. In some cases, the one or more additives include CaCl 2 and ethanol. CaCl 2 It can be added at a concentration of about 1 mM to about 100 mM. In some cases, CaCl 2 is added at a concentration of about 10 mM. Ethanol may be added at a concentration of about 1% v / v to about 10% v / v. In some cases, ethanol is added at a concentration of about 2% v / v.

[0087] In various aspects, the methods provided herein produce a higher ratio of alpha-cyclodextrin to beta-cyclodextrin, gamma-cyclodextrin or both. For example, in some cases, the ratio of alpha-cyclodextrin to beta-cyclodextrin, gamma-cyclodextrin or both provided by the methods provided herein is at least 2: 1, at least 3: 1, at least 4: 1, at least 5: 1, at least 6: 1, at least 7: 1, at least 8: 1, at least 9: 1, at least 10: 1, at least 20: 1, at least 30: 1, at least 40: 1, at least 50: 1, at least 60: 1, at least 70: 1, at least 80: 1, at least 90: 1, at least 100: 1 or greater. In embodiments, the ratio of alpha-cyclodextrin to beta-cyclodextrin provided by the methods provided herein is at least 1.25: 1. For example, the ratio can be at least 1.5: 1, at least 1.75: 1, at least 2: 1, at least 2.25: 1, at least 2.5: 1, at least 2.75: 1, at least 3: 1 or greater. In embodiments, the ratio of alpha-cyclodextrin to gamma-cyclodextrin provided by the methods provided herein is at least 2: 1. For example, the ratio can be at least 3: 1, at least 5: 1, at least 10: 1, at least 20: 1, at least 30: 1, at least 40: 1, at least 50: 1, at least 75: 1, at least 100: 1 or greater. In embodiments, the ratio of alpha-cyclodextrin to both beta-cyclodextrin and gamma-cyclodextrin provided by the methods provided herein is at least 1.25: 1. For example, the ratio can be at least 1.3: 1, at least 1.4: 1, at least 1.5: 1, at least 1.6: 1, at least 1.7: 1, at least 1.8: 1, at least 1.9: 1, at least 2: 1, at least 3: 1 or greater.

[0088] This disclosure outlines throughout the text a method for obtaining robust enzyme activity in each step to obtain a higher yield of α-cyclodextrin than is currently possible. In some embodiments, a first enzymatic step of converting sucrose to amylose (e.g., as described herein) is performed for a first time period, thereby enabling catalytic conversion of sucrose to amylose, and then a second enzymatic step of converting amylose to α-cyclodextrin (e.g., as described herein) is performed for a second time period, thereby enabling catalytic conversion of amylose to α-cyclodextrin. In some embodiments, the first enzymatic reaction (e.g., converting sucrose to amylose, e.g., as described herein) and the second enzymatic reaction (e.g., converting amylose to α-cyclodextrin, e.g., as described herein) are performed in the same container (e.g., a one-pot synthesis method).

[0089] In some embodiments, the first time period is at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 85 minutes, at least 90 minutes, at least 105 minutes, at least 120 minutes, at least 135 minutes, at least 150 minutes, at least 165 minutes, at least 180 minutes, at least 195 minutes, at least 210 minutes, at least 225 minutes, at least 240 minutes, at least 255 minutes, at least 270 minutes, at least 285 minutes or at least 300 minutes. In some embodiments, the second time period is at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 85 minutes, at least 90 minutes, at least 105 minutes, at least 120 minutes, at least 135 minutes, at least 150 minutes, at least 165 minutes, at least 180 minutes, at least 195 minutes, at least 210 minutes, at least 225 minutes, at least 240 minutes, at least 255 minutes, at least 270 minutes, at least 285 minutes or at least 300 minutes. In some embodiments, the first time period is shorter than the second time period. In some embodiments, the first time period is longer than the second time period. In some embodiments, the length of the first time period is the same or substantially the same as the second time period. In some embodiments, sucrose is added to the reaction vessel in batches. In some embodiments, the enzyme used in the first enzymatic reaction step (e.g., sucrose is converted into amylose, for example, as described herein) is added once at the beginning of the reaction period, and then added again after a period of time has passed to accelerate the catalytic activity. In some embodiments, sucrose is added once at the beginning of the reaction period, and then added again after a period of time has passed to supplement sucrose. In some embodiments, the enzyme participating in the first enzymatic reaction step (e.g., sucrose is converted into amylose, for example, as described herein) and the enzyme participating in the second enzymatic reaction step (e.g., amylose is converted into alpha-cyclodextrin) are added simultaneously in the same reaction vessel. In some embodiments, the enzyme participating in the first enzymatic reaction step (e.g., sucrose is converted into amylose, for example, as described herein) and the enzyme participating in the second enzymatic reaction step (e.g., amylose is converted into alpha-cyclodextrin) are added at different times (e.g., the former before the latter).

[0090] In some embodiments, the sucrose concentration is maximized to achieve efficient conversion to amylose. In some embodiments, the initial concentration of sucrose in the reaction is at least about 50 g / L. In some embodiments, the initial concentration of sucrose in the reaction is at least about 100 g / L. In some embodiments, the initial concentration of sucrose in the reaction is at least about 150 g / L. In some embodiments, the initial concentration of sucrose in the reaction is at least about 200 g / L. In some embodiments, the initial concentration of sucrose in the reaction is at least about 250 g / L. In some embodiments, the initial concentration of sucrose in the reaction is at least about 300 g / L. In some embodiments, the initial concentration of sucrose in the reaction is at least about 350 g / L.

[0091] In some embodiments, reaction time is an important consideration for obtaining the maximum yield of alpha-cyclodextrin. In some embodiments, the production of alpha-cyclodextrin may be accompanied by the decomposition of the product into glucose, maltose and other sugars. Therefore, it is very important to obtain it without allowing alpha-cyclodextrin to decompose. In some embodiments, the total reaction (e.g., method steps (a) and method steps (b)) is carried out for no more than 12 hours. In some embodiments, the total reaction (e.g., method steps (a) and method steps (b)) is carried out for no more than 8 hours. In some embodiments, the total reaction is carried out for no more than 7 hours. In some embodiments, the total reaction is carried out for no more than 6 hours. In some embodiments, the total reaction is carried out for no more than 5 hours. In some embodiments, the total reaction is carried out for no more than 4 hours. In some embodiments, the total reaction is carried out for no more than 3 hours. In some embodiments, the total reaction is carried out for no more than 2 hours. In some embodiments, the total reaction is carried out for no more than 1 hour.

[0092] Temperature is an important consideration for maximizing the yield of α-cyclodextrin. In some embodiments, one or more enzymatic reactions are carried out at about 30°C to about 55°C, for example, about 40°C to about 50°C. In some embodiments, one or more enzymatic reactions are carried out at about 40°C. In some embodiments, one or more enzymatic reactions are carried out at about 41°C. In some embodiments, one or more enzymatic reactions are carried out at about 42°C. In some embodiments, one or more enzymatic reactions are carried out at about 43°C. In some embodiments, one or more enzymatic reactions are carried out at about 44°C. In some embodiments, one or more enzymatic reactions are carried out at about 45°C. In some embodiments, one or more enzymatic reactions are carried out at about 46°C. In some embodiments, one or more enzymatic reactions are carried out at about 47°C. In some embodiments, one or more enzymatic reactions are carried out at about 48°C. In some embodiments, one or more enzymatic reactions are carried out at about 49°C. In some embodiments, one or more enzymatic reactions are carried out at about 50°C. Preferably, one or more reactions are carried out at about 45°C.

[0093] In some embodiments, the enzymatic reaction of step (a) is carried out at about 40°C to about 55°C, for example, about 45°C to about 50°C. In some embodiments, the enzymatic reaction of step (b) is carried out at about 40°C to about 50°C. Step (a) and step (b) can be carried out at different temperatures, or preferably, step (a) and step (b) are carried out at about the same temperature. When step (a) involves the use of a single enzyme (e.g., amylosucrase), the enzymatic reaction of step (a) is preferably carried out at about 45°C. In this embodiment, the enzymatic reaction of step (b) is preferably also carried out at about 45°C. When step (a) involves the use of at least two enzymes (e.g., sucrose phosphorylase and α-glucan phosphorylase), the enzymatic reaction of step (a) is preferably carried out at about 45°C or about 50°C. In this embodiment, the enzymatic reaction of step (b) is preferably also carried out at about 45°C or about 50°C, respectively.

[0094] In a one-pot synthesis, the enzyme mixture should function maximally, although the optimal temperature for each enzyme may be slightly different.

[0095] In some embodiments, the reaction is carried out at a pH of 5.0 to 9.0, such as 6.0 to 8.0, such as 6.5 to 7.5. In some embodiments, the reaction is carried out at a pH of 6.0. In some embodiments, the reaction is carried out at a pH of 7.0. In some embodiments, the reaction is carried out at a pH of 8.0.

[0096] In some embodiments, one or more enzymatic reactions are carried out in a reaction mixture comprising a buffer. Any suitable buffer known in the art can be used. For example, the buffer can be selected from the group consisting of sodium citrate, disodium hydrogen phosphate and Tris-HCl. The concentration of the buffer in the mixture can be from about 50mM to about 200mM. In a preferred embodiment, the concentration of the buffer is about 100mM.

[0097] In some embodiments, the reaction is carried out in a vessel having a vessel volume of about 1 mL to about 1,000,000 L. For example, the reaction can be carried out in a vessel having a vessel volume of about 100 mL to about 10 L, such as a vessel volume of about 500 mL or about 10 L.

[0098] In some embodiments, the total reaction volume is from about 1 mL to about 1,000,000 L. For example, the total reaction volume can be from about 100 mL to about 10 L, for example, the total reaction volume is about 500 mL or about 5 L. In some embodiments, the total reaction volume is less than the container volume. For example, a reaction carried out in a container having a container volume of about 10 L can use a total reaction volume of about 5 L.

[0099] In some embodiments, the reaction is carried out in a stirred tank reactor (STR), a loop reactor, a plug flow reactor, a single or multi-stage continuous stirred tank reactor, or any other suitable reactor known in the art. In some embodiments, the reaction is carried out in a stirred tank reactor, wherein the reaction is stirred at about 100 to about 200 rpm, such as about 160 rpm.

[0100] In some embodiments, one or more enzymatic reactions are carried out in a reaction mixture comprising an organic solvent (preferably toluene). The reaction mixture preferably also comprises water. Without wishing to be bound by any theory described herein, the inventors have found that the addition of an organic solvent can unexpectedly increase the yield of alpha-cyclodextrin obtained by the enzymatic reaction. For example, compared with the yield obtained by the enzymatic reaction without using an organic solvent, the addition of an organic solvent can increase the yield of alpha-cyclodextrin by at least about 5%, for example, at least about 10%, for example, at least about 15%, for example, at least about 20%, for example, at least about 50%, for example, at least about 100%, for example, at least about 150%, for example, at least about 200%, for example, at least about 250%, for example, at least about 300%, for example, at least about 350%, for example, at least about 400%. It is believed that the addition of an organic solvent can reduce the solubility of alpha-cyclodextrin in the reaction mixture, thereby causing alpha-cyclodextrin precipitation, which reduces the concentration of alpha-cyclodextrin in the reaction mixture, thereby increasing the yield of alpha-cyclodextrin. This can prevent the enzyme from decomposing alpha-cyclodextrin.

[0101] In some embodiments, the amount of organic solvent (preferably toluene) in the reaction mixture is about 0.1% to about 40% v / v of the reaction mixture, such as about 1% to about 35% v / v, such as about 5% to about 25% v / v.

[0102] In some embodiments, an organic solvent is introduced at the beginning or during the enzymatic reaction of step (a). In some preferred embodiments, an organic solvent is introduced at the beginning or during the enzymatic reaction of step (b). For example, in embodiments where the total reaction (e.g., method step (a) and method step (b)) is performed for no more than 8 hours, an organic solvent may be introduced about 1 hour after the enzymatic reaction (b) begins.

[0103] In some embodiments, the enzyme used in step (a) is amylosucrase. In some embodiments, the initial concentration of amylosucrase in the reaction mixture is about 1 to about 30 U / mL, such as about 5 to about 25 U / mL, such as about 8 to about 25 U / mL.

[0104] In some embodiments, the enzyme mixture used in step (a) comprises sucrose phosphorylase and α-glucan phosphorylase. In some embodiments, the starting concentration of sucrose phosphorylase in the reaction mixture is about 1 to about 30 U / mL, such as about 5 to about 25 U / mL, such as about 8 to about 25 U / mL. In some embodiments, the starting concentration of α-glucan phosphorylase in the reaction mixture is about 1 to about 30 U / mL, such as about 5 to about 25 U / mL, such as about 8 to about 25 U / mL.

[0105] In some embodiments, the enzyme is provided in a whole cell lysate, preferably wherein the ratio of the starting concentration of the enzyme in step (b) (measured by volume of the whole cell lysate) to the enzyme in step (a) is from about 1:1 to about 50:1, such as from about 2:1 to about 50:1, such as from about 5:1 to about 40:1, such as from about 10:1 to about 30: 1. In a preferred embodiment, the ratio is about 20:1.

[0106] In certain embodiments, any enzymatic reaction provided herein (e.g., a first enzymatic reaction for converting sucrose into amylose and / or a second enzymatic reaction for converting amylose into alpha-cyclodextrin) can occur in a microbial host cell. In some cases, the microbial cell is a bacterial cell. In some cases, the bacterial cell is Escherichia coli. For example, the microbial host cell can include one or more heterologous nucleic acid molecules encoding one or more enzymes provided herein. The microbial host cell can express one or more enzymes provided herein. In some cases, sucrose and / or one or more enzymatic reaction intermediates can be supplied to the microbial host cell. For example, sucrose can be supplied to the microbial host cell, and the conversion of sucrose to alpha-cyclodextrin can occur in the microbial host cell.

[0107] In some embodiments, one or more enzymes used in the enzymatic reaction provided herein can be fixed on a resin. For example, the enzyme can be covalently attached to the resin. Alternatively, the enzyme can be non-covalently attached to the resin. For example, the enzyme can be attached to the Ni resin by a His tag. For example, the enzyme of (a) can be a variant amylosucrase (for example, wherein the variant amylosucrase can include the amino acid sequence according to SEQ ID NO:3 or be composed of it) and the enzyme can be fixed on a resin. Alternatively or additionally, the enzyme of (b) can be a variant cyclodextrin glucanotransferase and the enzyme can be fixed on a resin. Optionally, the enzyme of (a) or the enzyme mixture and the enzyme of (b) are fixed on the same resin.

[0108] The immobilized resin enzyme can be reused in the methods described herein. However, the inventors have found that the yield of α-cyclodextrin tends to decrease when the immobilized resin enzyme is reused, which may be due to the leaching of the enzyme from the resin during use, resulting in a lower enzyme conversion rate. Therefore, it is necessary to improve the stability of the enzyme on the resin to prevent the leaching of the enzyme, because this will allow the immobilized resin enzyme to be reused more frequently and / or have a higher enzymatic conversion rate, thereby improving the yield of the reaction.

[0109] The present inventors have found that the stability of the enzyme can be improved by using freeze-dried enzyme, spray-dried enzyme and / or introducing stabilizing compounds.

[0110] In some embodiments, the enzyme is provided in a cytoplasm or a whole cell lysate. For example, the cytoplasm of the recombinant cell comprising the expression enzyme can be suspended in a buffer (e.g., sodium citrate buffer), cracked and centrifuged to provide a whole cell lysate comprising the enzyme. Cell lysis methods are known in the art. For example, cells can be lysed by homogenization, chemical lysis, ultrasonic treatment, freezing / thawing, lyase, acid lysis and / or alkaline lysis. In a preferred embodiment, cells are lysed by homogenization.

[0111] In some embodiments, the cytoplasm or whole cell lysate also comprises a stabilizing compound. In some embodiments, the stabilizing compound is selected from the group consisting of the following: PEG, maltose, sorbitol, sucrose, glucose, mannitol, lactose, milk powder, starch and combinations thereof. In some embodiments, the addition amount of the stabilizing compound is about 0.1% w / v to about 10% w / v of the cytoplasm or whole cell lysate, for example, about 0.5% w / v to about 5% w / v. For example, the stabilizing compound is added with 0.5% w / v, 1.0% w / v or 5% w / v of the cytoplasm or whole cell lysate. In a preferred embodiment, the stabilizing compound is mannitol, sorbitol, sucrose or a combination thereof.

[0112] In some embodiments, the cell paste or cell lysate can be freeze-dried. For example, the cell paste or cell lysate can be freeze-dried for two days. Freeze-drying methods are known in the art.

[0113] The inventors have found that adding a stabilizing compound (as described above) to a cell paste or whole cell lysate improves the stability of the enzyme compared to a cell paste or whole cell lysate without the stabilizing compound, and that freeze-drying a cell paste or whole cell lysate (as described above) improves the stability of the enzyme compared to a cell paste or whole cell lysate that has not been freeze-dried. Prior to use in the methods described herein, the cell paste or cell lysate may be resuspended and shaken to redissolve.

[0114] In some embodiments, the methods described herein produce compositions comprising at least 2 g / L α-cyclodextrin. In some embodiments, the methods produce compositions comprising at least 3 g / L α-cyclodextrin, at least 4 g / L α-cyclodextrin, at least 5 g / L α-cyclodextrin, at least 6 g / L α-cyclodextrin, at least 7 g / L α-cyclodextrin, at least 8 g / L α-cyclodextrin, at least 9 g / L α-cyclodextrin, at least 10 g / L α-cyclodextrin, at least 12 g / L α-cyclodextrin, at least 15 g / L α-cyclodextrin, at least 20 g / L α-cyclodextrin, at least 30 g / L α-cyclodextrin, at least 40 g / L α-cyclodextrin, at least 50 g / L α-cyclodextrin or at least 60 g / L α-cyclodextrin. In a preferred embodiment, the methods produce compositions comprising at least 10 g / L α-cyclodextrin.

[0115] In some embodiments, the percent yield of α-cyclodextrin is at least about 10%, such as at least about 20%, such as at least about 30%, such as at least about 40%, or such as at least about 50%, such as at least about 60%, wherein the percent yield is calculated by dividing the total amount of α-cyclodextrin produced in the methods described herein by the maximum theoretical amount of α-cyclodextrin that can be produced from the starting sucrose reagent.

[0116] Also provided herein is a composition comprising cyclodextrin, wherein the cyclodextrin comprises α-cyclodextrin and may optionally further comprise β-cyclodextrin, γ-cyclodextrin or any combination thereof, and the amount and / or concentration of the α-cyclodextrin contained in the composition comprising cyclodextrin is greater than β-cyclodextrin, γ-cyclodextrin or both. Preferably, the composition is obtained by the method provided herein. Preferably, the composition does not comprise β-cyclodextrin and / or γ-cyclodextrin. Preferably, the ratio of α-cyclodextrin to β-cyclodextrin, γ-cyclodextrin or both in the composition is at least 2: 1, at least 3: 1, at least 4: 1, at least 5: 1, at least 6: 1, at least 7: 1, at least 8: 1, at least 9: 1, at least 10: 1, at least 20: 1, at least 30: 1, at least 40: 1, at least 50: 1, at least 60: 1, at least 70: 1, at least 80: 1, at least 90: 1, at least 100: 1 or greater.

[0117] In a preferred embodiment, the present invention provides a method for producing a composition comprising cyclodextrins, the method comprising: (a) contacting sucrose with an enzyme or enzyme mixture capable of converting sucrose into amylose under conditions that allow sucrose to be converted into amylose, thereby producing amylose; (b) contacting the amylose produced in (a) with a cyclodextrin glucanotransferase, thereby producing a composition comprising cyclodextrins, wherein the cyclodextrin glucanotransferase in (b) is a wild-type enzyme capable of converting amylose into cyclodextrins that is capable of producing more β-cyclodextrin, γ-cyclodextrin or both than β-cyclodextrin, γ-cyclodextrin or both. The invention relates to a method for preparing a cyclodextrin-containing composition comprising an α-cyclodextrin variant enzyme in an amount and / or concentration of α-cyclodextrin, wherein the cyclodextrin-containing composition comprises α-cyclodextrin and may optionally further comprise β-cyclodextrin, γ-cyclodextrin or any combination thereof, wherein the ratio of α-cyclodextrin to β-cyclodextrin, γ-cyclodextrin or both in the composition is at least 2:1, wherein steps (a) and (b) are performed simultaneously, wherein steps (a) and (b) are performed at about 45° C. to about 55° C., wherein steps (a) and (b) are performed at a pH of about 7.0 to about 7.5, wherein steps (a) and (b) are performed in a solution comprising water, ethanol, CaCl 2 and an optionally present organic solvent (preferably toluene) in a reaction mixture, and wherein the total reaction is carried out for no more than 8 hours.

[0118] Also provided herein is α-cyclodextrin. Preferably, α-cyclodextrin is obtained by the method provided herein.

[0119] Also provided herein is the use of sucrose as a starting material to make α-cyclodextrin. Also provided herein is the use of sucrose in a method of producing α-cyclodextrin, wherein the method does not use starch.

[0120] Also provided herein is the use of any one of the enzymes or enzyme mixtures described herein capable of converting sucrose into amylose for converting sucrose into amylose.

[0121] Also provided herein is the use of any of the variant enzymes described herein capable of converting amylose to cyclodextrin for converting amylose to cyclodextrin and / or for producing a greater amount and / or concentration of α-cyclodextrin than β-cyclodextrin, γ-cyclodextrin, or both.

[0122] Also provided herein is the use of any one of the enzymes or enzyme mixtures described herein for the production of alpha-cyclodextrin, wherein the production does not require starch as a starting material.

[0123] Also provided herein is any enzyme or enzyme mixture described herein. For example, provided herein is an enzyme comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 1-42. Also provided herein is an enzyme comprising or consisting of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity, to an amino acid sequence of any one of SEQ ID NOs: 1-42.

[0124] Preferably, the enzyme is a variant amylosucrase comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 3-16 or 42. Also provided herein is an enzyme comprising or consisting of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 3-16 or 42.

[0125] Preferably, the enzyme is a variant sucrose phosphorylase comprising or consisting of the amino acid sequence of SEQ ID NO: 20. Also provided herein is an enzyme comprising or consisting of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 20.

[0126] Preferably, the enzyme is a variant α-glucan phosphorylase comprising or consisting of the amino acid sequence of SEQ ID NO: 24. Also provided herein is an enzyme comprising or consisting of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 24.

[0127] Preferably, the enzyme is a variant cyclodextrin glucanotransferase comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 29-41. Also provided herein is an enzyme comprising or consisting of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity, to an amino acid sequence of any one of SEQ ID NOs: 29-41.

[0128] Also provided herein are enzyme compositions comprising one or more enzymes described herein.

[0129] Also provided herein are genes encoding any of the variant enzymes described herein.

[0130] Also provided herein are vectors encoding any of the variant enzymes described herein.

[0131] Also provided herein are recombinant host cells comprising any one of the genes, vectors or enzymes described herein.

[0132] Also provided herein is the use of an organic solvent, preferably toluene, for increasing the yield of α-cyclodextrin obtained in a process for producing α-cyclodextrin, such as α-cyclodextrin obtained by any of the processes described herein.

[0133] This article also provides CaCl 2 and / or ethanol for increasing the yield of α-cyclodextrin relative to β-cyclodextrin and γ-cyclodextrin.

[0134] This article also provides CaCl 2 and / or the use of ethanol for increasing the yield of α-cyclodextrin obtained in a process for producing α-cyclodextrin, such as α-cyclodextrin obtained by any of the processes described herein.

[0135] Generally, the term "sequence identity" refers to the exact correspondence of nucleotide-nucleotide or amino acid-amino acid of two polynucleotides or polypeptide sequences respectively. Generally, the technology used to determine sequence identity includes determining the nucleotide sequence of the polynucleotide and / or determining the amino acid sequence encoded by it, and comparing these sequences with a second nucleotide or amino acid sequence. It can be compared by determining the percent consistency of two or more sequences (polynucleotides or amino acids). The percent consistency of two sequences (whether nucleic acid sequences or amino acid sequences) is the number of exact matches between the two aligned sequences divided by the length of the longer sequence, multiplied by 100. The percent consistency can also be determined, for example, by using the advanced BLAST computer program (including version 2.2.9) available from the National Institutes of Health to compare sequence information. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87: 2264-2268 (1990), and as discussed in Altschul et al., J. Mol. Biol., 215: 403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993); and Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997). The program can be used to determine the percent identity over the entire length of the compared proteins. Default parameters are provided to optimize searches using short query sequences, such as using the blastp program. The program also allows the use of SEG filters to mask fragments of the query sequence, as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). The desired degree of sequence identity ranges from about 70% to 100% and integer values ​​therebetween. In general, the present disclosure encompasses sequences having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% sequence identity to any of the sequences provided herein.

[0136] As used herein, the term "about" generally refers to a range of 15% greater or less than the stated numerical value in the context of a particular use. For example, "about 10" would include a range of 8.5 to 11.5.

[0137] As used herein, the term "or" is not exclusively used to encompass "or" and "and." For example, unless otherwise stated, "A or B" includes "A but not B," "B but not A," and "A and B."

[0138] As used herein, "a," "an," and "the" may include plural referents unless expressly and unambiguously limited to one referent.

[0139] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0140] Numbering Implementation Plan

[0141] The following embodiments describe non-limiting arrangements of the combinations of features disclosed herein. Other arrangements of the combinations of features are also contemplated. In particular, each of these numbered embodiments is considered to be dependent on or related to each previous or subsequent numbered embodiment, regardless of their listing order.

[0142] Embodiment 1: A method for producing a composition containing cyclodextrin, the method comprising: (a) contacting sucrose with an enzyme or enzyme mixture capable of converting sucrose into amylose under conditions that allow sucrose to be converted into amylose, thereby producing amylose; (b) contacting the amylose produced in (a) with an enzyme capable of converting amylose into cyclodextrin under conditions that allow amylose to be converted into cyclodextrin, thereby producing a composition containing cyclodextrin, wherein the enzyme capable of converting amylose into cyclodextrin in (b) is a variant enzyme, and relative to the wild-type enzyme capable of converting amylose into cyclodextrin, the variant enzyme is capable of producing a greater amount and / or concentration of α-cyclodextrin than β-cyclodextrin, γ-cyclodextrin or both, wherein the composition containing cyclodextrin contains α-cyclodextrin, and may optionally also contain β-cyclodextrin, γ-cyclodextrin or any combination thereof, and the amount and / or concentration of α-cyclodextrin contained in the composition containing cyclodextrin is greater than that of β-cyclodextrin, γ-cyclodextrin or both.

[0143] Embodiment 2: The method of embodiment 1, wherein the enzyme of (a) is amylosucrase, or the enzyme mixture of (a) comprises amylosucrase.

[0144] Embodiment 3: The method of embodiment 2, wherein the amylosucrase is a variant amylosucrase comprising at least one amino acid variant relative to the wild-type amylosucrase.

[0145] Embodiment 4: The method of Embodiment 3, wherein the variant amylosucrase is capable of producing a greater amount and / or concentration of amylose from sucrose relative to the wild-type amylosucrase.

[0146] Embodiment 5: The method of embodiment 3 or 4, wherein the wild-type amylosucrase is the Carbonysone Cellulomonas T26 amylosucrase.

[0147] Embodiment 6: The method of embodiment 5, wherein the wild-type amylosucrase comprises or consists of the amino acid sequence of SEQ ID NO: 1.

[0148] Embodiment 7: The method of embodiment 3 or 4, wherein the wild-type amylosucrase is a Neisseria polysaccharidase amylosucrase.

[0149] Embodiment 8: The method of Embodiment 7, wherein the wild-type amylosucrase comprises or consists of the amino acid sequence of SEQ ID NO: 2.

[0150] Embodiment 9: The method of any one of embodiments 3-8, wherein the variant amylosucrase comprises or consists of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0151] Embodiment 10: The method of any one of Embodiments 3-9, wherein the at least one amino acid variant comprises at least one amino acid substitution relative to the wild-type amylosucrase.

[0152] Embodiment 11: The method of Embodiment 10, wherein the at least one amino acid substitution comprises an amino acid substitution at amino acid position 234 relative to the wild-type amylosucrase having the amino acid sequence of SEQ ID NO:2.

[0153] Embodiment 12: The method of embodiment 11, wherein the amino acid substitution at position 234 is selected from the group consisting of: R234Q, R234G, R234A, R234S, R234M, R234C, R234K, R234I, R234D, R234Y, R234W, R234E, R234L and R234H.

[0154] Embodiment 13: The method of Embodiment 1, wherein the enzyme mixture of (a) comprises at least two enzymes that together or in combination are capable of converting sucrose to amylose.

[0155] Embodiment 14: The method of Embodiment 13, wherein the enzyme mixture comprises sucrose phosphorylase.

[0156] Embodiment 15: The method of Embodiment 14, wherein the sucrose phosphorylase is capable of converting sucrose into glucose-1-phosphate.

[0157] Embodiment 16: The method of Embodiment 15, wherein the contacting of (a) further comprises contacting the sucrose with a sucrose phosphorylase under conditions that allow conversion of the sucrose to glucose-1-phosphate.

[0158] Embodiment 17: The method of any one of Embodiments 14-16, wherein the sucrose phosphorylase is selected from the group consisting of Bifidobacterium longum sucrose phosphorylase, Leuconostoc mesenteroides sucrose phosphorylase, and Streptococcus mutans sucrose phosphorylase.

[0159] Embodiment 18: The method of any one of embodiments 14-17, wherein the sucrose phosphorylase comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 17-20 or an amino acid sequence having at least about 70% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 17-20.

[0160] Embodiment 19: The method of any one of Embodiments 13-18, wherein the enzyme mixture comprises α-glucan phosphorylase.

[0161] Embodiment 20: The method of Embodiment 19, wherein the α-glucan phosphorylase is capable of converting glucose-1-phosphate into amylose.

[0162] Embodiment 21: The method of Embodiment 20, wherein the contacting of (a) further comprises contacting glucose-1-phosphate with α-glucan phosphorylase under conditions that allow conversion of glucose-1-phosphate to amylose.

[0163] Embodiment 22: The method of any one of Embodiments 19-21, wherein the α-glucan phosphorylase is selected from the group consisting of potato α-glucan phosphorylase, Sulfolobus solanum strain 7 α-glucan phosphorylase and Corynebacterium heatherum DSM 20145 α-glucan phosphorylase.

[0164] Embodiment 23: The method of any one of embodiments 19-22, wherein the α-glucan phosphorylase comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 21-24 or an amino acid sequence having at least about 70% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 21-24.

[0165] Embodiment 24: The method of any one of Embodiments 1-23, wherein the enzyme capable of converting amylose to cyclodextrin in (b) comprises a variant cyclodextrin glucanotransferase.

[0166] Embodiment 25: The method of Embodiment 24, wherein the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to the wild-type cyclodextrin glucanotransferase.

[0167] Embodiment 26: The method of Embodiment 25, wherein the wild-type cyclodextrin glucanotransferase is Paenibacillus macerans cyclodextrin glucanotransferase.

[0168] Embodiment 27: The method of Embodiment 26, wherein the wild-type cyclodextrin glucanotransferase comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 25-28.

[0169] Embodiment 28: The method of any one of Embodiments 24-27, wherein the variant cyclodextrin glucanotransferase comprises or consists of an amino acid sequence having at least about 70% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 25-28.

[0170] Embodiment 29: The method of any one of Embodiments 25-28, wherein the at least one amino acid variant comprises at least one amino acid substitution relative to a wild-type cyclodextrin glucanotransferase.

[0171] Embodiment 30: The method of Embodiment 29, wherein the at least one amino acid substitution comprises an amino acid substitution at amino acid position 146 relative to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO:28.

[0172] Embodiment 31: The method of Embodiment 30, wherein the amino acid substitution at position 146 is selected from the group consisting of: R146A and R146P.

[0173] Embodiment 32: The method of Embodiment 29, wherein the at least one amino acid substitution comprises an amino acid substitution at amino acid position 147 relative to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO:28.

[0174] Embodiment 33: The method of Embodiment 32, wherein the amino acid substitution at position 147 is selected from the group consisting of: D147P and D147A.

[0175] Embodiment 34: The method of embodiment 29, wherein the at least one amino acid substitution includes an amino acid substitution at amino acid position 146 relative to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO:28; and an amino acid substitution at amino acid position 147 relative to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO:28.

[0176] Embodiment 35: The method of Embodiment 34, wherein the amino acid substitution at position 146 is selected from the group consisting of: R146A and R146P.

[0177] Embodiment 36: The method of Embodiment 34 or 35, wherein the amino acid substitution at position 147 is selected from the group consisting of: D147P and D147A.

[0178] Embodiment 37: The method of Embodiment 29, wherein the at least one amino acid substitution comprises an amino acid substitution at amino acid position 372 relative to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 26 or 28.

[0179] Embodiment 38: The method of Embodiment 37, wherein the amino acid substitution at position 372 is D372K.

[0180] Embodiment 39: The method of embodiment 29, wherein the at least one amino acid substitution comprises an amino acid substitution at amino acid position 89 relative to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 26 or 28.

[0181] Embodiment 40: The method of Embodiment 39, wherein the amino acid substitution at position 89 is Y89R.

[0182] Embodiment 41: The method of embodiment 29, wherein the at least one amino acid substitution includes an amino acid substitution at position 372 relative to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 26 or 28; and an amino acid substitution at position 89 relative to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 26 or 28.

[0183] Embodiment 42: The method of Embodiment 41, wherein the amino acid substitution at position 372 is D372K.

[0184] Embodiment 43: The method of Embodiment 41 or 42, wherein the amino acid substitution at position 89 is Y89R.

[0185] Embodiment 44: The method of any one of Embodiments 1-43, wherein the contacting of (a), the contacting of (b), or both, further comprises adding at least one additive during (a), (b), or both, wherein the additive increases the yield of α-cyclodextrin relative to β-cyclodextrin, γ-cyclodextrin, or both.

[0186] Embodiment 45: The method of Embodiment 44, wherein at least one additive is CaCl 2 .

[0187] Embodiment 46: The method of Embodiment 45, wherein CaCl 2 Add at a concentration of about 1 mM to about 100 mM.

[0188] Embodiment 47: The method of any of Embodiments 44-46, wherein the at least one additive is ethanol.

[0189] Embodiment 48: The method of Embodiment 47, wherein the ethanol is added at a concentration of about 1% v / v to about 10% v / v.

[0190] Embodiment 49: The method of any of Embodiments 1-48, wherein the contacting of (a) and the contacting of (b) occur sequentially.

[0191] Embodiment 50: The method of any of Embodiments 1-48, wherein the contacting of (a) and the contacting of (b) occur simultaneously or substantially simultaneously.

[0192] Embodiment 51: The method of any one of Embodiments 1-50, wherein the amylose produced in (a) is not purified or isolated prior to the contacting of (b).

[0193] Embodiment 52: The method of any one of Embodiments 1-51, wherein the contacting of (a), the contacting of (b), or both, is performed in vitro.

[0194] Embodiment 53: The method of Embodiment 52, wherein the contacting of (a), the contacting of (b), or both, is performed in a container, vial, jar, test tube, well, plate, or package.

[0195] Embodiment 54: The method of Embodiment 52 or 53, wherein the enzyme or at least one enzyme in the enzyme mixture of (a), the variant enzyme of (b), or both are purified enzymes, isolated enzymes, or both.

[0196] Embodiment 55: The method of any one of Embodiments 52-54, wherein the enzyme or at least one enzyme in the enzyme mixture of (a), the variant enzyme of (b), or both are recombinantly produced enzymes.

[0197] Embodiment 56: The method of any one of Embodiments 1-51, wherein the contacting of (a), the contacting of (b), or both, is performed in vivo.

[0198] Embodiment 57: The method of Embodiment 56, wherein the contacting of (a), the contacting of (b), or both, is performed in a recombinant host cell.

[0199] Embodiment 58: The method of Embodiment 57, wherein the recombinant host cell comprises a heterologous nucleic acid encoding the enzyme or at least one enzyme of the enzyme mixture of (a), the variant enzyme of (b), or both.

[0200] Embodiment 59: The method of Embodiment 57 or 58, wherein the recombinant host cell is a microbial cell.

[0201] Embodiment 60: The method of Embodiment 59, wherein the microbial cells are bacterial cells.

[0202] Embodiment 61: The method of any one of Embodiments 1-60, wherein the ratio of α-cyclodextrin to β-cyclodextrin in the composition comprising cyclodextrin is at least 2:1.

[0203] Embodiment 62: The method of any one of Embodiments 1-61, wherein the ratio of α-cyclodextrin to γ-cyclodextrin in the composition comprising cyclodextrin is at least 2:1.

[0204] Example

[0205] Example 1. Variant cyclodextrin glucanotransferases are capable of increasing the production of α-cyclodextrin relative to β-cyclodextrin and γ-cyclodextrin.

[0206] This example demonstrates that cyclodextrin glucanotransferases are able to increase the production of α-cyclodextrin from amylose relative to β-cyclodextrin, γ-cyclodextrin, or both. In this example, several different cyclodextrin glucanotransferases ("PMcgt2", having an amino acid sequence according to SEQ ID NO: 28; "PMcgt2[AP]", having an amino acid sequence according to SEQ ID NO: 33; "PMcgt2[PA]", having an amino acid sequence according to SEQ ID NO: 34; "PMcgt2[PP]", having an amino acid sequence according to SEQ ID NO: 35; and "PMcgt2[KR]", having an amino acid sequence according to SEQ ID NO: 41) were expressed in E. coli and then separated from a mixture of insoluble cell debris by centrifugation. The cyclodextrin glucanotransferases were exposed to soluble starch (30 g / L) at 45° C. for 1 hour in 100 mM sodium citrate buffer (pH 6.0). The amounts of α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin were measured by HPLC. Figure 4 As demonstrated in Table 6 below, only certain mutant cyclodextrin glucanotransferases (PMcgt2, PMcgt2[AP], PMcgt2[PA], PMcgt2[PP], and PMcgt2[KR]) are able to produce a higher ratio of α-cyclodextrin to γ-cyclodextrin. Figure 4 As further demonstrated in Table 6 below, some of the tested cyclodextrin glucanotransferases (PMcgt2[PA] and PMcgt2[PP]) were also able to produce a higher ratio of α-cyclodextrin to β-cyclodextrin, with PMcgt2[PP] performing best.

[0207] Table 6. Summary of the ratios of α-cyclodextrin to β-cyclodextrin and γ-cyclodextrin produced using various cyclodextrin glucanotransferases.

[0208]

[0209] Example 2. One-pot synthesis of α-cyclodextrin from sucrose

[0210] In this example, α-cyclodextrin is produced from sucrose using a dual enzyme system (i.e., method step (a) is a single enzyme method (e.g., as described herein), and method step (b) is a single enzyme method (e.g., as described herein)). Amylosucrase R234Q (having an amino acid sequence according to SEQ ID NO: 3) and cyclodextrin glucanotransferase (having an amino acid sequence according to SEQ ID NO: 35) are expressed in E. coli and then separated from a mixture of cell debris. 200 μL of amylosucrase (SEQ ID NO: 3) and different amounts of cyclodextrin glucanotransferase (SEQ ID NO: 35; 30 μL, 50 μL, and 100 μL) are then exposed to 250 g / L sucrose at different pHs (pH 6.0, pH 7.0, and pH 8.0) at 50° C. in 0.1 M sodium citrate buffer. The α-cyclodextrin levels at different time points (1 hour, 2 hours, and 3 hours) are measured by HPLC. Figure 5 It was demonstrated that a one-pot synthesis reaction can produce α-cyclodextrin from sucrose under various pH conditions.

[0211] Example 3. Various additives can enhance the production of α-cyclodextrin in a one-pot synthesis reaction

[0212] In this example, α-cyclodextrin is produced from sucrose using a dual enzyme system (i.e., method step (a) is a single enzyme method (e.g., as described herein), and method step (b) is a single enzyme method (e.g., as described herein)). Amylosucrase R234Q (having an amino acid sequence according to SEQ ID NO: 3) and cyclodextrin glucanotransferase (having an amino acid sequence according to SEQ ID NO: 35) are expressed in E. coli and then separated from a mixture of cell debris. 1 mL of amylosucrase (SEQ ID NO: 3) and 1 mL of cyclodextrin glucanotransferase (SEQ ID NO: 35) are then exposed to 300 g / L sucrose in 0.1 M sodium citrate buffer at 45° C. at pH 6.5. Various additives (10 mM CaCl 2 , 2% ethanol (v / v), or 10 mM CaCl 2 and 2% ethanol (v / v). The α-cyclodextrin levels at different time points (2 hours, 3 hours, 4 hours and 5 hours) were measured by HPLC.

[0213] Figure 6 Prove that adding ethanol or CaCl 2 The combination of ethanol and sucrose can increase the production of α-cyclodextrin from sucrose in a one-pot synthesis reaction. CK refers to the reaction without adding CaCl 2In this reaction, the enzyme converts sucrose into products under the basic conditions of 0.1 M citric acid (pH 6.5).

[0214] Example 4. Freeze drying of amylosucrase cell slurry and cell lysate containing stabilizing compounds.

[0215] Lysates and whole cell pastes of amylosucrase (having an amino acid sequence according to SEQ ID NO: 3) containing different stabilizing compounds were freeze dried. In more detail, 0.5%, 1.0% or 5.0% w / v of PEG, maltose, sorbitol, sucrose, glucose, mannitol, lactose, milk powder, starch or β-cyclodextrin were added to 1 mL of lysate or cell paste. The mixture was then freeze dried for two days.

[0216] The resulting material was resuspended in 1 mL of water and shaken at 1200 rpm for 30 minutes at room temperature (about 25° C.) to allow dissolution. The retained enzyme activity of the resulting solution was measured as described below.

[0217] Amylosucrase enzyme activity

[0218] 33 μL of amylosucrase cell-free lysate (or whole cell paste) was added to 2.67 mL of a solution of sucrose (100 g / L) in sodium citrate buffer (0.1 M, pH 7, 40°C). The solution was shaken at 1200 rpm for 1 hour. The starch concentration was quantified by spectrophotometric analysis. One unit of activity (U / mL) was defined as the amount of enzyme required to produce 1 g / L of amylose per minute at pH 7 and 40°C. The activity was compared with that of non-lyophilized cell lysate (or whole cell paste, respectively), and the results were as follows: Fig. 7A and Figure 7B shown.

[0219] As shown in Figure 7, the addition of stabilizing compounds to amylosucrase prior to freeze drying increased the stability of the enzyme. In particular, the addition of 5% sucrose, 0.5% mannitol, and 0.5% sorbitol was shown to increase the retained enzyme activity of amylosucrase cell lysate and whole cell paste.

[0220] The examples are only used to illustrate various embodiments of the present invention and are not intended to limit the present invention in any way. The examples of the present invention and the methods described herein currently represent preferred embodiments, are exemplary, and are not intended to limit the scope of the present invention. Those skilled in the art will appreciate that changes therein and other uses within the spirit of the present invention defined by the scope of the claims will be contemplated.

[0221] Although preferred embodiments of the present invention have been shown and described herein, such embodiments are provided by way of example only, which will be apparent to those skilled in the art. Various modifications, variations and substitutions will now be made by those skilled in the art without departing from the present invention. It should be understood that various alternatives to the inventive embodiments described herein may be used to implement the present invention. This means that the appended claims define the scope of the present invention and methods and structures within the scope of these claims and their equivalents are therefore encompassed.

Claims

1. A method for producing a composition comprising cyclodextrin, the method comprising: (a) contacting sucrose with an enzyme or enzyme mixture capable of converting sucrose to amylose under conditions allowing the conversion of the sucrose to amylose, thereby producing amylose; (b) contacting the amylose produced in (a) with an enzyme capable of converting amylose into cyclodextrin under conditions that allow the amylose to be converted into cyclodextrin, thereby producing the composition comprising cyclodextrin, wherein the enzyme capable of converting amylose into cyclodextrin in (b) is a variant enzyme, which is capable of producing a greater amount and / or concentration of α-cyclodextrin than β-cyclodextrin, γ-cyclodextrin or both relative to a wild-type enzyme capable of converting amylose into cyclodextrin, wherein the cyclodextrin-containing composition comprises α-cyclodextrin, and may optionally further comprise β-cyclodextrin, γ-cyclodextrin or any combination thereof, and The cyclodextrin-containing composition comprises α-cyclodextrin in an amount and / or concentration greater than β-cyclodextrin, γ-cyclodextrin, or both.

2. The method of claim 1, wherein the enzyme of (a) is amylosucrase, or the enzyme mixture of (a) comprises amylosucrase.

3. The method of claim 2, wherein the amylosucrase is a variant amylosucrase comprising at least one amino acid variant relative to a wild-type amylosucrase, e.g., wherein the variant amylosucrase is capable of producing a greater amount and / or concentration of amylose from sucrose relative to the wild-type amylosucrase.

4. The method of claim 3, wherein the wild-type amylosucrase is: (i) carbon source Cellulomonas T26 amylosucrase, for example, wherein the wild-type amylosucrase comprises or consists of the amino acid sequence of SEQ ID NO: 1; or (ii) a polysaccharide Neisseria amylosucrase, for example, wherein the wild-type amylosucrase comprises or consists of the amino acid sequence of SEQ ID NO:

2.

5. The method of any one of claims 3-4, wherein the variant amylosucrase comprises or consists of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

6. The method of any one of claims 3 to 5, wherein the at least one amino acid variant comprises at least one amino acid substitution relative to wild-type amylosucrase, Preferably, wherein the at least one amino acid substitution comprises an amino acid substitution at amino acid position 234 relative to the wild-type amylosucrase having the amino acid sequence of SEQ ID NO: 2, Preferably, wherein the amino acid substitution at position 234 is selected from the group consisting of R234Q, R234G, R234A, R234S, R234M, R234C, R234K, R234I, R234D, R234Y, R234W, R234E, R234L and R234H, and Preferably, the amino acid substitution at position 234 is R234Q.

7. The method of claim 1, wherein the enzyme mixture of (a) comprises at least two enzymes that together or in combination are capable of converting sucrose to amylose.

8. The method of claim 7, wherein the enzyme mixture comprises sucrose phosphorylase, Preferably, the sucrose phosphorylase is capable of converting sucrose into glucose-1-phosphate, and Preferably, wherein the contacting of (a) further comprises contacting the sucrose with the sucrose phosphorylase under conditions that allow conversion of the sucrose to glucose-1-phosphate.

9. The method of claim 8, wherein the sucrose phosphorylase is selected from the group consisting of Bifidobacterium longum sucrose phosphorylase, Leuconostoc mesenteroides sucrose phosphorylase, and Streptococcus mutans sucrose phosphorylase.

10. The method of any one of claims 8-9, wherein the sucrose phosphorylase comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 17-20, or an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity, to the amino acid sequence of any one of SEQ ID NOs: 17-20.

11. The method of any one of claims 7 to 10, wherein the enzyme mixture comprises α-glucan phosphorylase, Preferably, wherein the α-glucan phosphorylase is capable of converting the glucose-1-phosphate into amylose, and Preferably, wherein the contacting of (a) further comprises contacting the glucose-1-phosphate with the α-glucan phosphorylase under conditions that allow the glucose-1-phosphate to be converted into amylose.

12. The method of claim 11, wherein the α-glucan phosphorylase is selected from the group consisting of potato α-glucan phosphorylase, Sulfolobus solanum strain 7 α-glucan phosphorylase, and Corynebacterium heatherum DSM 20145 α-glucan phosphorylase.

13. The method of any one of claims 11-12, wherein the α-glucan phosphorylase comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 21-24, or an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity, to the amino acid sequence of any one of SEQ ID NOs: 21-24.

14. The method of any one of claims 1-13, wherein the enzyme capable of converting amylose to cyclodextrin in (b) comprises a variant cyclodextrin glucanotransferase.

15. The method of claim 14, wherein the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to a wild-type cyclodextrin glucanotransferase.

16. The method of claim 15, wherein the wild-type cyclodextrin glucanotransferase is Paenibacillus macerans cyclodextrin glucanotransferase, preferably wherein the wild-type cyclodextrin glucanotransferase comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 25-28.

17. The method of any one of claims 14-16, wherein the variant cyclodextrin glucanotransferase comprises or consists of an amino acid sequence having at least about 70% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 25-28.

18. The method of any one of claims 14 to 17, wherein the at least one amino acid variant comprises at least one amino acid substitution relative to a wild-type cyclodextrin glucanotransferase, preferably wherein the at least one amino acid substitution comprises: (i) an amino acid substitution at amino acid position 146 relative to a wild-type cyclodextrin glucanotransferase having an amino acid sequence of SEQ ID NO: 28, Preferably, wherein the amino acid substitution at position 146 is selected from the group consisting of: R146A and R146P; or (ii) an amino acid substitution at amino acid position 147 relative to a wild-type cyclodextrin glucanotransferase having an amino acid sequence of SEQ ID NO: 28, Preferably, wherein the amino acid substitution at position 147 is selected from the group consisting of: D147P and D147A; or (iii) an amino acid substitution at amino acid position 146 relative to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 28; and an amino acid substitution at amino acid position 147 relative to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 28, Preferably, wherein the amino acid substitution at position 146 is selected from the group consisting of: R146A and R146P, and / or preferably, wherein the amino acid substitution at position 147 is selected from the group consisting of: D147P and D147A; or (iv) an amino acid substitution at amino acid position 372 relative to a wild-type cyclodextrin glucanotransferase having an amino acid sequence of SEQ ID NO: 26 or 28, Preferably, wherein the amino acid substitution at position 372 is D372K; or (v) an amino acid substitution at amino acid position 89 relative to a wild-type cyclodextrin glucanotransferase having an amino acid sequence of SEQ ID NO: 26 or 28, Preferably, wherein the amino acid substitution at position 89 is Y89R; or (vi) an amino acid substitution at position 372 relative to a wild-type cyclodextrin glucanotransferase having an amino acid sequence of SEQ ID NO: 26 or 28; and an amino acid substitution at position 89 relative to a wild-type cyclodextrin glucanotransferase having an amino acid sequence of SEQ ID NO: 26 or 28, Preferably, wherein the amino acid substitution at position 372 is D372K, And / or preferably, wherein the amino acid substitution at position 89 is Y89R.

19. The method of any one of claims 1-18, wherein the contacting of (a), the contacting of (b), or both further comprises adding at least one additive during (a), (b), or both, wherein the additive increases the yield of α-cyclodextrin relative to β-cyclodextrin, γ-cyclodextrin, or both.

20. The method of claim 19, wherein: (i) the at least one additive is CaCl2, Preferably, wherein the CaCl2 is added at a concentration of about 1 mM to about 100 mM; and / or (ii) the at least one additive is ethanol, Preferably, wherein the ethanol is added at a concentration of about 1% v / v to about 10% v / v.

21. The method of any one of claims 1-20, wherein the contacting of (a) and the contacting of (b) occur sequentially, or wherein the contacting of (a) and the contacting of (b) occur simultaneously or substantially simultaneously.

22. The method of any one of claims 1-21, wherein the amylose produced in (a) is not purified or isolated prior to the contacting of (b).

23. The method of any one of claims 1-22, wherein the contacting of (a), the contacting of (b), or both, is performed in vitro.

24. The method of claim 23, wherein the contacting of (a), the contacting of (b), or both, is performed in a container, vial, jar, test tube, well, plate, or package.

25. The method of claim 23 or 24, wherein the enzyme of (a) or at least one of the enzymes in the enzyme mixture, the variant enzyme of (b), or both are purified enzymes, isolated enzymes, or both, Preferably, the enzyme or at least one enzyme in the enzyme mixture of (a), the variant enzyme of (b), or both are recombinantly produced enzymes.

26. The method of any one of claims 1-22, wherein the contacting of (a), the contacting of (b), or both, is performed in vivo.

27. The method of claim 26, wherein the contacting of (a), the contacting of (b), or both, is performed in a recombinant host cell, Preferably, wherein the recombinant host cell comprises a heterologous nucleic acid encoding the enzyme or at least one enzyme in the enzyme mixture of (a), the variant enzyme of (b), or both, Preferably, wherein the recombinant host cell is a microbial cell, Preferably, the microbial cells are bacterial cells.

28. The method of any one of claims 1-27, wherein the ratio of α-cyclodextrin to β-cyclodextrin in the cyclodextrin-containing composition is at least 2:

1.

29. The method of any one of claims 1-28, wherein the ratio of α-cyclodextrin to γ-cyclodextrin in the cyclodextrin-containing composition is at least 2:1, preferably at least 100:

1.

30. The method of any one of claims 1-29, wherein the enzyme or at least one enzyme in the enzyme mixture of (a), the variant enzyme of (b), or both are produced in a Pichia cell.

31. The process of any one of claims 1 to 30, wherein the contacting of (a) and / or the contacting of (b) is performed in a reaction mixture comprising an organic solvent, preferably toluene.

32. The method of any one of claims 1-31, wherein the enzyme or at least one enzyme in the enzyme mixture of (a), the variant enzyme of (b), or both are immobilized on a resin.

33. The method of any one of claims 1-32, wherein the enzyme of (a) or at least one enzyme of the enzyme mixture, the variant enzyme of (b), or both are provided in a cell slurry or a whole cell lysate.

34. The method of claim 33, wherein the cell paste or whole cell lysate further comprises a stabilizing compound selected from the group consisting of PEG, maltose, sorbitol, sucrose, glucose, mannitol, lactose, milk powder, starch, and combinations thereof, Preferably, the stabilizing compound is selected from the group consisting of mannitol, sorbitol, sucrose and combinations thereof.

35. The method of any one of claims 1-34, wherein the ratio of α-cyclodextrin to β-cyclodextrin, γ-cyclodextrin, or both in the composition is at least 2:1, wherein steps (a) and (b) are performed simultaneously, wherein steps (a) and (b) are performed at about 45°C to about 55°C, wherein steps (a) and (b) are performed at a pH of about 7.0 to about 7.5, wherein steps (a) and (b) are performed in a reaction mixture comprising water, ethanol, CaCl2, and optionally an organic solvent (preferably toluene), and wherein the total reaction is performed for no more than 8 hours.

36. A composition comprising cyclodextrin, wherein the cyclodextrin comprises α-cyclodextrin and may optionally further comprise β-cyclodextrin, γ-cyclodextrin, or any combination thereof, and wherein the composition comprising cyclodextrin comprises an amount and / or concentration of α-cyclodextrin that is greater than β-cyclodextrin, γ-cyclodextrin, or both, and wherein the composition is obtained by the method of any one of claims 1-35.

37. The composition of claim 36, wherein the ratio of α-cyclodextrin to β-cyclodextrin in the composition comprising cyclodextrin is at least 2:1, preferably at least 100:1, And / or wherein the ratio of α-cyclodextrin to γ-cyclodextrin in the cyclodextrin-containing composition is at least 2:1, preferably at least 100:

1.

38. The composition of claim 36 or 37, wherein the percent yield of alpha-cyclodextrin is at least about 10%, preferably at least about 60%.

39. Use of sucrose for producing α-cyclodextrin, wherein the production method is the method according to any one of claims 1 to 35.

40. Use of one or more enzymes of SEQ ID NO: 1 to SEQ ID NO: 42 for producing α-cyclodextrin, Preferably, the manufacturing method is the method according to any one of claims 1-35.

41. An enzyme comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 1-42, or an enzyme comprising or consisting of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity, to the amino acid sequence of any one of SEQ ID NOs: 1-42.

42. The enzyme of claim 41, wherein the enzyme is: (i) a variant amylosucrase comprising or consisting of the amino acid sequence of any one of SEQ ID NO: 3-16 or 42; (ii) a variant sucrose phosphorylase comprising or consisting of the amino acid sequence of SEQ ID NO: 20; (iii) a variant α-glucan phosphorylase comprising or consisting of the amino acid sequence of SEQ ID NO: 24; or (iv) a variant cyclodextrin glucanotransferase comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 29-41.

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