US 20130244920A1 (19) United States (12) Patent Application Publication (10) Pub. N0.: US 2013/0244920 A1 Lee et al. (43) Pub. Date: Sep. 19, 2013

(54) WATER SOLUBLE COMPOSITIONS (52) US. Cl. INCORPORATING ENZYMES, AND METHOD USPC ...... 510/392; 264/299 OF MAKING SAME (57) ABSTRACT (76) Inventors: David M. Lee, CroWn Point, IN (US); Jennifer L‘ Sims’ Lowell’ IN (Us) Disclosed herein are Water soluble compositions, such as ?lms, including a mixture of a ?rst Water-soluble resin, an (21) Appl' NO': 13/422’709 enzyme, and an enzyme stabilizer Which comprises a func (22) Filed: Man 16, 2012 tional substrate for the enzyme, methods of making such compositions, and methods of using such compositions, e.g. Publication Classi?cation to make packets containing functional ingredients. The enzymes can include proteases and mixtures of proteases (51) Int. Cl. With other enzymes, and the compositions provide good C11D 3/386 (2006.01) retention of enzyme function following ?lm processing and B29C 39/02 (2006.01) storage. US 2013/0244920 A1 Sep. 19,2013

WATER SOLUBLE COMPOSITIONS preheated to a temperature less than 77° C., optionally in a INCORPORATING ENZYMES, AND METHOD range ofabout 66° C. to about 77° C., or about 74° C.; drying OF MAKING SAME the Water from the cast mixture over a period of less than 24 hours, optionally less than 12 hours, optionally less than 8 FIELD OF THE DISCLOSURE hours, optionally less than 2 hours, optionally less than 1 [0001] The present disclosure relates generally to Water hour, optionally less than 45 minutes, optionally less than 30 soluble ?lms. More particularly, the disclosure relates to minutes, optionally less than 20 minutes, optionally less than Water soluble ?lms comprising a mixture of a ?rst Water 10 minutes, for example in a range of about 6 minutes to about soluble resin, an enzyme, and an enzyme stabilizer Which 10 minutes, or 8 minutes, to form a Water soluble ?lm. comprises a functional substrate for the enzyme. [0009] Another aspect of the disclosure is a method of forming a Water-soluble ?lm, Wherein the Water soluble ?lm BACKGROUND is cast from a Water- soluble mixture prepared according to the steps of: [0002] Water-soluble ?lms are Well knoWn in the art. providing a mixture of Water soluble resin, Water, and any Water-soluble ?lms have many applications, such as packag optional additives excluding enzymes; boiling the mixture for ing materials, Wherein the ?lm itself can make up a Water 30 minutes; degassing the mixture in an oven at a temperature soluble packet. These compositions have the advantage that ofat least 400 C.; optionally in a range of40° C. to 70° C., eg they are easy to dose, handle, transport and store. about 65° C.; adding at least one enzyme, optionally at least [0003] Water-soluble packets are Well suited for single dose one plasticizer, and optionally additional Water to the mixture applications Wherein the packet encloses a laundry detergent, at a temperature of 65° C. or less; and stirring the mixture, bleach or other laundry additives, fabric care, dishWashing, optionally Without vortex, until the mixture appears substan hard surface cleaning, beauty care, skin care, other personal tially uniform in color and consistency; optionally for a time care, or food product compositions. Often enclosed in Water period in a range of 30 minutes to 90 minutes, optionally at soluble packets are active agents that may become unstable in least 1 hour; and casting the mixture promptly after the time the presence of other active agents, or may cause other ingre period of stirring, e.g., Within 4 hours, or 2 hours, or Within 1 dients to become unstable, thereby limiting What active hour or less. agents can be prepackaged together in a single dose form. [0010] Optionally, the Water-soluble ?lms described herein [0004] The activity of enzymes in previously-knoWn ?lms can be thermoformable, for example into a pouch. tended to be loW and to decrease With storage time. This loss of activity tended to be especially severe When the enzyme [0011] While the present disclosure provides ?lm composition included a protease, as the protease can break examples, the compositions can take other forms and shapes, doWn itself or other enzymes. In an attempt to compensate for including those having thicknesses substantially in excess of the decrease in enzymatic activity, a large concentration of What Would normally be considered a ?lm. enzyme had been included in ?lm compositions, and this [0012] For the compositions and methods described herein, approach Was not alWays successful in producing a ?lm With optional features, including but not limited to components, at least a moderate activity. Accordingly, it is highly desirable compositional ranges thereof, substituents, conditions, and to produce a ?lm that maintains a high level of enzyme steps, are contemplated to be selected from the various activity Without requiring starting With a high concentration aspects, embodiments, and examples provided herein. of enzyme (i.e., high enzyme percent recovery after process [0013] Further aspects and advantages Will be apparent to ing and storage). those of ordinary skill in the art from a revieW of the folloWing [0005] Active agents often used as additives in laundry and detailed description. While the compositions and methods are dishWashing applications include enzymes such as protease, susceptible of embodiments in various forms, the description amylase, lipase, and oxido-reductase enzymes, including but hereafter includes speci?c embodiments With the understand not limited to oxido-reductase enzymes that catalyze the for ing that the disclosure is illustrative, and is not intended to mation of bleaching agents. Therefore, a Water-soluble ?lm limit the invention to the speci?c embodiments described containing enzymes that maintain a high level of activity, herein. especially in the presence of protease, Wouldbe advantageous for use in Water-soluble single dose packets enclosing deter DETAILED DESCRIPTION gents or other laundry or dishWashing additives. [0014] Disclosed herein are Water-soluble compositions SUMMARY (e.g., ?lms) including a Water-soluble mixture of a ?rst Water soluble resin, an enzyme, and an enzyme stabilizer Which is a [0006] One aspect of the disclosure is a Water-soluble com functional substrate for the enzyme. Optionally, the Water position including a Water-soluble mixture of a ?rst Water soluble ?lms include tWo or more types of enzymes. The soluble resin, an enzyme, and an enzyme substrate. The com Water-soluble ?lms of one aspect of the disclosure can be position can take the form of a ?lm, for example. particularly advantageous in that they can be designed such [0007] Another aspect of the disclosure is a Water-soluble that a high percentage of enzymes remain active folloWing composition (e.g., a ?lm) including a Water-soluble mixture ?lm formation. The Water-soluble ?lms according to the dis of a Water- soluble resin comprising acrylamido -2 -methylpro closure can optionally be designed such that a high percent panesulfonate, and an enzyme, Wherein the enzyme activity age of enzymes remain active When the ?lm is stored at recovery is at least 87% folloWing ?lm formation. temperatures in a range of about 5° C. to about 45° C. for at [0008] Another aspect of the disclosure is a method of least 2 Weeks, or in a range of about 2 Weeks to 2 years, e. g., forming a Water-soluble ?lm, including the steps of providing 3 months. The Water-soluble ?lms of the disclosure herein can a Water soluble mixture according to the disclosure dissolved have one or more other, optional advantages including unex in Water; casting the Water soluble mixture onto a surface pectedly high recovery of enzyme activity in ?lms containing US 2013/0244920 A1 Sep. 19, 2013

a protease and a second enzyme, therrnoformable (e. g., into a acetate groups are alloWed to remain after the hydrolysis of pouch or packet), a solid form for delivery of enzymes to a polyvinyl acetate, the PVOH polymer then being knoWn as substrate that is dust free. partially hydrolyzed, it is more Weakly hydrogen-bonded and [0015] The ?lms, packets, and their methods of manufac less crystalline and is soluble in cold Wateriless than about ture and use are contemplated to include embodiments 50° F. (10° C.). An intermediate cold/hot Water soluble ?lm including any combination of one or more of the additional can include, for example, intermediate partially-hydrolyzed optional elements, features, and steps further described beloW PVOH (e. g., With degrees of hydrolysis of about 94% to about (including those shoWn in any ?gures), unless stated other 98%), and is readily soluble only in Warm Water4e.g., rapid Wise. dissolution at temperatures of about 40° C. and greater. Both [0016] As used herein, the term “comprising” indicates the fully and partially hydrolyzed PVOH types are commonly potential inclusion of other agents, elements, steps, or fea referred to as PVOH homopolymers although the partially tures, in addition to those speci?ed. hydrolyzed type is technically a vinyl alcohol-vinyl acetate [0017] As used herein and unless speci?ed otherWise all copolymer. measurements of viscosity in centipoise (cP) are of a 4% [0021] The degree of hydrolysis of the PVOH included in solution at 20° C. the Water-soluble ?lms of the present disclosure can be about [0018] As used herein, the “enzyme percent recovery” 75% to about 99%. As the degree of hydrolysis is reduced, a refers to the percentage of enzymes that remain active folloW ?lm made from the resin Will have reduced mechanical ing one or more processing steps or storage time, e. g. folloW strength but faster solubility at temperatures beloW about 20° ing the Water-soluble composition (e.g., ?lm) processing C. As the degree of hydrolysis increases, a ?lm made from the steps (e.g. mixing, casting, and drying) and for a period of resin Will tend to be mechanically stronger and the thermo storage of about 1 month at about 5° C. Accordingly, as used formability Will tend to decrease. The degree of hydrolysis of herein, the “recovery of enzyme activity” or “enzyme activity the PVOH can be chosen such that the Water-solubility of the recovery” refers to the activity of the enzymes in the Water resin is temperature dependent, and thus the solubility of a soluble composition maintained folloWing a process or stor ?lm made from the resin and additional ingredients is also age time as compared to the original enzyme activity, eg the in?uenced. In one class of embodiments the ?lm is cold enzyme activity measured in a representative sample of the Water-soluble. A cold Water-soluble ?lm, soluble in Water at a raW material enzyme composition that Was added to a ?lm. temperature of less than 10° C., can include PVOH With a Thus, for example the enzyme activity recovery can be the degree of hydrolysis in a range of about 75% to about 90%, or ratio of enzyme activity following processing steps to make in a range of about 80% to about 90%, or in a range of about the composition, eg a ?lm, and for a period of storage of 85% to about 90%. In another class of embodiments the ?lm about one month at about 5° C. to the enzyme activity of the is hot Water-soluble. A hot Water-soluble ?lm, soluble in original enzyme composition added to the ?lm. Contem Water at a temperature of at least about 60° C., can include plated processing steps contemplated include each of the PVOH With a degree of hydrolysis of at least about 98%. steps described herein, individually or in combinations, for [0022] Other ?lm-forming resins for use in addition to or in example but not limited to a heating step, a dissolution step, a an alternative to PVOH can include, but are not limited to, mixing step (e.g., a Wet mixing step), and a drying step (e.g. modi?ed polyvinyl alcohols, polyacrylates, Water-soluble optionally With application of heat). Enzyme activity can be acrylate copolymers, polyacryamides, polyvinyl pyrrolidone, measured by any suitable method, so long as the same or pullulan, Water-soluble natural polymers including, but not equivalent methods are used for the enzyme raW material limited to, guar gum, xanthan gum, carrageenan, and starch, measurement and the enzyme-loaded, processed composition Water-soluble polymer derivatives including, but not limited (e.g., enzyme-containing ?lm) measurement. In one type of to, ethoxylated starch and hydroxypropylated starch, poly embodiment, the enzyme activity can be determined by a (sodium acrylamido-2-methylpropane sulfonate), poly colorometric method. Colorometric measurements for pro monomethylmaleate, copolymers thereof, and combinations tease, amylase, and lipase activity are described beloW in of any of the foregoing. In one class of embodiments, the connection With the experiments. ?lm-forming resin is a terpolymer consisting of vinyl alcohol, [0019] As used herein and unless speci?ed otherWise, the vinyl acetate, and sodium acrylamido-2-methylpropane terms “Wt. %” and “Wt %” are intended to refer to the com sulfonate. Unexpectedly, Water-soluble ?lms based on a vinyl position of the identi?ed element in “dry” (non Water) parts alcohol, vinyl acetate, and sodium acrylamido-2-methylpro by Weight of the entire ?lm (When applicable) or parts by panesulfonate terpolymer have demonstrated a high percent Weight of the entire composition enclosed Within a pouch recovery of enzyme activity, both With and Without the inclu (When applicable). As used herein and unless speci?ed oth sion of an enzyme substrate in the Water-soluble ?lm. erWise, the term “phr” is intended to refer to the composition [0023] The Water-soluble resin can be included in the of the identi?ed element in parts per one hundred parts Water Water-soluble ?lm in any suitable amount, for example an soluble polyvinyl alcohol (PVOH) resin. amount in a range of about 35 Wt % to about 90 Wt %. The [0020] Water-soluble ?lms, optional ingredients for use preferred Weight ratio of the amount of the Water-soluble therein, and methods of making the same are Well knoWn in resin as compared to the combined amount of all enzymes, the art. In one class of embodiments, the Water-soluble ?lm enzyme stabilizers, and secondary additives can be any suit includes PVOH. PVOH is a synthetic resin generally pre able ratio, for example a ratio in a range of about 0.5 to about pared by the alcoholysis, usually termed hydrolysis or saponi 5, or about 1 to 3, or about 1 to 2. ?cation, of polyvinyl acetate. Fully hydrolyzed PVOH, [0024] Water-soluble resins for use in the ?lms described Wherein virtually all the acetate groups have been converted herein (including, but not limited to PVOH resins) can be to alcohol groups, is a strongly hydrogen-bonded, highly characterized by any suitable viscosity for the desired ?lm crystalline polymer Which dissolves only in hot Wateri properties, optionally a viscosity in a range of about 5.0 to greater than about 140° F. (60° C.). If a suf?cient number of about 30.0 cP, or about 10.0 cP to about 25 cP. The viscosity US 2013/0244920 A1 Sep. 19, 2013

of a PVOH resin is determined by measuring a freshly made 3-phosphate dehydrogenase (NAD+), D-xylulose reductase, solution using a Brook?eld LV type viscometer With UL L-xylulose reductase, D-arabinitol 4-dehydrogenase, L-ara adapter as described in British Standard EN ISO 15023-2: binitol 4-dehydrogenase, L-arabinitol 2-dehydrogenase, 2006 Annex E Brook?eld Test method. It is international L-iditol 2-dehydrogenase, D-iditol 2-dehydrogenase, galac practice to state the viscosity of 4% aqueous polyvinyl alco titol 2-dehydrogenase, mannitol-l-phosphate 5-dehydroge hol solutions at 200 C. All PVOH viscosities speci?ed herein nase, inositol 2-dehydrogenase, glucuronate reductase, glu in cP should be understood to refer to the viscosity of 4% curonolactone reductase, aldehyde reductase, UDP-glucose aqueous polyvinyl alcohol solution at 200 C., unless speci?ed 6-dehydrogenase, histidinol dehydrogenase, quinate dehy otherWise. drogenase, shikimate dehydrogenase, glyoxylate reductase, [0025] It is Well known in the art that the viscosity of a L-lactate dehydrogenase, D-lactate dehydrogenase, glycerate PVOH resin is correlated With the Weight average molecular dehydrogenase, 3-hydroxybutyrate dehydrogenase, 3-hy Weight (MW) of the same PVOH resin, and often the viscosity droxyisobutyrate dehydrogenase, mevaldate reductase, is used as a proxy fOI‘MW. Thus, the Weight average molecular Weight of the Water-soluble resin optionally can be in a range mevaldate reductase (NADPH), hydroxymethylglutaryl of about 35,000 to about 190,000, or about 80,000 to about CoA reductase (NADPH), 3-hydroxyacyl-CoA dehydroge 160,000. The molecular Weight of the resin need only be nase, acetoacetyl-CoA reductase, malate dehydrogenase, suf?cient to enable it to be molded by suitable techniques to malate dehydrogenase (oxaloacetate-decarboxylating), form a thin plastic ?lm. malate dehydrogenase (decarboxylating), malate dehydroge nase (oxaloacetate-decarboxylating) (NADP+), isocitrate [0026] Enzymes dehydrogenase (NAD+), isocitrate dehydrogenase (NADP+), [0027] It is contemplated that any desired types of enZymes phosphogluconate 2-dehydrogenase, phosphogluconate can be used in the ?lms described herein. dehydrogenase (decarboxylating), L-gulonate 3-dehydroge [0028] For example, such enZymes include enZymes cat nase, L-arabinose 1-dehydrogenase, glucose 1-dehydroge egoriZed in any one of the six conventional EnZyme Commis nase, galacto se l-dehydrogenase, glucose-6-pho sphate dehy sion (EC) categories, i.e., the oxidoreductases of EC 1 (Which drogenase, 30t-hydroxysteroid dehydrogenase (B-speci?c), catalyZe oxidation/reduction reactions), the transferases of 3(or 17)[3-hydroxysteroid dehydrogenase, 30t-hydroxy EC 2 (Which transfer a functional group, e.g., a methyl or cholanate dehydrogenase, 30t(or 20[3)-hydroxysteroid dehy phosphate group), the hydrolases of EC 3 (Which catalyZe the drogenase, allyl-alcohol dehydrogenase, lactaldehyde reduc hydrolysis of various bonds), the lyases of EC 4 (Which cleave tase (NADPH), ribitol 2-dehydrogenase, fructuronate various bonds by means other than hydrolysis and oxidation), reductase, tagaturonate reductase, 3-hydroxypropionate the isomerases of EC 5 (Which catalyZe isomeriZation dehydrogenase, 2-hydroxy-3-oxopropionate reductase, 4-hy changes Within a molecule) and the ligases of EC 6 (Which droxybutyrate dehydrogenase, estradiol 17 [3-dehydrogenase, join tWo molecules With covalent bonds). Examples of such testosterone 17[3-dehydrogenase, testosterone 17[3-dehydro enZymes include dehydrogenases and oxidases in EC 1, tran genase (NADP+), pyridoxine 4-dehydrogenase, W-hydroxy saminases and kinases in EC 2, lipases, cellulases, amylases, decanoate dehydrogenase, mannitol 2-dehydrogenase, glu mannanases, and peptidases (a.k.a. proteases or proteolytic conate 5-dehydrogenase, alcohol dehydrogenase [NAD(P)+], enZymes) in EC 3, decarboxylases in EC 4, isomerases and glycerol dehydrogenase (NADP+), octanol dehydrogenase, mutases in EC 5 and synthetases and synthases of EC 6. (R)-aminopropanal dehydrogenase, (S,S)-butanediol dehy [0029] Oxidoreductases drogenase, lactaldehyde reductase, methylglyoxal reductase [0030] Oxidoreductases include, but are not limited to: (NADH-dependent), glyoxylate reductase (NADP+), isopro those acting on the CH4OH group of donors; those acting on panol dehydrogenase (NADP+), hydroxypyruvate reductase, the aldehyde or oxo group of donors; those acting on the malate dehydrogenase (NADP+), D-malate dehydrogenase CH4CH group of donors; those acting on the CHiNH2 (decarboxylating), dimethylmalate dehydrogenase, 3-isopro group of donors; those acting on the CHiNH group of pylmalate dehydrogenase, ketol-acid reductoisomerase, donors; those acting on NADH or NADPH; those acting on homoisocitrate dehydrogenase, hydroxymethylglutaryl-CoA other nitrogenous compounds as donors; those acting on a reductase, aryl-alcohol dehydrogenase, aryl-alcohol dehy sulfur group of donors; those acting on a heme group of drogenase (NADP+), oxaloglycolate reductase (decarboxy donors; those acting on diphenols and related substances as lating), tartrate dehydrogenase, glycerol-3-phosphate dehy donors; those acting on a peroxide as acceptor; those acting drogenase [NAD(P)+], phosphoglycerate dehydrogenase, on hydrogen as donor; those acting on single donors With diiodophenylpyruvate reductase, 3-hydroxybenZyl-alcohol incorporation of molecular oxygen (oxygenases); those act dehydrogenase, (R)-2-hydroxy-fatty-acid dehydrogenase, ing on paired donors, With incorporation or reduction of (S)-2-hydroxy-fatty-acid dehydrogenase, 3-oxoacyl-[acyl molecular oxygen; those acting on superoxide radicals as carrier-protein] reductase, acylglycerone-phosphate reduc acceptor; those oxidiZing metal ions; those acting on CH or tase, 3-dehydrosphinganine reductase, L-threonine 3-dehy CH2 groups; those acting on iron-sulfur proteins as donors; drogenase, 4-oxoproline reductase, all-trans-retinol those acting on reduced ?avodoxin as donor; those acting on dehydrogenase (NAD+), pantoate 4-dehydrogenase, pyri phosphorus or arsenic in donors; those acting on XiH and doxal 4-dehydrogenase, carnitine 3-dehydrogenase, indole YiH to form an XiY bond; and those acting on halogen in lactate dehydrogenase, 3-(imidaZol-5-yl)lactate dehydroge donors. nase, indanol dehydrogenase, L-xylose 1-dehydrogenase, [0031] The oxidoreductases Which act on the CH4OH apiose 1-reductase, ribose 1-dehydrogenase (NADP+), group of donors can include, but are not limited to, those With D-arabinose l-dehydrogenase, D-arabinose 1-dehydroge NAD+ or NADP+ as acceptor (including alcohol dehydroge nase [NAD(P)+], glucose 1-dehydrogenase (NAD+), glucose nase, alcohol dehydrogenase (NADP+), homoserine dehy 1-dehydrogenase (NADP+), galactose 1-dehydrogenase drogenase, (R,R)-butanediol dehydrogenase, glycerol dehy (NADP+), aldose 1-dehydrogenase, D-threo-aldose 1-dehy drogenase, propanediol-phosphate dehydrogenase, glycerol drogenase, sorbose 5-dehydrogenase (NADP+), fructose US 2013/0244920 A1 Sep. 19, 2013

5-dehydrogenase (NADP+), 2-deoXy-D-gluconate 3-dehy sabinol dehydrogenase, diethyl 2-methyl-3-oxosuccinate drogenase, 2-dehydro-3-deoXy-D-gluconate 6-dehydroge reductase, 30t-hydroxyglycyrrhetinate dehydrogenase, nase, 2-dehydro-3-deoXy-D-gluconate 5-dehydrogenase, 15-hydroxyprostaglandin-l dehydrogenase (NADP+), 15-hy L-idonate 2-dehydrogenase, L-threonate 3-dehydrogenase, droxyicosatetraenoate dehydrogenase, N-acylmannosamine 3-dehydro-L-gulonate 2-dehydrogenase, mannuronate 1-dehydrogenase, ?avanone 4-reductase, 8-oxocoformycin reductase, GDP-mannose 6-dehydrogenase, dTDP-4-dehy reductase, tropinone reductase ll, hydroxyphenylpyruvate drorhamnose reductase, dTDP-6-deoXy-L-talose 4-dehydro reductase, 12[3-hydroxysteroid dehydrogenase, 30t(17[3-hy genase, GDP-6-deoXy-D-talose 4-dehydrogenase, UDP-N droxysteroid dehydrogenase (NAD+), N-acetylhexosamine acetylglucosamine 6-dehydrogenase, ribitol-5-phosphate 1-dehydrogenase, 6-endo-hydroxycineole dehydrogenase, 2-dehydrogenase, mannitol 2-dehydrogenase (NADP+), sor carveol dehydrogenase, methanol dehydrogenase, cyclohex bitol-6-phosphate 2-dehydrogenase, 15-hydroxyprostaglan anol dehydrogenase, pterocarpin synthase, codeinone reduc din dehydrogenase (NAD+), D-pinitol dehydrogenase, tase (NADPH), salutaridine reductase (NADPH), D-arabini sequoyitol dehydrogenase, perillyl-alcohol dehydrogenase, tol 2-dehydrogenase, galactitol-1 -pho sphate 3 [3-hydroXy-A5-steroid dehydrogenase, 1 1[3-hydroxysteroid 5-dehydrogenase, tetrahydroxynaphthalene reductase, (S) dehydrogenase, 160t-hydroxysteroid dehydrogenase, estra carnitine 3-dehydrogenase, mannitol dehydrogenase, ?uo diol 170t-dehydrogenase, 200t-hydroxysteroid dehydroge ren-9-ol dehydrogenase, 4-(hydroXymethyl)benZene nase, 21-hydroxysteroid dehydrogenase (NAD’'), 21 -hydroX sulfonate dehydrogenase, 6-hydroxyhexanoate ysteroid dehydrogenase (NADP+), 30t-hydroXy-5[3 dehydrogenase, 3-hydroXypimeloyl-CoA dehydrogenase, androstane-17-one 30t-dehydrogenase, sepiapterin sulcatone reductase, sn-glycerol-1-phosphate dehydroge reductase, ureidoglycolate dehydrogenase, glycerol 2-dehy nase, 4-hydroXythreonine-4-phosphate dehydrogenase, 1,5 drogenase (NADP+), 3-hydroxybutyryl-CoA dehydroge anhydro-D-fructose reductase, L-idonate 5-dehydrogenase, nase, UDP-N-acetylmuramate dehydrogenase, 70t-hydrox 3-methylbutanal reductase, dTDP-4-dehydro-6-deoxyglu ysteroid dehydrogenase, dihydrobunolol dehydrogenase, cose reductase, 1-deoXy-D-Xylulose-5-phosphate reductoi cholestanetetraol 26-dehydrogenase, erythrulose reductase, somerase, 2-(R)-hydroXypropyl-CoM dehydrogenase, 2-(S) cyclopentanol dehydrogenase, hexadecanol dehydrogenase, hydroXypropyl-CoM dehydrogenase, 3-keto-steroid 2-alkyn-1-ol dehydrogenase, hydroxycyclohexanecarboxy reductase, GDP-L-fucose synthase, (R)-2-hydroxyacid dehy late dehydrogenase, hydroxymalonate dehydrogenase, 2-de drogenase, Vellosimine dehydrogenase, 2,5-didehydroglu hydropantolactone reductase (A-speci?c), 2-dehydropan conate reductase, (+)-trans-carveol dehydrogenase, serine toate 2-reductase, 3B-hydroxy-40t 3-dehydrogenase, 3B-hydroxy-5B-steroid dehydrogenase, methylcholestenecarboxylate 3-dehydrogenase 3[3-hydroXy-50t-steroid dehydrogenase, (R)-3-hydroxyacid (decarboxylating), 2-oxoadipate reductase, L-rhamnose ester dehydrogenase, (S)-3-hydroxyacid ester dehydroge 1-dehydrogenase, cycloheXane-1,2-diol dehydrogenase, nase, GDP-4-dehydro-6-deoXy-D-mannose reductase, D-Xylose 1-dehydrogenase, 120t-hydroxysteroid dehydroge quinate/shikimate dehydrogenase, methylglyoxal reductase nase, glycerol-3-phosphate 1-dehydrogenase (NADP+), (NADPH-dependent), S-(hydroxymethyl)glutathione dehy 3-hydroXy-2-methylbutyryl-CoA dehydrogenase, D-Xylose drogenase, 3"-deamino-3"-oxonicotianamine reductase, 1-dehydrogenase (NADP+), cholest-5-ene-3[3,70t-diol 3 [3-de isocitrateihomoisocitrate dehydrogenase, D-arabinitol hydrogenase, geraniol dehydrogenase, carbonyl reductase dehydrogenase (NADP+), xanthoxin dehydrogenase, sorbose (NADPH), L-glycol dehydrogenase, dTDP-galactose 6-de reductase, 4-phosphoerythronate dehydrogenase, 2-hy hydrogenase, GDP-4-dehydro-D-rhamnose reductase, pros droxymethylglutarate dehydrogenase, 1,5-anhydro-D-fruc taglandin-F synthase, prostaglandin-E2 9-reductase, indole tose reductase (1,5-anhydro-D-mannitol-forming), chloro 3-acetaldehyde reductase (NADH), indole-3-acetaldehyde phyll(ide) b reductase, momilactone-A synthase, reductase (NADPH), long-chain-alcohol dehydrogenase, dihydrocarveol dehydrogenase, limonene-1,2-diol dehydro 5-amino-6-(5-phosphoribosylamino)uracil reductase, genase, 3-hydroxypropionate dehydrogenase (NADP+), coniferyl-alcohol dehydrogenase, cinnamyl-alcohol dehy malate dehydrogenase [NAD(P)+], NADP-retinol dehydro drogenase, 15-hydroXyprostaglandin-D dehydrogenase genase, D-arabitol-phosphate dehydrogenase, 2,5-diamino (NADP+), 1 5 -hydroxypro staglandin dehydrogenase 6-(ribosylamino)-4(3H)-pyrimidinone 5'-phosphate reduc (NADP+), (+)-bomeol dehydrogenase, (S)-usnate reductase, tase, diacetyl reductase [(R)-acetoin forming], diacetyl aldose-6-phosphate reductase (NADPH), 7[3-hydroxysteroid reductase [(S)-acetoin forming], UDP-glucuronic acid dehy dehydrogenase (NADP+), 1,3-propanediol dehydrogenase, drogenase (UDP-4-keto-hexauronic acid decarboxylating), uronate dehydrogenase, IMP dehydrogenase, tropinone S-(hydroxymethyl)mycothiol dehydrogenase, D-xylose reductase l, (—)-menthol dehydrogenase, (+)-neomenthol reductase, phosphonoacetaldehyde reductase (NADH), sul dehydrogenase, 3(or 17)0t-hydroxysteroid dehydrogenase, fopropanediol 3-dehydrogenase, (S)-sulfolactate dehydroge 3[3(or 20a)-hydroxysteroid dehydrogenase, long-chain-3 nase, (S)-1-phenylethanol dehydrogenase, 2-hydroXy-4-car hydroXyacyl-CoA dehydrogenase, 3-oXoacyl-[acyl-carrier boxymuconate semialdehyde hemiacetal dehydrogenase, protein] reductase (NADH), 30t-hydroxysteroid dehydroge sulfoacetaldehyde reductase, germacreneA alcohol dehydro nase (A-speci?c), 2-dehydropantolactone reductase genase, and 1'-cis-retinol dehydrogenase); or With a cyto (B-speci?c), gluconate 2-dehydrogenase, farnesol dehydro chrome as acceptor (including mannitol dehydrogenase (cy genase, benZyl-2-methyl-hydroxybutyrate dehydrogenase, tochrome), L-lactate dehydrogenase (cytochrome), D-lactate morphine 6-dehydrogenase, dihydrokaempferol 4-reductase, dehydrogenase (cytochrome), D-lactate dehydrogenase (cy 6-pyruvoyltetrahydropterin 2'-reductase, Vomifoliol 4'-dehy tochrome c-553), polyvinyl alcohol dehydrogenase (cyto drogenase, (R)-4-hydroxyphenyllactate dehydrogenase, chrome), methanol dehydrogenase (cytochrome c), and alco isopiperitenol dehydrogenase, mannose-6-phosphate 6-re hol dehydrogenase (cytochrome c)); or With oxygen as ductase, chlordecone reductase, 4-hydroxycyclohexanecar acceptor (including malate oxidase, glucose oxidase, hexose boxylate dehydrogenase, (—)-bomeol dehydrogenase, (+) oxidase, cholesterol oxidase, aryl-alcohol oxidase, L-gulono US 2013/0244920 A1 Sep. 19,2013

lactone oxidase, galactose oxidase, pyranose oxidase, L-sor hyde dehydrogenase, 3(>t,70t,l20t-trihydroxycholestan-26-al hose oxidase, pyridoxine 4-oxidase, alcohol oxidase, cat 26-oxidoreductase, glutamate-5-semialdehyde dehydroge echol oxidase (dimeriZing), (S)-2-hydroxy-acid oxidase, nase, hexadecanal dehydrogenase (acylating), forrnate dehy ecdysone oxidase, choline oxidase, secondary-alcohol oxi drogenase (NADP+), cinnamoyl-CoA reductase, formalde dase, 4-hydroxymandelate oxidase, long-chain-alcohol oxi hyde dehydrogenase, 4-trimethylammoniobutyraldehyde dase, glycerol-3-phosphate oxidase, thiamin oxidase, dehydrogenase, long-chain-aldehyde dehydrogenase, hydroxyphytanate oxidase, nucleoside oxidase, N-acylhex 2-oxoaldehyde dehydrogenase (NADP+), long-chain-fatty osamine oxidase, polyvinyl-alcohol oxidase, D-arabinono-l, acyl-CoA reductase, pyruvate dehydrogenase (NADP+), oxo 4-lactone oxidase, Vanillyl-alcohol oxidase, nucleoside oxi glutarate dehydrogenase (NADP+), 4-hydroxyphenylacetal dase (H2O2-forming), and D-mannitol oxidase, alditol dehyde dehydrogenase, y-guanidinobutyraldehyde oxidase); or With a disul?de as acceptor (including Vitamin dehydrogenase, butanal dehydrogenase, phenylglyoxylate K-epoxide reductase (Warfarin-sensitiVe) or Vitamin-K-ep dehydrogenase (acylating), glyceraldehyde-3-phosphate oxide reductase (Warfarin-insensitiVe)); or With a quinone or dehydrogenase (NAD(P)+) (phosphorylating), 5-carboxym similar compound as acceptor (including quinoprotein glu ethyl-2-hydroxymuconic-semialdehyde dehydrogenase, cose dehydrogenase, glycerol-3-phosphate dehydrogenase, 4-hydroxymuconic semialdehyde dehydrogenase, 4-formyl malate dehydrogenase (quinone), alcohol dehydrogenase benZenesulfonate dehydrogenase, 6-oxohexanoate dehydro (quinone), formate dehydrogenase-N, cyclic alcohol dehy genase, 4-hydroxybenZaldehyde dehydrogenase, salicylalde drogenase (quinone), and quinate dehydrogenase (quinine)); hyde dehydrogenase, Vanillin dehydrogenase, coniferyl or With other, knoWn, acceptors (including alcohol dehydro aldehyde dehydrogenase, ?uoroacetaldehyde genase (aZurin) and glucose-6-phosphate dehydrogenase (co dehydrogenase, glutamyl-tRNA reductase, succinyl enZyme-F42O)); or With other acceptors (including choline glutamate-semialdehyde dehydrogenase, erythrose-4-phos dehydrogenase, 2-hydroxyglutarate dehydrogenase, glucon phate dehydrogenase, sulfoacetaldehyde dehydrogenase, ate 2-dehydrogenase (acceptor), dehydrogluconate dehydro abietadienal dehydrogenase, malonyl CoA reductase (mal genase, D-2-hydroxy-acid dehydrogenase, lactateimalate onate semialdehyde-forming), succinate-semialdehyde transhydrogenase, pyridoxine 5-dehydrogenase, glucose dehydrogenase (acetylating), 3,4-dehydroadipyl-CoA semi dehydrogenase (acceptor), fructose 5-dehydrogenase, sor aldehyde dehydrogenase (NADP+), 2-formylbenZoate dehy bose dehydrogenase, glucoside 3-dehydrogenase, glycolate drogenase, succinate-semialdehyde dehydrogenase dehydrogenase, cellobiose dehydrogenase (acceptor), alkan (NADP+), long-chain acyl-[acyl-carrier-protein] reductase, l-ol dehydrogenase (acceptor), D-sorbitol dehydrogenase sulfoacetaldehyde dehydrogenase (acylating), and [3-apo-4' (acceptor), glycerol dehydrogenase (acceptor), hydroxyacid carotenal oxygenase); or With a cytochrome as acceptor (in oxoacid transhydrogenase, 3-hydroxycyclohexanone dehy cluding formate dehydrogenase (cytochrome), formate dehy drogenase, (R)-pantolactone dehydrogenase (?avin), glu drogenase (cytochrome-c-553), and carbon-monoxide cose-fructose oxidoreductase, pyranose dehydrogenase dehydrogenase (cytochrome-b-56l)); or With oxygen as (acceptor), 2-oxoacid reductase, (S)-mandelate dehydroge acceptor (including aldehyde oxidase, pyruvate oxidase, nase, L-sorbose l-dehydrogenase, formate dehydrogenase oxalate oxidase, glyoxylate oxidase, pyruvate oxidase (CoA (acceptor), soluble quinoprotein glucose dehydrogenase, acetylating), indole-3-acetaldehyde oxidase, pyridoxal oxi NDMA-dependent alcohol dehydrogenase, and NDMA-de dase, aryl-aldehyde oxidase, retinal oxidase, 4-hydroxyphe pendent methanol dehydrogenase). nylpyruvate oxidase, and abscisic aldehyde oxidase); or With a disul?de as acceptor (including pyruvate dehydrogenase [0032] The oxidoreductases Which act on the aldehyde or (acetyl-transferring), oxoglutarate dehydrogenase (succinyl oxo group of donors can include, but are not limited to, those transferring), and 3-methyl-2-oxobutanoate dehydrogenase With NAD+ or NADP+ as acceptor (including forrnate dehy drogenase, aldehyde dehydrogenase (NAD+), aldehyde dehy (2 -methylpropanoyl-transferring)); or With a quinone or simi drogenase (NADP+), aldehyde dehydrogenase [NAD(P)+], lar compound as acceptor (including pyruvate dehydrogenase benZaldehyde dehydrogenase (NADP+), betaine-aldehyde (quinone)); or With an iron-sulfur protein as acceptor (includ dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase ing pyruvate synthase, 2-oxobutyrate synthase, 2-oxoglut (NADP+), acetaldehyde dehydrogenase (acetylating), aspar arate synthase, carbon-monoxide dehydrogenase (ferre tate-semialdehyde dehydrogenase, glyceraldehyde-3-phos doxin), aldehyde ferredoxin oxidoreductase, glyceraldehyde phate dehydrogenase (phosphorylating), glyceraldehyde-3 3-phosphate dehydrogenase (ferredoxin), 3-methyl-2 phosphate dehydrogenase (NADP+) (phosphorylating), oxobutanoate dehydrogenase (ferredoxin), indolepyruvate malonate-semialdehyde dehydrogenase, succinate-semialde ferredoxin oxidoreductase, and oxalate oxidoreductase); or hyde dehydrogenase [NAD(P)+], glyoxylate dehydrogenase With other acceptors (including carbon-monoxide dehydro (acylating), malonate-semialdehyde dehydrogenase (acety genase (acceptor), aldehyde dehydrogenase (pyrroloquino lating), aminobutyraldehyde dehydrogenase, glutarate-semi line-quinone), formaldehyde dismutase, formylmethanofu aldehyde dehydrogenase, glycolaldehyde dehydrogenase, ran dehydrogenase, carboxylate reductase, and aldehyde lactaldehyde dehydrogenase, 2-oxoaldehyde dehydrogenase dehydrogenase (FAD-independent)). (NAD+), succinate-semialdehyde dehydrogenase (NAD+), [0033] The oxidoreductases Which act on the CH4CH 2-oxoisovalerate dehydrogenase (acylating), 2,5-dioxovaler group of donors can include, but are not limited to, those With ate dehydrogenase, methylmalonate-semialdehyde dehydro NAD+ or NADP+ as acceptor (e.g., dihydropyrimidine dehy genase (acylating), benZaldehyde dehydrogenase (NAD+), drogenase (NAD+), dihydropyrimidine dehydrogenase aryl-aldehyde dehydrogenase, aryl-aldehyde dehydrogenase (NADP+), A4-3-oxosteroid 5[3-reductase, cortisone ot-reduc (NADP+), L-aminoadipate-semialdehyde dehydrogenase, tase, cucurbitacin A23-reductase, fumarate reductase aminomuconate-semialdehyde dehydrogenase, (R)-dehy (NADH), meso-tartrate dehydrogenase, acyl-CoA dehydro dropantoate dehydrogenase, retinal dehydrogenase, genase (NADP+), enoyl-[acyl-carrier-protein] reductase N-acetyl-y-glutamyl-phosphate reductase, phenylacetalde (NADH), enoyl-[acyl-carrier-protein] reductase (NADPH, US 2013/0244920 A1 Sep. 19, 2013

B-speci?c), 2-coumarate reductase, prephenate dehydroge protein as acceptor (e.g., 6-hydroxynicotinate reductase, nase, prephenate dehydrogenase (NADP+), dihydroorotate 15,16-dihydrobiliverdin:ferredoxin oxidoreductase, phyco dehydrogenase (NAD+), dihydroorotate dehydrogenase erythrobilinzferredoxin oxidoreductase, phytochromobilin: (NADP+), [3-nitroacrylate reductase, 3-methyleneoxindole ferredoxin oxidoreductase, phycocyanobilinzferredoxin oxi reductase, kynurenate-7,8-dihydrodiol dehydrogenase, cis-1, doreductase, phycoerythrobilin synthase, ferredoxin: 2-dihydrobenZene-1,2-diol dehydrogenase, trans-1,2-dihy protochlorophyllide reductase (ATP-dependent), benZoyl drobenZene-1,2-diol dehydrogenase, 7-dehydrocholesterol CoA reductase, and 4-hydroXybenZoyl-CoA reductase) or reductase, cholestenone Sot-reductase, biliverdin reductase, With ?avin as acceptor (e.g., butyryl-CoA dehydrogenase or 1 ,6-dihydroxycycloheXa-2,4-diene-1-carboxylate dehydro 4,4'-diapophytoene desaturase), or With other, knoWn accep tors (e.g., dihydroorotate dehydrogenase (fumarate)), or With genase, dihydrodipicolinate reductase, 2-hexadecenal reduc other acceptors (e.g., succinate dehydrogenase, acyl-CoA tase, 2,3-dihydro-2,3-dihydroXybenZoate dehydrogenase, dehydrogenase, 3-oxosteroid 1-dehydrogenase, 3-oXo-50t cis -1 ,2 -dihydro -1 ,2 -dihydroxynaphthalene dehydro genase, steroid 4-dehydrogenase, 3-oXo-5[3-steroid 4-dehydroge progesterone Set-reductase, 2-enoate reductase, maleylac nase, glutaryl-CoA dehydrogenase, 2-furoyl-CoA dehydro etate reductase, protochlorophyllide reductase, 2,4-dienoyl genase, isoValeryl-CoA dehydrogenase, 2-methylacyl-CoA CoA reductase (NADPH), phosphatidylcholine desaturase, dehydrogenase, long-chain-acyl-CoA dehydrogenase, cyclo geissoschiZine dehydrogenase, cis-2-enoyl-CoA reductase hexanone dehydrogenase, isoquinoline l-oxidoreductase, (NADPH), trans-2-enoyl-CoA reductase (NADPH), enoyl quinoline 2-oxidoreductase, quinaldate 4-oxidoreductase, [acyl-carrier-protein] reductase (NADPH, A-speci?c), 2-hy quinoline-4-carboxylate 2-oxidoreductase, (R)-benZylsucci droxy-6-oxo-6-phenylhexa-2,4-dienoate reductase, xanth nyl-CoA dehydrogenase, coproporphyrinogen dehydroge ommatin reductase, 12-oxophytodienoate reductase, nase, all-trans-retinol 13,14-reductase, 2-amino-4-deoxy arogenate dehydrogenase, trans-2-enoyl-CoA reductase chorismate dehydrogenase, carvone reductase, all-trans-‘Q (NAD+), 2'-hydroxyiso?avone reductase, biochanin-A carotene desaturase, l-hydroxycarotenoid 3,4-desaturase, reductase, ot-santonin 1,2-reductase, 15-oxoprostaglandin phytoene desaturase (neurosporene-forming), phytoene 13-oxidase, cis-3,4-dihydrophenanthrene-3,4-diol dehydro desaturase (‘Q-carotene-forming), phytoene desaturase (3,4 genase, 2'-hydroxydaidZein reductase, 2-methyl-branched didehydrolycopene-forming), and phytoene desaturase (ly chain-enoyl-CoA reductase, (3S,4R)-3,4-dihydroxycyclo copene-forming)). heXa-1,5-diene-1 ,4-dicarboxylate dehydrogenase, precorrin 6A reductase, cis-2,3-dihydrobiphenyl-2,3-diol [0034] The oxidoreductases Which act on the CHiNH2 dehydrogenase, phloroglucinol reductase, 2,3-dihydroxy-2, group of donors can include, but are not limited to, those With 3-dihydro-p-cumate dehydrogenase, dibenZothiophene dihy NAD+ or NADP+ as acceptor (e.g., alanine dehydrogenase, drodiol dehydrogenase, terephthalate 1,2-cis-dihydrodiol glutamate dehydrogenase, glutamate dehydrogenase [NAD dehydrogenase, pimeloyl-CoA dehydrogenase, 2,4-dichlo (P)+], glutamate dehydrogenase (NADP+), L-amino-acid robenZoyl-CoA reductase, phthalate 4,5-cis-dihydrodiol dehydrogenase, serine 2-dehydrogenase, Valine dehydroge dehydrogenase, 5,6-dihydroXy-3-methyl-2-oxo-1,2,5,6-tet nase (NADP+), leucine dehydrogenase, glycine dehydroge rahydroquinoline dehydrogenase, cis-dihydroethylcatechol nase, L-erythro-3,S-diaminohexanoate dehydrogenase, 2,4 dehydrogenase, cis- 1 ,2-dihydroXy-4 -methylcycloheXa-3 ,5 - diaminopentanoate dehydrogenase, glutamate synthase diene- 1 -carboxylate dehydrogenase, 1,2-dihydroxy-6-meth (NADPH), glutamate synthase (NADH), lysine dehydroge ylcycloheXa-3,S-dienecarboxylate dehydrogenase, Zeatin nase, diaminopimelate dehydrogenase, N-methylalanine reductase, Al4-sterol reductase, A24(24l)-sterol reductase, dehydrogenase, lysine 6-dehydrogenase, tryptophan dehy A24-sterol reductase, 1,2-dihydrovomilenine reductase, drogenase, phenylalanine dehydrogenase, and aspartate 2-alkenal reductase, divinyl chlorophyllide a 8-Vinyl-reduc dehydrogenase), or With a chyochrome as acceptor (e.g. gly tase, precorrin-2 dehydrogenase, anthocyanidin reductase, cine dehydrogenase (cytochrome)), or With oxygen as accep arogenate dehydrogenase (NADP+), arogenate dehydroge tor (e. g. D-aspartate oxidase, L-amino-acid oxidase, D-amino nase [NAD(P)+], red chlorophyll catabolite reductase, (+) acid oxidase, amine oxidase, pyridoxal 5'-phosphate pulegone reductase, (—)-isopiperitenone reductase, gera synthase, D-glutamate oxidase, ethanolamine oxidase, nylgeranyl diphosphate reductase acrylyl-CoA reductase putrescine oxidase, L-glutamate oxidase, cyclohexylamine (NADPH), crotonyl-CoA carboXylase/reductase, crotonyl oxidase, protein-lysine 6-oxidase, L-lysine oxidase, CoA reductase, 3-(cis-5,6-dihydroxycycloheXa-1,3-dien-1 D-glutamate(D-aspartate) oxidase, L-aspartate oxidase, gly yl)propanoate dehydrogenase, tRNA-dihydrouridinel 6/17 cine oxidase, L-lysine 6-oxidase, primary-amine oxidase, synthase [NAD(P)+], tRNA-dihydrouridine47 synthase diamine oxidase, and 7-chloro-L-tryptophan oxidase), or With [NAD(P)+], tRNA-dihydrouridine2O/2Ol7 synthase [NAD a disul?de as acceptor (e.g. glycine dehydrogenase (decar (P)’'], and tRNA-dihydrouridine2o synthase [NAD(P)+]) or boxylating)), or With a quinine or similar compound as accep With a cytochrome as acceptor, e. g. L-galactonolactone dehy tor (e.g. D-amino acid dehydrogenase (quinone)) or With an drogenase, or With oxygen as acceptor (e.g., coproporphy iron-sulfur protein as acceptor (e.g. glutamate synthase rinogen oxidase, protoporphyrinogen oxidase, bilirubin oxi (ferredoxin)), or With a copper protein as acceptor (e.g. dase, acyl-CoA oxidase, dihydrouracil oxidase, methylamine dehydrogenase (amicyanin) and aralkylamine tetrahydroberberine oxidase, secologanin synthase, tryp dehydrogenase (aZurin)), and With other acceptors (e.g tophan otB-oxidase, pyrroloquinoline-quinone synthase, and D-amino-acid dehydrogenase, taurine dehydrogenase, and L-galactonolactone oxidase), or With a quinine or related glycine dehydrogenase (cyanide-forming)). compound as acceptor (e.g., succinate dehydrogenase [0035] The oxidoreductases Which act on the CHiNH (ubiquinone), dihydroorotate dehydrogenase (quinone), pro group of donors can include, but are not limited to, those With toporphyrinogen IX dehydrogenase (menaquinone), fuma NAD+ or NADP+ as acceptor (e.g., pyrroline-2-carboxylate rate reductase (menaquinone), 15-cis-phytoene desaturase, reductase, pyrroline-S-carboxylate reductase, dihydrofolate and 9,9'-dicis-

(NADP+), formyltetrahydrofolate dehydrogenase, saccha With NAD+ or NADP+ as acceptor (e.g., nitrate reductase ropine dehydrogenase (NAD+, L-lysine-forming), saccha (NADH), nitrate reductase [NAD(P)H], nitrate reductase ropine dehydrogenase (NADP+, L-lysine-forming), saccha (NADPH), nitrite reductase [NAD(P)H], hyponitrite reduc ropine dehydrogenase (NAD+, L-glutamate-forming), tase, aZobenZene reductase, GMP reductase, nitroquinoline saccharopine dehydrogenase (NADP+, L-glutamate-form N-oxide reductase, hydroxylamine reductase (NADH), ing), D-octopine dehydrogenase, l-pyrroline-5-carboxylate 4-(dimethylamino)phenylaZoxybenZene reductase, N-hy dehydrogenase, methylenetetrahydrofolate dehydrogenase droxy-2-acetamido?uorene reductase, preQl reductase, and (NAD+), D-lysopine dehydrogenase, alanopine dehydroge nitric oxide reductase [NAD(P), nitrous oxide-forming]), nase, ephedrine dehydrogenase, D-nopaline dehydrogenase, With a cytochrome as acceptor (e.g. nitrite reductase (NO methylenetetrahydrofolate reductase [NAD(P)H], Al-piperi forming), nitrite reductase (cytochrome; ammonia-forming), deine-Z-carboxylate reductase, strombine dehydrogenase, trimethylamine-N-oxide reductase (cytochrome c), nitrous tauropine dehydrogenase, N5-(carboxyethyl)omithine syn oxide reductase, and nitric oxide reductase (cytochrome c)), thase, thiomorpholine-carboxylate dehydrogenase, [3-alan With oxygen as acceptor (e.g., nitroalkane oxidase, acetylin opine dehydrogenase, 1,2-dehydroreticulinium reductase doxyl oxidase, factor-independent urate hydroxylase, (NADPH), opine dehydrogenase, ?avin reductase (NADPH), hydroxylamine oxidase, and 3-aci-nitropropanoate oxidase), berberine reductase, Vomilenine reductase, pteridine reduc With a quinone or similar compound as acceptor (e.g. nitrate tase, 6,7-dihydropteridine reductase, ?avin reductase reductase (quinone) and nitric oxide reductase (NADH), FAD reductase (NADH), FMN reductase (menaquinol)), With a nitrogenous group as acceptor (e.g. (NADPH), FMN reductase [NAD(P)H], 8-hydroxy-5-dea nitrite dismutase), With an iron-sulfur protein as acceptor (e. g, za?avinzNADPH oxidoreductase, ribo?avin reductase [NAD ferredoxininitrite reductase and ferredoxininitrate reduc (P)H], and FMN reductase (NADH)), With oxygen as accep tase), and With other acceptors (e.g., hydroxylamine reduc tor (e.g., sarcosine oxidase, N-methyl-L-amino-acid oxidase, tase, nitrate reductase, and hydrazine oxidoreductase). N6-methyl-lysine oxidase, (S)-6-hydroxynicotine oxidase, [0038] The oxidoreductases Which act on a sulfur group of (R)-6-hydroxynicotine oxidase, L-pipecolate oxidase, dim donors can include, but are not limited to, those With NAD+ or ethylglycine oxidase, dihydrobenZophenanthridine oxidase, NADP+ as acceptor (e.g., sul?te reductase (NADPH), hypo Nl -acetylpolyamine oxidase, polyamine oxidase (propane-l, taurine dehydrogenase, dihydrolipoyl dehydrogenase, 2-oxo 3-diamine-forming), Nl -acetylspermidine oxidase (propane propyl-CoM reductase (carboxylating), cystine reductase, l,3-diamine-forming), spermine oxidase, non-speci?c glutathione-disul?de reductase, protein-disul?de reductase, polyamine oxidase, and L-saccharopine oxidase), With a dis thioredoxin-disul?de reductase, CoA-glutathione reductase, ul?de as acceptor (e.g. pyrimidodiaZepine synthase), With a asparagusate reductase, trypanothione-disul?de reductase, quinine or similar compound as acceptor (e.g. electron-trans bis-y-glutamylcystine reductase, CoA-disul?de reductase, ferring-?avoprotein dehydrogenase), With an iron-sulfur pro mycothione reductase, glutathione amide reductase, and dim tein as acceptor (e.g. methylenetetrahydrofolate reductase ethylsulfone reductase), With a cytochrome as acceptor (e.g., (ferredoxin)), With a ?avin as acceptor (e.g. dimethylamine sul?te dehydrogenase, thiosulfate dehydrogenase sul?de-cy dehydrogenase, and trimethylamine dehydrogenase), and tochrome-c reductase (?avocytochrome c), and dimethyl sul With other acceptors (e.g., sarcosine dehydrogenase, dimeth ?dezcytochrome c2 reductase), With oxygen as acceptor (e.g. ylglycine dehydrogenase, L-pipecolate dehydrogenase, nico sul?te oxidase, thiol oxidase, glutathione oxidase, meth tine dehydrogenase, methylglutamate dehydrogenase, sper anethiol oxidase, prenylcysteine oxidase, and famesylcys midine dehydrogenase, proline dehydrogenase, teine lyase), With a disul?de as acceptor (e.g., glutathionei methylenetetrahydromethanopterin dehydrogenase, 5,10 homocystine transhydrogenase, protein-disul?de reductase methylenetetrahydromethanopterin reductase, cytokinin (glutathione), glutathioneiCoA-glutathione transhydroge dehydrogenase, and D-proline dehydrogenase). nase, glutathioneicystine transhydrogenase, enZyme-thiol [0036] The oxidoreductases Which act on NADH or transhydrogenase (glutathione-disul?de), phosphoadenylyl NADPH can include, but are not limited to, those With NAD+ sulfate reductase (thioredoxin), adenylyl-sulfate reductase or NADP+ as acceptor (e.g., NAD(P)+ transhydrogenase (glutathione), adenylyl-sulfate reductase (thioredoxin), pep (B-speci?c), and NAD(P)+ transhydrogenase (AB-speci?c)), tide-methionine (S)iS-oxide reductase, peptide-methionine With a heme protein as acceptor (e.g. cytochrome-h5 reduc (R)iS-oxide reductase, L-methionine (S)iS-oxide reduc tase, NADPHihemoprotein reductase, NADPH4cyto tase, and L-methionine (R)iS-oxide reductase), With a qui chrome-c2 reductase, and leghemoglobin reductase), With nine or similar compound as acceptor (e.g., glutathione dehy oxygen as acceptor (e.g. NAD(P)H oxidase), With a quinine drogenase (ascorbate), thiosulfate dehydrogenase (quinone), or similar compound as acceptor (e.g., NAD(P)H dehydroge dimethylsulfoxide reductase, and sul?dezquinone reductase), nase (quinone), NADHzubiquinone reductase (H+-translocat With a nitrogenous group as acceptor, With an iron-sulfur ing), monodehydroascorbate reductase (NADH), NADPH: protein as acceptor (e.g. sul?te reductase (ferredoxin) and quinone reductase, p-benZoquinone reductase (NADPH), ferredoxinzthioredoxin reductase), With other, knoWn accep 2-hydroxy-l,4-benZoquinone reductase, NADHzubiquinone tors (e.g. CoB4CoM heterodisul?de reductase and sulfure reductase (Na+-transporting), NADHzubiquinone reductase doxin), and With other acceptors (e. g. sul?te reductase, ade (non-electrogenic), and NADPH dehydrogenase (quinone)), nylyl-sulfate reductase, and hydrogen sul?te reductase). With a nitrogenous group as acceptor (e.g. trimethylamine-N [0039] The oxidoreductases Which act on a heme group of oxide reductase), With an iron- sulfur protein as acceptor, With donors can include, but are not limited to, those With oxygen a ?avin as acceptor, and With other acceptors (e.g, NADPH as acceptor (e.g., cytochrome-c oxidase), With a nitrogenous dehydrogenase, NADH dehydrogenase, NADH dehydroge group as acceptor (e.g. nitrate reductase (cytochrome)), and nase (quinone)). With other acceptors (e.g. iron4cytochrome-c reductase). [0037] The oxidoreductases Which act on other nitrogenous [0040] The oxidoreductases Which act on diphenols and compounds as donors can include, but are not limited to, those related substances as donors can include, but are not limited US 2013/0244920 A1 Sep. 19,2013

to, those With NAD+ or NADP+ as acceptor (e.g. trans genase, lignostilbene (xB-dioxygenase, linoleate diol syn acenaphthene-l ,2-diol dehydrogenase), With a cytochrome as thase, linoleate ll-lipoxygenase, 4-hydroxymandelate acceptor (e.g. L-ascorhate4cytochrome-h5 reductase and synthase, 3-hydroxy-4-oxoquinoline 2,4-dioxygenase, 3-hy ubiquinol4cytochrome-c reductase), With oxygen as accep droxy-2-methyl-quinolin-4-one 2,4-dioxygenase, chlorite tor (e.g, catechol oxidase, laccase, L-ascorbate oxidase, O2-lyase, acetylacetone-cleaving enZyme, 9-cis-epoxycaro o-aminophenol oxidase, 3-hydroxyanthranilate oxidase, rifa tenoid dioxygenase, indoleamine 2,3-dioxygenase, acireduc mycin-B oxidase, photosystem II, ubiquinol oxidase (H+ tone dioxygenase (Ni2+-requiring), acireductone dioxyge transporting), ubiquinol oxidase, and menaquinol oxidase nase [iron(ll)-requiring], sulfur oxygenase/reductase, 1,2 (H+-transporting)), and With other acceptors (e.g., plasto dihydroxynaphthalene dioxygenase, gallate dioxygenase, quinoliplastocyanin reductase, ribosyldihydronicotina linoleate 9S-lipoxygenase, and torulene dioxygenase), With mide dehydrogenase (quinone), and Violaxanthin de-epoxi incorporation of one atom of oxygen (internal monooxyge dase). nases or internal mixed function oxidases) (e.g., arginine [0041] The oxidoreductases Which act on peroxide as 2-monooxygenase, lysine 2-monooxygenase, tryptophan acceptor can include, but are not limited to, peroxidases (e.g., 2-monooxygenase, lactate 2-monooxygenase, Renilla-lu NADH peroxidase, NADPH peroxidase, fatty-acid peroxi ciferin 2-monooxygenase, Cypridina-luciferin 2-monooxy dase, cytochrome-c peroxidase, catalase, peroxidase, iodide genase, Pholinus-luciferin 4-monooxygenase (ATP-hydrol peroxidase, glutathione peroxidase, chloride peroxidase, ysing), WaZasenia-luciferin 2-monooxygenase, L-ascorbate peroxidase, phospholipid-hydroperoxide glu phenylalanine 2-monooxygenase, apo-[3-carotenoid-l4',13' tathione peroxidase, manganese peroxidase, lignin peroxi dioxygenase, Oplophorus-luciferin 2-monooxygenase, 3,4 dase, peroxiredoxin, Versatile peroxidase, glutathione amide dihydroxyphenylalanine oxidative deaminase, nitronate dependent peroxidase, bromide peroxidase, dye decoloriZing monooxygenase, dichloroarcyria?avin A synthase, peroxidase, prostamide/prostaglandin F20‘ synthase, catalase dino?agellate luciferase, and 2-oxuglutarate dioxygenase peroxidase) and those With H2O2 as acceptor, one oxygen (ethylene-forming)), inositol oxygenase, and tryptophan atom of Which is incorporated into the product (eg unspe 2'-dioxygenase. ci?c peroxygenase, myeloperoxidase, plant seed peroxyge [0044] The oxidoreductases Which act on paired donors, nase, and fatty-acid peroxygenase). With incorporation or reduction of molecular oxygen can [0042] The oxidoreductases Which act on hydrogen as include, but are not limited to, those With ascorbate as one donor can include, but are not limited to, those With NAD+ or donor, With reduced pteridine as one donor, With 2-oxoglut NADP+ as acceptor (e.g., hydrogen dehydrogenase, hydro arate as one donor, and incorporation of one atom each of gen dehydrogenase (NADP+), and hydrogenase (NAD+, oxygen into both donors (e.g. y-butyrobetaine dioxygenase, ferredoxin)), With a cytochrome as acceptor (e.g. cyto procollagen-proline dioxygenase, pyrimidine-deoxynucleo chrome-c3 hydrogenase), With a quinine or similar compound side 2'-dioxygenase, procollagen-lysine 5-dioxygenase, as acceptor (e.g., hydrogenzquinone oxidoreductase), With an thymine dioxygenase, procollagen-proline 3-dioxygenase, iron-sulfur protein as acceptor (e.g. ferredoxin hydrogenase), trimethyllysine dioxygenase, ?avanone 3-dioxygenase, pyri With other knoWn acceptors (e.g., coenZyme F420 hydroge midine-deoxynucleoside l'-dioxygenase, hyoscyamine (6S) nase, 5,l0-methenyltetrahydromethanopterin hydrogenase, dioxygenase, gibberellin-44 dioxygenase, gibberellin 2[3-di and Melhanosarcina-phenazine hydrogenase), and With oxygenase, 6[3-hydroxyhyoscyamine epoxidase, gibberellin other acceptors (e.g. hydrogenase (acceptor)). 3[3-dioxygenase, peptide-aspartate [3-dioxygenase, taurine [0043] The oxidoreductases Which act on single donors dioxygenase, phytanoyl-CoA dioxygenase, leucocyanidin With incorporation of molecular oxygen (oxygenases) can oxygenase, deacetoxyvindoline 4-hydroxylase, clavaminate include, but are not limited to, those With incorporation of tWo synthase, ?avone synthase, ?avonol synthase, 2'-deoxymugi atoms of oxygen (e.g., catechol 1,2-dioxygenase, catechol neic-acid 2'-dioxygenase, mugineic-acid 3-dioxygenase, 2,3-dioxygenase, protocatechuate 3,4-dioxygenase, gentisate deacetoxycephalosporin-C hydroxylase, [histone-H3] 1,2-dioxygenase, homogentisate 1,2-dioxygenase, 3-hy lysine-36 demethylase, proline 3-hydroxylase, hypoxia-in droxyanthranilate 3,4-dioxygenase, protocatechuate 4,5-di ducible factor-proline dioxygenase, hypoxia-inducible fac oxygenase, 2,5-dihydroxypyridine 5,6-dioxygenase, 7,8-di tor-asparagine dioxygenase, thebaine 6-O-demethylase, hydroxykynurenate 8,8a-dioxygenase, tryptophan 2,3 codeine 3-O-demethylase, DNA oxidative demethylase, and dioxygenase, linoleate l3S-lipoxygenase, ascorbate 2,3 2-oxoglutarate/L-arginine monooxygenase/decarboxylase dioxygenase, 2,3-dihydroxybenZoate 3,4-dioxygenase, 3,4 (succinate-forming)), With NADH or NADPH as one donor, dihydroxyphenylacetate 2,3-dioxygenase, and incorporation of tWo atoms of oxygen into one donor 3-carboxyethylcatechol 2,3-dioxygenase, indole 2,3-dioxy (e.g., anthranilate 1,2-dioxygenase (deaminating, decarboxy genase, sulfur dioxygenase, cysteamine dioxygenase, cys lating), benZene 1,2-dioxygenase, 3-hydroxy-2-methylpy teine dioxygenase, caffeate 3,4-dioxygenase, 2,3-dihydroxy ridinecarboxylate dioxygenase, 5-pyridoxate dioxygenase, indole 2,3-dioxygenase, quercetin 2,3-dioxygenase, 3,4 phthalate 4,5-dioxygenase, 4-sulfobenZoate 3,4-dioxyge dihydroxy-9, l O-secoandrosta- l ,3,5(l 0)-triene-9, l 7-dione nase, 4-chlorophenylacetate 3,4-dioxygenase, benZoate 1,2 4,5-dioxygenase, peptide-tryptophan 2,3-dioxygenase, 4-hy dioxygenase, toluene dioxygenase, naphthalene 1,2-dioxy droxyphenylpyruvate dioxygenase, 2,3-dihydroxybenZoate genase, 2-chlorobenZoate 1,2-dioxygenase, 2,3-dioxygenase, stiZolobate synthase, stiZolobinate syn 2-aminobenZenesulfonate 2,3-dioxygenase, terephthalate thase, arachidonate l2-lipoxygenase, arachidonate l5-li 1,2-dioxygenase, 2-hydroxyquinoline 5,6-dioxygenase, poxygenase, arachidonate 5-lipoxygenase, pyrogallol 1,2 nitric oxide dioxygenase, biphenyl 2,3-dioxygenase, 3-phe oxygenase, chloridaZon-catechol dioxygenase, nylpropionate dioxygenase, pheophorbide a oxygenase, ben hydroxyquinol 1,2-dioxygenase, l-hydroxy-2-naphthoate Zoyl-CoA 2,3-dioxygenase, and carbaZole 1,9a-dioxyge 1,2-dioxygenase, biphenyl-2,3-diol 1,2-dioxygenase, arachi nase), With NADH or NADPH as one donor, and donate 8-lipoxygenase, 2,4'-dihydroxyacetophenone dioxy incorporation of one atom of oxygen (e.g., salicylate l-mo US 2013/0244920 A1 Sep. 19,2013

nooxygenase, 4-hydroxybenZoate 3-monooxygenase, deoxocathasterone 23-monooxygenase, FAD-dependent melilotate 3-monooxygenase, imidaZoleacetate 4-monooxy urate hydroxylase, 6-hydroxynicotinate 3-monooxygenase, genase, orcinol 2-monooxygenase, phenol 2-monooxyge angelicin synthase, geranylhydroquinone 3"-hydroxylase, nase, ?aVin-containing monooxygenase, kynurenine 3-mo isoleucine N-monooxygenase, Valine N-monooxygenase, nooxygenase, 2,6-dihydroxypyridine 3-monooxygenase, 5-epiaristolochene 1,3-dihydroxylase, costunolide synthase, trans -cinnamate 4-monooxygenase, benZoate 4-monooxyge premnaspirodiene oxygenase, chlorophyllide-a oxygenase, nase, calcidiol l-monooxygenase, trans-cinnamate 2-mo germacrene A hydroxylase, phenylalanine N-monooxyge nooxygenase, cholestanetriol 26-monooxygenase, cyclopen nase, tryptophan N-monooxygenase, Vitamin D3 24-hy tanone monooxygenase, cholesterol 70t-monooxygenase, droxylase, 3-(3-hydroxyphenyl)propanoate hydroxylase, 4-hydroxyphenylacetate l-monooxygenase, taxifolin 8-mo 7-methylxanthine demethylase, [3-carotene 3-hydroxylase, nooxygenase, 2,4-dichlorophenol 6-monooxygenase, ?a pyrrole-2-carboxylate monooxygenase, dimethyl-sul?de Vonoid 3'-monooxygenase, cyclohexanone monooxygenase, monooxygenase, and squalene monooxygenase, With 3-hydroxybenZoate 4-monooxygenase, 3-hydroxybenZoate reduced ?avin or ?avoprotein as one donor, and incorporation 6-monooxygenase, methane monooxygenase (soluble), of one atom of oxygen (e.g., unspeci?c monooxygenase, phosphatidylcholine l2-monooxygenase, 4-aminobenZoate alkanal monooxygenase (FMN-linked), alkanesulfonate l-monooxygenase, 3,9-dihydroxypterocarpan 60t-monooxy monooxygenase, tryptophan 7-halogenase, anthranilate genase, 4-nitrophenol 2-monooxygenase, leukotriene-B4 3-monooxygenase (FAD), 4-hydroxyphenylacetate 3-mo 20-monooxygenase, 2-nitrophenol 2-monooxygenase, nooxygenase, and nitrilotriacetate monooxygenase), With albendaZole monooxygenase, 4-hydroxybenZoate 3-mo reduced iron-sulfur protein as one donor, and incorporation of nooxygenase [NAD(P)H], leukotriene-E4 20-monooxyge one atom of oxygen (e.g., camphor 5-monooxygenase, cam nase, anthranilate 3-monooxygenase (deaminating), 5-O-(4 phor 1,2-monooxygenase, alkane l-monooxygenase, steroid coumaroyl)-D-quinate 3'-monooxygenase, llB-monooxygenase, corticosterone l8-monooxygenase, methyltetrahydroprotoberberine l4-monooxygenase, anhy cholesterol monooxygenase (side-chain-cleaving), choline drotetracycline monooxygenase, nitric-oxide synthase, monooxygenase, and steroid lSB-monooxygenase), With anthraniloyl-CoA monooxygenase, tyrosine N-monooxyge reduced pteridine as one donor, and incorporation of one atom nase, questin monooxygenase, 2-hydroxybiphenyl 3-mo of oxygen (e.g., phenylalanine 4-monooxygenase, tyrosine nooxygenase, (—)-menthol monooxygenase, (S)-limonene 3-monooxygenase, anthranilate 3-monooxygenase, tryp 3-monooxygenase, (S)-limonene 6-monooxygenase, (S)-li tophan 5-monooxygenase, alkylglycerol monooxygenase, monene 7-monooxygenase, pentachlorophenol monooxyge and mandelate 4-monooxygenase), With reduced ascorbate as nase, 6-oxocineole dehydrogenase, iso?avone 3'-hydroxy one donor, and incorporation of one atom of oxygen (e.g, lase, 4'-methoxyiso?avone 2'-hydroxylase, ketosteroid dopamine [3-monooxygenase, peptidylglycine monooxyge monooxygenase, protopine 6-monooxygenase, dihydrosan nase, and aminocyclopropanecarboxylate oxidase), With guinarine lO-monooxygenase, dihydrochelirubine 12-mo another compound as one donor, and incorporation of one nooxygenase, benZoyl-CoA 3-monooxygenase, L-lysine atom of oxygen (e.g., monophenol monooxygenase, CMP 6-monooxygenase (NADPH), 27-hydroxycholesterol N-acetylneuraminate monooxygenase, and methane 70t-monooxygenase, 2-hydroxyquinoline 8-monooxyge monooxygenase (particulate)), With oxidation of a pair of nase, 4 -hydroxyquinoline 3 -monooxygenase, 3 -hydroxyphe donors resulting in the reduction of molecular oxygen to tWo nylacetate 6-hydroxylase, 4-hydroxybenZoate l-hydroxy molecules of Water (e.g., stearoyl-CoA 9-desaturase, acyl lase, 2-hydroxycyclohexanone 2-monooxygenase, quinine [acyl-carrier-protein] desaturase, linoleoyl-CoA desaturase, 3-monooxygenase, 4-hydroxyphenylacetaldehyde oxime As-fatty-acid desaturase, All-fatty-acid desaturase, A12 monooxygenase, alkene monooxygenase, sterol l4-dem fatty-acid desaturase, and (S)-2-hydroxypropylphosphonic ethylase, N-methylcoclaurine 3'-monooxygenase, methyl acid epoxidase), With 2-oxoglutarate as one donor, and the sterol monooxygenase, tabersonine l6-hydroxylase, other dehydrogenated (e.g. deacetoxycephalosporin-C syn 7-deoxyloganin 7-hydroxylase, Vinorine hydroxylase, taxane thase), With NADH or NADPH as one donor, and the other l0[3-hydroxylase, taxane l30t-hydroxylase, ent-kaurene oxi dehydrogenated (e.g., (S)-stylopine synthase, (S)-cheilanthi dase, ent-kaurenoic acid oxidase, (R)-limonene 6-monooxy foline synthase, berbamunine synthase, salutaridine syn genase, magnesium-protoporphyrin IX monomethyl ester thase, (S)-canadine synthase, lathosterol oxidase, bi?aviolin (oxidative) cyclase, Vanillate monooxygenase, precorrin-3B synthase, pseudobaptigenin synthase), and others including synthase, 4-hydroxyacetophenone monooxygenase, glyceol prostaglandin-endoperoxide synthase, kynurenine 7,8-hy lin synthase, 2-hydroxyiso?avanone synthase, licodione syn droxylase, heme oxygenase, progesterone monooxygenase, thase, ?avonoid 3',5'-hydroxylase, iso?avone 2'-hydroxylase, steroid l70t-monooxygenase, steroid 2l-monooxygenase, Zeaxanthin epoxidase, deoxysarpagine hydroxylase, pheny estradiol 6[3-monooxygenase, 4-androstene-3,l7-dione lacetone monooxygenase, (+)-abscisic acid 8'-hydroxylase, monooxygenase, progesterone l lot-monooxygenase, lithocholate 6[3-hydroxylase, 70t-hydroxycholest-4-en-3 -one 4-methoxybenZoate monooxygenase (O-demethylating), l20t-hydroxylase, 5 [3 -cholestane-30t,70t-diol l20t-hydroxy plasmanylethanolamine desaturase, phylloquinone lase, taurochenodeoxycholate 60t-hydroxylase, cholesterol monooxygenase (2,3-epoxidiZing), Latia-luciferin 24-hydroxylase, 24-hydroxycholesterol 70t-hydroxylase, monooxygenase (demethylating), ecdysone 20-monooxyge 25-hydroxycholesterol 70t-hydroxylase, senecionine N-oxy nase, 3-hydroxybenZoate 2-monooxygenase, steroid 90t-mo genase, psoralen synthase, 8-dimethylallylnaringenin 2'-hy nooxygenase, 2-hydroxypyridine 5-monooxygenase, juglone droxylase, (+)-menthofuran synthase, monocyclic monoter 3-monooxygenase, linalool 8-monooxygenase, deoxyhypu pene ketone monooxygenase, epi-isoZiZaene sine monooxygenase, myristoyl-CoA ll-(E) desaturase, 5-monooxygenase, limonene 1,2-monooxygenase, abietadi myristoyl-CoA ll-(Z) desaturase, Alz-fatty acid dehydroge ene hydroxylase, abietadienol hydroxylase, geranylgeraniol nase, monoprenyl iso?avone epoxidase, thiophene-2-carbo l8-hydroxylase, methanesulfonate monooxygenase, 3-epi-6 nyl-CoA monooxygenase, [3-carotene l5,l5'-monooxyge US 2013/0244920 A1 Sep. 19,2013

nase, taxadiene 50t-hydroxylase, cholesterol 25-hydroxylase, a disul?de as acceptor (e.g., D-proline reductase (dithiol), ammonia monooxygenase, 5,6-dimethylbenZimidaZole syn glycine reductase, sarcosine reductase, and betaine reduc thase, all-trans-8'-apo-[3-carotenal l5,l5'-oxygenase, Zeax tase), and With other acceptors (e.g., [3-cyclopiaZonate dehy anthin 7,8-dioxygenase, [3-amyrin 24-hydroxylase, diapoly drogenase). copene oxygenase, and carotene e-monooxygenase. [0052] The oxidoreductases Which act on halogen in donors [0045] The oxidoreductases Which act on superoxide as can include, but are not limited to, those With NAD+ or acceptor can include, but are not limited to, superoxide dis NADP+ as acceptor (e.g. iodotyrosine deiodinase). mutase and superoxide reductase. [0053] Other oxidoreductases can include, but are not lim [0046] The oxidoreductases Which act on metal ions can ited to, chlorate reductase, pyrogallol hydroxyltransferase, include, but are not limited to, those With NAD+ or NADP+ as sulfur reductase, [formate-C-acetyltransferase]activating acceptor (e.g., mercury(ll) reductase, diferric-transferrin enZyme, tetrachloroethene reductive dehalogenase, selenate reductase, aquacobalamin reductase, cob(ll)alamin reduc reductase, thyroxine 5'-deiodinase, thyroxine 5-deiodinase, tase, aquacobalamin reductase (NADPH), cyanocobalamin and photosystem I). reductase (cyanide-eliminating), ferric-chelate reductase [0054] Suitable oxidoreductases include Various sugar oxi (NADH), [methionine synthase] reductase, and ferric-chelate dases, laccases, peroxidases and haloperoxidases. reductase (NADPH)), With oxygen as acceptor (e.g., ferroxi [0055] Glycosylases dase), With quinone or similar compound as acceptor (e.g. [0056] Glycosylases include glycosidases, i.e. enZymes ascorbate ferrireductase (transmembrane)), With ?avin as hydrolyZing O- and S-glycosyl compounds, including but not acceptor (e.g. cob(ll)yrinic acid a, c-diamide reductase), With limited to ot-amylase, [3-amylase, glucan 1,4-0t-glucosidase, other acceptors (e.g. ironzrusticyanin reductase). cellulase, endo-l,3(4)-[3-glucanase, inulinase, endo-l,4-[3 [0047] The oxidoreductases Which act on CH or CH2 xylanase, oligo-l,6-glucosidase, dextranase, chitinase, groups can include, but are not limited to, those With NAD+ or polygalacturonase, lysoZyme, exo-ot-sialidase, ot-glucosi NADP+ as acceptor (e.g., CDP-4-dehydro-6-deoxyglucose dase, [3-glucosidase, ot-galactosidase, [3-galactosidase, reductase, 4-hydroxy-3-methylbut-2-enyl diphosphate ot-mannosidase, [3-mannosidase, [3-fructofuranosidase, 0t,0t reductase, leucoanthocyanidin reductase, xanthine dehydro trehalase, [3-glucuronidase, endo-l,3-[3-xylanase, amylo-l,6 genase, and nicotinate dehydrogenase), With a cytochrome as glucosidase, hyaluronoglucosaminidase, hyaluronoglucu acceptor (e.g. nicotinate dehydrogenase (cytochrome)), With ronidase, xylan 1,4-[3-xylosidase, [3-D-fucosidase, glucan oxygen as acceptor (e.g., pteridine oxidase, xanthine oxidase, endo-l,3-[3-D-glucosidase, ot-L-rhamnosidase, pullulanase, and 6-hydroxynicotinate dehydrogenase), With a disul?de as GDP-glucosidase, [?-L-rhamnosidase, fucoidanase, glucosyl acceptor (e.g., ribonucleoside-diphosphate reductase, and ceramidase, galactosylceramidase, galactosylgalactosylglu ribonucleoside-triphosphate reductase), With a quinine or cosylceramidase, sucrose ot-glucosidase, ot-N-acetylgalac similar compound as acceptor (e.g., phenylacetyl-CoA dehy tosaminidase, ot-N-acetylglucosaminidase, ot-L-fucosidase, drogenase and caffeine dehydrogenase), With an iron-sulfur [3-L-N-acetylhexosaminidase, [3-N-acetylgalactosaminidase, protein as acceptor (e.g., (E)-4-hydroxy-3 -methylbut-2-enyl cyclomaltodextrinase, (x-N-arabinofuranosidase, glucurono diphosphate synthase), and With other acceptors (e.g., 4-me syl-disulfoglucosamine glucuronidase, isopullulanase, glu thylphenol dehydrogenase (hydroxylating), ethylbenZene can 1,3-[3-glucosidase, glucan endo-l,3-0t-glucosidase, glu hydroxylase, 3(>t,70t, l 20t-trihydroxy-5 [3 -cholestanoyl-CoA can 1 ,4-0t-maltotetraohydrolase, mycodextranase, 24-hydroxylase, uracil/thymine dehydrogenase, and bile glycosylceramidase, l,2-(x-L-?1cosidase, 2,6-[3-fructan 6-le acid 70t-dehydroxylase). Vanbiohydrolase, levanase, quercitrinase, galacturan 1,4-0t [0048] The oxidoreductases Which act on iron-sulfur pro galacturonidase, isoamylase, glucan 1,6-0t-glucosidase, glu teins as donors can include, but are not limited to, those With can endo-l,2-[3-glucosidase, xylan 1,3-[3-xylosidase, NAD+ or NADP+ as acceptor (e.g., ruhredoxiniNAD+ licheninase, glucan 1,4-[3-glucosidase, glucan endo-l,6-[3 reductase, ferredoxiniNADP+ reductase, ferredoxini glucosidase, L-iduronidase, mannan l,2-(l,3)-0t-mannosi NAD+ reductase, and ruhredoxiniNAD(P)+ reductase), dase, mannan endo-l,4-[3-mannosidase, fructan [3-fructosi With H+ as acceptor, and With dinitrogen as acceptor (e.g., dase, [3-agarase, exo-poly-ot-galacturonosidase, nitrogenase). K-carrageenase, glucan 1,3-0t-glucosidase, 6-phospho-[3-ga [0049] The oxidoreductases Which act on reduced ?a lactosidase, 6-phospho-[3-glucosidase, capsular-polysaccha Vodoxin as donor can include, but are not limited to, those ride endo-l ,3-0t-galactosidase, [3-L-arabinosidase, arabinoga With dinitrogen as acceptor (e.g., nitrogenase (?avodoxin)). lactan endo-l ,4- [3-galactosidase, cellulose 1,4-[3 - [0050] The oxidoreductases Which act on phosphorus or cellobiosidase (non-reducing end), peptidoglycan [3-N arsenic in donors can include, but are not limited to, those acetylmuramidase, 0t,0t-phosphotrehalase, glucan 1,6-0t With NAD(P)+ as acceptor (e.g. phosphonate dehydroge isomaltosidase, dextran 1,6-a-isomaltotriosidase, mannosyl nase), With a cytochrome as acceptor (e.g., arsenate reductase glycoprotein endo-[3-N-acetylglucosaminidase, endo-ot-N (cytochrome c)), With disul?de as acceptor (e.g., arsenate acetylgalactosaminidase, glucan 1,4-0t-maltohexaosidase, reductase (glutaredoxin), methylarsonate reductase, and arabinan endo-l,5-(x-L-arabinanase, mannan l,4-mannobio mycoredoxin), With a copper protein as acceptor (e. g. arsen sidase, mannan endo-l,6-0t-mannosidase, blood-group-sub ate reductase (aZurin)), and With other acceptors (e. g. arsenate stance endo-l ,4-[3-galactosidase, keratan-sulfate endo-l ,4-[3 reductase (donor)). galactosidase, steryl-[3-glucosidase, strictosidine [0051] The oxidoreductases Which act on XiH and YiH [3-glucosidase, mannosyl-oligosaccharide glucosidase, pro to form an XiY bond can include, but are not limited to, tein-glucosylgalactosylhydroxylysine glucosidase, lactase, those With oxygen as acceptor (e.g., isopenicillin-N synthase, endogalactosaminidase, l,3-(x-L-?1cosidase, 2-deoxyglu columbamine oxidase, reticuline oxidase, sulochrin oxidase cosidase, mannosyl-oligosaccharide 1,2-0t-mannosidase, [(+)-bisdechlorogeodin-forming], sulochrin oxidase [(—) manno syl-oligo saccharide 1,3 -l ,6-0t-mannosidase, bisdechlorogeodin-forming], and aureusidin synthase), With branched-dextran exo-l,2-0t-glucosidase, glucan 1,4-0t-mal US 2013/0244920 A1 Sep. 19, 2013

totriohydrolase, amygdalin [3-glucosidase, prunasin [3-glu ric diester hydrolases including phosphlipase C, cosidase, vicianin [3-glucosidase, oligoxyloglucan [3-glycosi phospholipase D, phosphoinositide phospholipase C, glyco dase, polymannuronate hydrolase, maltose-6'-phosphate sylphosphatidylinositol phospholipase D, and N-acetylphos glucosidase, endoglycosylceramidase, 3-deoxy-2-octu phatidylethanolamine-hydrolysing phospholipase D) and losonidase, raucaffricine [3-glucosidase, coniferin [3-glucosi glycosylases, including glycosidases, i.e. enzymes hydrolys dase, 1,6-(x-L-?1cosidase, glycyrrhiZinate [3-glucuronidase, ing O- and S-glycosyl compounds, for example amylases endo-ot-sialidase, glycoprotein endo-ot-1,2-mannosidase, (including alpha-amylase, beta-amylase, and isoamylase), xylan ot-1,2-glucuronosidase, chitosanase, glucan 1,4-0t maltohydrolase, difructose-anhydride synthase, neopullula cellulases, and mannanases. nase, glucuronoarabinoxylan endo-1,4-[3-xylanase, mannan [0061] Suitable lipases and cutinases include those of bac exo-1,2-1,6-0t-mannosidase, ot-glucuronidase, lacto-N-bio terial or fungal origin. Chemically modi?ed or protein engi sidase, 4-0t-D-{(1Q4)-0t-D-glucano}trehalose trehalohydro neered mutants are included. Examples include lipase from lase, limit dextrinase, poly(ADP-ribose) glycohydrolase, Thermomyces, e.g., from T lanuginasus (previously named 3-deoxyoctulosonase, galactan 1,3-[3-galactosidase, [3-ga Humicala lanuginasa) as described in EP 258 068 (and US. lactofuranosidase, thioglucosidase, [3-primeverosidase, oli Pat. No. 4,810,414) and EP 305 216 (and US. Pat. Nos. goxyloglucan reducing-end-speci?c cellobiohydrolase, 5,766,912; 5,874,558; 5,965,384; 7,517,668; 5,536,661 and xyloglucan-speci?c endo-[3-1,4-glucanase, mannosylglyco 5,863,759), cutinase from Humicola, e.g. H. insolens as protein endo-[3-mannosidase, fructan [3-(2,1)-fructosidase, described in W0 96/ 13580, a Pseudomanas lipase, e.g., from fructan [3-(2,6)-fructosidase, xyloglucan-speci?c exo-[3-1,4 P alcaligenes orP pseudoalcaligenes (EP 218 272, US. Pat. glucanase, oligosaccharide reducing-end xylanase, L-carrag Nos. 5,766,912; 5,863,759; 5,874,558; 5,965,384; 7,517,668 eenase, ot-agarase, (x-neoagaro-oligosaccharide hydrolase, and 5,536,661), R cepacia (EP 331 376 and US. Pat. No. [3-apiosyl-[3-glucosidase, lt-carrageenase, 1,6-0t-D-mannosi 5,290,694), P slulzeri (GB 1,372,034), P ?uorescens, dase, galactan endo-1,6-[3-galactosidase, exo-1,4-[3-D-glu Pseudomanas sp. strain SD 705 (WO 95/06720, US. Pat. No. cosaminidase, heparanase, baicalin-[3-D-glucuronidase, hes 5,827,718, WO 96/27002, and US. Pat. No. 5,942,431), R peridin 6-O-0t-L-rhamnosyl-[3-D-glucosidase, protein wisconsinensis (WO 96/12012), a Bacillus lipase, e.g., from l-GlcNAcase, mannosylglycerate hydrolase, rhamnogalactu B. sublilis (Dartois et al., 1993, Biochemica et Biophysica ronan hydrolase, unsaturated rhamnogalacturonyl hydrolase, Acla, 1131: 253-360), B. slearolhermophilus (JP 64/744992) rhamnogalacturonan galacturonohydrolase, rhamnogalactur or B. pumilus (WO 91/16422 and US. Pat. No. 5,427,936). onan rhamnohydrolase, [3-D-glucopyranosyl abscisate [3-glu Other examples are lipase variants such as those described in cosidase, cellulose 1,4-[3-cellobiosidase (reducing end), (x-D WO 92/05249, WO 94/01541, EP 407 225, EP 260105,WO xyloside xylohydrolase, and [3-porphyranase. 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO [0057] Glycosylases also include hydrolyZing N-glycosyl 95/14783, WO 95/22615, WO 97/04079, WO 97/07202, WO compounds, including but not limited to purine nucleosidase, 00/060063, WO2007/087508, WO 2009/109500 and US. inosine nucleosidase, uridine nucleosidase, AMP nucleosi Pat. Nos. 5,892,013; 5,869,438; 5,976,855; 6,020,180; 6,074, dase, NAD+ nucleosidase, NAD(P)+ nucleosidase, adenosine 863; 5,658,871; 4,760,025; 5,155,033; 5,182,204; 5,185,258; nucleosidase, ribosylpyrimidine nucleosidase, adenosylho 5,204,015; 5,244,791; 5,264,366; 5,310,675; 5,316,941; mocysteine nucleosidase, pyrimidine-5'-nucleotide nucleosi 5,346,823; 5,352,594; 5,371,008; 5,371,190; 5,411,873; dase, [3-aspartyl-N-acetylglucosaminidase, inosinate nucle 5,441,882; 5,472,855; 5,652,136; 5,700,676; 5,763,257; osidase, 1-methyladeno sine nucleosidase, NMN 5,801,038; 5,939,315; 5,955,340; 5,972,682; 6,465,235; and nucleosidase, DNA-deoxyinosine glycosylase, methylthio RE34,606. Preferred commercially available lipase enzymes adenosine nucleosidase, deoxyribodipyrimidine endonucle include LipolaseTM, Lipolase UltraTM, and LipexTM; osidase, ADP-ribosylarginine hydrolase, DNA-3-methylad LecitaseTM, LipolexTM; LipocleanTM, LipoprimeTM (No enine glycosylase l, DNA-3-methyladenine glycosylase ll, voZymes A/ S). Other commercially available lipases include rRNA N-glycosylase, DNA-formamidopyrimidine glycosy Lumafast (Genencor lnt lnc); Lipomax (Gist-Brocades/Ge lase, ADP-ribosyl-[dinitrogen reductase] hydrolase, N-me nencor lnt Inc) and Bacillus sp lipase from Solvay. thyl nucleosidase, futalosine hydrolase, uracil-DNA glycosy [0062] Suitable amylases (O. and/or [3) include those of lase, double-stranded uracil-DNA glycosylase, and thymine bacterial or fungal origin. Chemically modi?ed or protein DNA glycosylase, and hydrolyZing S-glycosyl compounds. engineered mutants are included. Amylases include, for [0058] Hydrolases example, ot-amylases obtained from Bacillus, e.g., a special [0059] Hydrolases of EC 3 include, but are not limited to: strain of Bacillus licheniformis, described in more detail in those acting on ester bonds; glycosylases, those acting on GB 1,296,839. Examples of useful amylases are the variants ether bonds; those acting on peptide bonds (peptidases/pro described in WO 94/02597, WO 94/18314, WO 96/23873, teases); those acting on carbon-nitrogen bonds, other than WO 97/43424, US. Pat. Nos. 5,824,532; 5,849,549; 6,297, peptide bonds; those acting on acid anhydrides; those acting 037; 6,093,562; 6,297,038; 6,867,031; and US Publication on carbon-carbon bonds; those acting on halide bonds; those Nos. 2002/0098996; 2003/0064908; 2004/0253676; 2005/ acting on phosphorus-nitrogen bonds; those acting on sulfur 0059131; 2005/0250664; 2006/0035323; 2009/0280527; nitrogen bonds; those acting on carbon-phosphorus bonds; 2010/0099597; 2010/0099598; and 2011/0177990, espe those acting on sulfur-sulfur bonds; and those acting on car cially the variants With substitutions in one or more of the bon-sulfur bonds. folloWing positions: 15, 23, 105, 106, 124, 128, 133, 154, [0060] The hydrolases of EC 3 Which act on ester bonds, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, can include, but are not limited to, carboxylic ester hydrolases 391, 408, and 444. Commercially available amylases are (for example lipases including triacylglycerol lipase, phos DuramylTM, TermamylTM, FungamylTM and BANTM (No pholipase A1, phospholipase A2, lysophospholipase, acylg voZymes A/ S), RapidaseTM and PurastarTM (from Genencor lycerol lipase, galactolipase, lipoprotein lipase; and phospho International Inc.) US 2013/0244920 A1 Sep. 19, 2013

[0063] Suitable cellulases include those of bacterial or fun ypeptidase T, carboxypeptidase Taq, carboxypeptidase U, gal origin. Chemically modi?ed or protein engineered glutamate carboxypeptidase ll, metallocarboxypeptidase D, mutants are included. Suitable cellulases include cellulases and angiotensin-converting enZyme 2); ccysteine-type car from the genera Bacillus, Pseudomonas, Humicola, boxypeptidases, including cathepsin X; omega peptidases Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases (including acylaminoacyl-peptidase, peptidyl-glycinami produced from Humicola insolens, Myceliophlhora thermo dase, pyroglutamyl-peptidase I, beta-aspartyl-peptidase, phila and Fusarium oxysporum disclosed in US. Pat. Nos. pyroglutamyl-peptidase ll, N-formylmethionyl-peptidase, 4,435,307; 5,648,263; 5,691,178; 5,776,757 and WO gamma-glutamyl hydrolase, gamma-D-glutamyl-meso-di 89/09259. Especially suitable cellulases are the alkaline or aminopimelate peptidase I, and ubiquitinyl hydrolase 1); neutral cellulases having color care bene?ts. Examples of serine endopeptidases (including chymotrypsin, chymot such cellulases are cellulases described in EP 0 495 257, EP rypsin C, metridin, trypsin, thrombin, coagulation factor Xa, 0 531372,WO 96/11262,WO 96/29397, WO 98/08940, US. Pat. Nos. 5,520,838; 5,443,750; 5,668,073; 5,948,672; 6,423, plasmin, enteropeptidase, acrosin, alpha-Lytic endopepti 524; 5,919,691; 6,071,735; 6,001,639; 6,387,690; 6,855,531; dase, glutamyl endopeptidase, cathepsin G, coagulation fac 7,226,773; and US Publication Nos. 2001/0036910; 2003/ tor Vlla, coagulation factor lXa, cucumisin, prolyl oligopep 01 19167; 2003/0054539; 2005/0070003; 2008/0145912; and tidase, coagulation factor Xla, brachyuran, plasma kallikrein, 2010/0107342. Other examples are cellulase variants such as tissue kallikrein, pancreatic elastase, leukocyte elastase, those described in WO 94/07998, EP 0 531 315, WO coagulation factor Xlla, chymase, complement subcompo 95/24471, WO 98/12307, PCT/DK98/00299, US. Pat. Nos. nent Cl’, complement subcomponent C“, classical-comple 5,457,046; 5,686,593; 5,763,254; 5,792,641; 6,114,296; ment-pathWay C3/C5 convertase, complement factor I, 5,457,046; 5,912,157; 6,117,664; 7,993,898; 8,017,372; and complement factor D, alternative-complement-pathWay US Publication Nos. 2003/0092097; 2005/0009166; 2008/ C3/C5 convertase, cerevisin, hypodermin C, lysyl endopep 0206836; 2009/0170747 and 2011/0250674. Commercially tidase, endopeptidase La, gamma-renin, venombin AB, available cellulases include CelluZymeTM, and CareZymeTM leucyl endopeptidase, tryptase, scutelarin, kexin, subtilisin, (NovoZymes A/ S), ClaZinaseTM, and Puradax HATM (Genen oryZin, peptidase K, thermomycolin, thermitase, endopepti dase So, t-plasminogen activator, protein C (activated), pan cor lntemational Inc.), and KAC-500(B)TM (Kao Corpora creatic endopeptidase E, pancreatic elastase ll, lgA-speci?c tion). serine endopeptidase, u-plasminogen activator, venombin A, [0064] Peptidases/Proteases furin, myeloblastin, semenogelase, granZyme A, granZyme [0065] The hydrolases of EC 3 Which act on peptide bonds B, streptogrisinA, streptogrisin B, glutamyl endopeptidase ll (peptidases/proteases) can include, but are not limited to ami oligopeptidase B, limulus clotting factor C, limulus clotting nopeptidases (including leucyl aminopeptidase, membrane factor B, limulus clotting enZyme, repressor LeXA, signal alanyl aminopeptidase, cystinyl aminopeptidase, tripeptide peptidase I, togavirin, ?avivirin, endopeptidase Clp, propro aminopeptidase, prolyl aminopeptidase, arginyl aminopepti tein convertase 1, proprotein convertase 2, snake venom fac dase, glutamyl aminopeptidase, Xaa-Pro aminopeptidase, tor V activator, lactocepin, assembling, hepacivirin, sper bacterial leucyl aminopeptidase, clostridial aminopeptidase, mosin, sedolisin, Xanthomonalisin, C-terminal processing cytosol alanyl aminopeptidase, lysyl aminopeptidase, Xaa peptidase, physarolisin, mannan-binding lectin-associated Trp aminopeptidase, tryptophanyl aminopeptidase, methio serine protease-2, rhomboid protease, hepsin, peptidase Do, nyl aminopeptidase, D-stereospeci?c aminopeptidase, ami HtrA2 peptidase, matriptase, C5a peptidase, aqualysin 1, nopeptidase Ey, aspartyl aminopeptidase, aminopeptidase I, site-1 protease, pestivirus NS3 polyprotein peptidase, equine PepB aminopeptidase, aminopeptidase S, beta-peptidyl ami arterivirus serine peptidase, infectious pancreatic necrosis nopeptidase, and intermediate cleaving peptidase 55); dipep birnavirus Vp4 peptidase, SpolVB peptidase, stratum cor tidases (including Xaa-Arg dipeptidase. Xaa-methyl-His neum chymotryptic enZyme, kallikrein 8, kallikrein 13, and dipeptidase, Glu-Glu dipeptidase, Xaa-Pro dipeptidase, Met oviductin); cysteine endopeptidases(including cathepsin B, Xaa dipeptidase, non-stereospeci?c dipeptidase, cytosol non papain, ?cain, chymopapain, asclepain, clostripain, strepto speci?c dipeptidase, membrane dipeptidase, beta-Ala-His pain, actimidain, cathepsin L, cathepsin H, cathepsin T, gly dipeptidase, dipeptidase E, and D-Ala-D-Ala dipeptidase); cyl endopeptidase, cancer procoagulant, cathepsin S, picor Dipeptidyl-peptidases and tripeptidyl-peptidases (including nain 3C, picornain 2A, caricain, ananain, stem bromelain, dipeptidyl-peptidase I, dipeptidyl-peptidase ll, dipeptidyl fruit bromelain, legumain, histolysain, caspase-1, gingipain peptidase Ill, dipeptidyl-peptidase IV, dipeptidyl-dipepti R, cathepsin K, adenain, bleomycin hydrolase, cathepsin F, dase, tripeptidyl-peptidase I, tripeptidyl-peptidase ll, Xaa cathepsinV, cathepsin O, nuclear-inclusion-a endopeptidase, Pro dipeptidyl-peptidase, and prolyltripeptidyl helper-component proteinase, L-peptidase, gingipain K, sta aminopeptidase); peptidyl-dipeptidases (including peptidyl phopain, separase, V-cath endopeptidase, cruZipain, calpain dipeptidase A, peptidyl-dipeptidase B, peptidyl-dipeptidase 1, calpain-2, calpain-3, caspase-2, caspase-3, caspase-4, Dcp, and cyanophycinase); serine-type carboxypeptidases caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, (including lysosomal Pro-Xaa carboxypeptidase, serine-type caspase-10, caspase-11,peptidase 1 (mite), calicivirin, Zingi D-Ala-D-Ala carboxypeptidase, carboxypeptidase C, and pain, Ulp1 peptidase, SARS coronavirus main proteinase, carboxypeptidase D); metallocarboxypeptidases (including sortase A, and sortase B); aspartic endopeptidases (including carboxypeptidase A, carboxypeptidase B, lysine carboxypep pepsinA, pepsin B, gastricsin, chymosin, cathepsin D, nepen tidase, Gly-Xaa carboxypeptidase, alanine carboxypepti thesin, renin, HIV-1 retropepsin, Pro-opiomelanocortin con dase, muramoylpentapeptide carboxypeptidase, carboxypep verting enZyme, aspergillopepsin I, aspergillopepsin ll, peni tidase E, glutamate carboxypeptidase, carboxypeptidase M, cillopepsin, rhiZopuspepsin, endothiapepsin, mucorpepsin, muramoyltetrapeptide carboxypeptidase, Zinc D-Ala-D-Ala candidapepsin, saccharopepsin, rhodotorulapepsin, acrocy carboxypeptidase, carboxypeptidase A2, membrane Pro-Xaa lindropepsin, polyporopepsin, pycnoporopepsin, scytali carboxypeptidase, tubulinyl-Tyr carboxypeptidase, carbox dopepsin A, scytalidopepsin B, cathepsin E, barrierpepsin, US 2013/0244920 A1 Sep. 19, 2013

signal peptidase II, plasmepsin I, plasmepsin II, phytepsin, synthase, and oxido-reductase enzymes, including oxido-re yapsin 1, thermopsin, prepilin peptidase, nodavirus endopep ductase enzymes that catalyze the formation of bleaching tidase, memapsin 1, memapsin 2, HIV-2 retropepsin, plasmi agents. no gen activator Pla, omptin, human endogenous retrovirus K [0068] It is contemplated that an enzyme for use in a com endopeptidase, and HycI peptidase); metalloendopeptidases position described herein can come from any suitable source (including atrolysin A, microbial collagenase, leucolysin, or combination of sources, for example bacterial, fungal, interstitial collagenase, neprilysin, envelysin, IgA-speci?c plant, or animal sources. In one type of embodiment, a mix metalloendopeptidase, procollagen N-endopeptidase, thimet ture of tWo or more enzymes Will come from at least tWo oligopeptidase, neurolysin, stromelysin 1, meprin A, procol different types of sources. For example, a mixture of protease lagen C-endopeptidase, peptidyl-Lys metalloendopeptidase, and lipase can come from a bacterial (protease) and fungal astacin, stromelysin 2, matrilysin, gelatinase A, vibriolysin, (lipase) sources. pseudolysin, thermolysin, bacillolysin, aureolysin, coccol [0069] In one type of embodiment, it is contemplated that ysin, mycolysin, [3-lytic metalloendopeptidase, peptidyl-Asp the enzyme for use in a composition described herein is not metalloendopeptidase, neutrophil collagenase, gelatinase B, amylase. In another type of embodiment, When only one type leishmanolysin, saccharolysin, gametolysin, deuterolysin, of enzyme is included in the composition, the enzyme is not serralysin, atrolysin B, atrolysin C, atroxase, atrolysin E, amylase. In yet another type of embodiment, When tWo or atrolysin F, adamalysin, horrilysin, ruberlysin, bothropasin, more types of enzymes are included in the composition the bothrolysin, ophiolysin, trimerelysin I, trimerelysin II, tWo or more enzymes are not amylase. In still another type of mucrolysin, pitrilysin, insulysin, O-sialoglycoprotein embodiment, When tWo or more types of enzymes are endopeptidase, russellysin, mitochondrial intermediate pep included in the composition, one type of enzyme is amylase tidase, dactylysin, nardilysin, magnolysin, meprin B, mito and another type of enzyme is not amylase. chondrial processing peptidase, macrophage elastase, chori [0070] Optionally, an enzyme for use herein, including but olysin L, choriolysin H, tentoxilysin, bontoxilysin, not limited to any enzyme class or member described herein, oligopeptidase A, endothelin-converting enzyme, ?brolase, is one Which Works in alkaline pH conditions, e. g. for use in jararhagin, fragilysin, lysostaphin, ?avastacin, snapalysin, detergent applications including laundry detergent and/or gpr endopeptidase, pappalysin-1, membrane-type matrix dish detergent, for example but not limited to a pH in a range metalloproteinase- 1, ADAM10 endopeptidase, ADAMTS-4 of about 7 to about 12, or greater than 7 to about 12, or about endopeptidase, anthrax lethal factor endopeptidase, Ste24 8 to about 12, or about 8 to about 11. Optionally, an enzyme endopeptidase, S2P endopeptidase, ADAM 17 endopepti for use herein, including but not limited to any enzyme class dase, and ADAMTS13 endopeptidase); and threonine or member described herein, is one Which Works in a tem endopeptidases (including proteasome endopeptidase com perature in a range of about 50 C. to about 450 C. plex and HslUiHslV peptidase). [0071] It is Well knoWn in the art that the quantity of enzyme used in any application is a function of the activity of [0066] Suitable proteases include those of animal, veg the speci?c enzyme. While the activity of the enzyme is the etable or microbial origin. Microbial origin is preferred. important parameter, it is readily appreciated that this is not a Chemically modi?ed or protein engineered mutants are unit of measure that can be easily used in production. There included. The protease may be a serine protease or a metal fore, it is knoWn in the art to characterize the enzyme accord loprotease, preferably an alkaline microbial protease or a ing to the activity per gram of the dry enzyme mixture. The trypsin-like protease. Examples of alkaline proteases are sub dry enzyme mixture may be of high purity and essentially tilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and 100% active enzyme or it may contain fermentation byprod ucts. Therefore a commercially available enzyme mixture subtilisin 168 (described in WO 89/06279, US. Pat. Nos. may have a Wide range of purity but can still be used in a 6,506,589; 6,808,913; 6,835,821; and US Publication Nos. formulation based on a reported activity per unit Weight. As 2003/0148495; 2003/0175933; 2003/0186378 and 2005/ used herein, the phrase “enzyme mixture” Will refer to the dry 0003986). Examples of trypsin-like proteases are trypsin mixture of active enzyme and optional residual fermentation (e. g., of porcine or bovine origin) and the Fusarium protease products. The total amount of active enzyme (or optionally, described in WO 89/06270, WO 94/25583, and US. Pat. Nos. mixture of tWo or more enzymes) included in the Water 5,288,627 and 5,693,520. Examples of useful proteases are soluble ?lm can be at least about 0.1 phr, optionally in a range the variants described in WO 92/19729, WO 98/20115, WO of about 0.1 to about 20 phr, or to about 2 phr, 4 phr, 5 phr, 10 98/20116, WO 98/34946, and US. Pat. Nos. 5,858,757; phr, or 15 phr, including such individual values and any 6,300,116; 7,098,017; 6,159,731; and US Publication No. combination of ranges formed by such values. In embodi 2002/0102702, especially the variants With substitutions in ments that include both a protease and a different, second one or more ofthe folloWing positions: 27, 36, 57, 76, 87, 97, enzyme, the protease activity to second enzyme activity ratio 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235, and 274. Preferred commercially available protease enzymes can be in any suitable range, for example in a rangeof about 0.2:1 to about 10:1, or about 0.25:1 to about 10:1, or about include AlcalaseTM, SavinaseTM, PrimaseTM, DuralaseTM 0.2:1 to about 3: 1, for example. The handling of dry poWders EsperaseTM, and KannaseTM (Novozymes A/ S), MaxataseTM, of enzyme is not alWays desirable, particularly due to the MaxacalTM, MaxapemTM ProperaseTM, PurafectTM, Purafect safety hazards of handling enzyme dusts. Frequently enzyme OxPTM, FN2TM, and FN3TM (Genencor International Inc.) mixtures are obtained as liquids With the enzyme dissolved in [0067] Enzymes for use in laundry and dishWashing appli a carrier solvent. Enzyme solutions are characterized by the cations can include one or more of protease, amylase, lipase, enzyme activity per unit Weight (or volume) alloWing for a dehydrogenase, transaminase, kinase, cellulase, mannanase, speci?c Weight or volume of enzyme mixture per batch. Suit peptidase, decarboxylase, isomerase, mutase, synthetase, able active enzymes are commercially available as solutions US 2013/0244920 A1 Sep. 19, 2013

from suppliers including Novozymes, Genencor Int Inc, Kao soluble compositions can depend upon one or more variables, Corporation and Gist-Brocades. including, but not limited to the Weight of the substrate, molar [0072] Enzyme Substrate concentration, steric availability, and processing tempera [0073] Water-soluble ?lms according to the present disclo tures. Suitable enzyme amounts in the composition and suit sure can further include an enzyme stabilizer Which is a able substrate to enzyme ratios can be determined by routine functional substrate for one or more enzymes in the enzyme experimentation, for example using the enzyme activity mea composition. Embodiments of enzyme substrates, When surement technique described herein. Thus, for example included in Water-soluble ?lms according to the present dis When a glycerol monooleate is used as a substrate With a closure, have been shoWn to result in a percent recovery of lipase, the glycerol monooleate can be provided in a ratio of enzymes in a range of about 20% to 100% recovery. In one the Weight of the glycerol monooleate to the enzyme activity. type of embodiment, the inclusion of enzyme substrate can Alternatively, for example, When a glycerol dioleate is used as result in at least 70% recovery of enzyme activity. That is, at a substrate With a lipase the glycerol dioleate can be provided least 70% of enzyme activity Was maintained folloWing ?lm in a ratio of half the Weight of the glycerol dioleate to the formation. In another aspect, inclusion of an enzyme sub enzyme activity (i.e., tWo available binding sites), hoWever strate can result in an increase in enzyme recovery of at least more may be needed if one of the binding sites is sterically 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, inaccessible. Similarly, in the case of a protein-protease inter 100%, or more, for example up to 250% or more, relative to action, for example, it is believed that With a given Weight ?lms With no enzyme substrate included, as demonstrated in ratio of protein to enzyme activity, a loW molecular Weight the Examples beloW. protein may provide better protection than a medium molecu [0074] Enzyme substrates Will be evident to the person of lar Weight protein (i.e., there Will be more proteins available ordinary skill in the art in vieW of the present disclosure. For for interaction With a loW molecular Weight protein). In the example, substrates can be selected from those donor and case of a high molecular Weight protein, the protection acceptor classes described above in connection With the types offered may be better than that of a medium molecular Weight of enzymes. As speci?c examples, substrates for amylases protein for a given Weight ratio of protein to enzyme activity Will include complex sugars and starches, substrates for (i.e., the larger protein may be sterically available to more lipases Will include lipids, for example glycerol monooleate, than one enzyme). substrates for proteases Will include proteins, for example a [0076] In one class of embodiments, the enzyme substrate soy protein, substrates for cellulases Will include celluloses, Will not provide any other function in the ?lm other than to for example methyl cellulose, and substrates for mannanases serve as a substrate for enzyme activity. Thus, for example, in Will include mannan, for example, a guar gum. one type of embodiment, the enzyme substrate Will not act as [0075] The amount of the enzyme substrate incorporated a surfactant. In another non-exclusive type of embodiment, into the ?lm can be any suitable amount to provide the the enzyme substrate Will not act as a plasticizer. enzyme With functional substrate material during ?lm pro [0077] In another class of embodiments, the enzyme sub cessing and storage. Therefore, the amount of enzyme sub strate may be the type that Would provide secondary func strate to be incorporated into the ?lm is an amount effective for increasing the stability of the enzyme and thus increasing tionality to the ?lm other than to serve as a substrate for the recovery of enzyme activity, folloWing processing, for enzyme activity, and in this class of embodiments the enzyme substrate can be included in amounts less than those neces example ?lm formation. In one type of embodiment, option sary for the secondary function, or in excess of those neces ally the amount of enzyme substrate is not in great excess of sary to provide the secondary function. Thus, for example, the amount effective for increasing stability of the enzyme; While starches can affect ?lm performance as compounding for example, the amount of enzyme substrate in embodiments agents, a starch can be included in a ?lm herein in an amount can be no more than 1.5 times the effective amount, or no that Would not have a practical effect on the mechanical more than 2 times the effective amount, or no more than 3 properties of the ?lm, but in an amount Which Would be times the effective amount. In the alternative, the composition suf?cient to act as an enzyme stabilizer in functioning as a can contain an excess of enzyme substrate to ensure suf?cient quantity for the enzyme loading in the composition, subject to substrate for enzyme activity. the excess quantity not compromising other desired proper [0078] Without intending to be bound by any particular ties of the composition (e.g., ?lm strength). For example, the theory, it is believed that the enzyme substrate improves enzyme substrate can be incorporated into the ?lm in an enzyme activity recovery by one or both of tWo mechanisms. amount of at least about 1phr or at least 2 phr, optionally in a First, it is believed that When the enzyme is provided a sub range of about 2 to about 8 phr. In other embodiments, the strate upon Which to act, and thus the enzyme Will be com amount of enzyme substrate can be incorporated into the plexing With the substrate, the enzyme is less likely to dena composition or ?lm in an amount of 1 phr or less, or 2 phr or ture by one or more of various mechanisms (e.g., heat, acid, less, or 2.5 phr or less, or 3 phr or less, or 3.5 phr or less, or 4 alkali, radiation). Second, it is believed that When an enzyme phr or less, or 4.5 phr orless, or 5 phr or less, or 5.5 phr or less, is provided With a substrate upon Which to act, and thus the or 6 phr or less, or 6.5 phr or less, or 7 phr or less, or 7.5 phr enzyme Will be complexing With the substrate, the enzyme is or less, or 8 phr or less, or 8.5 phr or less, or 9 phr or less. The less likely to be subject to lysis by protease enzymes because ratio of substrate to enzyme mixture can be any suitable the binding site(s) for lysis are occupied or otherWise steri amount consistent With the disclosure herein, for example at cally hindered. Thus, for example, in Example 6 beloW, the least about 05:10, or at least about 201.0, in a range of lipase (a fungal enzyme) apparently has no inherent resis about 05:10 to about 2.0:1.0, or at least about 05:10 to tance to the protease enzyme (a bacterial enzyme), and so about 10: 1, for example. Without intending to be bound by recovery of lipase enzyme activity Was undetectable. HoW any particular theory, it is believed that the amount of sub ever, providing a lipase substrate in the ?lm led to a surprising strate that is needed to stabilize the enzymes in the Water and substantial recovery of lipase enzyme activity. US 2013/0244920 A1 Sep. 19,2013

[0079] The Water-soluble ?lms according to the present bubbles. Suitable defoamers for use in Water-soluble ?lms disclosure may include other optional additive ingredients according to the present disclosure include, but are not lim including, but not limited to, plasticizers, surfactants, ited to, hydrophobic silicas, for example silicon dioxide or defoamers, ?lm formers, antiblocking agents, caustic sodas, fumed silica in ?ne particle sizes, including Foam Blast® internal release agents, anti-yelloWing agents, colorants, defoamers available from Emerald Performance Materials, including but not limited to dyes and pigments, and other including Foam Blast® 327, Foam Blast® UVD, Foam functional ingredients, including but not limited to carriers Blast® 163, Foam Blast® 269, Foam Blast® 338, Foam and/ or encapsulates containing perfume components, bleach Blast® 290, Foam Blast® 332, Foam Blast® 349, Foam ing agents, or bleach catalysts, for example in amounts suit Blast® 550 and Foam Blast® 339, Which are proprietary, able for their intended purpose. non-mineral oil defoamers. In embodiments, defoamers can [0080] Water is recognized as a very ef?cient plasticizer for be used in an amount of 0.5 phr, or less, for example, 0.05 phr, PVOH and other polymers; including but not limited to Water 0.04 phr, 0.03 phr, 0.02 phr, or 0.01 phr. Preferably, signi? soluble polymers, hoWever, the volatility of Water makes its cant amounts of silicon dioxide Will be avoided, in order to utility limited since polymer ?lms need to have at least some avoid stress Whitening. resistance (robustness) to a variety of ambient conditions [0084] Processes for making Water-soluble articles, includ including loW and high relative humidity. Glycerin is much ing ?lms, include casting, bloW-molding, extrusion and less volatile than Water and has been Well established as an bloWn extrusion, are knoWn in the art. One contemplated class effective plasticizer for PVOH and other polymers. Glycerin of embodiments is characterized by the Water-soluble ?lm or other such liquid plasticizers by themselves can cause described herein being formed by casting, for example, by surface “sWeating” and greasiness if the level used in the ?lm admixing the ingredients described herein With Water to cre formulation is too high. This can lead to problems in a ?lm ate an aqueous mixture, for example a solution With option such as unacceptable feel to the hand of the consumer and ally dispersed solids, applying the mixture to a surface, and evenblocking of the ?lm on the roll or in stacks of sheets if the drying off Water to create a ?lm. Similarly, other composi sWeating is not mitigated in some manner, such as poWdering tions can be formed by drying the mixture While it is con?ned of the surface. This could be characterized as over plasticiza in a desired shape. tion. HoWever, if too little plasticizer is added to the ?lm the [0085] While the present disclosure provides ?lm ?lm may lack suf?cient ductility and ?exibility for many end examples, the compositions can take other forms and shapes, uses, for example to be converted into a ?nal use format such including those having thicknesses substantially in excess of as pouches. What Would normally be considered a ?lm. Composition [0081] Plasticizers for use in Water-soluble ?lms of the thicknesses (i.e. at their thinness point or average thickness), present disclosure include, but are not limited to, sorbitol, for ?lm and non-?lm embodiments, can be in any desired glycerol, diglycerol, propylene glycol, ethylene glycol, dieth range, incluging values and ranges of at least 0.1 pm at least yleneglycol, triethylene glycol, tetraethyleneglycol, polyeth 10 um at least 50 pm at least 100 pm at least 1 mmm, at least ylene glycols up to MW 400, 2 methyl 1, 3 propane diol, lactic 2 mm, at least 3 mm, at least 4 um at least 5 mm, at least 10 acid, monoacetin, triacetin, triethyl citrate, l,3-butanediol, mm, andinarangeof0.l umto 100 umor0.l umto l000p.m trimethylolpropane (TMP), polyether triol, and combinations for example. thereof. As less plasticizer is used, the ?lm can become more [0086] In one contemplated class of embodiments, the brittle, Whereas as more plasticizer is used the ?lm can lose Water soluble ?lm is formed by casting a Water-soluble mix tensile strength. Plasticizers can be included in the Water ture Wherein the Water-soluble mixture is prepared according soluble ?lms in an amount in a range of about 25 phr to about to the steps of: 50 phr, or from about 30 phr to about 45 phr, or from about 32 (a) providing a mixture of Water soluble resin, Water, and any phr to about 42 phr, for example. optional additives excluding enzymes; [0082] Surfactants for use in Water-soluble ?lms are Well (b) boiling the mixture for 30 minutes; knoWn in the art. Optionally, surfactants are included to aid in (c) degassing the mixture in an oven at a temperature of at the dispersion of the resin solution upon casting. Suitable least 400 C.; optionally in a range of 400 C. to 70° C., eg surfactants for Water-soluble ?lms of the present disclosure about 650 C.; include, but are not limited to, dialkyl sulfosuccinates, lacty (d) adding at least one enzyme, optionally at least one plas lated fatty acid esters of glycerol and propylene glycol, lac ticizer, and optionally additional Water to the mixture at a tylic esters of fatty acids, sodium alkyl sulfates, polysorbate temperature of 650 C. or less; and 20, polysorbate 60, polysorbate 65, polysorbate 80, alkyl (e) sti?ing the mixture, optionally Without vortex, until the polyethylene glycol ethers, lecithin, acetylated fatty acid mixture appears substantially uniform in color and consis esters of glycerol and propylene glycol, sodium lauryl sulfate, tency; optionally for a time period in a range of 30 minutes to acetylated esters of fatty acids, myristyl dimethylamine 90 minutes, optionally at least 1 hour; and oxide, trimethyl talloW alkyl ammonium chloride, quaternary casting the mixture promptly after the time period of stirring ammonium compounds, salts thereof and combinations of (e.g. Within 4 hours, or 2 hours, or Within 1 hour or less). If the any of the forgoing. Too little surfactant can sometimes result enzyme is added to the mixture too early, eg with the sec in a ?lm having holes, Whereas too much surfactant can result ondary additives or resin, the level of enzyme activity recov in the ?lm having a greasy or oily feel from excess surfactant ered may be loW. Without intending to be bound by any present on the surface of the ?lm. Thus, surfactants can be particular theory, it is believed that boiling of the mixture With included in the Water-soluble ?lms in an amount of less than the enzyme leads to the enzyme denaturing and storing in about 2 phr, for example less than about 1 phr, or less than solution for extended periods of time also leads to a reduction about 0.5 phr, for example. in recovered enzyme activity. [0083] One type of secondary component contemplated for [0087] In one class of embodiments, high enzyme activity use is a defoamer. Defoamers can aid in coalescing of foam is maintained in the Water soluble ?lms according to the US 2013/0244920 A1 Sep. 19, 2013

present disclosure by drying the ?lms quickly under moderate superimposed on the second compartment, Which is in turn to mild conditions. As used herein, drying quickly refers to a superimposed on the ?rst compartment in a sandWich con drying time of less than 24 hours, optionally less than 12 ?guration. Alternatively the second and third compartments hours, optionally less than 8 hours, optionally less than 2 may be superimposed on the ?rst compartment. HoWever it is hours, optionally less than 1 hour, optionally less than 45 also equally envisaged that the ?rst, second and optionally minutes, optionally less than 30 minutes, optionally less than third and subsequent compartments may be attached to one 20 minutes, optionally less than 10 minutes, for example in a another in a side by side relationship. The compartments may range of about 6 minutes to about 10 minutes, or 8 minutes.As be packed in a string, each compartment being individually used herein, moderate to mild conditions refer to drying tem separable by a perforation line. Hence each compartment may peratures of less than 170° F. (77° C.), optionally in a range of be individually tom-off from the remainder of the string by about 150° F. to about 170° F. (about 66° C. to about 77° C.), the end-user, e.g. 165° F. (74° C.). As the drying temperature increases, the [0091] In some embodiments, multi-compar‘tment pouches enzymes tend to denature faster, Whereas as the drying tem and/or packets include three compartments consisting of a perature decreases, the drying time increases, thus exposing large ?rst compartment and tWo smaller compartments. The the enzymes to solution for an extended period of time. second and third smaller compartments are superimposed on [0088] The ?lm is useful for creating a packet to contain a the ?rst larger compartment. The size and geometry of the composition, for example laundry or dishWashing composi compartments are chosen such that this arrangement is tions, thereby forming a pouch. The ?lm described herein can achievable. The geometry of the compartments may be the also be used to make a packet With tWo or more compartments same or different. In some embodiments the second and made of the same ?lm or in combination With ?lms of other optionally third compartment each has a different geometry polymeric materials. Additional ?lms can, for example, be and shape as compared to the ?rst compartment. In these obtained by casting, bloW-molding, extrusion orbloWn extru embodiments, the second and optionally third compartments sion of the same or a different polymeric material, as knoWn are arranged in a design on the ?rst compartment. The design in the art. In one type of embodiment, the polymers, copoly may be decorative, educative, or illustrative, for example to mers or derivatives thereof suitable for use as the additional illustrate a concept or instruction, and/or used to indicate ?lm are selected from polyvinyl alcohols, polyvinyl pyrroli origin of the product. In some embodiments, the ?rst com done, polyalkylene oxides, polyacrylic acid, cellulose, cellu partment is the largest compartment having tWo large faces lose ethers, cellulose esters, cellulose amides, polyvinyl sealed around the perimeter, and the second compartment is acetates, polycarboxylic acids and salts, polyaminoacids or smaller covering less than about 75%, or less than about 50% peptides, polyamides, polyacrylamide, copolymers of of the surface area of one face of the ?rst compartment. In maleic/acrylic acids, polysaccharides including starch and embodiments in Which there is a third compartment, the gelatin, natural gums such as xanthan, and carrageenans. For aforementioned structure may be the same but the second and example, polymers can be selected from polyacrylates and third compartments cover less than about 60%, or less than Water-soluble acrylate copolymers, methylcellulose, car about 50%, or less than about 45% of the surface area of one boxymethylcellulose sodium, dextrin, ethylcellulose, face of the ?rst compartment. hydroxyethyl cellulose, hydroxypropyl methylcellulose, [0092] The pouches and/or packets of the present disclo maltodextrin, polymethacrylates, and combinations thereof, sure may comprise one or more different ?lms. For example, or selected from polyvinyl alcohols, polyvinyl alcohol in single compartment embodiments, the packet may be made copolymers and hydroxypropyl methyl cellulose (HPMC), from one Wall that is folded onto itself and sealed at the edges, and combinations thereof. or alternatively, tWo Walls that are sealed together at the [0089] The pouches and/or packets of the present disclo edges. In multiple compartment embodiments, the packet sure comprise at least one sealed compartment. Thus the may be made from one or more ?lms such that any given pouches may comprise a single compartment or multiple packet compartment may comprise Walls made from a single compartments. The pouches may have regions With and With ?lm or multiple ?lms having differing compositions. In one out enzymes. In embodiments including multiple compart embodiment, a multi-compartment pouch comprises at least ments, each compartment may contain identical and/or dif three Walls: an outer upper Wall; an outer loWer Wall; and a ferent compositions. In turn, the compositions may take any partitioning Wall. The outer upper Wall and the outer loWer suitable form including, but not limited to liquid, solid and Wall are generally opposing and form the exterior of the combinations thereof (eg a solid suspended in a liquid). In pouch. The partitioning Wall is interior to the pouch and is some embodiments, the pouches comprise a ?rst, second and secured to the generally opposing outer Walls along a seal third compartment, each of Which respectively contains a line. The partitioning Wall separates the interior of the multi different ?rst, second and third composition. In some compar‘tment pouch into at least a ?rst compartment and a embodiments, the compositions may be visually distinct as second compartment. In one class of embodiments, the par described in European Patent Application Number titioning Wall may be the only enzyme containing ?lm 091616920 (?led Jun. 2, 2009 and assigned to the Procter & thereby minimizing the exposure of the consumer to the Gamble Company), published as EP 2258820 (and counter enzymes. part US Patent Application Publication No. 2010/ 0305020). [0093] Pouches and packets may be made using any suit [0090] The compartments of multi-compar‘tment pouches able equipment and method. For example, single compart and/ or packets may be of the same or different size(s) and/or ment pouches may be made using vertical form ?lling, hori volume(s). The compartments of the present multi-compart zontal form ?lling, or rotary drum ?lling techniques ment pouches can be separate or conjoined in any suitable commonly knoWn in the art. Such processes may be either manner. In some embodiments, the second and/ or third and/ or continuous or intermittent. The ?lm may be dampened, and/ subsequent compartments are superimposed on the ?rst com or heated to increase the malleability thereof. The method partment. In one embodiment, the third compartment may be may also involve the use of a vacuum to draW the ?lm into a US 2013/0244920 A1 Sep. 19, 2013

suitable mold. The vacuum drawing the ?lm into the mold can heated directly by passing it under a heating element or be applied for about 0.2 to about 5 seconds, or about 0.3 to through hot air, prior to feeding it onto a surface or once on a about 3, or about 0.5 to about 1.5 seconds, once the ?lm is on surface. Alternatively, it may be heated indirectly, for the horiZontal portion of the surface. This vacuum canbe such example by heating the surface or applying a hot item onto the that it provides an under-pressure in a range of 10 mbar to ?lm. The ?lm can be heated using an infrared light. The ?lm 1000 mbar, or in a range of 100 mbar to 600 mbar, for may be heated to a temperature of at least 500 C., for example example. about 50 to about 150° C., about 50 to about 120° C., about 60 [0094] The molds, in Which packets may be made, can have to about 130° C., about 70 to about 120° C., or about 60 to any shape, length, Width and depth, depending on the required about 90° C. dimensions of the pouches. The molds may also vary in siZe [0101] Alternatively, the ?lm can be Wetted by any suitable and shape from one to another, if desirable. For example, the means, for example directly by spraying a Wetting agent volume of the ?nal pouches may be about 5 ml to about 300 (including Water, a solution of the ?lm composition, a plasti ml, or about 10 to 150 ml, or about 20 to about 100 ml, and that ciZer for the ?lm composition, or any combination of the the mold siZes are adjusted accordingly. foregoing) onto the ?lm, prior to feeding it onto the surface or [0095] In one embodiment, the packet includes a ?rst and a once on the surface, or indirectly by Wetting the surface or by second sealed compartment. The second compartment is in a applying a Wet item onto the ?lm. generally superposed relationship With the ?rst sealed com partment such that the second sealed compartment and the [0102] Once a ?lm has been heated and/ or Wetted, it may be draWn into an appropriate mold, preferably using a vacuum. ?rst sealed compartment share a partitioning Wall interior to the pouch. The ?lm can be thermoformed With a draW ratio of at least about 1.5, for example, and optionally up to a draW ratio of 2, [0096] In one embodiment, the packet including a ?rst and for example. The ?lling of the molded ?lm can be accom a second compartment further includes a third sealed com plished by utiliZing any suitable means. In some embodi partment. The third sealed compartment is in a generally ments, the most preferred method Will depend on the product superposed relationship With the ?rst sealed compartment form and required speed of ?lling. In some embodiments, the such that the third sealed compartment and the ?rst sealed molded ?lm is ?lled by in-line ?lling techniques. The ?lled, compartment share a partitioning Wall interior to the pouch. open packets are then closed forming the pouches, using a [0097] In various embodiments, the ?rst composition and second ?lm, by any suitable method. This may be accom the second composition are selected from one of the folloW plished While in horiZontal position and in continuous, con ing combinations: liquid, liquid; liquid, poWder; liquid, gel; stant motion. The closing may be accomplished by continu liquid, paste; poWder, poWder; poWder, liquid; poWder, gel; ously feeding a second ?lm, preferably Water-soluble ?lm, poWder; paste; gel, liquid; gel, poWder; gel, gel; gel, paste; over and onto the open packets and then preferably sealing the paste, liquid; paste, poWder; paste, gel; and paste, paste. ?rst and second ?lm together, typically in the area betWeen [0098] In various embodiments, the ?rst, second and third the molds and thus betWeen the packets. compositions are selected from one of the folloWing combi nations: [0103] Any suitable method of sealing the packet and/or the poWder, liquid, liquid; poWder, liquid, poWder; poWder, liq individual compartments thereof may be utiliZed. Non-limit uid, gel; poWder, liquid, paste; poWder, poWder, liquid; poW ing examples of such means include heat sealing, solvent der, poWder, poWder; poWder, poWder, gel; poWder, poWder, Welding, solvent or Wet sealing, and combinations thereof. paste; poWder, gel, liquid; poWder, gel, poWder; poWder, gel, The Water- soluble packet and/ or the individual compartments gel; poWder, gel, paste; poWder, paste, liquid; poWder, paste, thereof can be heat sealed at a temperature of at least 200° F. poWder; poWder, paste, gel; poWder, paste, paste; liquid, liq (93° C.), for example in a range of about 220° F. (about 105° uid, liquid; liquid, liquid, poWder; liquid, liquid, gel; liquid, C.) to about 290° F. (about 145° C.), or about 230° F. (about liquid, paste; liquid, poWder, liquid; liquid, poWder, poWder; 110° C.) to about 280° F. (about 140° C.). Typically, only the liquid, poWder, gel; liquid, poWder, paste; liquid, gel, liquid; area Which is to form the seal is treated With heat or solvent. liquid, gel, poWder; liquid, gel, gel; liquid, gel, paste; liquid, The heat or solvent can be applied by any method, typically paste, liquid; liquid, paste, poWder; liquid, paste, gel; liquid, on the closing material, and typically only on the areas Which paste, paste; gel, liquid, liquid; gel, liquid, poWder; gel, liq are to form the seal. If solvent or Wet sealing or Welding is uid, gel; gel, liquid, paste; gel, poWder, liquid; gel, poWder, used, it may be preferred that heat is also applied. Preferred poWder; gel, poWder, gel; gel, poWder, paste; gel, gel, liquid; Wet or solvent sealing/Welding methods include selectively gel, gel, poWder; gel, gel, gel; gel, gel, paste; gel, paste, liquid; applying solvent onto the area betWeen the molds, or on the gel, paste, poWder; gel, paste, gel; gel, paste, paste; paste, closing material, by for example, spraying or printing this liquid, liquid; paste, liquid, poWder; paste, liquid, gel; paste, onto these areas, and then applying pressure onto these areas, liquid, paste; paste, poWder, liquid; gel, poWder, poWder; gel, to form the seal. Sealing rolls and belts as described above poWder, gel; gel, poWder, paste; paste, gel, liquid; paste, gel, (optionally also providing heat) can be used, for example. poWder; paste, gel, gel; paste, gel, paste; paste, paste, liquid; [0104] The formed pouches may then be cut by a cutting paste, paste, poWder; paste, paste, gel; and paste, paste, paste. device. Cutting can be accomplished using any knoWn [0099] In one embodiment, the single compartment or plu method. It may be preferred that the cutting is also done in rality of sealed compartments contains a composition. The continuous manner, and preferably With constant speed and plurality of compartments may each contain the same or a preferably While in horiZontal position. The cutting device different composition. The composition is selected from a can, for example, be a sharp item, or a hot item, or a laser, liquid, poWder, gel, paste or combination thereof. Whereby in the latter cases, the hot item or laser ‘burns’ [0100] Heat can be applied to the ?lm in the process com through the ?lm/ sealing area. monly knoWn as thermoforming. The heat may be applied [0105] The different compartments of a multi-compart using any suitable means. For example, the ?lm may be ment pouches may be made together in a side-by-side style US 2013/0244920 A1 Sep. 19, 2013

wherein the resulting, cojoined pouches may or may not be boxylic acids and primary amines Were produced. The pro separated by cutting. Alternatively, the compartments can be duced amines reacted under alkaline conditions With tri made separately. nitrobenZene-sulfonic acid (TNBS) to form a colored [0106] In some embodiments, pouches may be made complex. The colored complex Was detected at 405 nm. The according to a process including the steps of: reaction betWeen the primary amines and TNBs Was assumed [0107] a) forming a ?rst compartment (as described above); to be faster than the cleavage of DMC. Therefore, the reaction [0108] b) forming a recess Within some or all of the closed in the presence of excess substrate can be assumed to be a compartment formed in step (a), to generate a second molded function of the enZyme concentration only. compartment superposed above the ?rst compartment; [0127] Protease activity Was determined relative to an [0109] c) ?lling and closing the second compartments by enZyme standard. The standard Was a sample of the raW means of a third ?lm; material protease composition that Was used to prepare the [0110] d) sealing the ?rst, second and third ?lms; and ?lm composition according to the Examples herein. The [0111] e) cutting the ?lms to produce a multi-compartment result is reported in convenient units of enZyme activity, for pouch. example katals (Eur. J. Biochemistry, Vol 97, 319-320, 1979). [0112] The recess formed in step (b) may be achieved by A set of standards Was prepared through serial dilutions using applying a vacuum to the compartment prepared in step (a). an enZyme of knoWn activity. The standards Were buffered to [0113] In some embodiments, second, and/or third com pH 8.3, Were stirred for 9 minutes at 400 C., and the absor partment(s) can be made in a separate step and then combined bance at 405 nm Was measured. The absorbance Was cor With the ?rst compartment as described in European Patent rected to account for a blank, and a calibration curve (linear) Application Number 081014425 or WO 2009/152031 (?led Was obtained by plotting the activity versus the corrected Jun. 13, 2008 and assigned to the Procter & Gamble Com absorbance. The calibration curve Was accepted if r2 Was Pany) greater than 0.995. A sample of enZyme-containing ?lm [0114] In other embodiments, pouches may be made according to the corresponding Example Was Weighed and according to a process including the steps of: dissolved in a standard solution of DMC and TNBS, buffered [0115] a) forming a ?rst compartment, optionally using to pH 8.3, and Was stirred for 9 minutes at 400 C. The absor heat and/or vacuum, using a ?rst ?lm on a ?rst forming bance at 405 nm Was measured at 400 C. Using the absor machine; bance value, the activity per gram of ?lm Was determined. [0116] b) ?lling the ?rst compartment With a ?rst compo Each ?lm composition Was tested in multiple replicates, and sition; the average value is reported herein. [0117] c) on a second forming machine, deforming a sec ond ?lm, optionally using heat and vacuum, to make a second Colorimetric Measurement of Amylase Activity and optionally third molded compartment; [0118] d) ?lling the second and optionally third compart [0128] The substrate 4,6-ethylidene-(G7)-p-nitrophenyl ments; (G1)-0t-D-maltoheptaosid (ethylidene-G7pNP) Was hydro [0119] e) sealing the second and optionally third compart lyZed by ot-amylase to G2pNP, G3pNP and G4pNP. These ment using a third ?lm; fragments Were totally hydrolyZed to p-nitrophenol (pNP) [0120] f) placing the sealed second and optionally third and glucose by an ot-glucodiase. The intensity of the formed compartments onto the ?rst compartment; pNP Was proportional to the ot-amylase activity of the sample. [0121] g) sealing the ?rst, second and optionally third com The pNP Was detected at 405 nm partments; and [0129] ot-amylase activity Was determined relative to an [0122] h) cutting the ?lms to produce a multi-compartment enZyme standard. The standard Was a sample of the raW pouch. amylase composition that Was used to prepare the ?lm com [0123] The ?rst and second forming machines may be position according to the Examples herein. Standards Were selected based on their suitability to perform the above pro prepared at a pH of 7.35, Were stirred for 4 minutes at 27° C., cess. In some embodiments, the ?rst forming machine is and the absorbance Was measured at 405 nm A calibration preferably a horizontal forming machine, and the second curve (linear) Was obtained, as described above. A sample of forming machine is preferably a rotary drum forming enZyme-containing ?lm of the corresponding Example Was machine, preferably located above the ?rst forming machine. Weighed and dissolved in a standard solution of ethylidene [0124] It should be understood that by the use of appropri G7pNP and ot-glucodiase, buffered to pH 7.35 and Was stirred ate feed stations, it may be possible to manufacture multi for 4 minutes at 27° C. The absorbance at 405 nm Was mea compartment pouches incorporating a number of different or sured at 27° C. Using the absorbance value, the activity per distinctive compositions and/or different or distinctive poW gram of ?lm Was determined. Each ?lm composition Was der, liquid, gel or paste compositions. tested in multiple replicates, and the average value is reported [0125] The Water-soluble ?lms in accordance With the dis herein. closure can be better understood in light of the folloWing examples, Which are merely intended to illustrate the Water Colorimetric Measurement of Lipase Activity soluble ?lms and Water-soluble packets and are not meant to [0130] The lipase used is knoWn to hydrolyZe p-nitrophe limit the scope thereof in any Way. nol-valerate (pNP-val). The hydrolysis cleaved the ester bond betWeen valerate and p-nitrophenol (pNP). The intensity of EXAMPLES the formed pNP Was proportional to the lipase activity of the sample. The pNP Was detected at 405 nm. Colorimetric Measurement of Protease Activity [0131] Lipase activity Was determined relative to an [0126] The protease used is knoWn to hydrolyZe N,N-dim enZyme standard. The standard Was a sample of the raW lipase ethylcasein (DMC). By the hydrolysis of peptide bonds, car composition that Was used to prepare the ?lm composition. US 2013/0244920 A1 Sep. 19, 2013

Standards Were prepared at a pH of 7.70, Were stirred for 4.2 from the casting bed, cut into sheets of about 8.5 in by 12 in minutes at 400 C., and the absorbance Was measured at 405 (21.59 cm by 30.48 cm) and sealed into airtight bags for nm A calibration curve (linear) Was obtained, as described storage. The individual sheets Were either placed into reseal above. A sample ofenzyme-containing ?lm of the corre able zipper-style plastic bags, or into foil pouches With air sponding Example Was Weighed and dissolved in a standard gently pressed out, then heat sealed and stored in a 50 C. solution of pNP-val, buffered to pH 7.70 and Was stirred for refrigerator until tested for activity. 4.2 minutes at 400 C. The absorbance at 405 nm Was mea [0135] The percent recoveries of the enzyme activity in the sured at 400 C. Using the absorbance value, the activity per PVOH-based Water-soluble ?lms Were determined as gram of ?lm Was determined. Each ?lm composition Was described above. The percent recovery of the enzymes is the tested in multiple replicates, and the average value is reported percentage of the enzymes that remained active folloWing the herein. combination of mixing, casting, drying and storage pro Example 1 cesses. The Water-soluble ?lm that contained no enzyme sub strate, ?lm 1, demonstrated a 44% recovery of protease activ [0132] A set of Water-soluble ?lms Were prepared With the ity. The Water- soluble ?lm that contained a protease substrate, ingredients identi?ed in the table and description beloW, in ?lm 2, demonstrated a 72% recovery of protease activity. In the amounts shoWn. Unless speci?ed otherWise, amounts are this example, the inclusion of the protease substrate lead to a in phr. 63% increase in recovered protease activity.

Example 2

Chemical Component Name Description 1 2 [0136] A set of Water-soluble ?lms Were prepared With the ingredients identi?ed in the table and description beloW, in PVOH resin PVOH 88% 100 100 the amounts shoWn. Unless speci?ed otherWise, amounts are hydrolyzed, viscosity in phr. 23.0-27.0 Alpha- Amylase 458 45 8 amylase Enzyme (amylase Mixture Chemical activity/100 Component Name Description 3 4 parts PVOH resin) PVOH resin VOH/MA PVOH >99% 100 100 Protease Protease 759 759 copolymer hydrolyzed; (protease Enzyme viscosity activity/100 Mixture 17.0-23.0; parts PVOH 8.8-10.0 Wt % resin) MA Pro-Fam 974 Soy protein Protease 0.00 2.56 Alpha- Amylase 45 8 45 8 (phr) isolate substrate amylase Enzyme Protease 44 72 (amylase Mixture activity (% activity/100 recovery) parts PVOH Amylase 1 15 1 16 resin) activity (% Protease Protease 759 759 recovery) (protease Enzyme activity/100 Mixture parts PVOH [0133] Each ?lm also included about 35 phr of plasticizers, resin) 390 phr of Water, and minor amounts of one or more process Pro-Fam 974 Soy protein Protease 0.00 2.60 (phr) isolate substrate ing aids Which are not believed to affect enzyme recovery, Protease 41 84 e.g., antiblocking agents, surfactants, caustic soda, ?lm form activity (% ers, defoaming agents, antiyelloWing agents, etc, in an recovery) amount totaling less than about 2.5 phr. The Water soluble Amylase 106 1 1 6 activity (% ?lm according to formula 1 Was prepared as folloWs. In a recovery) beaker, the processing aids and Water Were agitated for 10 minutes. The Water-soluble resin Was added to the mixture and the mixture Was heated. The solution Was left to boil for [0137] Each ?lm also included about 35 phr of plasticizers, 30 minutes. The solution Was degassed overnight in an oven at 400 phr of Water, and minor amounts of one or more process 650 C. The enzymes, plasticizers, and additional Water Were ing aids Which are not believed to affect enzyme recovery, mixed into the solution at a temperature of no higher than 650 e.g., antiblocking agents, surfactants, caustic soda, ?lm form C. The mixture Was stirred for 1 hour and the mixture accord ers, defoaming agents, antiyelloWing agents, etc, in an ing to formula 1 Was immediately cast. The Water soluble ?lm amount totaling less than about 6 phr. The procedure of according to formula 2 Was prepared by the same procedure, Example 1 Was folloWed. The percent recoveries of the With the exception of the addition of the soy protein protease enzyme activity in the vinyl alcohol/methyl acrylate copoly substrate. The soy protein Was introduced into the system mer-based Water-soluble ?lms Were determined as described With the processing aids and Water. above. The Water-soluble ?lm that contained no enzyme sub [0134] The ?lm Was cast using a doctor blade onto a heated strate, ?lm 3, demonstrated a 41% recovery of protease activ casting bed and dried for approximately 8 minutes at about ity. The Water- soluble ?lm that contained a protease substrate, 740 C. The resulting Water soluble ?lm Was then removed ?lm 4, demonstrated an 84% recovery of protease activity. In