Patent Application
20250129315
The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on Aug. 26, 2024, is named Parent 081906-1458189_253210US_SL.xml and is 104,434 bytes in size.
The present disclosure generally relates to compositions and methods for hydrolyzing smoke-associated volatile phenols from phenolic glycosides, and more specifically to one or more glycosidases that have utility in hydrolyzing volatile phenols from products such as wine.
Many wine regions such as Australia, North America, South America, and Europe are periodically ravaged by devastating wildfires, seemingly exacerbated by prolonged droughts, intense heatwaves, and years of uncontrolled forest growth. These fires have significant detrimental impacts on wines produced from smoke-exposed fruit imparting negative smoke aromas and flavors to wine. This “smoke taint” occurs when grape berries exposed to wildfire smoke absorb the volatile phenols (VPs) produced from lignin combustion. Wines produced from these smoke-exposed grapes acquire undesirable smoky aromas, often described as ‘burnt wood’, ‘ashtray’, ‘burning rubber’, and ‘smoked meat’. These persistent aromas and flavors can be sufficiently high in concentration that resultant wines are considered unmarketable.
Due to the detrimental effect of smoke exposure on flavor, strategies to mitigate the impact of smoke taint are necessary. First, a decision must be made as to whether or not to harvest smoke-affected fruits. However, the decision to harvest the fruit may not be straightforward. Low or high concentrations of free volatile phenols and/or bound phenols glycosides may give a clear answer, intermediate levels may be difficult to interpret due to uncertainty regarding the different thresholds at which the products of the fruit become smoke tainted. In addition, small-scale fermentations take time and resources and may not be representative of the presence of volatile phenols in the final product after aging and storage.
Current methods for quantifying phenolic glycosides also present several challenges including the requirement of expensive capital equipment, limited accuracy due the molecular complexity of the glycosides, and the utilization of harsh reagents.
During wine processing and fermentation, current strategies used to mitigate smoke taint include excluding leaf material, keeping fruit cool, and minimizing the time fermentations are in contact with the skin tissue. These strategies often have limited effectiveness and are unlikely to reduce the concentration of volatile phenols below the flavor detection threshold. Methods for remediation of finished, smoke-tainted wine include treating wine with activated carbon, molecularly imprinted polymers, cyclodextrin or cellulose polymers, yeast products such as yeast lees, phenols-converting enzymes or organisms, treating with reverse osmosis or filtration, diluting wine with non-tainted wine, and adding tannins or oak chips to mask smoke sensory notes. Each of these strategies have significant challenges and limitations. Interaction with an affinity media, for example, often removes color, flavor, and desirable aroma compounds from the fermented beverages. Reverse osmosis also removes desirable aromas but also does not fully remove glycosides, resulting in the recurrence of smoke taint will return over time as the glycosides are hydrolyzed. Dilution of wine with non-tainted wine requires a high volume of non-tainted wine, and the addition of tannin or oak to the fermented beverage may produce a very different wine from the one intended.
The present disclosure provides compositions for hydrolyzing smoke associated volatile phenols from a phenolic glycoside. In one aspect, the composition includes a glucoside and/or a gentiobioside hydrolyzing enzyme with an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1-72. In one aspect, the composition includes a rutinosidase having an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 73-78. In one aspect, the glucoside and/or the gentiobioside hydrolyzing enzyme is CbBg1B-1 (MBR2796233.1, SEQ ID NO: 1), OscbBglB (MBQ3381008.1, SEQ ID NO: 2), CbBg1B-2 (MBQ3268742.1, SEQ ID NO: 3), GiBg1B (SEQ ID NO: 4), IgBg1B (SEQ ID NO: 5), CrumBgl-2 (SEQ ID NO: 6), CrumBgl-7 (SEQ ID NO: 7), CrumBgl-6 (SEQ ID NO: 8), CrumBgl-8 (SEQ ID NO: 9), CrumBgl-1 (SEQ ID NO: 10), CrumBgl-4 (SEQ ID NO: 11), CrumBgl-5 (SEQ ID NO: 12), CrumBgl-3 (SEQ ID NO: 13), CbBg1B-3 (MBQ6595599.1, SEQ ID NO: 14), TcBg1B (WP 088862624, SEQ ID NO: 15), VsBg1B (KJR72531, SEQ ID NO: 16), TgBglB (WP 062370819.1, SEQ ID NO: 17), TaBg1B-1 (RLG75229.1, SEQ ID NO: 18), laBg1B (ADM27756.1, SEQ ID NO: 19), TaBg18-2 (RLG79985.1, SEQ ID NO: 20), CmBg1B (WP 012185712, SEQ ID NO: 21), TuBg1B (WP 013680114.1, SEQ ID NO: 22), CmBglB (PSN97385, SEQ ID NO: 23), FcBg1B (WP 090223355, SEQ ID NO: 24), FtBg1B (WP 069292479, SEQ ID NO: 25), FgBg1B (WP 072757753, SEQ ID NO: 26), SaciBgl (P14288, SEQ ID NO: 27), CmaqBgl (A8MBRO, SEQ ID NO: 28), TvoiBgl (SEQ ID NO: 29), PfurBgl (E7FHY4, SEQ ID NO: 30), TgorBgl (SEQ ID NO: 31), FnodBgl (A7HNB8, SEQ ID NO: 32), TafrBgl (B7IGM4, SEQ ID NO: 33), LcasBgl (SEQ ID NO: 34), SequBgl (SEQ ID NO: 35), CbeiBgl (C8W8S6, SEQ ID NO: 36), CaurBgl (A9WDK4, SEQ ID NO: 37), BdenBgl (SEQ ID NO: 38), SrocBgl (SEQ ID NO: 39), CaceBgl (Q97M15, SEQ ID NO: 40), SterBgl (DIAQN8, SEQ ID NO: 41), LrrhaBgl (Q297J1, SEQ ID NO: 42), BthuBgl (SEQ ID NO: 43), BamyBgl (SEQ ID NO: 44), LlacBgl (Q9CFLO, SEQ ID NO: 45), Ent7Bg1 (SEQ ID NO: 46), GkauBgl-2 (Q5KXG4, SEQ ID NO: 47), GeoYBgl (SEQ ID NO: 48), GkauBgl-3 (Q5KUY7, SEQ ID NO: 49), PchrBgl (Q25BW5, SEQ ID NO: 50), SdegBg1-1 (Q21EMI, SEQ ID NO: 51), HsapCyBgl (Q9H227, SEQ ID NO: 52), RratCyBgl (SEQ ID NO: 53), CcanCyBgl (A0A8B7TQ98, SEQ ID NO: 54), CporCyBgl (P97265, SEQ ID NO: 55), OpriCyBgl (SEQ ID NO: 56), CasinPRI (A0A2R6RAC3, SEQ ID NO: 57), CcelBgl (B815U2, SEQ ID NO: 58), TnonBgl (SEQ ID NO: 59), TcurBgl (D1A786, SEQ ID NO: 60), TbisBgl (D6Y5B2, SEQ ID NO: 61), DdesBgl (CICXP6, SEQ ID NO: 62), CflaBgl (D5ULE7, SEQ ID NO: 63), BbreBgl (P94248, SEQ ID NO: 64), TfusBgl (SEQ ID NO: 65), TterBgl (DICGH4, SEQ ID NO: 66), SdegBg1-2 (Q21KX3, SEQ ID NO: 67), VvulBgl (Q7MG41, SEQ ID NO: 68), HoreBgl (B8CYA8, SEQ ID NO: 69), CtheBgl (P26208, SEQ ID NO: 70), BacGBgl (AOAIIOZQD8 9BACL, SEQ ID NO: 71), and/or BhalBgl (Q9KBK3, SEQ ID NO: 72). In one aspect, the glucoside and/or the gentiobioside hydrolyzing enzyme is CbBglB-1 (MBR2796233.1; SEQ ID NO: 1). In one aspect, the rutinosidase is selected from AoryRut (SEQ ID NO: 73), CtroEXG (SEQ ID NO: 74); CmalEXG (SEQ ID NO: 75); AcreRut (SEQ ID NO: 76); and/or AniRut (SEQ ID NO: 77). In one aspect, the rutinosidase comprises the amino acid sequence of SEQ ID NO. 78. In one aspect, the composition includes the glucoside and/or the gentiobioside hydrolyzing enzyme CbBg1B-1 (MBR2796233.1; SEQ ID NO: 1); and the rutinosidase AoryRut (SEQ ID NO: 73). In one aspect, the composition includes 0.001 mg/ml to 50 mg/ml of the glucoside and/or the gentiobioside hydrolyzing enzyme. In one aspect, the composition includes about 0.01 mg/ml to 5 mg/ml of the glucoside and/or the gentiobioside hydrolyzing enzyme. In one aspect, the composition includes 0.001 mg/ml to 50 mg/ml of the rutinosidase. In one aspect, the composition includes 0.01 mg/ml to 5 mg/ml of the rutinosidase.
The present disclosure provides compositions for hydrolyzing smoke associated volatile phenols from a phenolic glycoside. In one aspect, the composition includes a glucoside and/or a gentiobioside hydrolyzing enzyme with an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1-72. In one aspect, the composition includes a rutinosidase having an amino acid sequence with a mutation at one or more of position 141, 190, 279, 307, 38, 39, 41, 87, 94, 145, 156, 168, 181, 183, 184, 214, 270, 276, 297, 324, 328, and/or 342 of SEQ ID NO: 73. In one embodiment, the mutation is at one or more of position 141, 190, and/or 279 of SEQ ID NO: 73. In one aspect, the mutation is at one or more of position 141, 190, and/or 307. In one aspect, the mutation comprises one or more of T141V, M190I, Q307N, T297V, Q38D, F39W, G41N, G87N, T94N, T1411, T145V, Y156F, V168M, S181Y, Q183W, S184F. T214A, N270R, L276K, R279H, M324W, S328T, and/or A342F relative to SEQ ID NO: 73. In one aspect, the mutations are T141V, M190I, and/or R279H, relative to SEQ ID NO: 73. In one aspect, the mutation comprises one or more of T141V, M190I, and/or Q307N relative to SEQ ID NO: 73 and wherein the composition comprises SEQ ID NO: 78. In one aspect, the glucoside and/or the gentiobioside hydrolyzing enzyme is CbBg1B-1 (MBR2796233.1, SEQ ID NO: 1), OscbBg1B (MBQ3381008.1, SEQ ID NO: 2), CbBg1B-2 (MBQ3268742.1, SEQ ID NO: 3), GiBg1B (SEQ ID NO: 4), TpBglB (SEQ ID NO: 5), CrumBgl-2 (SEQ ID NO: 6), CrumBgl-7 (SEQ ID NO: 7), CrumBgl-6 (SEQ ID NO: 8), CrumBgl-8 (SEQ ID NO; 9), CrumBgl-1 (SEQ ID NO: 10), CrumBgl-4 (SEQ ID NO: 11), CrumBgl-5 (SEQ ID NO: 12), CrumBgl-3 (SEQ ID NO: 13), CbBg1B-3 (MBQ6595599.1, SEQ ID NO: 14), kcg/B (WP 088862624, SEQ ID NO: 15), VsBg1B (KJR72531, SEQ ID NO: 16), TgBg1B (WP 062370819.1, SEQ ID NO: 17), TaBg/IB-(RLG75229.1, SEQ ID NO: 18), laBg1B (ADM27756.1, SEQ ID NO: 19), TaBg1B-2 (RLG79985.1, SEQ ID NO: 20), CmBg1B (WP 012185712, SEQ ID NO: 21), TuBg1B (WP 013680114.1, SEQ ID NO: 22), CmBg1B (PSN97385, SEQ ID NO: 23), FcBg1B (WP 090223355, SEQ ID NO: 24), FtBg1B (WP 069292479, SEQ ID NO: 25), FgBg1B (WP 072757753, SEQ ID NO: 26), SaciBgl (P14288, SEQ ID NO: 27), CmaqBgl (A8MBRO, SEQ ID NO: 28), TvolBgl (SEQ ID NO: 29), PfurBgl (E7FHY4, SEQ ID NO: 30), TgorBgl (SEQ ID NO: 31), FnodBgl (A7HNB8, SEQ ID NO: 32), TafrBgl (B7IGM4, SEQ ID NO: 33), LcasBgl (SEQ ID NO: 34), SequBgl (SEQ ID NO: 35), CbeiBgl (C8W8S6, SEQ ID NO: 36), CaurBgl (A9WDK4, SEQ ID NO: 37), BdenBgl (SEQ ID NO: 38), SrocBgl (SEQ ID NO: 39), CaceBgi (Q97M15, SEQ ID NO: 40), SterBgl (DIAQN8, SEQ ID NO: 41), LrhaBgl (Q29ZJ1, SEQ ID NO: 42), BthuBgl (SEQ ID NO: 43), BamyBgl (SEQ ID NO: 44), LlacBgl (Q9CFLO, SEQ ID NO: 45), Ent7Bg1 (SEQ ID NO: 46), GkauBgil-2 (Q5KXG4, SEQ ID NO: 47), GeoYBgl (SEQ ID NO: 48), GkauBgl-3 (Q5KUY7, SEQ ID NO: 49), PchrBgl (Q25BW5, SEQ ID NO: 50), SdegBgI-1 (Q21EMI, SEQ ID NO: 51), HsapCyBgl (Q911227, SEQ ID NO: 52), RratCyBgl (SEQ ID NO: 53), CcanCyBgl (A0A8B7TQ98, SEQ ID NO: 54), CporCyBgl (P97265, SEQ ID NO: 55), OpriCyBgl (SEQ ID NO: 56), CasinPRI (A0A2R6RAC3, SEQ ID NO: 57), CcelBgl (B8I5U2, SEQ ID NO: 58), TnonBgl (SEQ ID NO: 59), TcurBgl (Dl A786, SEQ ID NO: 60), TbisBg) (D6Y5B2, SEQ ID NO: 61), DdesBgl (CICXP6, SEQ ID NO: 62), CflaBgl (D5ULE7, SEQ ID NO: 63), BbreBgl (P94248, SEQ ID NO: 64), TfusBgl (SEQ ID NO: 65), TterBgl (D1CGH4, SEQ ID NO: 66), SdegBgl-2 (Q21KX3, SEQ ID NO: 67), VvulBgl (Q7MG41, SEQ ID NO: 68), HoreBgi (B8CYA8, SEQ ID NO: 69), CtheBgl (P26208, SEQ ID NO: 70), BacGBgl (A0A110ZQD8 9BACL, SEQ ID NO: 71), and/or BhalBgl (Q9KBK3, SEQ ID NO: 72). In one aspect, the glucoside and/or the gentiobioside hydrolyzing enzyme is CbBglB-1 (MBR2796233.1; SEQ ID NO: 1). In one aspect, the glucoside and/or the gentiobioside hydrolyzing enzyme is CbBglB-1 (MBR2796233.1; SEQ ID NO: 1); and the rutinosidase comprising an amino acid sequence of SEQ ID NO: 78. In one aspect, the composition includes 0.001 mg/ml to 50 mg/ml of the glucoside and/or the gentiobioside hydrolyzing enzyme. In one aspect, the composition includes about 0.01 mg/ml to 5 mg/ml of the glucoside and/or the gentiobioside hydrolyzing enzyme. In one aspect, the composition includes 0.001 mg/ml to 50 mg/ml of the rutinosidase. In one aspect, the composition includes 0.01 mg/ml to 5 mg/ml of the rutinosidase.
The present disclosure provides isolated polypeptides having a mutation at one or more of positions 141, 190, 279, 307, 38, 39, 41, 87, 94, 145, 156, 168, 181, 183, 184, 214, 270, 276, 297, 324, 328, and/or 342 of SEQ ID NO: 73. In one aspect, the mutation is at one or more of position 141, 190, and/or 279 of SEQ ID NO: 73. In one aspect, the mutation is at one or more of position 141, 190, and/or 307 of SEQ ID NO: 73. In one aspect, the mutation includes one or more of T141V, M190I, Q307N, T297V, Q38D, F39W, G41N, G87N, T94N, T1411, T145V, Y156F, V168M, S181Y, Q183W, S184F, T214A, N270R, L276K, R279H, M324W, S328T, and/or A342F relative to SEQ ID NO: 73. In one aspect, the mutation includes T141V, M190I, and/or R279H relative to SEQ ID NO: 73. In one aspect, the mutation includes one or more of T141V, M190I, and/or Q307N relative to SEQ ID NO: 73, wherein the polypeptide comprises SEQ ID NO: 78.
In one embodiment, the present disclosure provides a method of hydrolyzing smoke associated volatile phenols from phenolic glycoside in a fruit product or a fermented product. The method includes incubating the fruit product or a fermented product thereof with the composition of the disclosure. In one aspect, the fruit product or the fermented product thereof is smoke-exposed. In one aspect, the incubation is performed for about 4 hours. In one aspect, the incubation is performed at about 37 degrees C., in one aspect, the method includes removing the smoke-associated volatile phenols and/or the phenolic glycoside from the fruit product or the fermented product thereof, using filtration with activated carbon, reverse osmosis with activated carbon, yeast lees, cell walls, an enzyme, a cyclodextrin polymer and/or a molecularly imprinted polymer. In one aspect, the fruit product is derived from a fruit such as grape, an apple, a blueberry, a blackberry, a raspberry, a currant, a strawberry, a cherry, a pear, a peach, a nectarine, an orange, a pineapple, a mango, and a passionfruit. In one aspect, the fruit product is a fruit homogenate, a fruit juice, a fruit pulp, a fruit skin, a fruit peel, a fruit seed, a fruit concentrate, or combinations thereof. In one aspect, the fermented fruit product is a fermented beverage. In one aspect, the fermented beverage is table wine, dessert wine, fortified wine, sparkling wine, beer, spirits, cider, mead, liqueurs, sake, or brandy. In one aspect, the table wine is red wine, a white wine, or a rose wine. In one aspect, the red wine is Cabernet Sauvignon, Alicante Henri Bouschet, Barbera, Bobal, Cabernet Franc, Carignan, Cinsaut, Malbec, Douce noir, Gamay, Grenache, Isabella, Merlot, Montepulciano, Mourvedre, Pinot noir, Sangiovese, Syrah, Tempranillo, Zinfandel, Aglianico, Blaufrankisch, Bordo, Carmenere, CastelAo, Concord, Corvina Veronese, Criolla Grande, Croatina, Dolcetto, Dornfelder, Marufo, Mencia, Black Muscat, and/or Nebbiolo. In one aspect, the rose wine is Provence Rose Fresh, Grenache Rose, Sangiovese Rose, Syrah Rose, Zinfandel Rose, and/or Cabernet Sauvignon Rose. In one aspect, the white wine is Chardonnay, Sauvignon Blanc, Pinot Grigio, Moscato, Riesling, and/or Chenin Blanc.
In one embodiment, the present disclosure provides a method of quantifying a volatile phenol and/or a phenolic glycoside in a fruit product or a fermented product thereof. In one aspect, the method includes incubating the fruit product or a fermented product thereof with the composition of the disclosure and measuring the levels of the volatile phenol and/or a phenolic glycoside using mass spectrometry. In one aspect, the mass spectrometry is gas chromatography mass spectrometry or liquid chromatography mass spectrometry. In one aspect, the fruit product or the fermented product thereof is smoke-exposed. In one aspect, the incubation is performed for about 4 hours. In one aspect, the incubation is performed at about 37 degrees C. In one aspect, the fruit product is derived from a fruit such as grape, an apple, a blueberry, a blackberry, a raspberry, a currant, a strawberry, a cherry, a pear, a peach, a nectarine, an orange, a pineapple, a mango, and a passionfruit. In one aspect, the fruit product is a fruit homogenate, a fruit juice, a fruit pulp, a fruit skin, a fruit peel, a fruit seed, a fruit concentrate, or combinations thereof. In one aspect, the fermented fruit product is a fermented beverage. In one aspect, the fermented beverage is table wine, dessert wine, fortified wine, sparkling wine, beer, spirits, cider, mead, liqueurs, sake, or brandy. In one aspect, the table wine is red wine, a white wine, or a rose wine. In one aspect, the red wine is Cabernet Sauvignon, Alicante Henri Bouschet, Barbera, Bobal, Cabernet Franc, Carignan, Cinsaut, Malbec, Douce noir, Gamay, Grenache, Isabella, Merlot, Montepulciano, Mourvedre, Pinot noir, Sangiovese, Syrah, Tempranillo, Zinfandel, Aglianico, Blaufrankisch, Bordo, Carmenere, CastelAo, Concord, Corvina Veronese, Criolla Grande, Croatina, Dolcetto, Dornfelder, Marufo, Mencia, Black Muscat, and/or Nebbiolo. In one aspect, the rose wine is Provence Rose Fresh, Grenache Rose, Sangiovese Ross, Syrah Rose, Zinfandel Rose, and/or Cabernet Sauvignon Rosé. in one aspect, the white wine is Chardonnay, Sauvignon Blanc, Pinot Grigio, Moscato, Riesling, and/or Chenin Blanc.
In one embodiment, the present disclosure provides a cell engineered to express (i) a glucoside and/or a gentiobioside hydrolyzing enzyme having an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 1-72; and/or a rutinosidase having an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 73-77. In one aspect, the cell expresses the glucoside and/or the gentiobioside hydrolyzing enzyme CbBg1B-1 (MBR2796233.1; SEQ ID NO: 1); and the rutinosidase AoryRut (SEQ ID NO: 73).
In one embodiment, the present disclosure provides a cell engineered to express a polypeptide with a mutation at one or more of position 141, 190, 279, 307, 38, 39, 41, 87, 94, 145, 156, 168, 181, 183, 184, 214, 270, 276, 297, 324, 328, and/or 342 of SEQ ID NO: 73.
In one embodiment, the present disclosure provides a cell engineered to express (i) a glucoside and/or a gentiobioside hydrolyzing enzyme having an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 1-72; and/or a rutinosidase with a mutation at one or more of position 141, 190, 279, 307, 38, 39, 41, 87, 94, 145, 156, 168, 181, 183, 184, 214, 270, 276, 297, 324, 328, and/or 342 of SEQ ID NO: 73. In one aspect, the mutation is at one or more of position 141, 190, and/or 279 of SEQ ID NO: 73. In one aspect, the mutation is at one or more of position 141, 190, and/or 307 of SEQ ID NO: 73. In one aspect, the mutation includes one or more of T141V, M190I, Q307N, T297V, Q38D, F39W, G41N, G87N, T94N, T1411, T145V, Y156F, V168M, S181Y, Q183W, S184F, T214A, N270R, L276K, R279H, M324W, S328T, and/or A342F relative to SEQ ID NO: 73. In one aspect, the mutation includes T141V, M190I, and/or R279H relative to SEQ ID NO: 73. In one aspect, the mutation includes one or more of T141V, M190I, and/or Q307N relative to SEQ ID NO: 73. In one aspect, the rutinosidase includes an amino acid sequence of SEQ ID NO: 78.
The present disclosure also provides methods of hydrolyzing smoke-associated phenols from phenolic glycoside from a fruit fermentation apparatus and/or a fruit fermentation container. In one aspect, the method includes incubating the fruit fermentation apparatus and/or the fruit fermentation container with the composition or polypeptides described herein. In one aspect, the fruit fermentation apparatus and/or the fruit fermentation container can be a crusher, a destemmer, a fermentation vessel, a press, a pump, an airlock, a fermentation lock, a hydrometer, a refractometer, a thermometer, a primary fermenter, a secondary fermenter, a bottle, a barrel, a demijohn, a keg, a fermentation bucket, or a cork.
The present disclosure also provides for methods resulting in compositions having levels (e.g., elevated levels) of smoke-associated volatiles products as described herein, as well as compositions resulting from the methods described herein. In some embodiments, the composition comprises a fruit-derived beverage (e.g., as described herein) and levels (e.g., elevated levels) of smoke-associated volatiles (e.g., compared to starting levels in smoke-associated fruit): guaiacol, 4-methylguaiacol, 4-ethylguaiacol, p-cresols, m-cresols, o-cresols, phenol, 4-ethylphenol, syringol, and/or 4-methylsyringol, at levels above 37.0 μg/L (e.g., up to 50, 100, or 200 μg/L), 6.2 μg/L (e.g., up to 20, 50, 100, or 200 μg/L), 0.5 μg/L (e.g., up to 10, 50, 100, or 200 μg/IL), 16.3 μg/L (e.g., up to 50, 100, or 200 μg/L), 26.2 μg/L (e.g., up to 50, 100, or 200 μg/L), 23.5 μg/L (e.g., up to 50, 100, or 200 μg/L), 79.1 μg/L (e.g., up to 100 or 200 μg/L), 6.2 μg/L (e.g., up to 20, 50, 100, or 200 μg/L), 51.2 μg/L, (e.g., up to 100 or 200 μg/L), 4.1 μg/L (e.g., up to 10, 20, 50, 100, or 200 μg/L), respectively. In some embodiments, the disclosure provides a composition comprising a fruit-derived beverage and having levels of smoke-associated volatiles from: guaiacol, 4-methylguaiacol, 4-ethylguaiacol, p-cresols, m-cresols, o-cresols, phenol, 4-ethylphenol, syringol, and/or 4-methylsyringol, at levels above 2.2 μg/L (e.g., up to 10, 25, 50, 100, or 200 μg/L), 0.3 μg/L (e.g., up to 10, 25, 50, 100, or 200 μg/L), 0.1 μg/L (e.g., up to 10, 25, 50, 100, or 200 μg/L), 1.1 μg/L (e.g., up to 10, 25, 50, 100, or 200 μg/L), 1.1 μg/L (e.g., up to 10, 25, 50, 100, or 200 μg/L), 1.6 μg/L (e.g., up to 10, 25, 50, 100, or 200 μg/L), 7.4 μg/L (e.g., up to 510, 25, 0, 100, or 200 μg/L), 0.3 μg/L (e.g., up to 10, 25, 50, 100, or 200 μg/L), 31.1 μg/L (e.g., up to 50, 100, or 200 μg/L), 0.3 μg/L (e.g., up to 10, 25, 50, 100, or 200 μg/L), respectively.
In some embodiments, the pH of the beverage is between 2-5 (e.g., 2.5-4.0, 2.8-4.0 or 3.0-4.0).
The present disclosure provides compositions and methods for hydrolyzing volatile phenols from phenolic glycosides. Specifically, certain glucosidases, gentiobiosidases and rutinosidases and combinations thereof hydrolyze smoke associated volatile phenols from phenolic glycosides. Further, novel methods of quantifying levels of volatile phenols are disclosed.
When fruits such as grapes are exposed to wildfire smoke, certain smoke-related volatile phenols (VPs) can be transferred into the fruit. Once inside the fruit, the VPs can be converted into phenolic glycosides through glycosylation. These phenolic glycosides can be particularly problematic from a winemaking standpoint as they can lead to defects in aroma and flavor. Current methods for quantifying and/or eliminating these phenolic glycosides present several challenges including the requirement of expensive capital equipment, limited accuracy due the molecular complexity of the glycosides, and the utilization of harsh reagents. There is therefore a need in the art for composition and methods for hydrolyzing smoke-related phenolic glycosides to facilitate both their quantification and removal from wines.
Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.
All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the invention, it will be understood that modifications and variations are encompassed within the spirit and scope of the instant disclosure. The preferred methods and materials are now described.
As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.
As used herein, the term “about” in association with a numerical value is meant to include any additional numerical value reasonably close to the numerical value indicated. For example, and based on the context, the value may vary up or down by 5-10%. For example, for a value of about 100, means 90 to 110 (or any value between 90 and 110).
In some embodiments, the present disclosure provides compositions for hydrolyzing smoke-associated phenolics from a phenolic glycoside.
In some embodiments, volatile phenolics are produced from lignin combustion in wildfires. Such volatile phenolics can be absorbed by fruit exposed to wildfire smoke. In some embodiments, hydrolysis of a non-volatile phenolic glycoside results in the production of one or more volatile phenols. In some embodiments, the fruit is grape berries.
Non-limiting examples of volatile phenols include, guaiacol (also herein VP1), 4-methylguaiacol (also herein VP2), 4-ethylguaiacol (VP3), cresol-p (VP4), cresol-m (VP5), cresol-o (VP6), phenol (VP7), 4-ethylphenol (VP-8), syringol (VP-9), and/or 4-methylsyringol (VP-10).
As used herein, the term “phenolic glycosides” refers to a sugar moiety bound to a phenol. In one embodiment, the phenolic glycosides are non-volatile, i.e., they are in a form that does not evaporate into a gas form under particular conditions. In one embodiment, the phenolic glycosides can be associated with smoke taint. Examples of phenolic glycoside associated with smoke taint include, without limitation, glucosides, gentiobiosides, and/or rutinosides.
In one embodiment, the phenolic glycosides can include any of the volatile phenols described herein bound to any of the glycosides described herein. In one embodiment, the phenolic glycoside is a compound of Formula I:
wherein R1, R2, R3 and R4 are as shown in Table 1. In one aspect, R1, R2, R3, and R4 in Formula I determine the identity of phenolic glycoside.
In one embodiment, the phenolic glycoside is a compound of Formula II:
wherein R1, R2, R3 and R4 are as shown in Table 1. In one aspect, R1, R2, R3, and R4 in Formula II determine the identity of phenolic glycoside.
In one embodiment, the phenolic glycoside is a compound of Formula III:
wherein R1, R2, R3 and R4 are as shown in Table 1. In one aspect, R1, R2, R3, and R4 in Formula III determine the identity of phenolic glycoside.
In one embodiment, as described herein, compound 1a refers to guaiacol glucoside, compound 1b refers to guaiacol gentiobioside, and/or compound 1c refers to guaiacol rutinoside. In one embodiment, as described herein, compound 2a refers to 4-methylguaiacol glucoside, compound 2b refers to 4-methylguaiacol gentiobioside, and/or compound 2c refers to 4-methylguaiacol rutinoside. In one embodiment, as described herein, compound 3a refers to 4-ethylguaiacol glucoside, compound 3b refers to 4-ethylguaiacol gentiobioside, and/or compound 3c refers to 4-ethylguaiacol rutinoside. In one embodiment, as described herein, compound 4a refers to cresol-p glucoside, compound 4b refers to cresol-p gentiobioside, and/or compound 4c refers to cresol-p rutinoside. In one embodiment, as described herein, compound 5a refers to cresol-m glucoside, compound 5b refers to cresol-m gentiobioside, and/or compound 5c refers to cresol-m rutinoside. In one embodiment, as described herein, compound 6a refers to cresol-o glucoside, compound 6b refers to cresol-o gentiobioside, and/or compound 6c refers to cresol-o rutinoside. In one embodiment, as described herein, compound 7a refers to phenol glucoside, compound 7b refers to phenol gentiobioside, and/or compound 7c refers to phenol rutinoside. In one embodiment, as described herein, compound 8a refers to 4-ethylphenol glucoside, compound 8b refers to 4-ethylphenol gentiobioside, and/or compound 8c refers to 4-ethylphenol rutinoside. In one embodiment, as described herein, compound 9a refers to syringol glucoside, compound 9b refers to syringol gentiobioside, and/or compound 9c refers to syringol rutinoside. In one embodiment, as described herein, compound 10a refers to 4-methylsyringol glucoside, compound 10b refers to 4-methylsyringol gentiobioside, and/or compound 10c refers to 4-methylsyringol rutinoside.
In some embodiments, the compositions of the disclosure can hydrolyze smoke-associated volatile phenolics from one or more phenolic glycosides. In some embodiments, the compositions of the disclosure include glycosidase enzymes. In some embodiments, the compositions of the disclosure catalyze removal (release) of a glucose moiety from a glucoside associated with smoke taint. In some embodiments, the compositions of the disclosure can catalyze removal (release) of at least one glucose moiety from a gentiobioside associated with smoke taint. In some embodiments, the glycosidase can catalyze removal (release) of a glucose moiety and/or a rhamnose from a rutinoside associated with smoke taint.
In some embodiments, the glycosidase is a glycosidase 1 (GH1) enzyme. In some embodiments, GH1s catalyze the hydrolysis of β1-4 bonds. In some embodiments, the glycosidase is glycosidase derived from archaea, eubacteria, and/or eukaryotes. In one embodiment, the glycosidase is derived from Oscillospiraceae bacterium, Clostridia bacterium, Thermococcus celer, Vulcanisaeta sp. AZ3, Thermococcus guaymasensis, Thermoprotei archaeon, Ignisphaera aggregans DSM 17230, Caldivirga maquilingensis, Thermoproteus uzoniensis, candidatus Marsarchaeota G2 archaeon ECH_B_3, Fervidobacterium changbaicum, Fervidobacterium thailandense, Fervidobacterium gondwanense, Sulfolobus acidocaldarius DSM 639, Vulcanisaeta distributa DSM 14429, Pyrococcus furiosus, Fervidobacterium nodosum, Thermosipho africanus, Lancefieldella parvula, Chloroflexus aurantdacus, Clostridium acetobutylicum, Sebaldella termitidis, Lactococcus lactis subsp. Lactis, Geobacillus kaustophilus, Phanerodontia chrysosporium, Homo sapiens, Castor canadensis, Cavia porcellus, Actinidia chinensis var. chinensis, Ruminiclostridium cellulolyticum, Thermomonospora curvata, Thermobispora bispora, Deinococcus deserti, Cellulomonas flavigena, Bifidobacterium breve, Thermobaculum terrenum, Saccharophagus degradans, Vibrio vulnificus, Halothermothrix orenii, Acetivibrio thermocellus, Cohnella sp. OV330, and/or Halalkalibacterium halodurans, In some embodiments, the glycosidase is a GH5 subfamily 23 glycosidase.
In one embodiment, the glycosidase is a rutinosidase (also herein a 6-O-a-L-rhamnopyranosyl-b-D-glucosidase). In one embodiment, the rutinosidase derived from Acremonium sp, Actinoplanes missouriensis, Aspergillus niger, Candida tropicalis, Candida maltosa and/or Aspergillus oryzae RIB40.
In some embodiments, the glycosidase can be a glucoside hydrolyzing enzyme and/or a gentiobioside hydrolyzing enzyme. In one embodiment, the glucoside hydrolyzing enzyme and/or a gentiobioside hydrolyzing enzyme can include one or more enzymes from Table 2. In one embodiment, the compositions of the disclosure can include a glucoside hydrolyzing enzyme and/or a gentiobioside hydrolyzing enzyme having about 506%, 55%, 60% e, 65%, 70%, 75%, 80/%, 85%, 90° %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9916, or 100% to the sequences in Table 2. In one embodiment, the sequences in Table 2 can further be mutated to tune the enzymatic activity of the sequences.
Clostridia
bacterium
Oscillospiraceae
bacterium
Clostridia
bacterium
Clostridia
bacterium
Thermococcus
celer
Vulcanisaeta
Thermococcus
guaymasensis
Thermoprotei
archaeon
Ignisphaer
aggregans
Thermoprotei
archaeon
Caldivirga
maquilingensis
Thermoproteus
uzomiensis
Candidatus
Marsarchaeota
Fervidobacterium
changbaicum
Fervidobacterium
thailandens
Fervidobacterium
gondwanense
Sulfolobus
acidocaldarius
Vulcanisaeta
distributa
Pyrococcus
furiosus
Fervidobacterium
nodosum
Thermosipho
africanus
Lancefieldella
parvula
Chloroflexus
aurantiacum
Clostridium
acetobutylicum
Sebaldella
termitidis
Sebaldella
termitidis
Lactococcus
lactis
Lactis
Geobacillus
kaustophilus
Geobacillus
kaustophilus
Phanerodontia
chrysosporium
Saccharophagus
degradans
Homo
sapiens
Castor
canadensis
Cavia
porcellus
Actinidia
chinensis
chinensis
Ruminiclostridium
cellulolyticum
Thermomonospora
curvata
Thermobispora
bispora
Deinococcus
deserti
Cellulomonas
flavigena
Bifidobacterium
breve
Thermobaculum
terrenum
Saccharophagus
degradans
Vibrio
vulnificus
Halothermothrix
orenii
Acetivibrio
thermocellus
Cohnella
Halalkalibacterium
halodurans
In some embodiments, the glycosidase can be a rutinosidase. In one embodiment, rutinosidase can include one or more enzymes from Table 3. In one embodiment, the compositions of the disclosure can include a rutinosidase having about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% to the sequences in Table 3. In one embodiment, the sequences in Table 3 can further be mutated to tune the enzymatic activity of the sequences. In some embodiments, the rutinosidase is AoryRu derived from UniProt ID: A0A1S9DRB1. In some embodiments, the rutinosidase is CtroEXG derived from UniProt ID: C5ME42. In some embodiments, the rutinosidase is CmalEXG derived from UniProt ID: M3IJY9. In some embodiments, the rutinosidase is AcreRut derived from UniProt ID: A0A286JZ59. In some embodiments, the rutinosidase is AniRut derived from UniProt ID: A0A6B9UJ04. In some embodiments, rutinosidases of the disclosure derived from UniProt sequences described herein does not include the native leader sequence or signal peptide sequence.
Aspergillus
oryzae
Candida
tropicalis
Candida
maltosa
Acremonium
Aspergillus
niger
In one embodiment, the compositions of the disclosure can include a rutinosidase which has an amino acid sequence with a mutation at one or more positions. In one embodiment, the compositions of the disclosure can include a rutinosidase which has an amino acid sequence with a mutation at one or more positions of SEQ ID NO: 73. In some embodiments, the mutation can be a conservative or a non-conservative amino acid mutation. In one embodiment, the compositions of the disclosure can include a rutinosidase which has an amino acid sequence with a mutation at one or more of position 141, 190, 279, 307, 38, 39, 41, 87, 94, 145, 156, 168, 181, 183, 184, 214, 270, 276, 297, 324, 328, and/or 342 of SEQ ID NO: 73. In one embodiment, the composition can include a rutinosidase with a mutation at one or more positions such as, but not limited to position 141, 190, and/or 279 of SEQ ID NO: 73. In one embodiment, the composition can include a rutinosidase of SEQ ID NO: 78. In one embodiment, the composition can include a rutinosidase with a mutation at one or more positions such as, but not limited to position 141, 190, and/or 307 of SEQ ID NO: 73. In one embodiment the mutations include one or more of T141V, M190I, Q307N, T297V, Q38D, F39W, G41N, G87N, T94N, T1411, T145V, Y156F, V168M, S181Y, Q183W, S184F, T214A, N270R, L276K, R279H, M324W, S328T, and/or A342F relative to SEQ ID NO: 73. In one embodiment, the mutations can include one or more of T141V, M190I, and/or R279H relative to SEQ ID NO: 73. In one embodiment, the mutations can include one or more of T141V, M190I, and/or Q307N relative to SEQ ID NO: 73.
In one embodiment, the compositions of the disclosure can include glycosidases at a concentration of about 0.001 mg/mL, 0.002 mg/ml, 0.003 mg/ml, 0.004 mg/ml, 0.005 mg/mL, 0.006 mg/ml, 0.007 mg/ml, 0.008 mg/ml, 0.009 mg/ml, 0.01 mg/mL, 0.02 mg/mL, 0.03 mg/ml, 0.04 mg/mL, 0.05 mg/ml, 0.06 mg/ml, 0.07 mg/ml, 0.08 mg/mL, 0.09 mg/ml, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 2 mg/ml, 2.5 mg/ml, 3 mg/ml, 3.5 mg/ml, 4 mg/ml, 4.5 mg/ml, 5 mg/ml, 5.5 mg/ml, 6 mg/ml, 6.5 mg/ml, 7 mg/ml, 7.5 mg/ml, 8 mg/ml, 8.5 mg/ml, 9 mg/ml, 9.5 mg/ml, 10 mg/ml, 10.5 mg/ml, 11 mg/ml, 11.5 mg/ml, 12 mg/ml, 12.5 mg/ml, 13 mg/ml, 13.5 mg/ml, 14 mg/ml, 14.5 mg/ml, 15 mg/ml, 15.5 mg/ml, 16 mg/ml, 16.5 mg/ml, 17 mg/ml, 17.5 mg/ml, 18 mg/ml, 18.5 mg/ml, 19 mg/ml, 19.5 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml or more. In one embodiment, the compositions of the disclosure can include glycosidases at a concentration of about 0.1 to 1 mg/ml, 0.2 mg/ml to 1.2 mg/ml, 0.4 mg/ml to 5 mg/ml, 0.5 to 5 mg/ml, 1 to 10 mg/ml, 5 to 15 mg/ml, 10 to 20 mg/ml, 15 to 25 mg/ml, 20 to 30 mg/ml, 25 to 35 mg/ml, 30 to 40 mg/ml, 35 to 45 mg/ml, or 40 to 50 mg/ml, or more.
In one embodiment, the compositions of the disclosure can include glycosidases at a concentration of about 0.001 mg/ml to 50 mg/ml, for example, 0.001 mg/ml to 0.01 mg/ml, 0.005 mg/ml to 0.05 mg/ml, 0.01 mg/ml to 0.1 mg/ml, 0.05 mg/ml to 0.5 mg/ml, 0.1 to 1 mg/ml, 0.2 mg/ml to 1.2 mg/ml, 0.4 mg/ml to 5 mg/ml, 0.5 to 5 mg/ml, 1 to 10 mg/ml, 5 to 15 mg/ml, 10 to 20 mg/ml, 15 to 25 mg/ml, 20 to 30 mg/ml, 25 to 35 mg/ml, 30 to 40 mg/ml, 35 to 45 mg/ml, or 40 to 50 mg/ml, or more.
In one embodiment, the compositions of the disclosure can include glucoside and/or the gentiobioside hydrolyzing enzymes at a concentration of about 0.001 mg/mL, 0.002 mg/ml, 0.003 mg/ml, 0.004 mg/ml, 0.005 mg/mL, 0.006 mg/ml, 0.007 mg/ml, 0.008 mg/ml, 0.009 mg/ml, 0.01 mg/mL, 0.02 mg/mL, 0.03 mg/ml, 0.04 mg/mL, 0.05 mg/ml, 0.06 mg/ml, 0.07 mg/ml, 0.08 mg/mL, 0.09 mg/ml, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 2 mg/ml, 2.5 mg/ml, 3 mg/ml, 3.5 mg/ml, 4 mg/ml, 4.5 mg/ml, 5 mg/ml, 5.5 mg/ml, 6 mg/ml, 6.5 mg/ml, 7 mg/ml, 7.5 mg/ml, 8 mg/ml, 8.5 mg/ml, 9 mg/ml, 9.5 mg/ml, 10 mg/ml, 10.5 mg/ml, 11 mg/ml, 11.5 mg/ml, 12 mg/ml, 12.5 mg/ml, 13 mg/ml, 13.5 mg/ml, 14 mg/ml, 14.5 mg/ml, 15 mg/ml, 15.5 mg/ml, 16 mg/ml, 16.5 mg/ml, 17 mg/ml, 17.5 mg/ml, 18 mg/ml, 18.5 mg/ml, 19 mg/ml, 19.5 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml or more.
In one embodiment, the compositions of the disclosure can include glucoside and/or the gentiobioside hydrolyzing enzymes at a concentration of about 0.001 mg/ml to 50 mg/ml, for example, 0.001 mg/ml to 0.01 mg/ml, 0.005 mg/ml to 0.05 mg/ml, 0.01 mg/ml to 0.1 mg/ml, 0.05 mg/ml to 0.5 mg/ml, 0.1 to 1 mg/ml, 0.2 mg/ml to 1.2 mg/ml, 0.4 mg/ml to 5 mg/ml, 0.5 to 5 mg/ml, 1 to 10 mg/ml, 5 to 15 mg/ml, 10 to 20 mg/ml, 15 to 25 mg/ml, 20 to 30 mg/ml, 25 to 35 mg/ml, 30 to 40 mg/ml, 35 to 45 mg/ml, or 40 to 50 mg/ml, or more.
In one embodiment, the compositions of the disclosure can include rutinosidases at a concentration of about 0.001 mg/mL, 0.002 mg/ml, 0.003 mg/ml, 0.004 mg/ml, 0.005 mg/mL, 0.006 mg/ml, 0.007 mg/ml, 0.008 mg/ml, 0.009 mg/ml, 0.01 mg/mL, 0.02 mg/mL, 0.03 mg/ml, 0.04 mg/mL, 0.05 mg/ml, 0.06 mg/ml, 0.07 mg/ml, 0.08 mg/mL, 0.09 mg/ml, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 2 mg/ml, 2.5 mg/ml, 3 mg/ml, 3.5 mg/ml, 4 mg/ml, 4.5 mg/ml, 5 mg/ml, 5.5 mg/ml, 6 mg/ml, 6.5 mg/ml, 7 mg/ml, 7.5 mg/ml, 8 mg/ml, 8.5 mg/ml, 9 mg/ml, 9.5 mg/ml, 10 mg/ml, 10.5 mg/ml, 11 mg/ml, 11.5 mg/ml, 12 mg/ml, 12.5 mg/ml, 13 mg/ml, 13.5 mg/ml, 14 mg/ml, 14.5 mg/ml, 15 mg/ml, 15.5 mg/ml, 16 mg/ml, 16.5 mg/ml, 17 mg/ml, 17.5 mg/ml, 18 mg/ml, 18.5 mg/ml, 19 mg/ml, 19.5 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml or more.
In one embodiment, the compositions of the disclosure can rutinosidases at a concentration of about 0.001 mg/ml to 50 mg/ml, for example, 0.001 mg/ml to 0.01 mg/ml, 0.005 mg/ml to 0.05 mg/ml, 0.01 mg/ml to 0.1 mg/ml, 0.05 mg/ml to 0.5 mg/ml, 0.1 to 1 mg/ml, 0.2 mg/ml to 1.2 mg/ml, 0.4 mg/ml to 5 mg/ml, 0.5 to 5 mg/ml, 1 to 10 mg/ml, 5 to 15 mg/ml, 10 to 20 mg/ml, 15 to 25 mg/ml, 20 to 30 mg/ml, 25 to 35 mg/ml, 30 to 40 mg/ml, 35 to 45 mg/ml, or 40 to 50 mg/ml, or more.
In some embodiments, the compositions of the disclosure can include at least one glycosidase enzyme. As a non-limiting example, the glycosidases (also herein glycoside hydrolyzing enzyme) include an amino acid sequence of SEQ ID NO: 1-72. As an example, the glycosidases can include an amino acid sequence of SEQ ID NO: 4-13.
In one embodiment, the compositions of the disclosure can include at least one glucoside and/or gentiobioside hydrolyzing enzyme and at least one rutinosidase. In one embodiment, the compositions can include a glucoside and/or a gentiobioside hydrolyzing enzyme having an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 1-72; and a rutinosidase having an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 73-78. In one embodiment, the compositions of the disclosure can include the glucoside and/or a gentiobioside hydrolyzing enzyme of SEQ ID NO: 1 and the rutinosidase of SEQ ID NO: 78. In one embodiment, the compositions of the disclosure can include two, three, four, five, six, seven, eight, nine, ten or more glucoside and/or gentiobioside hydrolyzing enzyme. In one embodiment, the compositions of the disclosure can include two, three, four, five, six, seven, eight, nine, ten or more rutinosidase.
As a non-limiting example, the compositions of the disclosure can include the glucoside and/or the gentiobioside hydrolyzing enzyme CbBg1B-1 (MBR2796233.1; SEQ ID NO: 1) and the rutinosidase AoryRut (A0A1S9DRB1; SEQ ID NO: 73).
As a non-limiting example, the compositions of the disclosure can include the glucoside and/or a gentiobioside hydrolyzing enzyme CbBg1B-1 (MBR2796233.1; SEQ ID NO: 1) and the rutinosidase of SEQ ID NO: 78.
Also provided herein are polynucleotides encoding the glycosidases described herein.
In some embodiments, the present disclosure also provides cells engineered to express (i) a glucoside and/or a gentiobioside hydrolyzing enzyme with an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 1-72; and/or (ii) a rutinosidase with an amino acid sequence with at least 85% sequence identity to SEQ ID NO: 73-78. The cell may be a eukaryotic cell or a prokaryotic cell. In some embodiments, the prokaryotic cell may be a bacterial cell e.g., E. coli. In some embodiments, the eukaryotic cells may be yeast cells, insect cells, and/or mammalian cells.
In some embodiments, the present disclosure provides methods for hydrolyzing volatile phenolics from phenolic glycosides. In some embodiments, the methods are for hydrolyzing volatile phenolics from phenolic glycosides in a fruit product or a fermented product thereof.
In some embodiments, the methods of the disclosure can involve incubating the fruit product or a fermented product thereof with the compositions described herein. In some embodiments, the fruit product or the fermented fruit product can be smoke-exposed.
In some embodiments, the methods of the disclosure are performed for about 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more.
In some embodiments, the methods of the disclosure are performed at room temperature. In some embodiments, the methods of the disclosure are performed at about 37 degrees C. In some embodiments, the methods of the disclosure are performed at about 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C. or more. In one embodiment, the methods of the disclosure are performed at less than 37° C. In some embodiments, the methods of the disclosure are performed at greater than 37° C.
In some embodiments, the methods of the disclosure are performed at the pH of the fruit product or fermented product thereof. In some embodiments, the pH can be about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 24, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8. In some embodiments, the fruit product is derived from any fruit. In some embodiments, the fruit is a berry. Non-limiting examples of fruit include grapes, apples, blueberries, blackberries, raspberries, currants, strawberries, cherries, pears, peaches, nectarines, oranges, pineapples, mangoes, and/or passionfruit. In some embodiments, the fruit product may be derived from two or more different fruits. In some embodiments, the fruit is a grape. In some embodiments, the fruit product may be derived from one or more varieties of grapes. Non-limiting examples of grape varieties include, Cabernet Sauvignon, Alicante Henri Bouschet, Barbera, Bobal, Cabernet Franc, Carignan, Cinsaut, Malbec, Douce noir, Gamay, Grenache, Isabella, Merlot, Montepulciano, Mourvedre, Pinot noir, Sangiovese, Syrah, Tempranillo, Zinfandel, Aglianico, Blaufrankisch, Bordo, Carmenere, Castello, Concord, Corvina Veronese, Criolla Grande, Croatina, Dolcetto, Domfelder, Marufo, Mencia, Black Muscat, and/or Nebbiolo. In some embodiments, the fruit product can include fruit homogenate, a fruit juice, a fruit pulp, a fruit skin, a fruit peel, a fruit seed, a fruit concentrate, or combinations thereof.
In one embodiment, the methods of the disclosure can be applied to fermented fruit products. In one embodiment, the fruit product can be fermented after the methods of the disclosure are applied to the fruit product. In some embodiments, the fermented fruit product is a fermented beverage. In some embodiments, the fermented beverage is wine. In some embodiments, the wine can be table wine, dessert wine, fortified wine, sparkling wine, beer, spirits, cider, mead, liqueurs, sake, or brandy. In some embodiments, the table wine is red wine, white wine, a rose wine. In some embodiments, the red wine is Cabernet Sauvignon, Alicante Henri Bouschet, Barbera, Bobal, Cabernet Franc, Carignan, Cinsaut, Malbec, Douce noir, Gamay, Grenache, Isabella, Merlot, Montepulciano, Mourvedre, Pinot noir, Sangiovese, Syrah, Tempranillo, Zinfandel, Aglianico, Blaufrankisch, Bordo, Carmenere, Castello, Concord, Corvina Veronese, Criolla Grande, Croatina, Dolcetto, Dornfelder, Marufo, Mencia, Black Muscat, and/or Nebbiolo. In some embodiments, the white wine is Chardonnay, Sauvignon Blanc, Pinot Grigio, Moscato, Riesling, and/or Chenin Blanc. In some embodiments, the rose wine is Provence Rose Fresh, Grenache Rose, Sangiovese Rose, Syrah Rose, Zinfandel Rose, and/or Cabernet Sauvignon Rose.
In some embodiments, the methods described herein may involve removing one or more volatile phenols from apparatus and containers involved in the wine making process or fruit fermentation process. Examples of apparatus and containers involved in the wine making process or fruit fermentation process include crushers/destemmers, fermentation vessels (stainless steel tanks, oak barrels, concrete tanks), presses (basket press, bladder press), pumps, airlocks and fermentation locks, hydrometers, refractometers, thermometers, primary fermenters (plastic food-grade buckets, glass carboys), secondary fermenters (glass carboys, stainless steel vessels), bottles, barrels, demijohns, kegs, fermentation buckets, and corks.
Any of the methods described herein may involve removing one or more volatile phenols from the fruit product or fermented fruit product. In some embodiments, removing or reducing the level of volatile phenols in the fruit product or fermented fruit product involves subjecting the fruit product or fermented fruit product to one or more additional processes, such as filtering (e.g., reverse osmosis), contacting the fruit product or fermented fruit product with a fining agent or other adsorbant/affinity agent (e.g., molecularly imprinted polymer), or modifying the volatile phenols (e.g., chemical modification such as methylation).
In some embodiments, the methods involve subjecting the fruit product or fermented fruit product to a filtration process. Filtration methods suitable for removal of volatile phenols from a fermented product are known in the art. In some embodiments, the filtration process is reverse osmosis, which involves passing the fruit product or fermented fruit product through a membrane (filter) having a molecular weight cut-off sufficient to remove volatile phenols from the fermented product.
In some embodiments, the methods involve contacting the fruit product or fermented fruit product with a fining or affinity agent. Examples of these agents for removal of smoke taint include activated carbon, molecularly imprinted polymers and cyclodextrin polymers.
In some embodiments, removing or reducing the level of volatile phenols in the fruit product or fermented fruit product involves subjecting the fruit product or fermented fruit product to an enzymatic process to modify the volatile phenol, for example contacting the fermented product with an enzyme capable of removing the undesired phenol or converting the undesired volatile phenol into a neutral or more desirable form.
The present disclosure also provides methods of quantifying the volatile phenolic and/or a phenolic glycoside in a fruit product or a fermented fruit product. The methods can include incubating the fruit product or fermented fruit product with the compositions of the disclosure. The levels are of the volatile phenolic and/or phenolic glycoside are then measured using mass spectrometry. In some embodiments, the mass spectrometry can be gas chromatography mass spectrometry or liquid chromatography mass spectrometry.
Presented below are examples discussing the utility of compounds of the invention contemplated for the discussed applications. The following examples are provided to further illustrate the embodiments of the present invention but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.
Smoke-associated volatiles levels have been identified, for example, after treatment with the enzymes described herein, for example, as described below. See, e.g.,
To identify enzymes with the ability to cleave glycosidic bonds in bound volatile phenols (VPs), the sequence space of the glycosidase 1 (GH1) enzyme family was explored through genome mining in a gene sequence database, UniProt and NCBI GenBank.
The approach involved collecting and characterizing an assortment of representatives from the gene sequence database that would capture a considerable amount of sequence diversity within the targeted enzyme family. GH1s catalyze the hydrolysis of β1-4 bonds and the GH1 enzyme family is widely distributed in archaea, eubacteria, and eukaryotes. The GH1 family was chosen as the primary target because GH1s have diverse substrate specificities on both conjugated sugars and aglycons. Recently, a comprehensive examination of the functional variety within this group of enzymes further validates GH1 substrate promiscuity and its suitability for industrial purposes.
A total of approximately 80,000 genes presumably annotated as the GH1 family were visualized via sequence similarity network (SSN) based on their phylogenetic relationships, in which all sequences sharing 75% or more identity were grouped into a single meta node (Rep node). A set of 73 synthetic genes encoding naturally occurring proteins were procured (
Synthetic genes encoding the 73 proteins were purchased, cloned into a pET29b+ vector with a C-terminal 6× histidine tag (SEQ ID NO: 79), and overexpressed in E. coli. The corresponding proteins were purified by IMAC and analyzed by SDS-PAGE. The obtained enzymes underwent stepwise testing to evaluate the ability to release VPs and the activity was semi-quantitatively assessed based on the degree of substrate disappearance post-reaction by LC-MS (
Initial proof of concept studies were acetic acid buffer conditions at pH 3.5 with 4.5 mg/L guaiacol glucoside (compound 1a) as the substrate at 37° C. over a 24-hour period. 45/73 enzymes were found to be active towards compound 1a while the other 28 enzymes were either inactive or not expressed in a soluble form (
The enzymes were then tested under acetic acid buffer conditions at pH 3.5 and baseline Cabernet Sauvignon (no pH adjustment) and a 4-hour incubation time. The enzyme activity in both systems were compared because it is well known that the chemical compounds in wines, especially in red wines, such as ethanol, glucose, tannins, and metals can inhibit GHs, and the side-by-side comparison can provide the necessary information to determine whether the lack of activity in wine was due to inhibition. For guaiacol glucoside (compound 1a), 22 enzymes exhibited glycosidase activity out of which 15 were capable of completely catalyzing the release of guaiacol in an acetic acid buffer (
Inhibition in Cabernet Sauvignon was clearly observed for both substrates. Among the 12 enzymes that can fully utilize compound 1a in acetic acid buffer, 9 enzymes maintained complete functionality. However, in the case of compound 1b, only 3 enzymes completely catalyzed the release of guaiacol in Cabernet Sauvignon, namely Bg1b from Oscillospiraceae bacterium (ObBg1B), Bg1B-1 (CbBg1B-1) and Bg1B-2 (CbBg1B-1) from Clostridia bacterium. These three enzymes also demonstrated shared activity towards compound 1a, indicating a potential functional overlap in their ability to catalyze the release of volatile phenols. All three enzymes are from Clostridia bacteria class in ruminant gastrointestinal microbiome and share about 70% sequence identity to each other. This represents the first instance where these three enzymes have been characterized against smoke associated phenolic glycosides.
To select the best candidate among the three outstanding enzymes in the initial screening, the actives and substrate scopes of the enzymes were compared with fortification experiments. 8 commercially available P-D-glycosides namely guaiacol glucoside (compound 1a), guaiacol gentiobioside (compound 1b), guaiacol rutinoside (compound 1c), 4-methylguaiacol rutinoside (compound 2c), p-cresol rutinoside (compound 4c), phenol rutinoside (compound 7c), syringol gentiobioside (compound 9b), 4-methylsyringol gentiobioside (compound 10b) with diverse VP aglycons and sugar moieties were spiked in baseline Cabernet Sauvignon with a more realistic concentration of 40 μg/L at 37° C. for 4 hours. The conversion value is calculated by subtracting the final concentration of each VP in baseline wine from those after enzymatic hydrolysis, then dividing by the theoretical mass of each VP. The conversion rate is determined based on the concentration of VPs recovered through enzymatic hydrolysis, as quantified by GC-MS. Similar substrate scope and activity profiles were observed for ObBg1B and CbBglB-2. All three enzymes can utilize more than 80% of guaiacol glycosides namely compound 1a, compound 1b and compound 1e as expected and about 80% of compound 9b (
To evaluate performance of CbBg1B-1 in a previously validated sample of smoke-tainted wine, a direct comparison was performed between acid hydrolysis and CbBg1B-1 mediated enzyme hydrolysis from phenolic glycosides in a smoke-tainted Cabernet Sauvignon. Using the levels of phenolic glycosides generated by acid hydrolysis as a benchmark, we can calculate the ratio of each glycoside converted by enzymatic hydrolysis relative to acid hydrolysis. The ratio for each VP was calculated by dividing the total VP release measured after enzymatic hydrolysis by that of acid hydrolysis. A value greater than 100% would imply that enzymatic hydrolysis is more accurate of total VP in the matrix than acid hydrolysis, while a value less than 100% would suggest the opposite. Triplicate data were collected, and averages reported, all standard deviations were <10%. Enzymatic hydrolysis achieved less than 90% conversion for the majority of the measured VPs compared to acid hydrolysis, with the majority of VPs between 20% to 50% of the conversion yields observed in acid hydrolysis (
The 6-O-a-L-rhamnopyranosyl-b-D-glucosidases (rutinosidases; EC 3.2.1.168) belong to the GH5 subfamily 23 and specifically act on the flavonoid diglycosides, including compounds like quercetin 3-O-rutinoside, hesperetin 7-O-rutinoside, kaempferol-3-O-rutinoside, and naringenin 7-O-neohesperidoside. Notable rutinosidases have been reported from several species, including Acremonium sp. DSM 24697, Actinoplanes missouriensis, Aspergillus niger K2, and Aspergillus oryzae Rf1340. Advancements have been made recently in understanding the properties of these enzymes and the crystal structures of rutinosidase from Aspergillus niger K2 (AniRut), and rutinosidase from Aspergillus oryzae RIB40 (AoryRut) were deciphered to shed light on the substrate specificity. Remarkedly, AoryRut is capable of accommodating various flavonoids including both 7-O-linked and 3-O-linked flavonoids, possibly contributed by the flexible loop located at the substrate entrance. While there's considerable interest in its application within the food industry, the exploration of the enzymes' substrate scope beyond flavonoid glycosides remains limited. Genome mining was performed in non-exhaustive manner with a particular emphasis on identifying rutinosidase activity against 4-methylguaiacol rutinoside compound 2c among the collection of selected proteins.
GH5 SSN composed of about 67,000 genes was built and previously identified rutinosidases such as AoryRut and AniRut centered on group 5. A higher preference was assigned to enzymes situated in group 1 and group 5 to ensure that the chosen representatives spanned across a wide sequence space, while also leveraging the accessible knowledge base (
CbBg1B-1 is annotated as a GH1 enzyme family in which the enzymes typically exhibit exacting activity with the progressive release of monosaccharides from these linkages. AoryRut has been classified as a GH5 diglycosidase and can cleave the entire disaccharide moiety from the aglycone. The obtained activity profile of AoryRut underscores that AoryRut can serve as an effective complement to CbBg1B-1 for the purpose of maximizing the release of phenolic glycosides. When the enzyme cocktail of CbBg1B-1 and AoryRut was employed, the synergetic effects led to the additive enhancement on harnessing the full spectrum of glycosides (
To establish the optimal parameters for enzymatic hydrolysis, that directly affect the process of enzymatic hydrolysis two notable parameters were examined, namely, incubation time and enzyme loading. To fine-tune the incubation time, various reaction durations including 0.25 hours, 1 hour, 4 hours and 24 hours were tested. Time-course experiment indicated that the reaction achieved equilibrium in 4 hours and the extension of reaction time would not necessarily yield more VPs (
A comparative study of hydrolysis using glycosidase 2 (Rapidase Revelation Aroma), CbBg1B-1, and AoryRut was done (
To further corroborate the efficacy of the enzyme cocktail, a direct quantification strategy for VP glycosides in wine and berries was implemented. Nonsmoke-affected samples were mixed with known VP glycoside substrates and then conducted LC-MS/MS analysis both before and after subjecting them to enzymatic and acid hydrolysis. This method allowed the measurement of the conversion of VP glycosides accurately. The results confirmed that both acidic and enzymatic hydrolysis successfully converted all VP glycosides (
Enzymatic hydrolysis catalyzed by the enzyme cocktail after formulation optimization was then carried out in Cabernet Sauvignon wines and grape berries that were divided into two categories: smoke-impacted and non-smoke-impacted. Both acid hydrolysis and enzymatic hydrolysis demonstrated significantly higher total VPs concentrations in smoke-impacted wine and grape than those in non-smoke-impacted samples. Reflected by the total concentration of VPs, both wine and grape samples impacted by smoke contained significantly elevated concentrations of phenolic glycosides compared to those samples unaffected by smoke, and the results validated the potential of hydrolysis method for binary and qualitative assessments of smoke impact (
A detailed analysis was conducted to compare the differences between enzymatic hydrolysis and acid hydrolysis in wine samples. The enzymatic hydrolysis led to a higher conversion of half of the bound VPs in both smoke-impacted and non-smoke-impacted wines, albeit for different VPs (
To alleviate the economic consequences of producing smoke-affected wines, it is useful to determine the quantities of both free and bound VPs in grapes prior to fermentation. As part of this initiative, enzymatic hydrolysis of smoke-impacted Cabernet Sauvignon grapes and control grapes was studied. This allowed us to assess the method's compatibility with grapes, which are more challenging to accurately determine VPs under acid hydrolysis conditions. Following a similar trend as observed in smoke-impacted wine, total VPs in post-hydrolysis of smoke-impacted grape berries were considerably higher than control grape, and compound 9 persisted as the most abundant VP after hydrolysis in smoke-impacted grape berries (
Consistent with the performance in wine samples, enzymatic hydrolysis showed 150%-300% increase of conversion than acid hydrolysis for bound forms of compound 5, 6 and 7 (a, b, c) (
Utilizing enzymatic hydrolysis has the potential to bring several notable advantages. First, enzymatic hydrolysis surpasses acid hydrolysis in efficacy. Second, acid hydrolysis is well known to be sensitive to conditions and handling, making it difficult to standardize across laboratories. Conversely, enzymatic hydrolysis operates under milder conditions and avoids the use of harsh chemicals. This provides a safer work environment, a useful consideration in laboratory settings. Third, the reduced sample preparation such as pH titration, makes enzymatic hydrolysis an efficient choice for high-throughput. This high-throughput capability is particularly beneficial for grape growers and wine makers, allowing for prompt decision-making, especially during fire seasons. Fourth, the method is cost-effective and eliminates the need for high cost and low throughput LC-MS/MS based analytics.
The bacterial strain used for cloning was Escherichia coli DH5a; the pET29 (+b) plasmids containing the protein encoding genes were expressed in E. coli BLR (DE3). All genes were purchased as synthetic genes optimized for E. coli codon usage with infusion of 6-histidine at the C-terminus. The sequences of genes encoding all glycosidases in the present work are listed in Table 2 and Table 3.
Grape and wine samples. The grapes used for this study were sourced from Vitis vinifera L. cv. Cabernet Sauvignon from California with a significant smoke impact in 2020. And the high-smoke-impacted Cabernet Sauvignon were obtained from simulated smoke exposed vinifera L. cv. Cabernet Sauvignon.
SSN was built by EFI-EST web-tool and visualized in Cytoscape. The Interpro IPR001360 collection of GH1 enzyme sequences combined with JGI IMG Integrated Microbial Genomes & Microbiomes database annotated GH1 enzymes were used as the input for EFI-EST analysis of GH1 while Interpro IPR001547 annotated as rutinosidase were used as the input for GH5. For both of SSN, only Ref50 clusters were used. Sequence identity threshold of 45 was used as parameter for filtering the sequences into clusters in SSN and representative node networks with 70% identity were displayed.
E. coli was first grown overnight as the starter culture at 37° C. in Terrific Broth medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl) supplemented with Kanamycin (50 μg/mL final concentration) and MgSO4 (1 mM final concentration), The culture for protein expression was diluted by—50-fold to 500 mL from the starter culture. The cultures were then grown until OD600 to—0.6 at 37° C., and IPTG was supplemented to final concentration of 0.5 mM for induction at 16° C. for 24 h. At the end of induction, cells were centrifuged (4,700×g, 4° C., 10 min), supernatant was removed, cells were resuspended in 40 mL lysis buffer (50 mM HEPES, pH 7.0, 300 mM NaCl, 10% glycerol, 1 mM MgSO4, 15 mM imidazole), and sonicated for 2 min at 4° C. Lysed cells were centrifuged at 4,700×g at 4° C. for 30 min to remove cell debris. Supernatant was loaded on a gravity flow column with 1 mL of cobalt slurry, which was pre-balanced with 30 mL of wash buffer (50 mM HEPES, pH 7.0, 300 mM NaCl, 10% glycerol, 1 mM MgSO4, 15 mM imidazole). The cobalt resin was then washed three times with 10 mL wash buffer: proteins were eluted with 0.6 mL of elution buffer (50 mM HEPES, pH 7.0, 300 mM NaCl, 10% glycerol, 1 mM MgSO4, 1 mM TCEP, 200 mM imidazole). Protein samples were immediately buffer exchanged with spin concentrators into storage buffer (50 mM HEPES, pH 7.0, 300 mM NaCl, 10% glycerol, 1 mM MgSO4) and stored at 4° C. until activity characterization. Protein concentrations were determined using a spectrophotometer by measuring absorbance at 280 nm using their calculated extinction coefficients. The protein samples were further analyzed by 12% SDS-PAGE gel.
Purified enzymes were added into both buffer and baseline wine samples with substrates 1a, 1b and 2c spiked in. The reaction mixture was kept at 37° C. for 24 hours or 4 hours. After cooling down on ice, the reactions were quenched by adding to 50% volume of acetonitrile then centrifuged. The supernatant was subjected to activity assay.
Reverse-phase high-performance liquid chromatography and mass spectrometry (LC-MS) for analysis were carried. The gas temperature was 350° C., drying flow was 13.0 L/min, and capillary voltage was 4300 V. Each sample was analyzed in triplicate. The mobile phase consisted of the following gradient: 70% H2O with 0.1% formic acid as mobile phase A and 30% ACN with 0.1% formic acid as mobile phase B for 5 mins; 10% mobile phase A and 90% mobile phase B from 8 to 19 min; mobile phase A was decreased to 70% with 30% mobile phase B until 25 min. The HPLC flow rate was 0.5 mL/min and the injection volume was 3 μL. The parameter of the mass spectrum was adjusted accordingly for different glycosides as shown in
Sample prep for grape berries: Samples were removed from the freezer, then 65 g of berries were separated from cluster rachi, taking care to prevent berry cap and other non-berry debris from introduction into the sample container. Samples were thawed for 15-20 minutes at room temperature. 15 mL water was added to the sample, homogenized with a high-speed commercial blender for 1 min, paused for 1 min and then homogenized for a further 30 s.
Enzymatic hydrolysis: 4 g of the homogenized berry sample or 4 mL of wine were transferred into 20 mL GC vials purchased from Agilent. 16 μL of ethanolic d3-guaiacol (5 mg/L) internal standard was added to samples (final concentration of 20 pug/kg in berry homogenate or 20 μg/L in wine). Glycosidase enzymes were then added to the samples. For enzymatic hydrolysis of real-world samples, the final concentrations of 4 mg/mL and 1 mg/mL of CbGglB-1 and AoryRut were added, respectively. The reactions were conducted at 37° C. for 4 hours.
Acid hydrolysis: Samples were aliquoted into 20 mL glass tubes in 10 mL and the pH was adjusted to 1.0 with 4M HC 1 then spiked with 40 uL of ethanolic d3-guaiacol (5 mg/L) internal standard.
Samples were then transferred from the glass tubes to 17 mL Teflon tubes equipped with tightly fitted caps. Samples were incubated at 100° C. for 1 hour, then cooled over ice for 10 min before aliquoting 4 mL wine or 4 g grape homogenate into GC vials.
HS-SPME: Smart SPME arrow 1.1 mm DVB/CarbonWR/PDMS (Agilent 5610-5861) was used by PAL3 robotic autosampler for sample injections. The SPME headspace settings: predesorption time: 4 min and temperature: 250° C. Sample incubation time: 4 min. Sample vial penetration depth: 35 mm. Inlet penetration depth: 40 mm. Inlet penetration speed: 100 mm/s.
Sample vial penetration Speed: 35 mm/s. Sample extraction time: 9 min and extraction temperature: 60° C. Heatex stirrer speed: 1,000 rpm and temperature: 40° C. Sample desorption time: 3 min.
GC-MS: All samples in 20 mL GC-MS headspace vials ready to assay were added with 40% w/v NaCl. The GC-MS injection mode was splitless at 250° C. GC has a constant flow of 1.2 mL/min helium gas. The oven program was 120° C. (hold 1 min); 9° C./min to 250° C. (hold 0 min); 250° C./min to 280° C. (hold 0 min). The guard chip temperature was 200° C., bus temperature 280° C. and MSD transfer line 280° C.
All experiments were independently carried out in triplicate. The differences between samples were evaluated by student's t-test. The P values <0.05 indicates statistically significant difference.
Following the enzymatic hydrolysis reactions described in Examples 1-4, volatile phenols are removed from fruit products or fermented fruit products such as wine using methods known in the art. Volatile phenols can be removed by available techniques, such as using (i) activated carbon by filtration or reverse osmosis, (ii) using yeast lees or cells walls, (iii) using enzymes, (iv) using cellulose, (v) using cyclodextrins polymers, and/or (vi) using molecularly imprinted polymers.
The computational enzyme design software Rosetta suite, which includes algorithms for computational modeling and analysis of protein structures was applied. Residues distal to the active site (>8 Å) were targeted for mutations to avoid potential activity disruption due to engineering. Each position was designed by Rosetta using a position-specific substitution matrix (PSSM) constructed from sequence alignment of the entire rutinosidase enzyme family. Only mutations with a favorable PSSM score (0) were chosen as targets. The selected mutations were then subjected to in silico mutation and further evaluated using Rosetta score terms. The top 50 designs with the lowest total scores were selected as potential candidates for further evaluation. The structures of these 50 designs were built using Rosetta and visualized in PyMOL software. Evaluation involved chemical intuition to remove obviously unreasonable designs, focusing on those that presumptively increase protein packing (e.g., small residue to large residue, non-polar residues to polar residues to introduce new hydrogen bonds). Ultimately, 22 designs (MC4-MC25) were constructed and screened. Beneficial mutations for protein expression were then combined to obtain MC52-MC60 for further screening.
To identify AoryRut (SEQ ID NO: 73) was mutated and the resulting mutants were screened to identify mutations that increase expression and enzyme stability while maintaining enzymatic activity. Table 4 shows the mutants and combination of mutants selected for screening. The AoryRut mutants were introduced into Escherichia coli (E. coli) and expression of the enzymes was measured. Table 4 shows the expression level of the AoryRut mutants. AoryRut mutants MC8 (T141V), MC14 (S184F), MC15 (M190I), MC21 (Q307N), MC55 (T141V, T214A, Q307N), MC56 (T141V, M190I, Q307N), MC58 (M190I, T214A) showed expression greater than AoryRut. Among the different mutants screened, AoryRut mutant MC56 having mutations at positions T141V, M190I and Q307N showed highest expression in E. coli.
The stability of AoryRut mutant MC56 (SEQ ID NO. 78) and having mutations at positions T141V, M190I and Q307N relative to SEQ ID NO: 73 was analyzed. The results are shown in Table 5. The stability analysis showed that MC56 has greater stability than wild type AoryRut of SEQ ID NO: 73.
While stability and expression of AoryRut mutant MC56 were enhanced, the enzymatic activity of this mutant was maintained compared to wildtype (see
Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.
This application is a continuation of U.S. patent application Ser. No. 18/798,577, filed Aug. 8, 2024, which claims the benefit of the U.S. Provisional Application No. 63/531,757, filed Aug. 9, 2023, which application is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63531757 | Aug 2023 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 18798577 | Aug 2024 | US |
| Child | 19006003 | US |
