Method for producing beta-cyclodextrin

Abstract

Provided herein are methods of enzymatic production of beta-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 beta-cyclodextrin. In some cases, the methods produce a higher yield of [beta]-cyclodextrin relative to [alpha]-cyclodextrin, [gamma]-cyclodextrin, or both.

Description

Background Art

[0001] Cyclodextrins are a class of cyclic oligosaccharides composed of glucose cyclic oligomers. Cyclodextrins have 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 enzymatic conversion of starch. However, there are various disadvantages in the standard production method, including supply chain shortages, scalability, quality variations, purification, and commodity costs. Therefore, there is a need for an improved method for producing cyclodextrins that addresses these problems. Summary of the invention

[0002] There is an unmet need for methods of producing cyclodextrins that do not involve starch conversion. 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 β-cyclodextrin than α-cyclodextrin, γ-cyclodextrin, or both relative to a wild-type enzyme capable of converting amylose into cyclodextrin, wherein the composition comprising cyclodextrin comprises β-cyclodextrin, and may optionally further comprise α-cyclodextrin, γ-cyclodextrin, or any combination thereof, and the amount and / or concentration of β-cyclodextrin contained in the composition comprising cyclodextrin is greater than α-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 the amino acid sequence of SEQ ID NO:1. In some cases, the wild-type amylosucrase is a polysaccharide Neisseria (Neisseriapolysaccharea) amylosucrase. In some cases, the wild-type amylosucrase comprises the amino acid sequence of 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, 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, and the enzyme combination or together is capable of converting sucrose into amylose.In some cases, the enzyme mixture includes 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 includes any one of SEQ ID NO:17-20 amino acid sequence or with any one of SEQ ID NO:17-20 amino acid sequence has at least about 70% sequence identity, preferably at least about 90% sequence identity amino acid sequence or consisting of it. In some cases, the enzyme mixture includes alpha-glucan phosphorylase. In some cases, α-glucan phosphorylase is capable of converting glucose-1-phosphate into amylose. In some cases, the contact of (a) also includes 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 the following: potato (Solanum tuberosum) α-glucan phosphorylase, eastern sulfur lobules strain 7 (S.tokodaii strain 7) α-glucan phosphorylase and heather Corynebacterium DSM 20145 (C.callunae DSM20145) 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 with an amino acid sequence of any one of SEQ ID NO: 21-24 having at least about 70% sequence identity, preferably at least about 90% sequence identity or consisting of it. In some cases, the enzyme capable of converting amylose into cyclodextrin in (b) includes variant cyclodextrin glucanotransferase. In some cases, variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to wild-type cyclodextrin glucanotransferase. In some cases, wild-type cyclodextrin glucanotransferase is bacillus (Bacillus sp.) strain number 38-2 cyclodextrin glucanotransferase. In some cases, bacillus strain number 38-2 cyclodextrin glucanotransferase comprises SEQ ID NO:25 amino acid sequence or is composed of it. In some cases, wild-type cyclodextrin glucanotransferase is circulans bacillus (Bacillus circulans) strain 251 cyclodextrin glucanotransferase.In some cases, the cyclodextrin glucanotransferase of Bacillus circulans strain 251 comprises SEQ ID NO:26 or 27, for example, SEQ ID NO:27 amino acid sequence or consists thereof. In some cases, the variant cyclodextrin glucanotransferase 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 any one of SEQ ID NO:25-27. 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 31 relative to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO:27. In some cases, the amino acid substitution at amino acid position 31 is selected from the group consisting of: A31R, A31P and A31T. 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 amylose 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), the 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), the variant enzyme of (b), or both are produced in a Pichia cell, such as a Pichia pastoris cell. In some cases, the ratio of β-cyclodextrin to α-cyclodextrin in the composition comprising cyclodextrin is at least 2: 1. In some cases, the ratio of β-cyclodextrin to γ-cyclodextrin in the composition comprising cyclodextrin is at least 2: 1.

[0004] The present invention also provides a method for purifying β-cyclodextrin, which comprises the following steps:

[0005] i. providing a crude composition comprising β-cyclodextrin;

[0006] ii. obtaining a first precipitate comprising β-cyclodextrin from the crude composition;

[0007] iii. dissolving the first precipitate to obtain a first solution comprising β-cyclodextrin;

[0008] iv. filtering the first solution to obtain a second solution comprising β-cyclodextrin;

[0009] v. Crystallizing and / or precipitating the second solution to obtain a purified β-cyclodextrin composition.

[0010] Therefore, the present invention also provides a purified β-cyclodextrin composition.

[0011] In some cases, step ii comprises filtering the crude composition, centrifuging the crude composition, subjecting the crude composition to a sedimentation operation and / or washing with water to obtain a first precipitate. In some cases, step iii comprises dissolving the precipitate in water.

[0012] Incorporated by Reference

[0013] 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

[0014] 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:

[0015] Figure 1A-Figure 1C The structures of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin are depicted respectively.

[0016] 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.

[0017] 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.

[0018] Figure 3 Depicted is a non-limiting example of an enzymatic reaction for converting amylose to β-cyclodextrin according to embodiments of the present disclosure.

[0019] Figure 4Depicted is a non-limiting example of a one-pot enzymatic synthesis reaction for converting sucrose to β-cyclodextrin using wild-type amylosucrase and variant cyclodextrin glucanotransferase according to embodiments of the present disclosure.

[0020] Figure 5 Depicted is a non-limiting example of a one-pot enzymatic synthesis reaction for converting sucrose to β-cyclodextrin using a variant amylosucrase and a variant cyclodextrin glucanotransferase according to embodiments of the present disclosure.

[0021] Figure 6 Depicted is a non-limiting example of a one-pot enzymatic synthesis reaction for converting sucrose to β-cyclodextrin using sucrose phosphorylase, α-glucan phosphorylase, and cyclodextrin glucanotransferase according to embodiments of the present disclosure.

[0022] Figure 7 Describes the composition of purified β-cyclodextrin 1 H-NMR spectrum: The purification method of Example 4 was used to obtain a composition.

[0023] Figure 8 The HLPC-ELSD spectrum of the purified β-cyclodextrin composition is depicted. The composition was obtained using the purification method of Example 6.

[0024] Fig. 9 Describes the composition of purified β-cyclodextrin 1 H-NMR spectrum: The purification method of Example 6 was used to obtain a composition.

[0025] Fig.10 Depicts two purified β-cyclodextrin compositions 1 H-NMR spectrum: The purification method of Example 7 was used to obtain a composition.

[0026] 11 depicts a graph of the retained enzyme activity (%) of lyophilized whole cell lysate or whole cell slurry of amylosucrase (with or without additives) as discussed in Example 8. The % retained enzyme activity was measured by comparing the enzyme activity to non-lyophilized whole cell lysate or whole cell slurry.

[0027] Fig.12 Depicted is a graph of the retained enzyme activity (%) of lyophilized whole cell lysates of cyclodextrin glucanotransferase (with or without additives) as discussed in Example 8. The % retained enzyme activity was measured by comparing the enzyme activity to non-lyophilized whole cell lysate. DETAILED DESCRIPTION

[0028] 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 simple enzymatic synthesis of cyclodextrins using sucrose as a starting material.

[0029] Provided herein is a method for producing a composition comprising cyclodextrin. Also provided herein is an enzymatic synthesis method for β-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. Provided herein is a method for enzymatically converting sucrose into β-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 β-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).

[0030] Cyclodextrins are formed by the conjugation of glucose pyranose units through α-1,4 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 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."

[0031] β-cyclodextrin is widely used in the pharmaceutical industry. In order to improve the oral bioavailability and solubility of cyclodextrin, different β-cyclodextrin derivatives have been prepared. For example, modifying the hydroxyl group of cyclodextrin with hydroxypropyl can significantly improve the solubility of cyclodextrin. Some potential derivatives include randomly methylated β-cyclodextrin and branched β-cyclodextrin.

[0032] 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 a greater amount and / or concentration (e.g., wt%, mol% or w / v) of β-cyclodextrin than α-cyclodextrin, γ-cyclodextrin or both. In some cases, the composition comprising cyclodextrin comprises β-cyclodextrin, and may optionally also comprise α-cyclodextrin, γ-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 β-cyclodextrin greater than α-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).

[0033] Method step (a): Enzymatic conversion of sucrose into amylose

[0034] 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.

[0035] A single enzyme method for producing amylose from sucrose

[0036] In some aspects, the enzyme is amylosucrase. Figure 2A Describe 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 (NCBI accession number N868_11335). In some cases, a wild-type carbon source cellulomonas T26 amylosucrase can have the amino acid sequence of SEQ ID NO:1. In some cases, a wild-type amylosucrase can be a polysaccharide Neisseria amylosucrase (NCBI accession number AJ011781). In some cases, a wild-type polysaccharide Neisseria amylosucrase can have the amino acid sequence of SEQ ID NO:2. Table 1 below describes the non-limiting examples of wild-type amylosucrase (and its amino acid sequence) that can be used according to the method provided herein.

[0037] Table 1. Non-limiting examples of wild-type amylosucrases

[0038]

[0039]

[0040] 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.

[0041] In some cases, the variant amylosucrase comprises at least one amino acid variant relative to the wild-type carbon source Cellulomonas T26 amylosucrase (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 (SEQ ID NO: 2). In some cases, the variant amylosucrase comprises at least one amino acid variant relative to the wild-type polysaccharide Neisseria amylosucrase (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) with the amino acid sequence of the wild-type carbon source Cellulomonas T26 amylosucrase, preferably at least about 90% sequence identity. 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 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 to the amino acid sequence of 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), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:2.

[0042] 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 comprises relative to SEQ ID NO:2 amino acid sequence, amino acid replacement at amino acid position 234 or consists of it. 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, the amino acid substitution at amino acid position 234 is R234H relative to the amino acid sequence of SEQ ID NO: 2 (e.g., SEQ ID NO: 16 in Table 2). In some aspects, the variant amylosucrase comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 3-16 or 48 as depicted in Table 2, or 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 any one of SEQ ID NOs: 3-16 or 48 as depicted in Table 2. In a preferred embodiment, the variant amylosucrase comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 3-9 or 48 depicted in Table 2.

[0043] Table 2. Non-limiting examples of variant amylosucrases.

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051]

[0052] 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.

[0053] 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.

[0054] A dual-enzyme method for producing amylose from sucrose

[0055] 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.

[0056] 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.

[0057] Table 3. Non-limiting examples of sucrose phosphorylases

[0058]

[0059]

[0060] 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.

[0061] 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.

[0062] 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 comprises or consists of 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.

[0063] Table 4. Non-limiting examples of α-glucan phosphorylases

[0064]

[0065]

[0066]

[0067] 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), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:24, and comprises amino acid substitutions F39L, N135S, and T706I relative to SEQ ID NO:21.

[0068] 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.

[0069] Method step (b): Enzymatic conversion of amylose into β-cyclodextrin

[0070] 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 β-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 β-cyclodextrin than α-cyclodextrin, γ-cyclodextrin, or both, relative to a wild-type enzyme capable of converting amylose into cyclodextrin.

[0071] 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 β-cyclodextrin using a cyclodextrin glucanotransferase. Preferably, the cyclodextrin glucanotransferase produces β-cyclodextrin from amylose in an amount and / or concentration greater than that of α-cyclodextrin and / or γ-cyclodextrin.

[0072] 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 is capable of producing greater amounts and / or concentrations of β-cyclodextrin (relative to α-cyclodextrin and / or γ-cyclodextrin) by amylose.

[0073] In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to a wild-type Bacillus (strain number 38-2) cyclodextrin glucanotransferase (e.g., NCBI accession number M19880.1; SEQ ID NO: 25). In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to a wild-type Bacillus circulans strain 251 cyclodextrin glucanotransferase (e.g., NCBI accession number X78145.1; SEQ ID NO: 26 or 27). In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to a wild-type Bacillus circulans strain 251 cyclodextrin glucanotransferase of SEQ ID NO: 27. 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 SEQ ID NO:25. 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 SEQ ID NO: 26 or 27. 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 SEQ ID NO: 27.

[0074] 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 31 relative to the amino acid sequence of SEQ ID NO:27. In some cases, relative to the amino acid sequence of SEQ ID NO:27, the amino acid substitution at amino acid position 31 is A31R (e.g., SEQ ID NO:28 in Table 5). In some cases, relative to the amino acid sequence of SEQ ID NO:27, the amino acid substitution at amino acid position 31 is A31P (e.g., SEQ ID NO:29 in Table 5). In some cases, relative to the amino acid sequence of SEQ ID NO:27, the amino acid substitution at amino acid position 31 is A31T (e.g., SEQ ID NO:30 in Table 5). In some aspects, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence according to any one of SEQ ID NO:25-30 as depicted in Table 5.

[0075] 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: 31-34). In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to any one of SEQ ID NOs: 31-34. 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: 31-34.

[0076] 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 the amino acid sequence of SEQ ID NO: 34. In some cases, relative to the amino acid sequence of SEQ ID NO: 34, the amino acid substitution at amino acid position 146 is R146A (e.g., SEQ ID NO: 35 in Table 5). In some cases, relative to the amino acid sequence of SEQ ID NO: 34, the amino acid substitution at amino acid position 146 is R146P (e.g., SEQ ID NO: 36 in Table 5). In some cases, at least one amino acid substitution comprises an amino acid substitution at amino acid position 147 relative to the amino acid sequence of SEQ ID NO: 34. In some cases, relative to the amino acid sequence of SEQ ID NO: 34, the amino acid substitution at amino acid position 147 is D147A (e.g., SEQ ID NO: 37 in Table 5). In some cases, relative to the amino acid sequence of SEQ ID NO: 34, the amino acid substitution at amino acid position 147 is D147P (e.g., SEQ ID NO: 38 in Table 5). In some cases, at least one amino acid substitution comprises amino acid substitutions at amino acid positions 146 and 147 relative to the amino acid sequence of SEQ ID NO: 34. In some cases, the amino acid substitution at amino acid position 146 relative to the amino acid sequence of SEQ ID NO: 34 is R146A, and the amino acid substitution at amino acid position 147 relative to the amino acid sequence of SEQ ID NO: 34 is D147P (e.g., SEQ ID NO: 39 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: 34 is R146P, and the amino acid substitution at amino acid position 147 relative to the amino acid sequence of SEQ ID NO: 34 is D147A (e.g., SEQ ID NO: 40 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: 34 is R146P, and the amino acid substitution at amino acid position 147 relative to the amino acid sequence of SEQ ID NO: 34 is D147P (e.g., SEQ ID NO: 41 in Table 5).

[0077] 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: 32 or SEQ ID NO: 34. In some cases, the amino acid substitution at amino acid position 372 relative to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 34 is D372K (e.g., SEQ ID NO: 42 (relative to SEQ ID NO: 32) and SEQ ID NO: 45 (relative to SEQ ID NO: 34) 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: 32 or SEQ ID NO: 34. In some cases, the amino acid substitution at amino acid position 89 relative to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 34 is Y89R (e.g., SEQ ID NO: 43 (relative to SEQ ID NO: 32) and SEQ ID NO: 47 (relative to SEQ ID NO: 34) 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: 32 or SEQ ID NO: 34, and an amino acid substitution at amino acid position 89 relative to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 34. In some cases, the amino acid substitution at amino acid position 372 relative to the amino acid sequence of SEQ ID NO: 32 or 34 is D372K, and the amino acid substitution at amino acid position 89 relative to the amino acid sequence of SEQ ID NO: 32 or 34 is Y89R (e.g., SEQ ID NO: 44 (relative to SEQ ID NO: 32) and SEQ ID NO: 47 (relative to SEQ ID NO: 34) in Table 5).

[0078] In some aspects, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 31-47 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: 31-47 as depicted in Table 5.

[0079] In specific aspects, 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.

[0080] In another specific aspect, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence according to SEQ ID NO: 39, 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: 39.

[0081] In another specific aspect, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence according to SEQ ID NO: 40, 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: 40.

[0082] 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.

[0083] In another specific aspect, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence according to SEQ ID NO: 47, 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: 47.

[0084] Table 5. Non-limiting examples of cyclodextrin glucanotransferases

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098] 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: 25, preferably at least about 90% sequence identity. 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), preferably at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 26 or 27.

[0099] 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: 27, preferably at least about 90% sequence identity and an amino acid substitution at amino acid position 31 relative to SEQ ID NO: 27. 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: 27, preferably at least about 90% sequence identity, and an amino acid substitution A31R relative to SEQ ID NO: 27. 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: 27, preferably at least about 90% sequence identity and an amino acid substitution A31P relative to SEQ ID NO: 27. 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: 27, preferably at least about 90% sequence identity, and an amino acid substitution A31T relative to SEQ ID NO: 27.

[0100] 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: 34, preferably at least about 90% sequence identity and an amino acid substitution at amino acid position 146 relative to SEQ ID NO: 34. 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: 34, preferably at least about 90% sequence identity, and the amino acid substitution R146A relative to SEQ ID NO: 34. 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: 34, preferably at least about 90% sequence identity, and the amino acid substitution R146P relative to SEQ ID NO: 34.

[0101] 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: 34, preferably at least about 90% sequence identity and an amino acid substitution at amino acid position 147 relative to SEQ ID NO: 34. 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: 34, preferably at least about 90% sequence identity and amino acid substitution D147P relative to SEQ ID NO: 34. 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: 34, preferably at least about 90% sequence identity, and amino acid substitution D147A relative to SEQ ID NO: 34.

[0102] 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: 34, preferably at least about 90% sequence identity, an amino acid substitution at amino acid position 146 relative to SEQ ID NO: 34, and an amino acid substitution at amino acid position 147 relative to SEQ ID NO: 34. 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: 34, preferably at least about 90% sequence identity, an amino acid substitution R146A relative to SEQ ID NO: 34, and an amino acid substitution D147P relative to SEQ ID NO: 34. 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: 34, preferably at least about 90% sequence identity, an amino acid substitution R146P relative to SEQ ID NO: 34, and an amino acid substitution D147A relative to SEQ ID NO: 34.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: 34, preferably at least about 90% sequence identity, an amino acid substitution R146P relative to SEQ ID NO: 34, and an amino acid substitution D147P relative to SEQ ID NO: 34.

[0103] 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: 32 or 34, preferably at least about 90% sequence identity and an amino acid substitution at amino acid position 372 relative to SEQ ID NO: 32 or 34. 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: 32 or 34, preferably at least about 90% sequence identity and an amino acid substitution D372K relative to SEQ ID NO: 32 or 34.

[0104] 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: 32 or 34, preferably at least about 90% sequence identity and an amino acid substitution at amino acid position 89 relative to SEQ ID NO: 32 or 34. 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: 32 or 34, preferably at least about 90% sequence identity and an amino acid substitution Y89R relative to SEQ ID NO: 32 or 34.

[0105] 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: 32 or 34, preferably at least about 90% sequence identity, an amino acid substitution at amino acid position 372 relative to SEQ ID NO: 32 or 34, and an amino acid substitution at amino acid position 89 relative to SEQ ID NO: 32 or 34. 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: 32 or 34, preferably at least about 90% sequence identity, an amino acid substitution D372K relative to SEQ ID NO: 32 or 34, and an amino acid substitution Y89R relative to SEQ ID NO: 32 or 34.

[0106] 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 Bacillus (strain number 38-2). In some embodiments, cyclodextrin glucanotransferase is derived from Bacillus circulans strain 251. 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.

[0107] In various aspects, the methods provided herein produce a higher ratio of beta-cyclodextrin to alpha-cyclodextrin, gamma-cyclodextrin or both. For example, in some cases, the ratio of beta-cyclodextrin to alpha-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 a preferred embodiment, the ratio of beta-cyclodextrin to alpha-cyclodextrin provided by the methods provided herein is at least 10: 1. For example, the ratio can be 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 preferred embodiments, the methods provided herein provide a ratio of beta-cyclodextrin to gamma-cyclodextrin of at least 5: 1. For example, the ratio can be 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 preferred embodiments, the methods provided herein provide a ratio of beta-cyclodextrin to alpha-cyclodextrin and gamma-cyclodextrin of at least 3.5: 1. For example, the ratio can be 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 60:1, at least 70:1, at least 80:1, at least 90:1, at least 100:1, or more.

[0108] This disclosure outlines throughout the text a method for obtaining robust enzymatic activity in each step to obtain a higher yield of β-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 β-cyclodextrin (e.g., as described herein) is performed for a second time period, thereby enabling catalytic conversion of amylose to β-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 β-cyclodextrin, e.g., as described herein) are performed in the same container (e.g., a one-pot synthesis).

[0109] 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 beta-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 beta-cyclodextrin) are added at different times (e.g., the former before the latter).

[0110] 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.

[0111] In some embodiments, reaction time is an important consideration for obtaining the maximum yield of β-cyclodextrin. In some embodiments, the production of β-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 β-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.

[0112] Temperature is an important consideration for maximizing the yield of β-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.

[0113] 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.

[0114] In a one-pot synthesis, the enzyme mixture should function maximally, although the optimal temperature for each enzyme may be slightly different.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] The pH of the reaction mixture may be an important consideration for maximizing the yield of β-cyclodextrin. In some embodiments, one or more enzymatic reactions are carried out at a pH of about 6 to about 8, for example, the pH may be about 6.5 to about 7.5. In a preferred embodiment, one or more enzymatic reactions are carried out at a pH of about 7.0 to about 7.5. Preferably, step (a) is carried out at a pH of about 7.0 to about 7.5. Preferably, step (b) is carried out at a pH of about 7.0 to about 7.5. Step (a) and step (b) may be carried out at different pHs, but preferably step (a) and step (b) are carried out at approximately the same pH.

[0119] 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 reaction mixture can be from about 50mM to about 200mM, for example, about 100mM.

[0120] 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 β-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 β-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%, or for example, at least about 400%. It is believed that the addition of an organic solvent can reduce the solubility of β-cyclodextrin in the reaction mixture, thereby causing β-cyclodextrin precipitation, which reduces the concentration of β-cyclodextrin in the reaction mixture, thereby increasing the yield of β-cyclodextrin. This can prevent the enzyme from decomposing β-cyclodextrin.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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 beta-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 an 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 beta-cyclodextrin can occur in the microbial host cell.

[0127] 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. Or, the enzyme can be non-covalently attached to the resin. For example, the enzyme can be connected 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. Or or additionally, the enzyme of (b) can be a variant cyclodextrin glucanotransferase (for example, wherein the variant cyclodextrin glucanotransferase can include the amino acid sequence according to SEQ ID NO:28 or be composed of it) 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.

[0128] The immobilized resin enzyme can be reused in the methods described herein. However, the inventors have found that the yield of β-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 enzymatic 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.

[0129] 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 additives.

[0130] 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.

[0131] In some embodiments, the cytoplasm or whole cell lysate also comprises an additive. In some embodiments, the additive 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 additive is added in an amount of about 0.1% w / v to about 10% w / v of the cytoplasm or whole cell lysate, such as about 0.5% w / v to about 5% w / v. For example, the additive can be 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 additive is mannitol, sorbitol, sucrose or a combination thereof.

[0132] 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 within two days. Freeze-drying methods are known in the art.

[0133] The inventors have found that adding additives to the cell paste or whole cell lysate (as described above) improves the stability of the enzyme compared to the cell paste or whole cell lysate without the additive, and freeze-drying the cell paste or whole cell lysate (as described above) improves the stability of the enzyme compared to the 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 can be resuspended and shaken to redissolve.

[0134] In some embodiments, the methods described herein produce compositions comprising at least 18 g / L of β-cyclodextrin. In some embodiments, the methods produce compositions comprising at least 25 g / L of β-cyclodextrin, at least 30 g / L of β-cyclodextrin, at least 40 g / L of β-cyclodextrin, at least 50 g / L of β-cyclodextrin, or at least 60 g / L of β-cyclodextrin. In preferred embodiments, the methods described herein produce compositions comprising at least 50 g / L of β-cyclodextrin.

[0135] In some embodiments, the percent yield of β-cyclodextrin is at least about 10%, such as at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, or such as at least about 60%, wherein the percent yield is calculated by dividing the total amount of β-cyclodextrin produced in the methods described herein by the maximum theoretical amount of β-cyclodextrin that can be produced from the starting sucrose reagent.

[0136] Also provided herein is a composition comprising cyclodextrin, wherein the cyclodextrin comprises β-cyclodextrin and may optionally further comprise α-cyclodextrin, γ-cyclodextrin or any combination thereof, and wherein the composition comprising cyclodextrin comprises an amount and / or concentration of β-cyclodextrin greater than α-cyclodextrin, γ-cyclodextrin or both. Preferably, the composition is obtained by the method provided herein. In some cases, the composition does not comprise α-cyclodextrin and / or γ-cyclodextrin. Preferably, the composition comprises a ratio of β-cyclodextrin to α-cyclodextrin, a ratio of β-cyclodextrin to γ-cyclodextrin, or a ratio of both β-cyclodextrin to γ-cyclodextrin and β-cyclodextrin to α-cyclodextrin of 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 more. Preferably, the composition comprises a ratio of β-cyclodextrin to α-cyclodextrin, a ratio of β-cyclodextrin to γ-cyclodextrin, or a ratio of both β-cyclodextrin to γ-cyclodextrin and β-cyclodextrin to α-cyclodextrin of at least 10:1, e.g., 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 more.

[0137] In a preferred embodiment, the present invention provides a method for producing a composition comprising 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 a cyclodextrin glucanotransferase, thereby producing a composition comprising cyclodextrin, wherein the cyclodextrin glucanotransferase in (b) is a mutant enzyme capable of producing a greater amount and / or concentration of β-cyclodextrin than α-cyclodextrin, γ-cyclodextrin, or both relative to a wild-type enzyme capable of converting amylose into cyclodextrin; The invention relates to a method for preparing an enzyme comprising a cyclodextrin, wherein the composition comprising cyclodextrin comprises β-cyclodextrin, and may optionally further comprise α-cyclodextrin, γ-cyclodextrin, or any combination thereof, wherein the ratio of β-cyclodextrin to α-cyclodextrin, γ-cyclodextrin, or both in the composition is at least 10:1, wherein steps (a) and (b) are carried out simultaneously, wherein steps (a) and (b) are carried out at about 45° C. to about 55° C., wherein steps (a) and (b) are carried out at a pH of about 7.0 to about 7.5, wherein steps (a) and (b) are carried out in a reaction mixture comprising water and an organic solvent (preferably toluene), and wherein the total reaction is carried out for no more than 8 hours.

[0138] Also provided herein is β-cyclodextrin. Preferably, β-cyclodextrin is obtained by the method provided herein.

[0139] Also provided herein is the use of sucrose as a starting material to make beta-cyclodextrin. Also provided herein is the use of sucrose in a method of producing beta-cyclodextrin, wherein the method does not use starch.

[0140] 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.

[0141] 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 β-cyclodextrin than α-cyclodextrin, γ-cyclodextrin, or both.

[0142] Also provided herein is the use of any one of the enzymes or enzyme mixtures described herein for producing β-cyclodextrin, wherein the production does not require starch as a starting material.

[0143] Also provided herein are any enzyme or enzyme mixtures described herein. For example, provided herein are enzymes comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 1-48. Also provided herein are enzymes 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: 1-48.

[0144] 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 48. 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 48.

[0145] 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.

[0146] 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.

[0147] Preferably, the enzyme is a variant cyclodextrin glucanotransferase comprising or consisting of an amino acid sequence of any one of SEQ ID NOs: 28-30 or 35-47. 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: 28-30 or 35-47.

[0148] Also provided herein are enzyme compositions comprising one or more enzymes described herein.

[0149] Also provided herein are genes encoding any variant enzyme described herein. Also provided herein are vectors encoding any variant enzyme described herein. Also provided herein are recombinant host cells comprising any gene, vector or enzyme described herein.

[0150] Also provided herein is the use of an organic solvent, preferably toluene, for increasing the yield of β-cyclodextrin obtained in a process for producing β-cyclodextrin, such as β-cyclodextrin obtained by any of the processes described herein.

[0151] Purification methods

[0152] The present invention also provides a method for purifying β-cyclodextrin, which comprises the following steps:

[0153] i providing a crude composition comprising β-cyclodextrin;

[0154] ii. obtaining a first precipitate containing β-cyclodextrin from the crude composition, for example, by filtering the crude composition, centrifuging the crude composition, subjecting the crude composition to a sedimentation operation and / or washing with water;

[0155] iii. dissolving the first precipitate to obtain a first solution comprising β-cyclodextrin, for example by dissolving the first precipitate in water;

[0156] iv filtering the first solution to obtain a second solution comprising β-cyclodextrin; and

[0157] v Crystallizing and / or precipitating the second solution to obtain a purified β-cyclodextrin composition.

[0158] Step (ii) and / or (iv)

[0159] A filtration step (iv) may remove insoluble material.

[0160] In some embodiments, steps (ii) and / or (iv) comprise filtering and washing the material obtained by filtering, for example with water or alkaline water.

[0161] In some embodiments, step (iv) comprises filtering through a filter aid. In some embodiments, the filter aid comprises silica. An example of a suitable filter aid is 1% Available from Sigma-Aldrich. In order to reduce the overall filtration time in step (iv), it may be advantageous to use a filter aid.

[0162] The filtering step (iv) may be carried out at a temperature of about 4°C to about 25°C.

[0163] Dissolution step (iii)

[0164] In some embodiments, step (iii) comprises dissolving the first precipitate in an alkaline solution.The precipitate can be dissolved in NaOH, for example in 1 M NaOH, for example by adding multiple (eg, five) volumes of 1 M NaOH.

[0165] In some embodiments, step (iii) may include heating the solution until the β-cyclodextrin is dissolved. For example, this may require heating the solution to about 60°C or higher, such as to about 65°C or higher, such as to about 70°C or higher, such as to about 75°C or higher. The temperature of the solution may then be reduced prior to subsequent steps, such as by about 5°C or more.

[0166] Crystallization step (v)

[0167] Step (v) may comprise neutralizing the second solution, optionally wherein neutralizing comprises adding HCl. For example, neutralizing may comprise adding 6M HCl.

[0168] Step (v) may comprise adding crystalline β-cyclodextrin to the second solution.

[0169] In some embodiments, step (v) may also include heating the solution until the β-cyclodextrin is dissolved. For example, this may require heating the solution to about 60°C or higher, such as to about 65°C or higher, such as to about 70°C or higher, such as to about 75°C or higher. In a preferred embodiment, the solution is heated to about 75°C. The temperature of the solution may then be reduced, such as by about 5°C or more, before adding the crystalline β-cyclodextrin. In a preferred embodiment, the solution is heated to about 75°C and then reduced to about 70°C before adding the seed crystals.

[0170] In some embodiments, step (v) may include cooling the solution to below room temperature after seeding, for example, to about 20°C or less, about 15°C or less, about 10°C or less, or about 5°C or less. In a preferred embodiment, the solution is cooled to about 4°C. In some embodiments, the solution is cooled for about 1 to about 12 hours. In some embodiments, the solution is cooled for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours. In a preferred embodiment, the solution is cooled to about 4°C for about 4 hours.

[0171] The seeded solution can be maintained under conditions suitable for the formation of β-cyclodextrin crystals. For example, the solution can be maintained below room temperature, for example, at about 20°C or lower, about 15°C or lower, about 10°C or lower, or about 5°C or lower. In a preferred embodiment, the solution is maintained at about 4°C. In some embodiments, the solution is maintained at a temperature below room temperature for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, or at least about 12 hours. Preferably, the solution is maintained at about 4°C for 12 hours or more.

[0172] The crystallization step (v) may comprise a filtration step. The filtration step may comprise vacuum filtration.

[0173] In some embodiments, step (v) further comprises washing the composition with water.

[0174] In some embodiments, step (v) further comprises drying the composition, optionally wherein the composition is dried at about 45°C (eg, in a vacuum oven).

[0175] Precipitation step (v)

[0176] Step (v) may comprise neutralizing the second solution, optionally wherein neutralizing comprises adding HCl. For example, neutralizing may comprise adding about 6M HCl.

[0177] Step (v) may include adding an anti-solvent.Anti-solvent can increase the yield of purified β-cyclodextrin in the composition obtained by purification method.Anti-solvent is a solvent in which β-cyclodextrin is difficult to dissolve, such as a solvent in which β-cyclodextrin is insoluble at about 50°C and about 60°C.Anti-solvent can be a mixture of THF, AcN, EtOH, toluene, acetone or acetone and water (e.g., 10:90, or 20:80, or 30:70, or 40:60, or 50:50, or 60:40, or 70:30, or 80:20, or 90:10 acetone: water). In some embodiments, when the anti-solvent is a mixture of acetone and water, the acetone content in the mixture can be between 10-90%, between 20-80%, between 30-70%, between 40-60%, or about 50%. Preferably, the anti-solvent used is a mixture of acetone and water, for example a mixture of 50% acetone and 50% water.

[0178] In some embodiments, step (v) may include cooling the solution to below room temperature after adding the antisolvent, for example, cooling to about 20°C or less, about 15°C or less, about 10°C or less, or about 5°C or less. In a preferred embodiment, the solution is cooled to about 4°C. In some embodiments, the solution is cooled for about 1 to about 12 hours. In some embodiments, the solution is cooled for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours. In a preferred embodiment, the solution is cooled to about 4°C for about 4 hours.

[0179] The solution can be maintained under conditions suitable for the formation of beta-cyclodextrin precipitates. For example, the solution can be maintained below room temperature, for example, maintained at about 20°C or lower, about 15°C or lower, about 10°C or lower, or about 5°C. In a preferred embodiment, the solution is maintained at about 4°C. In some embodiments, the solution is maintained at a temperature below room temperature for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, or at least about 12 hours. Preferably, the solution is maintained at about 4°C for 12 hours or longer.

[0180] In some embodiments, the solution is cooled to about 4°C over about 4 hours and then maintained at about 4°C for about 12 hours.

[0181] The precipitation of step (v) may include a filtration step. The filtration step may include vacuum filtration.

[0182] In some embodiments, step (v) further comprises washing the composition with water.

[0183] In some embodiments, step (v) further comprises drying the composition, optionally wherein the composition is dried at about 45°C (eg, under vacuum).

[0184] Composition

[0185] Preferably, the crude composition of step (i) is obtained by any one of the enzymatic processes described and claimed herein.

[0186] In some embodiments, the crude composition is cooled prior to step (ii). For example, the crude composition can be cooled to room temperature and maintained for at least about 3 hours, and then cooled to about 4°C and maintained for at least about 3 hours.

[0187] Also provided herein is a purified β-cyclodextrin composition. The purified β-cyclodextrin composition can be obtained by any of the purification methods described and claimed herein. The purity of the β-cyclodextrin in the composition can be 75 wt % or higher, such as 80 wt % or higher, such as 85 wt % or higher, such as 90 wt % or higher, or such as 95 wt % or higher.

[0188] The purity of β-cyclodextrin can be determined by 1 H-NMR can be used to measure the anhydrous amount of β-cyclodextrin.

[0189] Preferably, the purified β-cyclodextrin composition consists essentially of β-cyclodextrin and optionally present water, and preferably consists of β-cyclodextrin and optionally present water, and preferably consists of β-cyclodextrin. The purified β-cyclodextrin composition may contain 2% by weight or less of toluene, for example, without toluene. The purified β-cyclodextrin composition may contain 1% by weight or less of sucrose, fructose and / or amylose, for example, without sucrose, fructose and / or amylose. The purified β-cyclodextrin composition may preferably contain 5% by weight or less, preferably 1% by weight or less of α-cyclodextrin and / or γ-cyclodextrin, for example, without α-cyclodextrin and / or γ-cyclodextrin.

[0190] The recovery of β-cyclodextrin from the purification methods described herein can be at least 50%, at least 60%, at least 70%, or at least 80%. In other words, the amount of β-cyclodextrin in the purified composition can be at least 50% (or at least 60%, at least 70%, or at least 80%) of the amount of β-cyclodextrin in the crude composition. The amount of β-cyclodextrin and any other components in the composition can be determined by 1 H-NMR (in wt%) or HLPC-ELSD (in g / L) measurements.

[0191] Glossary

[0192] 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 disclosure encompasses sequences having at least about 70%, 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 sequence provided herein.

[0193] 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.

[0194] 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."

[0195] As used herein, "a," "an," and "the" may include plural referents unless expressly and unambiguously limited to one referent.

[0196] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0197] Numbering Implementation Plan

[0198] 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.

[0199] 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 β-cyclodextrin than α-cyclodextrin, γ-cyclodextrin or both, wherein the composition containing cyclodextrin contains β-cyclodextrin, and may optionally also contain α-cyclodextrin, γ-cyclodextrin or any combination thereof, and the amount and / or concentration of β-cyclodextrin contained in the composition containing cyclodextrin is greater than α-cyclodextrin, γ-cyclodextrin or both.

[0200] Embodiment 2: The method of embodiment 1, wherein the enzyme of (a) is amylosucrase, or the enzyme mixture of (a) comprises amylosucrase.

[0201] 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.

[0202] 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.

[0203] Embodiment 5: The method of embodiment 3 or 4, wherein the wild-type amylosucrase is the Carbonysone Cellulomonas T26 amylosucrase.

[0204] Embodiment 6: The method of Embodiment 5, wherein the wild-type amylosucrase comprises the amino acid sequence of SEQ ID NO:1.

[0205] Embodiment 7: The method of embodiment 3 or 4, wherein the wild-type amylosucrase is a Neisseria polysaccharidase amylosucrase.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] Embodiment 13: The method of Embodiment 1, wherein the enzyme mixture of (a) comprises at least two enzymes that in combination or together are capable of converting sucrose to amylose.

[0212] Embodiment 14: The method of Embodiment 13, wherein the enzyme mixture comprises sucrose phosphorylase.

[0213] Embodiment 15: The method of Embodiment 14, wherein the sucrose phosphorylase is capable of converting sucrose into glucose-1-phosphate.

[0214] 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.

[0215] 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.

[0216] Embodiment 18: The method of any one of embodiments 14-17, wherein the sucrose phosphorylase comprises or consists of an 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.

[0217] Embodiment 19: The method of any one of Embodiments 13-18, wherein the enzyme mixture comprises α-glucan phosphorylase.

[0218] Embodiment 20: The method of Embodiment 19, wherein the α-glucan phosphorylase is capable of converting glucose-1-phosphate into amylose.

[0219] 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.

[0220] 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.

[0221] 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, preferably at least about 90% sequence identity, to the amino acid sequence of any one of SEQ ID NOs: 21-24.

[0222] 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.

[0223] 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.

[0224] Embodiment 26: The method of Embodiment 25, wherein the wild-type cyclodextrin glucanotransferase is the cyclodextrin glucanotransferase of Bacillus strain No. 38-2.

[0225] Embodiment 27: The method of Embodiment 26, wherein the Bacillus strain No. 38-2 cyclodextrin glucanotransferase comprises or consists of the amino acid sequence of SEQ ID NO:25.

[0226] Embodiment 28: The method of Embodiment 25, wherein the wild-type cyclodextrin glucanotransferase is Bacillus circulans strain 251 cyclodextrin glucanotransferase.

[0227] Embodiment 29: The method of Embodiment 28, wherein the Bacillus circulans strain 251 cyclodextrin glucanotransferase comprises or consists of the amino acid sequence of SEQ ID NO: 26 or 27, eg, SEQ ID NO: 27.

[0228] Embodiment 30: The method of any one of Embodiments 24-29, wherein the variant cyclodextrin glucanotransferase 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 any one of SEQ ID NOs: 25-27.

[0229] Embodiment 31: The method of any one of Embodiments 28-30, wherein the at least one amino acid variant comprises at least one amino acid substitution relative to a wild-type cyclodextrin glucanotransferase.

[0230] Embodiment 32: The method of Embodiment 31, wherein the at least one amino acid substitution comprises an amino acid substitution at amino acid position 31 relative to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO:27.

[0231] Embodiment 33: The method of Embodiment 32, wherein the amino acid substitution at amino acid position 31 is selected from the group consisting of: A31R, A31P and A31T.

[0232] Embodiment 34: The method of any of Embodiments 1-33, wherein the contacting of (a) and the contacting of (b) occur sequentially.

[0233] Embodiment 35: The method of any of Embodiments 1-33, wherein the contacting of (a) and the contacting of (b) occur simultaneously or substantially simultaneously.

[0234] Embodiment 36: The method of any one of Embodiments 1-35, wherein the amylose produced in (a) is not purified or isolated prior to the contacting of (b).

[0235] Embodiment 37: The method of any one of Embodiments 1-36, wherein the contacting of (a), the contacting of (b), or both, is performed in vitro.

[0236] Embodiment 38: The method of Embodiment 37, wherein the contacting of (a), the contacting of (b), or both, is performed in a container, vial, jar, test tube, well, plate, or package.

[0237] Embodiment 39: The method of Embodiment 37 or 38, 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.

[0238] Embodiment 40: The method of any one of Embodiments 37-39, 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.

[0239] Embodiment 41: The method of any one of Embodiments 1-40, wherein the contacting of (a), the contacting of (b), or both, is performed in vivo.

[0240] Embodiment 42: The method of Embodiment 41, wherein the contacting of (a), the contacting of (b), or both, is performed in a recombinant host cell.

[0241] Embodiment 43: The method of Embodiment 42, 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.

[0242] Embodiment 44: The method of Embodiment 42 or 43, wherein the recombinant host cell is a microbial cell.

[0243] Embodiment 45: The method of Embodiment 44, wherein the microbial cells are bacterial cells.

[0244] Embodiment 46: The method of any one of Embodiments 1-45, wherein the ratio of β-cyclodextrin to α-cyclodextrin in the composition comprising cyclodextrin is at least 2:1.

[0245] Embodiment 47: The method of any one of Embodiments 1-46, wherein the ratio of β-cyclodextrin to γ-cyclodextrin in the composition comprising cyclodextrin is at least 2:1.

[0246] Example

[0247] Example 1. One-pot synthesis of β- Cyclodextrin.

[0248] In this embodiment, amylosucrase (having an amino acid sequence according to SEQ ID NO: 2) and variant cyclodextrin glucanotransferase (having an amino acid sequence according to SEQ ID NO: 28) were expressed in Escherichia coli and then separated from an insoluble cell debris mixture by centrifugation. The two enzymes were exposed to sucrose at different concentrations (150 g / L, 200 g / L and 250 g / L). In this embodiment, the two enzymes were simultaneously exposed to substrate sucrose. The enzyme was added only once. All reactions were carried out at 45 ° C in 0.1 M sodium citrate buffer. The amount of α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin was measured by HPLC. Figure 4 It was demonstrated that a one-pot synthetic reaction (including amylosucrase and a variant cyclodextrin glucanotransferase) was able to produce β-cyclodextrin at concentrations greater than 18 g / L from sucrose.

[0249] Example 2. One-pot synthesis of β-cyclodextrin from sucrose using variant amylosucrase and variant cyclodextrin glucanotransferase dextrin.

[0250] In this example, different variant amylosucrases were combined with variant cyclodextrin glucanotransferases (having an amino acid sequence according to SEQ ID NO: 28) and exposed to sucrose. In more detail, the different variant amylosucrases used were "R234Q" (having an amino acid sequence according to SEQ ID NO: 3), "R234G" (having an amino acid sequence according to SEQ ID NO: 4), "R234NΔ8AA" (having an amino acid sequence according to SEQ ID NO: 48), "R234A" (having an amino acid sequence according to SEQ ID NO: 5), "R234C" (having an amino acid sequence according to SEQ ID NO: 8) and "R234" (having an amino acid sequence according to SEQ ID NO: 2). Both enzymes were exposed to substrate sucrose simultaneously. In this example, only one enzyme was added. All reactions were carried out at 45° C. in 0.1 M sodium citrate buffer. In all examples, the ratio of amylosucrase to cyclodextrin glucanotransferase was 20: 1. The amounts of α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin were measured by HPLC. Figure 5 It was demonstrated that a one-pot synthetic reaction (including a variant amylosucrase and a variant cyclodextrin glucanotransferase) was able to produce β-cyclodextrin at a concentration greater than 18 g / L from sucrose.

[0251] Example 3. One-pot synthesis of β-cyclodextrin from sucrose using a three-enzyme system.

[0252] In this example, β-cyclodextrin is produced from sucrose using a three-enzyme system (i.e., method step (a) is a two-enzyme method and method step (b) is a single enzyme method). Sucrose phosphorylase (having an amino acid sequence according to SEQ ID NO: 20), α-glucan phosphorylase (having an amino acid sequence according to SEQ ID NO: 24) and cyclodextrin glucanotransferase (having an amino acid sequence according to SEQ ID NO: 28) are expressed in E. coli and then separated from the insoluble cell debris mixture by centrifugation. In this example, all enzymes are exposed to the substrate sucrose at the same time. The enzymes are added only once. In this example, the amounts of sucrose phosphorylase, α-glucan phosphorylase and sucrose remain unchanged, while the amount of cyclodextrin glucanotransferase is varied, as is the reaction time. Figure 6 It is demonstrated that a one-pot synthesis reaction using three enzymes (sucrose phosphorylase having an amino acid sequence according to SEQ ID NO: 20, α-glucan phosphorylase having an amino acid sequence according to SEQ ID NO: 24, and cyclodextrin glucanotransferase having an amino acid sequence according to SEQ ID NO: 28) can produce β-cyclodextrin at a concentration of greater than 1 g / L from sucrose. All reactions were performed at pH 7.4, 0.1 M Tris-HCl, 50°C.

[0253] Example 4. Purification of β-cyclodextrin.

[0254] In this example, a crude composition comprising β-cyclodextrin was produced by a dual enzyme system comprising an amylosucrase (having an amino acid sequence according to SEQ ID NO: 3) and a cyclodextrin glucanotransferase (having an amino acid sequence according to SEQ ID NO: 28), as described herein. The crude composition was purified by the following method.

[0255] The total product titer of the crude composition was 64.2 g / L. A first precipitate was obtained by filtration. The first precipitate was then dissolved by adding 200 mL of 1 M NaOH to obtain a first solution. The first solution was filtered to remove insoluble material and then neutralized with about 30 mL of 6 M HCl to obtain a second solution. The second solution was heated to 60° C. to dissolve the β-cyclodextrin and then the temperature was reduced to 55° C. The solution was seeded by adding 0.5 g of crystalline β-cyclodextrin to the solution. After 30 minutes, the temperature was reduced to 4° C. over 4 hours and kept overnight for about 12 hours. A purified β-cyclodextrin composition was obtained.

[0256] pass 1 H-NMR evaluation of the composition, such as Figure 7 The composition consists only of β-cyclodextrin and water. 1 The purity of β-cyclodextrin was 90.5% as measured by H-NMR analysis.1 No other impurities (except water) were identified by H-NMR or HPLC-ELSD.

[0257] The recovery rate of β-cyclodextrin in the purification process was 80.1% (ie, the amount of purified β-cyclodextrin in the final composition was 80.1% of the amount of β-cyclodextrin in the crude composition). This amount was measured from the dry matter of the composition by HPLC.

[0258] This example demonstrates that the purification method of the present invention provides a purified β-cyclodextrin composition in which the β-cyclodextrin has an advantageously high purity. The recovery rate of the β-cyclodextrin is also improved.

[0259] Example 5. One-pot synthesis of β-cyclodextrin from sucrose using a dual enzyme system in a 10 LSTR.

[0260] In this example, β-cyclodextrin was produced from sucrose in a 5 L reaction using a dual enzyme system comprising amylosucrase (having the amino acid sequence according to SEQ ID NO: 3) and a cyclodextrin glucanotransferase (having the amino acid sequence according to SEQ ID NO: 28).

[0261] In more detail, 266g Na2HPO4 and 20.1g citric acid were added to a 10L stirred tank reactor (STR) containing 4.65L H2O. The solution was stirred until completely dissolved. A total of 1.25kg sucrose was added to the solution, then heated to 45°C, and the pH was adjusted to 7.0 by adding 6M HCl. When the temperature was reached, 330mL of whole cell lysate containing 10.4U / mL amylosucrase was added, followed by 17mL of whole cell lysate containing cyclodextrin glucanotransferase. 400mL of toluene was added after 1 hour. The reactants were stirred for another 6 hours at 160rpm, and then the reactor was cooled to 4°C and kept overnight for about 12 hours.

[0262] The precipitate was collected by vacuum filtration on a sintered funnel and washed with approximately 150 mL of H2O. The solid was then partially dried under vacuum to obtain 678 g of wet crude material (containing 52 wt% β-CD and 6 wt% toluene (by 1 H-NMR measurement), and the filter cake volume was about 990 cm 3 ). The final reaction titer was 67.9 g / L β-cyclodextrin.

[0263] This example demonstrates the scale-up of a one-pot synthesis of β-cyclodextrin from sucrose using a dual enzyme system to produce high yields of β-cyclodextrin.

[0264] Example 6. Purification of β-cyclodextrin from crude precipitate.

[0265] In this example, the crude composition produced by the enzymatic method in Example 5 was purified. Example 5 describes providing a crude composition comprising β-cyclodextrin and obtaining a first precipitate by vacuum filtration and washing with water.

[0266] The first precipitate was then dissolved in 1.7 L of 1 M NaOH to obtain a first solution, which was then passed through The second solution was filtered through a pad of 400 mL of 6 M HCl to obtain a second solution. The second solution was neutralized with about 400 mL of 6 M HCl. The second solution was then heated to 75 ° C until dissolved, and then the temperature was reduced to 70 ° C. 3 g of crystalline β-CD was then added to the second solution as a seed, and allowed to stand for 30 minutes, and then the solution was cooled to 4 ° C over 4 hours. The solution was kept at 4 ° C overnight for about 12 hours. The composition was then collected under vacuum and washed with H2O. The composition was then dried in a vacuum oven at 45 ° C. The HPLC-ELSD and 1 Purity was assessed by H-NMR, as Figure 8 and Fig. 9 shown.

[0267] The composition consists of β-cyclodextrin, water and a trace amount (0.23 wt%) of toluene. 1 No other components were observed by H-NMR. 1 The purity of β-cyclodextrin was 91.7 wt % by H-NMR. HPLC-ELSD or 1 No other impurities were identified by H-NMR. 302 g of β-cyclodextrin were obtained. Therefore, the recovery rate of β-cyclodextrin was 78%.

[0268] This example demonstrates that the purification method of the present invention provides a purified β-cyclodextrin composition with a high β-cyclodextrin recovery rate.

[0269] Example 7. Purification of β-cyclodextrin using acetone as anti-solvent.

[0270] 15 g of a crude composition comprising β-cyclodextrin is provided. 1 The purity was 87% according to H-NMR measurement.The first precipitate was obtained by filtering the crude composition.

[0271] The first precipitate was dissolved in 75 mL of 1 M NaOH to obtain a first solution, which was then filtered to remove insoluble material. A mixture of 10% acetone and 90% water was added, and the second solution was then neutralized with 6 M HCl and cooled to 4 °C for about 2 hours. The second solution was filtered by vacuum filtration, washed with water, and then washed with acetone. The material was dried to obtain 9.0 g of β-cyclodextrin, which was then 1 The purity was 93.7 wt % as measured by H-NMR. Therefore, the recovery rate of β-cyclodextrin was 64.6 %.

[0272] The above purification process was repeated except that a mixture of 50% acetone and 50% water was added. The material was dried to obtain 10.72 g of β-cyclodextrin. 1 The purity was 98.8 wt % as measured by H-NMR. Therefore, the recovery rate of β-cyclodextrin was 81.2 %.

[0273] pass 1 The composition was evaluated by H-NMR. The results are shown in the following table. Fig.10 NMR analysis confirmed that only β-cyclodextrin, water, toluene and acetone were present in the composition containing purified β-cyclodextrin.

[0274]

[0275] This example demonstrates that a purification process using acetone as an anti-solvent can produce a purified β-cyclodextrin composition wherein the β-cyclodextrin has an increased purity and wherein advantageously there is a high recovery of β-cyclodextrin.

[0276] Example 8. Freeze-drying of amylosucrase and cyclodextrin glucanotransferase cell paste and cells containing additives Lysate.

[0277] Lysates and whole cell pastes of amylosucrase (having an amino acid sequence according to SEQ ID NO: 3) and cyclodextrin glucanotransferase (having an amino acid sequence according to SEQ ID NO: 28) containing different additives were freeze dried. In more detail, 0.5%, 1% or 5% 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.

[0278] 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.

[0279] Amylosucrase enzyme activity

[0280] 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.11A and Fig. 11B shown.

[0281] Cyclodextrin glucanotransferase enzyme activity

[0282] 2 μL of cyclodextrin glucanotransferase cell-free lysate was added to 2.998 mL of a solution of soluble starch (10 g / L) in sodium citrate buffer (0.1 M, pH 7, 45°C). The solution was shaken at 1200 rpm for 1 hour. One unit of activity (U / mL) is defined as the amount of enzyme required to produce 1 mmol of β-cyclodextrin per minute at pH 7 and 45°C. This activity was compared with the cooled cell-free lysate that was not freeze-dried, and the results are shown in Figure 2. Fig.12 shown.

[0283] As shown in Figure 11- Fig.12 As shown, adding additives to amylosucrase and cyclodextrin transferase prior to freeze drying improves enzyme stability. In particular, the addition of 5% sucrose, 0.5% mannitol and 0.5% sorbitol was shown to improve the retained enzyme activity of amylosucrase and cyclodextrin glucanotransferase cell lysates and whole cell pastes.

[0284] Example 9. Effect of organic solvents on the synthesis of β-cyclodextrin from sucrose.

[0285] In this example, a variant amylosucrase (having an amino acid sequence according to SEQ ID NO: 3) was combined with a variant cyclodextrin glucanotransferase (having an amino acid sequence according to SEQ ID NO: 28) and exposed to sucrose. Both enzymes were exposed to the substrate sucrose simultaneously. In this example, only one enzyme was added. The reaction volume was 500 mL and standard conditions as described herein were followed. In the absence of an organic solvent, a concentration of about 18 g / L of β-cyclodextrin was obtained.

[0286] Then different examples were performed, with 40, 80 or 160 mL of toluene added to the reaction mixture, respectively. In each of these examples, the addition of toluene resulted in a significant increase in the yield of β-cyclodextrin. Specifically, when the enzymatic reaction was performed in a reaction mixture containing toluene, a β-cyclodextrin concentration of about 70 g / L was obtained.

[0287] The above 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 embodiments 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.

[0288] 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 β-cyclodextrin than α-cyclodextrin, γ-cyclodextrin, or both, relative to a wild-type enzyme capable of converting amylose into cyclodextrin, wherein the cyclodextrin-containing composition comprises β-cyclodextrin, and may optionally further comprise α-cyclodextrin, γ-cyclodextrin or any combination thereof, and The cyclodextrin-containing composition comprises β-cyclodextrin in an amount and / or concentration greater than α-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, For example, 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) a carbon source Cellulomonas T26 amylosucrase, for example, wherein the wild-type amylosucrase comprises the amino acid sequence of SEQ ID NO: 1; or (ii) a polysaccharide Neisseria amylosucrase, for example, wherein the wild-type amylosucrase comprises 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 in combination or together 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 (i) cyclodextrin glucanotransferase from Bacillus sp. strain No. 38-2, Preferably, the Bacillus strain No. 38-2 cyclodextrin glucanotransferase comprises or consists of the amino acid sequence of SEQ ID NO: 25, or (ii) Bacillus circulans strain 251 cyclodextrin glucanotransferase, Preferably, the Bacillus circulans strain 251 cyclodextrin glucanotransferase comprises or consists of the amino acid sequence of SEQ ID NO: 26 or 27, such as SEQ ID NO:

27.

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, preferably at least about 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 25-27.

18. The method of any one of section (ii) of claim 16 to claim 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 an amino acid substitution at amino acid position 31 relative to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 27, and Preferably, wherein the amino acid substitution at amino acid position 31 is selected from the group consisting of: A31R, A31P and A31T.

19. The method of any one of claims 1-18, wherein the contacting of (a) and the contacting of (b) occur (i) sequentially, or (ii) simultaneously or substantially simultaneously.

20. The method of any one of claims 1-19, wherein the amylose produced in (a) is not purified or isolated prior to the contacting of (b).

21. The method of any one of claims 1-20, wherein the contacting of (a), the contacting of (b), or both, is performed in vitro.

22. The method of claim 21, wherein the contacting of (a), the contacting of (b), or both, is performed in a container, vial, jar, test tube, well, plate, or package.

23. The method of claim 21 or 22, 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.

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

25. The method of claim 24, 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 of (a) or at least one enzyme in the enzyme mixture, the variant enzyme of (b), or both, Preferably, the recombinant host cell is a microbial cell, and Preferably, the microbial cells are bacterial cells.

26. The method of any one of claims 1-25, wherein the ratio of β-cyclodextrin to α-cyclodextrin in the cyclodextrin-containing composition is at least 2:1, preferably at least 100:

1.

27. The method of any one of claims 1-26, wherein the ratio of β-cyclodextrin to γ-cyclodextrin in the cyclodextrin-containing composition is at least 2:1, preferably at least 100:

1.

28. The method of any one of claims 1-27, wherein the enzyme or at least one enzyme of the enzyme mixture of (a), the variant enzyme of (b), or both are produced in a Pichia cell.

29. The process of any one of claims 1 to 28, wherein the contacting of (a) and / or the contacting of (b) is performed in a reaction mixture comprising an organic solvent, preferably toluene.

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 immobilized on a resin.

31. The method of any one of claims 1-30, 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.

32. The method of claim 31, wherein the cell paste or whole cell lysate further comprises an additive selected from the group consisting of: PEG, maltose, sorbitol, sucrose, glucose, mannitol, lactose, milk powder, starch, and combinations thereof, Preferably, the additive is selected from the group consisting of mannitol, sorbitol, sucrose and combinations thereof.

33. The method of any one of claims 1-32, wherein the ratio of β-cyclodextrin to α-cyclodextrin, γ-cyclodextrin, or both in the composition is at least 10: 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 and toluene, and wherein the total reaction is performed for no more than 8 hours.

34. A composition comprising cyclodextrin, wherein the cyclodextrin comprises β-cyclodextrin and may optionally further comprise α-cyclodextrin, γ-cyclodextrin, or any combination thereof, and wherein the composition comprising cyclodextrin comprises β-cyclodextrin in an amount and / or concentration greater than α-cyclodextrin, γ-cyclodextrin, or both, and wherein the composition is obtained by the method of any one of claims 1-33.

35. The composition of claim 34, wherein the ratio of β-cyclodextrin to α-cyclodextrin in the composition comprising cyclodextrin is at least 2:1, preferably at least 100:1, And / or wherein the ratio of β-cyclodextrin to γ-cyclodextrin in the cyclodextrin-containing composition is at least 2:1, preferably at least 100:

1.

36. The composition of claim 34 or 35, wherein the percent yield of β-cyclodextrin is at least about 10%, preferably at least about 60%.

37. Use of sucrose for producing β-cyclodextrin, wherein the production method is the method according to any one of claims 1 to 33.

38. Use of one or more enzymes of SEQ ID NO: 1 to SEQ ID NO: 48 for producing β-cyclodextrin, preferably wherein the production method is the method of any one of claims 1-33.

39. An enzyme comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 1-48, 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: 1-48.

40. The enzyme of claim 39, 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 48; (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: 28-30 or 35-47.

41. A method for purifying β-cyclodextrin, the method comprising the following steps: i. providing a crude composition comprising β-cyclodextrin; ii. obtaining a first precipitate comprising β-cyclodextrin from the crude composition; iii. dissolving the first precipitate to obtain a first solution comprising β-cyclodextrin; iv. filtering the first solution to obtain a second solution comprising β-cyclodextrin; v. Crystallizing and / or precipitating the second solution to obtain a purified β-cyclodextrin composition.

42. The method of claim 41, wherein the crude composition of step (i) is a composition obtained by the method of any one of claims 1-33.

43. A purified β-cyclodextrin composition, wherein the purified β-cyclodextrin composition is a composition obtained by the method of any one of claims 41 and 42.

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