Attorney Docket 1410-158253-PC DEGRADATION OF POLYVINYL ALCOHOL AND DERIVATIVES THEREOF USING AN ENZYMATIC CASCADE CROSS REFERENCE TO RELATED APPLICATION [0001] This application claims the benefit of U.S. Provisional Application No.63/422,784, filed on November 4, 2022. REFERENCE TO A SEQUENCE LISTING [0002] The Sequence Listing associated with this application is filed in electronic form via Patent Center and is hereby incorporated into the specification in its entirety. The name of the electronic file containing the Sequence Listing is “1410-158253-US_SL.“ The size of the .xml file is 8 KB, and the file was created on October 31, 2023. FIELD [0003] The present application is directed to degradation of polyvinyl alcohol and derivatives thereof, including polyvinyl alcohol-containing materials used for packaging for comestibles. BACKGROUND [0004] Packaging materials are used to contain and protect a variety of items during storage and transport. Materials such as cardboard, plastics, metal, and glass have historically been used for packaging. However, many packaging materials are deposited in a landfill after use. Non-renewable resources such as petroleum have been used to produce conventional non- biodegradable plastics. Though plastic-based packaging may be desirable due to low cost, packaging materials containing such plastics can persist after disposal for a considerable period of time in the environment. [0005] There has been increasing demand that renewable resources and/or materials that more readily degrade in the environment be used to produce packaging materials. For example, polyhydroxyalkanoate (PHA) films are made from renewable resources, including bacterial Attorney Docket 1410-158253-PC sources. Polyhydroxybutanoate (PHB) films are water-insoluble but easily degraded. Poly(2- hydroxypropanoic acid) (polylactic acid, PLA) films are degradable but have high moisture and oxygen permeability. Further, materials based on bio-renewable and easily degradable cellulose have found recent use. [0006] Polyvinyl alcohol (PVA) is another such degradable polymer. PVA is a water- soluble synthetic vinyl polymer with a carbon-carbon backbone and repeating 1,3-diols. Due to its versatile properties, PVA is used in many industrial applications such as fabric and paper sizing, fiber coating, and film for packaging materials. Although PVA is generally considered a biodegradable polymer, it has become a major pollutant of industrial wastewater due to slow degradation. The printing and dyeing industries, in particular, are major PVA emitters. Treatment of PVA wastewater with chemical or physical methods such as adsorption, filtration ultrasonic degradation, and Fenton’s reaction are cost intensive, less efficient and can cause secondary pollution. [0007] Though PVA is considered a biodegradable polymer, due to the high amount of PVA that is released into the environment and its relatively slow biodegradation, the polymer can still accumulate and impact the environment, such as by inducing hypoxia. Established physical-chemical degradation methods for PVA have several disadvantages such as high price, low efficiency, and secondary pollution. Biodegradation of PVA by microorganisms is slow and frequently involves pyrroloquinoline quinone (PQQ)-dependent enzymes, making it expensive due to the cost of the cofactor and unattractive for industrial applications. BRIEF DESCRIPTION OF THE DRAWINGS [0008] FIG. 1 is a diagram of an enzymatic cascade for the degradation of PVA according to one aspect of the invention. [0009] FIG. 2 is a graph illustrating the degradation of PVA using the Finley method. Depicted is the absorbance at 660 nm for PVA degradation samples. The enzyme cascade contains LK-ADH, CHMO M15, and TL-IM. For the negative control, lysate without overexpressed enzyme was used. Additionally, PVA was incubated with every enzyme from the cascade on its own. Attorney Docket 1410-158253-PC [0010] FIG. 3 is a chart showing the effect of single enzyme treatment versus the cascade on Aquapak Hydropol™ “WWS” PVA film. [0011] FIG. 4A and 4B is a graph illustrating the temperature and pH stability of each enzyme used in the cascade. FIG.4A): The residual activity of TL-IM, LK-ADH and CHMO M15 was determined after incubating the enzyme for one hour at the indicated temperatures. The assay was carried out at 25 °C. FIG.4B): The residual activity was determined after incubating the enzyme at the indicated pH. Before determining the activity, the pH was reset to pH 7. [0012] FIG. 5 is a graph illustrating the effect of the ratio of LK-ADH : CHMO M15 enzymes in the reaction at pH 7 and pH 8 at 30°C and 40°C as analyzed by the Finley method. [0013] FIG. 6A–6F is a series of graphs showing the effect of various conditions (pH (FIG. 6A), temperature (FIG. 6B), amount of co-factor (FIG.6C), reaction time for LK-ADH (FIG.6D), reaction time for CHMO M15 (FIG. 6E), and ratio of ADH to CHMO enzymes (FIG.6F) on the enzymatic degradation of PVA-4 film. [0014] FIG. 7 is the nucleic acid sequence of LK-ADH. [0015] FIG. 8 is the amino acid sequence of LA-ADH. [0016] FIG. 9 is the nucleic acid sequence of CHMO variant 15, with the nucleotide changes compared to wildtype CHMO indicated with underlined capital letters. The His tag and spacer in front of the sequence is shown with underlined italic letters. [0017] FIG. 10 is the amino acid sequence of CHMO variant 15, with the amino acid changes compared to wildtype CHMO indicated with underlined capital letters. The His tag and spacer in front of the sequence is shown with underlined italic letters. [0018] FIGS. 11A, 11B, and 11C illustrate an embodiment of a food sachet. [0019] Elements in the figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions and/or relative positioning of some of the elements in the figures may be exaggerated relative to other elements to help to improve understanding of various embodiments of the present invention. Also, common but well-understood elements that are useful or necessary in a commercially feasible embodiment Attorney Docket 1410-158253-PC are often not depicted in order to facilitate a less obstructed view of these various embodiments of the present invention. Certain actions and/or steps may be described or depicted in a particular order of occurrence while those skilled in the art will understand that such specificity with respect to sequence is not actually required. The terms and expressions used herein have the ordinary technical meaning as is accorded to such terms and expressions by persons skilled in the technical field as set forth above except where different specific meanings have otherwise been set forth herein. DETAILED DESCRIPTION [0020] Polyvinyl alcohol (PVA) is a water-soluble synthetic vinyl polymer with desirable physical properties, including thermostability, such that it is desirable for use in a variety of consumer goods, including packaging materials for comestibles. Though PVA is water-soluble, it biodegrades slowly, and can lead to PVA accumulation in the environment. Methods are provided herein for degrading polyvinyl alcohol (PVA) and derivatives thereof, such as acylated and/or succinylated PVA derivatives, using an enzymatic cascade. Advantageously, the enzymes in the cascade are generally non-toxic and environmentally friendly. [0021] Also provided herein are biodegradable packaging materials, such as for food products, that include PVA-based materials and a combination of enzymes effective to degrade the PVA-based materials in an enzymatic cascade. In one particular approach, the enzymes are effective to degrade PVA-based materials in a pyrroloquinoline quinone (PQQ) independent cascade. In another particular approach, the combination of enzymes provides for in situ cofactor recycling, which makes the enzymatic cascade particularly advantageous for industrial applications and use in packaging materials whereby the provision of only an initial amount of _{cofactor (NAD(P)} ^{+) is required to enable the degradation process to take place.} [0022] PVA can be made by polymerizing vinyl acetate and hydrolyzing the acetyl ester groups on polyvinyl acetate to introduce alcohol groups. PVA may be fully hydrolyzed, where virtually all acetate groups have been converted to alcohol groups. PVA may also be partially hydrolyzed, meaning that some acetate groups remain after hydrolysis. The reaction can be controlled to provide the desired degree of hydrolysis. Both fully hydrolyzed and partially Attorney Docket 1410-158253-PC hydrolyzed polymers are commonly referred to as “PVA”. For example, various PVA polymers can have hydrolysis degrees of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or hydrolysis degrees ranging from 70% to 100%, 75% to 99%, 80% to 98%, 85% to 97%, 90% to 96%, 91% to 95%, 93% to 95%, or 92% to 94%. It is thought that polyvinyl alcohols having higher degrees of hydrolysis have higher water solubilities when compared to polyvinyl alcohols having relatively lower degrees of hydrolysis. [0023] As used herein, for ease in describing the present method, the term “PVA” refers to both “unmodified” and “modified PVA” as defined herein unless the context is clear that only one type of PVA is being referred to. [0024] As used herein, “unmodified PVA” refers to fully hydrolyzed PVA polymers (including polymer units with hydroxyl groups) and partially hydrolyzed PVA polymers (including one or more polymer units with hydroxyl groups and one or more polymer units with acetate groups), as indicated below. The number and relative distribution of the polymer units may be selected to provide the desired molecular weight in the final product, such as a film suitable for use in packaging. [0025] The term “modified PVA” or “PVA derivative” or similar term refers to other modified forms of fully or partially hydrolyzed PVA, such as polyvinyl polymers including a polyvinyl chain comprising one or more of units that increase the lipophilicity of the polyvinyl polymer, such as one or more of a unit comprising an ester moiety, a unit comprising an ether moiety, a unit comprising a carboxylic acid moiety, and a unit comprising an ester moiety and a Attorney Docket 1410-158253-PC carboxylic acid moiety. In some aspects, a modified polyvinyl polymer can include units where one or more of a hydroxyl moiety and an acetate moiety of a polyvinyl alcohol have been modified with more or more reactants. [0026] In one aspect, the modified polyvinyl polymer can include a polyvinyl chain comprising one or more units according to formula (A) and optionally one or more units of formula (B) and optionally one or more units of formula (C): , where R represents any one or more of a moiety including a carboxylic acid group, and a moiety including a carboxylic acid ester group. The number of units is selected to provide the desired molecular weight for the resulting polymer. In one aspect, the (A) units, optional (B) units, and optional (C) units may be randomly distributed in the polymer. [0027] In another aspect, at least a fraction of Rs represent a moiety including a carboxylic acid ester group. [0028] In yet another aspect, at least a fraction of the Rs independently represent: , wherein X represents a divalent group. In the divalent group has 2 to 6 carbon atoms. Attorney Docket 1410-158253-PC [0029] In another aspect, at least a fraction of the Rs represent . [0030] In yet another aspect, R independently represents a moiety derived from succinic anhydride or vinyl acetate, and at least a fraction of the Rs represent a moiety derived from succinic anhydride. Exemplary succinylated and acetylated PVA polymers are illustrated below. For example, acetylated PVA polymers may include those with residual acetyl ester groups remaining after partial hydrolysis of polyvinyl acetate to form partially hydrolyzed PVA. [0031] include one or more crosslink units having the following formula (I): (I) Attorney Docket 1410-158253-PC [0032] In formula (I) above, X is a divalent group, and a polyvinyl chain of the modified polyvinyl polymer includes unit (D) indicated with brackets. The divalent group X can generally have 2 to 6 carbon atoms. In some forms, the polyvinyl chain of the modified polyvinyl polymer includes both unit (D) and unit (E) indicated with brackets. In other forms, the polyvinyl chain of the modified polyvinyl polymer includes unit (D), a polyvinyl chain of a separate polymer includes unit (E), and the polyvinyl chain of the separate polymer is derived from polyvinyl alcohol. In some aspects, one of the following conditions is met for a crosslink unit of one or more crosslink units: the polyvinyl chain of the modified polyvinyl polymer includes unit (E), or a polyvinyl chain of a separate polymer includes unit (E), and the polyvinyl chain of the separate polymer is derived from polyvinyl alcohol. In some forms, a modified polyvinyl polymer can include one or more units according to formula (II) and one or more units according to formula (I). [0033] In some aspects, a polyvinyl chain of a modified polyvinyl polymer can include one or more crosslink units having the following formula (II): [0034] In formula (II), a polymer includes unit (D) indicated with brackets. In some forms, the polyvinyl chain of the modified polyvinyl polymer includes both unit (D) and unit (E) indicated with brackets. In other forms, the polyvinyl chain of the modified polyvinyl polymer includes unit (D), a polyvinyl chain of a separate polymer includes unit (E), and the polyvinyl chain of the separate polymer is derived from polyvinyl alcohol. In some aspects, one of the following conditions is met for a crosslink Attorney Docket 1410-158253-PC unit of one or more crosslink units: the polyvinyl chain of the modified polyvinyl polymer includes unit (E), or a polyvinyl chain of a separate polymer includes unit (B), and the polyvinyl chain of the separate polymer is derived from polyvinyl alcohol. [0035] In one approach, the molecular weight of the PVA or modified PVA, prior to degradation with the enzymatic cascade can include any molecular weight, but for degradation of PVA-based packaging films in particular, the molecular weight may be from about 50,000 g/mol to about 250,000 g/mol, in another aspect about 50,000 g/mol to about 200,000 g/mol, in another aspect about 75,000 g/mol to about 175,000 g/mol, in another aspect about 75,000 g/mol to about 150,000 g/mol, and in another aspect about 75,000 g/mol to about 125,000 g/mol. [0036] In one aspect, the enzyme cascade and method provided herein are effective to degrade modified PVA and/or unmodified PVA in a one pot reaction and may advantageously contain an in vitro cofactor recycling system. [0037] In one approach, the method described herein includes degradation of PVA using a combination of at least a lipase, an alcohol dehydrogenase, and a Baeyer-Villiger monooxygenase. The present approach is a pyrroloquinoline quinone (PQQ)-independent enzymatic cascade. Further, instead of requiring addition of PQQ, the present method allows for in situ cofactor of NAD(P)H recycling while degrading PVA. Enzyme effective to remove the R groups from PVA [0038] When modified PVA (such as with succinyl side chains) is the starting material, ester bonds in the derivatized side chains are hydrolyzed to remove the side chains (e.g., R groups shown below as OAc or OSuc) to provide unmodified PVA.

Attorney Docket 1410-158253-PC [0039] any to act on ester may one particular approach, the enzyme is an esterase or a lipase. Suitable esterases include carboxylesterases, serine‐hydrolases, and other esterases in EC 3.1.1.1. Suitable lipases include those in EC 3.1.1.3. [0040] In one approach, the lipase is a 1,3-specific lipase, such as Lipase TL-IM from Novozymes (Bagsværd, Denmark) originating from Thermomyces lanuginosus. TL-IM accepts substrates with bulky side chains. Though TL-IM is an immobilized enzyme, non-immobilized enzymes may also be used. Enzyme effective to oxidize hydroxyl groups of the PVA to ketones [0041] When the starting material includes unmodified PVA (i.e., no step “1” was needed) or modified PVA has already been treated according to step 1 to provide unmodified PVA, the next step is to oxidize the hydroxyl groups to ketones.

Attorney Docket 1410-158253-PC [0042] An enzyme effective to oxidize the hydroxyl groups to ketones is used. If not included in the initial reaction components, cofactor NAD(P) ⁺ may be added. In a further aspect, the enzyme may be effective to utilize the cofactor NAD(P) ⁺ to form NAD(P)H. [0043] In one approach, the ketones may be formed by an oxidoreductase, such as an alcohol dehydrogenase (ADH). The enzyme selected should be able to be active on a large substrate such as the large molecular weight PVA polymers and derivatives. [0044] In a particular aspect, the alcohol dehydrogenase may be derived from Lactobacillus kefir (LK-ADH). Enzyme effective to oxidize ketones of the PVA to esters [0045] In the next step, the ketone groups are then oxidized to esters. An enzyme effective to oxidize ketone groups of the PVA to esters is needed. [0046] may a monooxygenase . In one particular aspect, the BVMO may be a cyclohexanone monooxygenase (CHMO), such as variant M15 originally obtained from Acinetobacter calcoaceticus. CHMO M15 utilizes co-factor NAD(P)H with formation of NAD(P) ⁺ . This leads to an advantageous in situ cofactor recycling. Attorney Docket 1410-158253-PC [0047] Other enzymes that are effective to oxidize the ketone groups to esters may also be used. In particular, an enzyme that is also effective to utilize the cofactor NAD(P)H to form NAD(P) ⁺ may be preferred in some approaches. Enzyme effective to hydrolyze ester bonds to create fragments of the PVA [0048] In the next step (and, optionally, the final step), the newly formed ester bond is cleaved by hydrolysis. An enzyme effective to hydrolyze the newly formed ester bonds of the PVA and create smaller fragments of the PVA is needed. [0049] In one approach, the enzyme may be the same enzyme as used for step 1 above. In principle, any hydrolase able to act on ester bonds may be used. In one particular approach, the enzyme is an esterase or a lipase. Suitable esterases include carboxylesterases, serine‐ hydrolases, and other esterases in EC 3.1.1.1. Suitable lipases include those in EC 3.1.1.3. Attorney Docket 1410-158253-PC [0050] In one approach, the lipase is a 1,3-specific lipase, such as Lipase TL-IM from Novozymes (Bagsværd, Denmark) originating from Thermomyces lanuginosus. TL-IM accepts substrates with bulky side chains. Though TL-IM is an immobilized enzyme, non-immobilized enzymes may also be used. [0051] In another approach, a different enzyme may be included than that used in step 1. Other exemplary enzymes include cutinases (EC class 3.1.1.74), as well as proteases and peptidases that are known to act on esters. For example, a Subtilisin (protease) or chymotrypsin may be used. Cascade [0052] By carrying out the cascade in this order, the method can generate small fragments (oligomers) of PVA. The small fragments of PVA produced by the present cascade and method are more accessible to further degradation, such as by exposure to environmental conditions (e.g., UV light, moisture, and microorganisms). [0053] Further, when the enzyme cascade is carried out in a manner effective to recycle the cofactor NAD(P)H, the cascade is self-sustaining through the described steps without further input of cofactors after the initial provision of the cofactor. This feature of the cascade makes it particularly advantageous for industrial applications and for inclusion in packaging materials. [0054] The enzymes used in the cascade can be added at the same time or sequentially, such as in a one-pot reaction vessel. It has been found that the enzymes may be added at the same time without any of the enzymes interfering with the activity of the other enzymes. [0055] An exemplary enzyme cascade includes four steps and is provided in FIG.1 for modified PVA but can also be applied as a three-step process for unmodified PVA (beginning at step 2 in FIG.1). As steps 1 and 4 may both utilize a lipase, the lipase may be the same or different enzymes. To reduce costs, it may be advantageous to utilize the same lipase for both steps of the process. Attorney Docket 1410-158253-PC [0056] Each enzyme of the exemplary process of FIG.1 has been well characterized and may be recombinantly expressed in Escherichia coli or other bacteria, yeast, fungi, or other suitable production host as known to a person skill in the art, and/or is commercially available. [0057] In some approaches, it has been found to be advantageous to include the enzyme effective to oxidize hydroxyl groups of the PVA and the enzyme effective to oxidize ketones in a ratio of about 1:10 to about 10:1, in another aspect 1:4 to about 4:1, in another aspect about 1:3 to about 3:1, in another aspect about 1:2 to about 2:1. In another aspect, the enzyme effective to oxidize hydroxyl groups of the PVA and the enzyme effective to oxidize ketones in a ratio of about 1:1. [0058] Treatment with the enzyme cascade results in the breakdown of PVA to smaller polymer fragments or oligomers. These smaller fragments are more accessible to further degradation, such as by microorganisms in the environment or ultraviolet light from the sun, or may serve as building blocks for upcycling of the resulting material. [0059] In one aspect, the enzyme cascade may be used in wastewater treatment to degrade PVA into small enough fragments that the products formed can be more readily degraded by ultraviolet light and microorganisms. [0060] In another aspect, the enzyme cascade can be incorporated into PVA-containing materials for increasing the rate of degradation of the materials. In one approach, the enzymes of the cascade can be incorporated into PVA-containing films used to prepare packaging materials. [0061] Packaging materials made with PVA can include a single layer of film or, in another aspect, two or more layers of film. Similarly, the PVA may be layered with other materials such that the PVA may be an inner layer, an outer layer, and/or an intermediate layer. Further, multiple layers of the PVA may be used by itself or in combination with other layers. [0062] In one approach, enzymes of the cascade may be incorporated into one or more film layers. In another approach, enzymes of the cascade may be incorporated between layers of film. In yet another approach, enzymes of the cascade may be incorporated into a coating Attorney Docket 1410-158253-PC applied to one or more layers of film. In some aspects, enzymes of the cascade are provided in dried form. The enzymes may also be added to the packaging materials in encapsulated form. [0063] In some aspects, packaging including PVA and the enzymes can contain food such as condiments (e.g., ketchup, mayonnaise, mustard, relish, ponzu sauce, oil, vinegar, tartar sauce, fry sauce, and soy sauce), salad dressing, cheese, vegetables, soup, or meat. Examples of food packaging include pouches and sachets. [0064] FIG. 11A illustrates an embodiment of a food sachet comprising a sachet body 2 having seals 4 on three sides. The sachet may be formed by folding a film and sealing three of the sides. FIG. 11B illustrates a cross-section of the food sachet in FIG.11A taken along line A— A. In FIG.11B, a first biodegradable film 6 comprises a modified polyvinyl polymer and contacts a comestible item 8 (e.g., ketchup) disposed inside the sachet body. A second biodegradable film 10 includes an unmodified polyvinyl alcohol polymer and forms an exterior of the sachet body. [0065] FIG. 11C illustrates an enlarged portion of the cross section of the embodiment of the food sachet shown in FIG.11B, taken at box B. An enzyme composition 12 is disposed between a film layer 6 and a film layer 10. Upon contact with environmental influences, such as one or more of moisture, soil, sunlight, and microorganisms, the enzyme composition aids the degradation of the discarded sachet. [0066] The enzymes of the cascade can be included in packaging including PVA or PVA derivatives to further enhance degradation of the packaging upon contact with environmental conditions, such as one or more of moisture, soil, and sunlight. [0067] Though it is desirable for enzymes of the cascade to increase degradation of PVA or PVA derivatives upon disposal of the packaging, the enzymes should not cause significant degradation of the PVA or PVA derivatives during the shelf life of a food product contained in the packaging. In one aspect, the packaging materials are able to contain an aqueous food product for a desired shelf life while also being biodegradable and/or water soluble upon disposal. Attorney Docket 1410-158253-PC EXAMPLES Example 1 [0068] PVA films bearing succinylated hydroxyl groups were obtained. The films were synthesized according to the general protocol shown in Scheme 1 below. (Scheme I) Attorney Docket 1410-158253-PC [0069] Varying amounts of succinic anhydride (2g, 4g, and 6g per 30g PVA) were used. In this example, the modified PVA is referred to as “PVA-2” (2 g succinic anhydride), “PVA-4” (4 g succinic anhydride), and “PVA-6” (6 g succinic anhydride). [0070] Solid succinic anhydride (2 g, 4 g, or 6 g) was added to a solution/suspension of PVOH-418 (30 g in ca.300 mL deionized water) and vigorously stirred at 80 °C for 5 hours. PVOH-418 (a medium hydrolyzed PVOH commercially available from Aquapak Polymers Ltd., Birmingham, UK). The set-up was comprised of a Schott glass bottle (500 mL volume containing a 2 cm magnetic stir bar) and an IKA hotplate with magnetic stirring. Within 1 hour, a clear solution was obtained. This material was then poured without prior cooling into plastic storage boxes (ca.40 x 20 cm) aiming for 25% of solution per box (usually 3 on this scale). After two days of evaporation in a ventilated space (i.e., a fume hood), films were peeled off and stored between paper to avoid curling. [0071] Thin polymer films were obtained that could be easily peeled and used for further analysis. The films had the following thicknesses (average gauge): PVA-2: 42 µm; PVA-4: 141 µm, and PVA-6: 95 µm. [0072] Infrared spectroscopy was used to confirm a distinct change in the carbonyl stretching region (i.e., 1600–1800 cm ^-1 ), which is in agreement with the introduction of succinate moieties; i.e., 1714 cm ^-1 for unmodified PVA vs 1703 cm ^-1 for succinate hybrid (data not shown). Example 2 Expression of Enzymes (LK-ADH and CHMO M15) [0073] Alcohol dehydrogenase from Lactobacillus kefir (LK-ADH) as well as the cyclohexanone monooxygenase (CHMO) variant M15 (Opperman, D. J. et al., Towards practical Baeyer–Villiger-monooxygenases: Design of cyclohexanone monooxygenase mutants with enhanced oxidative stability, ChemBioChem 11, 2589–2596 (2010)), originally from Acinetobacter calcoaceticus, were recombinantly expressed in E. coli BL21. [0074] The nucleic acid and amino acid sequences for LK-AKH (GenBank: MW808990) are provided in FIG.7 (SEQ ID NO.1) and FIG.8 (SEQ ID NO.2), respectively. Attorney Docket 1410-158253-PC [0075] The nucleic acid and amino acid sequences for CHMO variant 15 (GenBank: M19029.1), variant M15: M5I/M291I/C330S/C376L/M400L/M412L/M481A/C520V) are provided in FIG.9 (SEQ ID NO.3) and FIG.10 (SEQ ID NO.4), respectively. In FIG.9, nucleotide changes compared to wildtype CHMO are in capital letters and marked in underline; His tag and Spacer in front of the sequence are in italics and underlined. In FIG.10, amino acid changes compared to wildtype CHMO are marked in underline; His tag and Spacer in front of the protein sequence are in italics and underlined. [0076] E. coli BL21 cells were grown in lysogeny broth medium including 50 µg/L kanamycin overnight at 37°C and 220 rpm. The main culture was grown in terrific broth medium including 50 µg/L kanamycin and in the case of LK-ADH addition of 1 mM MgCl ₂ . Inoculation was done with 1% of the main culture. Cells containing the plasmids for CHMO M15 were induced by autoinduction by adding 0.2% lactose and 0.05% glucose to the culture medium. The main culture was incubated for 3 hours at 37°C and afterwards for 21 hours at 21 °C. [0077] Cells containing the plasmid for LK-ADH were grown at 37°C until an OD ₆₀₀ of 0.5 and afterwards the enzyme expression was induced by adding 1 mM IPTG to the culture. The subsequent incubation took place at 21°C for 21 hours. [0078] Afterwards, cells were harvested by centrifugation at 10,000 ×g for 5 min, pellets were washed with 50 mM sodium phosphate buffer and the obtained pellets were stored at - 20°C until further usage. [0079] The pellet of 50 mL main culture was resuspended in 10 mL 50 mM sodium phosphate buffer (pH 7) and lysed by sonication (3x1.5 min, 50% pulse) with SONOPULS HD 2070 (BANDELIN electronic GmbH & Co. KG, Berlin, DE). The soluble fraction from the crude cell lysate was separated by centrifugation at 10,000 ×g for 30 min followed by filtration through a 0.2 µm membrane before usage. [0080] Lipase Thermomyces lanuginosus immobilized on silica gel (Lipozyme TL-IM) was purchased from Novozymes (Bagsværd, Denmark). Attorney Docket 1410-158253-PC Example 3 Degradation of PVA by enzymatic cascade [0081] Degradation of PVA can be measured by staining with iodide. According to the Finley method, PVA forms a green complex with iodide in the presence of boric acid with an absorption maximum at 660 nm (see Finley, J. H. Spectrophotometric determination of polyvinyl alcohol in paper coatings. Anal. Chem.33, 1925–1927 (1961)). Changes in the staining of PVA due to degradation of the polymer can thus be detected by absorbance measurements. [0082] Lipase (from Thermomyces lanuginosus) immobilized on silica gel (Lipozyme TL-IM) was purchased from Novozymes (Bagsværd, Denmark). [0083] PVA-4 film prepared according to Example 1 was incubated with crude cell lysates containing LK-ADH and CHMO M15 prepared according to Example 2, as well as the commercially available immobilized TL-IM, in a one pot reaction. As a negative control, cell lysate without overexpressed enzyme was used to exclude an influence of the lysate on the polymer. [0084] Specifically, the PVA-4 film prepared according to Example 1 was dissolved at 50°C in 10 mM sodium phosphate buffer with a concentration of 40 mg/mL and the pH adjusted to 8.5. Dissolving the polymer took approximately 10 minutes. [0085] The PVA samples were separately incubated with each enzyme and also with the enzyme cascade: LK-ADH, CHMO M15, and lipase TL-IM. The final reaction contained 20 mg/mL PVA. [0086] 100 µL LK-ADH-lysate and 100 µL CHMO M15-lysate, 2 mg of immobilized lipase TL IM, and 1 mM NADP ⁺ were added to 250 µL of the PVA stock solution and filled up to 0.5 mL with 10 mM sodium phosphate buffer (pH 8.5). E. coli BL21 with an empty vector was used as a negative control. [0087] 100 µL of LK-ADH lysate contains approximately 5.3 U, 100 µL of CHMO M15 lysate contains approximately 0.9 U. “U” was defined as µmol of converted substrate per minute. The determination of U was done following the increase or decrease in NADP(H) using (R)-1-phenyl ethanol (LK-ADH) or cyclohexanone (CHMO M15) as substrates. Attorney Docket 1410-158253-PC [0088] About 20 mg/mL film was added to the buffer. Reactions were incubated in a time frame of 24–72 hours at 30°C and 1000 rpm. Fresh LK-ADH and CHMO M15 enzyme was added every 24 hours and in the case of the negative control, cell lysate from the expression of an empty vector. Reactions were performed in triplicates unless stated otherwise. [0089] For the determination of PVA concentration, a modified Finley method according to Mohamed et. al. was used (Biodegradation of poly (vinyl alcohol) by an orychophragmus rhizosphere-associated fungus Penicillium brevicompactum OVR-5, and its proposed PVA biodegradation pathway, World J. Microbiol. Biotechnol.38, 10 (2021)). This method colorimetrically quantifies the green complex formed by PVA with iodide in the presence of boric acid. Changes in the amount of PVA per sample due to degradation of the polymer can be detected by absorbance measurements. Analysis regarding the molecular weight of the remaining PVA after degradation was done by gel permeation chromatography (GPC). [0090] A potassium iodide solution was prepared from 2.5 g/L KI and 12.7 g/L I ₂ .40 µL of potassium iodide solution were mixed with 60 µL of boric acid (4 g/L) and 40 µL of sample. Using a microplate reader (Infinite® M200 pro, Tecan Group Ltd. (Männedorf, Switzerland)) the absorbance of every sample was measured at 660 nm. [0091] As can be seen in FIG.2, it was observed that the addition of the enzyme cascade to the reaction caused a decrease in absorbance at 660 nm compared to the negative control (cell lysate from the expression of an empty vector). [0092] The first indication of PVA degradation was already observed after 24 hours of incubating the polymer with the cascade. Lipase alone had the smallest effect on absorbance. LK-ADH and CHMO M15 alone caused a high decrease in absorbance, indicating that these enzymes already influenced the PVA outside of an enzymatic cascade. Hence, it can be concluded that oxidized PVA is less stable compared to untreated PVA. [0093] The largest difference in absorbance was observed when the whole cascade with all three enzymes was incubated with the polymer confirming that the cascade with all three enzymes most efficiently degrades the decorated PVA. Attorney Docket 1410-158253-PC Example 4 [0094] In this experiment, it was investigated whether the individual enzymes can degrade PVA outside of the cascade. Degradation of the samples were then evaluated using the modified Finley method of Example 3. [0095] For this purpose, a commercially available warm-water soluble (“WWS”) Hydropol™ PVA film sample from Aquapak Polymers Ltd. was used as a substrate. WWS has a molecular weight of 68,000 (Mw/Mn = 2.2). The WWS sample is an unmodified PVA (with some degree of hydrolysis). [0096] The WWS film from Aquapak was incubated with either lipase, alcohol dehydrogenase (“ADH”), or Baeyer-Villiger monooxygenase plus the respective cofactor. The reaction conditions and enzyme concentrations were as follows: 200 µL LK-ADH-lysate and 200 _{µL CHMO M15-lysate, 2 mg of immobilized lipase TL IM, and 1 mM NADP} ^{+ were added to 500} µL of the PVA stock solution and filled up to 1 mL with 50 mM sodium phosphate buffer (pH 7). The film was included at 10 mg/mL PVA for 24 hours incubation. [0097] Degradation of the samples were evaluated using the Finley method, as described in Example 3 at 670 nm. The results are shown in FIG.3. The absorption of WWS without addition of enzyme, after carrying out the coloring reaction, was comparable to WWS treated with lipase. Hence, the lipase itself had no effect on PVA since the lipase is only active in the last step of the cascade for unmodified PVA and therefore depends on prior reaction of both ADH and BVMO on PVA. [0098] For PVA that was treated with ADH, less absorption was detected, indicating that ADH acted on the polymer. In the cascade for undecorated PVA, ADH is responsible for the first step in degradation of PVA. Surprisingly, also BVMO seemed to affect PVA. A possible explanation for this could be that the PVA does not only contain hydroxyl groups but also ketones which can be oxidized to esters without the prior reaction of the ADH. [0099] The lowest absorbance was measured for the PVA treated with the whole enzyme cascade, indicating that at least two enzymes are needed for the degradation of PVA. Attorney Docket 1410-158253-PC Example 5 Characterization of enzymes regarding temperature and pH stability [00100] To better assess industrial application of the enzymatic cascade, characterization of the enzymes regarding temperature stability and pH stability was performed. [00101] Enzymes were incubated at different temperatures (4°C, 20°C, 30°C, 40°C, and 50°C) as well as at different pH values (pH 2, 3, 4, 5, 6, and 7) for one hour. [00102] In this assay the cell lysis was done in 1 mM NaPi + 100 mM NaCl, and the final reaction was performed in 100 mM NaPi + 80 mM NaCl. [00103] The pH of the enzyme solution was lowered to pH 6, pH 5, pH 4, pH 3, and pH 2 by addition of 100 mM NaPi (without NaCl) and incubated for 1 hour at room temperature. The final buffer concentration during the incubation was 10 mM NaPi and 90 mM NaCl. [00104] After incubation, the pH of the solution was brought back up to pH 7 with 1 M NaPi (pH 7). The final buffer concentration after the incubation was therefore 100 mM NaPi and 80 mM NaCl. [00105] Approximately 0.025 U (LK-ADH) and 0.045 U (CHMO M15) were used for the assay. [00106] After incubation, the residual activity of each enzyme was determined spectrophotometrically at 25°C and pH 7 using different substrates. [00107] In the case of the lipase TL-IM, the formation of para-nitrophenol (pNP) from para- nitrophenyl acetate (pNPA) was monitored. For this purpose, 2 mg of TL-IM were mixed to 900 µL of 50 mM sodium phosphate buffer (pH 7), followed by adding 100 µL pNPA (10 mM in dimethyl sulfoxide (DMSO)) to start the reaction with a final substrate concentration of 1 mM. After one minute of reaction, the amount of pNP (extinction coefficient: ε = 17,500 M ^-1 cm ^-1 ) was determined at 410 nm in a microplate reader. [00108] For LK-ADH and CHMO M15, the increase or decrease of NADPH levels was followed at 340 nm. Reactions were performed in a similar way as described for LK-ADH by Attorney Docket 1410-158253-PC Becker et. al., Appl. Microbiol. Biotechnol.105, 4189–4197 (2021). Prior to the reaction, LK-ADH lysate was diluted 1:10. (R)-1-phenylethanol (LK-ADH) and cyclohexanone (CHMO M15) were used as substrates.20 µL of (diluted) lysate were mixed with 0.5 mM NADP(H) and 1 mM of the respective substrate in a total volume of 200 µL. [00109] As can be seen in FIG.4A, incubating the lipase TL-IM at the indicated temperatures ranging from 4 to 50°C had no effect on its activity. TL-IM retained nearly 100% residual activity over the whole temperature range. In comparison, after one hour of incubation at 50°C, both LK-ADH and CHMO M15 activity decreased to 20%. Incubating LK-ADH at 40°C had no effect on the activity. CHMO M15, on the other hand, retained only 44% of its activity. The activity of CHMO M15 significantly decreased at temperatures over 30°C. These results are in agreement with the enzyme description by the TL-IM manufacturer (Novozymes). According to the manufacturer, TL-IM has a temperature optimum of 50–75°C, which supports the observation that TL-IM is more stable than LK-ADH and CHMO M15. Accordingly, it is presently believed that steps 2 and 3 of the four-step PVA degradation cascade are the most crucial. [00110] As can be seen in FIG.4B, the pH stability test revealed that all enzymes were stable for one hour in the pH range of 3 to 7. Incubation at pH 2 resulted in a lack of activity of LK- ADH while CHMO M15 and TL-IM activity were reduced to 63 and 68%, respectively. [00111] Accordingly, it was found that the reaction can be run efficiently at a temperature up to 40°C and a pH down to pH 3 for at least one hour. Example 6 [00112] To improve cofactor recycling during the cascade, reactions were investigated at varied enzyme ratios, particularly ratios of LK-ADH and CHMO M15. Ratios of 1:1, 1:2, 1:4, and 2:1 (LK-ADH : CHMO M15) were used. Temperatures of 30°C and 40°C, as well as pH 7 and pH 8, were all examined in a 50 mM sodium phosphate buffer. About 10 mg/mL PVA-4 film (from a 20 mg/mL stock solution) was added to the buffer. Attorney Docket 1410-158253-PC [00113] Reactions were incubated for 24 hours and then analyzed using the Finley method as described in Example 3. As shown in FIG.5, it was determined that a 1:1 ratio of LK-ADH and CHMO M15 performed best for PVA breakdown, regardless of pH or temperature. Comparing between samples with a ratio of 1:1, for the sample incubated at pH 7 and 30°C, the smallest absorbance was detected and, hence, the highest degradation of PVA. Example 7 [00114] In this further set of experiments, the reaction conditions were evaluated with respect to pH, temperature, reaction time, the ratio of ADH to CHMO and the amount of co- factor. The best conditions were found to be pH 8.5, 20 °C, 24 hours with 4 mM co-factor NADP ⁺ . The activity of LK-ADH was measured by following the increase of NADPH when converting (R)-1-phenyl ethanol. The activity of CHMO M15 was measured by following the decrease of NADPH when converting cyclohexanone. One Unit (U) was defined as the amount of substrate converted per minute by one µL of enzyme lysate. pH [00115] First, the pH of the reaction was varied in a range from 7 to 9 and, based on the results of Example 5, different temperatures for the cascade reaction were studied in a range from 20°C to 35°C. The PVA-4 film was added to the reaction mixture with the following conditions: 100 µL LK-ADH lysate, 100 µL CHMO M15 lysate, 2 mg TL-IM, 250 µL PVA (final concentration 20 mg/mL), filled up to 0.5 mL with 10 mM sodium phosphate buffer, pH 8.5. The reaction mixture was incubated for 24 hours at 20°C. [00116] The samples were then evaluated by the modified Finley method described in Example 3. As can be seen in FIG.6A and 6B, it was found that the enzymatic degradation of PVA-4 ideally takes place at pH 8.5 and 20 °C under the tested conditions. Time Dependent Enzyme Stability [00117] Further, the time dependent stability of LK-ADH and CHMO M15 was investigated at 20°C and pH 8.5 over 24 hours by following the increase or decrease of NADPH using the substrates (R)-1-phenyl ethanol (LK-ADH) or cyclohexanone (CHMO M15) (FIGS.6D and 6E). Attorney Docket 1410-158253-PC [00118] The activity of enzymes was tested in a 96-well plate. The total reaction volume was 200 µL containing 20 µL of enzyme lysate in the appropriate dilution (1:40 for LK-ADH, 1:5 for CHMO M15), 0.5 mM of co-factor (NADP+ for LK-ADH and NADPH for CHMO M15) as well as 2 mM of the substrates (R)-1-phenyl ethanol (LK-ADH) or cyclohexanone (CHMO M15) in 10 mM NaPi buffer. The reaction was started by the addition of the co-factor and the increase (LK- ADH) or decrease (CHMO M15) of NADPH measured on a Tecan Plate Reader at 340 nm. [00119] For LK-ADH and CHMO M15, the increase or decrease of NADPH levels was followed at 340 nm. Reactions were performed in a similar way as described for LK-ADH by Becker et. al., Appl. Microbiol. Biotechnol.105, 4189–4197 (2021). Prior to the reaction, LK-ADH lysate was diluted 1:10. (R)-1-phenylethanol (LK-ADH) and cyclohexanone (CHMO M15) were used as substrates.20 µL of (diluted) lysate were mixed with 0.5 mM NADP(H) and 1 mM of the respective substrate in a total volume of 200 µL. [00120] While CHMO M15 retained 94% of its activity for the duration of the experiment, the activity of LK-ADH decreased by 20% in 24 hours at 20°C. [00121] Accordingly, it was concluded that the degradation of PVA by the enzyme cascade can be performed at 20°C over 24 hours without a major loss of enzymatic activity. Amount of Co-factor [00122] Further, the optimal amount of co-factor for the reaction was investigated. Since the reaction contains an in situ co-factor recycling system and, in step 2, NADP ⁺ is firstly required by LK-ADH for the formation of ketones, only NADP ⁺ was added to the reaction using PVA-4 as the film. The reaction conditions were as follows: NADP+ was added at 0.5 mM, 1, mM, 2 mM, 4 mM, or 16 mM, 100 µL LK-ADH lysate, 100 µL CHMO M15 lysate, 2 mg TL-IM, 250 µL PVA (final concentration 20 mg/mL), filled up to 0.5 mL with 10 mM sodium phosphate buffer, with pH 8.5. The reaction was incubated at 24 hours at 20°C. [00123] The results are presented in FIG.6C. It was found that the efficiency of the reaction increases with increases of NADP ⁺ up to 4 mM. However, the reaction efficiency did not improve when more than 4 mM NADP ⁺ were added to the enzyme cascade. Attorney Docket 1410-158253-PC Enzyme Ratios [00124] Further ratios of ADH to CHMO were investigated for optimizing the reaction conditions for the degradation of PVA-4 film. The reaction conditions were as follows: 4 mM NADP ⁺ , 2 mg TL-IM, 250 µL PVA (final concentration 20 mg/mL), filled up to 0.5 mL with 10 mM sodium phosphate buffer, pH 8.5. The reaction mixture was incubated for 24 hours at 20°C. From the Finley method, carried out as described in Example 3, it was found that the enzyme ratios of 1:1 or 2:1 for ADH to CHMO are most favorable for the enzymatic reaction (FIG.6E). ^[00125] It is presently believed that the enzyme ratio and the amount of co-factor are most likely interdependent. The investigation of the enzyme ratio was performed with 4 mM NADP ⁺ whereas the investigation of the optimal amount of co-factor was carried out with a 1:1 LK- ADH to CHMO M15 ratio. [00126] In the stability testing of the enzymes, shown in Figure 6D and 6E, LK-ADH performed with a higher activity (ca. 0.053 U per µL) compared to CHMO M15 (0.009 U per µL) which could lead to the conclusion that LK-ADH will convert NADP ⁺ faster into NADPH than CHMO M15 converts NADPH into NADP ⁺ . [00127] However, the activity assay was carried out with the substrates (R)-1-phenylethanol and cyclohexanone and the activity for those substrates cannot be directly transferred to PVA. The enzyme ratios for PVA degradation were investigated with 2 mM NADP ⁺ (data not shown) and 4 mM NADP ⁺ which led to similar conclusions regarding the optimal enzyme ratio. [00128] Accordingly, the improved degradation reaction of PVA-4 with the proposed enzyme cascade contained 4 mM NADP ⁺ , LK-ADH, and CHMO M15 in a ratio of either 1:1 or 2:1 carried out at pH 8.5 and 20 °C over 24 hours. Example 8 [00129] The molecular mass of PVA before and after enzymatic treatment was also investigated using gel permeation chromatography (GPC). Unmodified PVA standards were purchased from PSS Polymer Standards Service GmbH (Mainz, Germany). The PVA standards were used to confirm the calibration of the GPC device. Attorney Docket 1410-158253-PC [00130] Unmodified PVA, PVA-2, PVA-4, and PVA-6 films were investigated for degradation with the enzyme cascade with either a 1:1 ratio of ADH:CHMO or 2:1 ratio of ADH:CHMO under the following conditions: 400 µL LK-ADH lysate and 400 µL CHMO M15 lysate (1:1 ratio) or 533 µL LK-ADH and 267 µL CHMO M15 (2:1 ratio), 8 mg TL-IM, 1000 µL PVA (final concentration 20 mg/mL), filled up to 2 mL with 10 mM sodium phosphate buffer, pH 8.5. The reaction mixture was incubated for 24 hours at 20°C. [00131] Determination of the molecular weight was carried out using gel permeation chromatography using Tosoh EcoSEC, HLC-8320GPC, equipped with a pre-column (PSS Suprema Pre-column 10 μ, 8x50 mm), and three columns (PSS Suprema 10 μ, 1000 Å/100 Å/30 Å, 8x300 mm). The eluent flux and temperature were set to 1 mL/min and 45 °C, respectively. Mn, and Mw were then determined using the instrument’s software. [00132] The polydispersity index (PDI), the number average molecular weight (M _n ), and the weight average molecular weight (M _w ) were calculated from the measurements, shown in Table 1. The average and the standard deviation were calculated from three replicates.

Attorney Docket 1410-158253-PC Table 1 LK-ADH to C _{HMO ratio} ^{Film Treatment Mn [g/mol] Mw [g/mol] PDI} [00133] PDI increased in most conditions tested when the PVA film was treated with enzymes, indicating a broadening of the molecular weight distribution in comparison to the untreated films, which in turn is an indicator for degradation of the polymers. Additionally, a decrease in M _n and M _w was shown in samples containing the enzyme cascade in comparison to Attorney Docket 1410-158253-PC the negative control. When PVA samples were treated with the enzyme cascade, both, Mn and M _w decreased in comparison to untreated PVA. As already observed with the Finley method, the GPC measurements confirmed that the enzyme ratio 1:1 or 2:1 result in a similar reduction of M _n and M _w and, hence, that changing the ratio of enzyme from 1:1 to 2:1 does not influence the reaction significantly. The largest decrease in M _n was observed in degradation reactions with film PVA-4. Independently of the LK-ADH to CHMO ratio, M _n decreased by 10% when PVA-4 was treated with enzyme. The largest decrease in M _w, however, was measured for unmodified PVA. [00134] When using a 2:1 LK-ADH to CHMO ratio, M _w decreased by 10% for unmodified PVA whereas for PVA-4 it only decreased by 7%. In general, the proportional decrease of M _w was lower in comparison to M _n in most cases. The exception to this is the PVA film without modification. Using an enzyme ratio of 2:1, M _w was reduced by 10% and M _n by 9% when the film was incubated with the enzyme cascade. In comparison to that, an enzyme ratio of 1:1 reduced M _w by only 4% and M _n by 7% indicating that the enzyme ratio of LK-ADH to CHMO M15 has a higher impact on PVA without modification in comparison to modified PVA. This could be due to the additional step in the reaction of modified PVA for hydrolyzing the ester bonds in the derivatized side chains of the polymer which delays the subsequent reactions of ADH and CHMO in comparison to unmodified PVA. Enhancement of enzyme stability of ADH and CHMO as well as prolonged reaction time may lead to similar yields for modified PVA as were gained for unmodified PVA. [00135] Furthermore, from a larger decrease of Mw in comparison to Mn it can be concluded that larger molecules were more readily degraded when an enzyme ratio of 2:1 was used for unmodified PVA. M _w is taking the weight of each polymer chain into account and hence the value is less influenced than M _n when smaller molecules are preferentially degraded. This in turn leads to the conclusion that larger amounts of LK-ADH are needed for the degradation of unmodified, larger weight PVA molecules. Even though LK-ADH was proven to tolerate a wide variety of substrates and to also accept bulky side chains ²² , the polymer is most likely challenging for the enzyme and an increase of the enzyme amount could hence help with degrading larger molecules. Enzyme engineering of LK-ADH towards acceptance of larger side changes could further improve the degradation rate of PVA. Attorney Docket 1410-158253-PC [00136] It is to be understood that the ranges provided herein include the stated range and any value or sub-range within the stated range. For example, a range of about 5 wt% to about 15 wt% should be interpreted to include not only the explicitly recited limits of range of about 5 wt% to about 15 wt%, but also to include individual values, such as 6.35 wt%, 7.5 wt%, 10 wt%, 12.75 wt%, 14 wt%, etc., and sub-ranges, such as about 7 wt% to about 10.5 wt%, about 8.5 wt% to about 12.7 wt%, about 9.75 wt% to about 14 wt%, etc. Furthermore, when “about” is utilized to describe a value, this is meant to encompass minor variations (up to +/- 10%) from the stated value. [00137] All percentages and ratios are calculated by weight unless otherwise indicated. All percentages and ratios are calculated based on the total weight of the compound or composition unless otherwise indicated. [00138] Reference throughout the specification to “an example,” “one example,” “another example,” “some examples,” “other examples,” and so forth, means that a particular element (e.g., feature, structure, and/or characteristic) described in connection with the example is included in at least one example described herein, and may or may not be present in other examples. In addition, it is to be understood that the described elements for any example may be combined in any suitable manner in the various examples unless the context clearly dictates otherwise. [00139] In describing and claiming the examples disclosed herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. [00140] While several examples have been described in detail, it is to be understood that the disclosed examples may be modified. Therefore, the foregoing description is to be considered non-limiting.