Acetolactate decarboxylase

Abstract

The present disclosure provides methods, compositions, apparatuses and kits comprising ALDC enzymes having a better stability and/or activity, and, optionally, the yield of ALDC enzymes which can be recovered from microorganisms is improved. In some embodiments, the present disclosure provides methods, apparatuses, compositions and kits for the use of metal ions to increase stability and/or activity, and which further can be used to recover the enzymes from microorganisms in improved yields.

1 . A composition comprising an acetolactate decarboxylase (ALDC) enzyme and zinc, wherein said zinc is present at a concentration of about 1 μM to about 200 mM. 
     
     
         2 . The composition of  claim 1 , wherein the zinc is present at a concentration of about 10 μM to about 150 mM, or about 20 μM to about 120 mM, or about 25 μM to about 100 mM, or about 25 μM to about 50 mM, or about 25 μM to about 20 mM, or about 25 μM to about 50 μM, or about 100 μM to about 20 mM, or about 250 μM to about 20 mM, or about 500 μM to about 20 mM, or about 1 mM to about 20 mM, or about 1 mM to about 10 mM, or about 1 mM to about 5 mM. 
     
     
         3 . The composition of  claim 2 , wherein the zinc
 (i) is present at a concentration of about 100 μM to about 10 mM; or   (ii) is present at a concentration of about 1 mM to about 5 mM.   
     
     
         4 . The composition of  claim 3 , wherein the molar ratio of zinc to ALDC enzyme is
 (i) higher than 1; or   (ii) 2:1 or higher; or   (iii) 10:1 or higher; or   (iv) 20:1 or higher; or   (v) 30:1 or higher; or   (vi) 60:1 or higher.   
     
     
         5 . The composition of  claim 4 , wherein said ALDC enzyme is an ALDC derivative. 
     
     
         6 . The composition of  claim 5 , wherein said ALDC derivative is an ALDC enzyme treated with glutaraldehyde. 
     
     
         7 . The composition of  claim 6 , wherein said ALDC enzyme is treated with glutaraldehyde at a concentration corresponding to about 0.1 to about 5 g of glutaraldehyde per g of pure ALDC enzyme. 
     
     
         8 . The composition of  claim 7 , wherein the activity of said ALDC enzyme is in the range of 950 to 2500 Units per mg of protein. 
     
     
         9 . The composition of  claim 8 , wherein the activity of said ALDC enzyme is in the range of 1000 to 2500 Units per mg of protein. 
     
     
         10 . The composition of  claim 9  further comprising at least one additional enzyme or enzyme derivative selected from the group consisting of acetolactate reductoisomerases, acetolactate isomerases, amylase, glucoamylase, hemicellulase, cellulase, glucanase, pullulanase, isoamylase, endo-glucanase and related beta-glucan hydrolytic accessory enzymes, xylanase, xylanase accessory enzymes (for example, arabinofuranosidase, ferulic acid esterase, and xylan acetyl esterase) and protease. 
     
     
         11 . The composition of  claim 1 , wherein the ALDC enzyme is from  Lactobacillus casei, Brevibacterium acetylicum, Lactococcus lactis, Leuconostoc lactis, Enterobacter aerogenes, Bacillus subtilis, Bacillus brevis, Lactococcus lactis  DX, or  Bacillus licheniformis.    
     
     
         12 . The composition of  claim 11 , wherein the ALDC enzyme is from  Bacillus brevis  or  Bacillus licheniformis.    
     
     
         13 . The composition of  claim 12 , wherein said ALDC enzyme has an amino acid sequence having at least 80% identity with any one selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8 or any functional fragment thereof. 
     
     
         14 . Use of the composition according to  claim 13  in beer and/or wine and/or cider and/or perry and/or sake fermentation. 
     
     
         15 . A method for increasing the activity and/or stability of an ALDC enzyme in a composition comprising ALDC wherein said method comprises the step of adding zinc to the composition so that said zinc is present in said composition at a concentration of about 1 μM to about 200 mM. 
     
     
         16 . The method of  claim 15 , wherein the zinc is present in said composition at a concentration of about 10 μM to about 150 mM, or about 20 μM to about 120 mM, or about 25 μM to about 100 mM, or about 25 μM to about 50 mM, or about 25 μM to about 20 mM, or about 25 μM to about 50 μM, or about 100 μM to about 20 mM, or about 250 μM to about 20 mM, or about 500 μM to about 20 mM, or about 1 mM to about 20 mM, or about 1 mM to about 10 mM, or about 1 mM to about 5 mM. 
     
     
         17 . The method of  claim 16 , wherein the zinc is present in said composition at a concentration of
 (i) about 100 μM to about 10 mM; or   (ii) about 1 mM to about 5 mM.   
     
     
         18 . The method of  claim 16 , wherein the molar ratio of zinc to ALDC enzyme is
 (i) higher than 1; or   (ii) 2:1 or higher; or   (iii) 10:1 or higher; or   (iv) 20:1 or higher; or   (v) 30:1 or higher; or   (vi) 60:1 or higher   
       in said composition. 
     
     
         19 . A method for increasing the activity and/or stability of an ALDC enzyme in a cultivation media comprising an ALDC producing host cell wherein said method comprises the step of adding zinc to the cultivation media as a supplement during the production of said ALDC enzyme by the ALDC producing host cell. 
     
     
         20 . The method of  claim 19 , wherein said zinc is added at a concentration of 1 μM to about 1 mM. 
     
     
         21 . The method of  claim 20 , wherein said zinc is added at a concentration of 25 μM to about 150 μM, or 60 μM to about 150 μM. 
     
     
         22 . The method of  claim 21 , wherein said host cell is a  Bacillus  host cell. 
     
     
         23 . The method of  claim 22 , wherein said  Bacillus  host cell is  Bacillus subtilis.    
     
     
         24 . A method for increasing the activity and/or stability of an ALDC enzyme in a fermentation media and/or maturation media comprising an ALDC producing host cell wherein said method comprises the step of adding zinc to the fermentation media and/or maturation media during a beer and/or wine and/or cider and/or perry and/or sake fermentation. 
     
     
         25 . The method of  claim 24 , wherein said zinc:
 (i) is added at a concentration of about 1 μM to about 300 μM, such as about 6 μM to about 300 μM; or   (ii) is added at a concentration of about 1 μM to about 50 μM; or   (iii) is added at a concentration of about 1 μM to about 25 μM, preferably about 6 μM to about 25 μM.   
     
     
         26 . The method of  claim 24 , wherein said zinc is added as a composition comprising ALDC and zinc, wherein said zinc is present in said composition at a concentration of 1 mM to about 5 mM. 
     
     
         27 . A cultivation media for an ALDC producing host cell comprising zinc at a concentration of about 1 μM to about 1 mM; preferably said cultivation media comprises an ALDC producing host cell. 
     
     
         28 . The cultivation media of  claim 27 , comprising zinc at concentration of about 25 μM to about 150 μM. 
     
     
         29 . The cultivation media of  claim 27 , wherein said zinc is added at a concentration of 60 μM to about 150 μM. 
     
     
         30 . A beer and/or wine and/or cider and/or perry and/or sake fermentation media and/or maturation media comprising a composition comprising an ALDC enzyme and zinc wherein said composition comprises zinc at a concentration of about 1 μM to about 200 mM. 
     
     
         31 . The beer and/or wine and/or cider and/or perry and/or sake fermentation media and/or maturation media of  claim 30 , wherein:
 (i) zinc is present in the composition at a concentration of about 1 μM to about 300 μM, such as about 6 μM to about 300 μM; or   (ii) zinc is present in the composition at a concentration of about 1 μM to about 50 μM, such as about 6 μM to about 50 μM, or about 6 μM to about 25 μM; or   (iii) the zinc and the ALDC enzyme are added in a composition, wherein zinc is present in the composition at a concentration of about 1 mM to about 20 mM, such as 1 mM to about 5 mM; or   (iv) the zinc and the ALDC enzyme are added in a composition, where the molar ratio of zinc to ALDC enzyme in the composition is higher than 1; or 2:1 or higher; or 10:1 or higher; or   20:1 or higher; or 30:1 or higher; or 60:1 or higher.   
     
     
         32 . The beer and/or wine and/or cider and/or perry and/or sake fermentation media and/or maturation media of  claim 31 , wherein the activity of said ALDC enzyme is in the range of 1000 to 2500 Units per mg of protein. 
     
     
         33 . The beer and/or wine and/or cider and/or perry and/or sake fermentation media and/or maturation media of  claim 32 , further comprising at least one additional enzyme or enzyme derivative selected from the group consisting of acetolactate reductoisomerases, acetolactate isomerases, amylase, glucoamylase, hemicellulase, cellulase, glucanase, pullulanase, isoamylase, endo-glucanase and related beta-glucan hydrolytic accessory enzymes, xylanase, xylanase accessory enzymes (for example, arabinofuranosidase, ferulic acid esterase, and xylan acetyl esterase) and protease. 
     
     
         34 . A composition comprising an ALDC enzyme, wherein said ALDC enzyme is in the range of 1000 to 2500 Units per mg of protein. 
     
     
         35 . The composition of  claim 34 , wherein the ALDC enzyme is from  Lactobacillus casei, Brevibacterium acetylicum, Lactococcus lactis, Leuconostoc lactis, Enterobacter aerogenes, Bacillus subtilis, Bacillus brevis, Lactococcus lactis  DX, or  Bacillus licheniformis.    
     
     
         36 . The composition of  claim 34 , wherein the ALDC enzyme is from  Bacillus brevis  or  Bacillus licheniformis.    
     
     
         37 . The composition of  claim 36 , wherein said ALDC enzyme has an amino acid sequence having at least 80% identity with any one selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8 or any functional fragment thereof. 
     
     
         38 . The composition of  claim 37 , wherein:
 (i) zinc is present at a concentration of about 1 μM to about 200 mM; or   (ii) the molar ratio of zinc to ALDC enzyme in the composition is higher than 1; or 2:1 or higher; or 10:1 or higher; or 20:1 or higher; or 30:1 or higher; or 60:1 or higher.   
     
     
         39 . A method for beer and/or wine and/or cider and/or perry and/or sake production comprising adding an ALDC enzyme and adding zinc to a media (such as a fermentation and/or a maturation media) for the beer and/or wine and/or cider and/or perry and/or sake during said beer and/or wine and/or cider and/or perry and/or sake production, so that said zinc is present in said media at a concentration of about 1 μM to about 300 μM, such as about 6 μM to about 300 μM. 
     
     
         40 . The method of  claim 39 , wherein said zinc is present in said media at a concentration of about 0.1 μM to about 50 μM, such as about 1 μM to about 50 μM, or about 6 μM to about 50 μM, or about 6 μM to about 25 μM. 
     
     
         41 . A method for beer and/or wine and/or cider and/or perry and/or sake production comprising adding a composition comprising an ALDC enzyme and zinc to a media (such as a fermentation and/or a maturation media) for the beer and/or wine and/or cider and/or perry and/or sake during said beer and/or wine and/or cider and/or perry and/or sake production, wherein
 (i) zinc is present in the composition at a concentration of about 1 mM to about 5 mM; or   (ii) the molar ratio of zinc to ALDC enzyme in the composition is higher than 1; or 2:1 or higher; or 10:1 or higher; or 20:1 or higher; or 30:1 or higher; or 60:1 or higher.   
     
     
         42 . The method of  claim 41 , wherein said ALDC enzyme and said zinc are added during a fermentation process or a maturation process. 
     
     
         43 . The method of  claim 42 , wherein said ALDC enzyme is added at a concentration of about 0.5 g to about 10 g per hectoliter of beer and/or wine and/or cider and/or perry and/or sake ferment. 
     
     
         44 . The method of  claim 43 , wherein said ALDC enzyme is added at a concentration of about 1 g to about 5 g per hectoliter of beer and/or wine and/or cider and/or perry and/or sake ferment. 
     
     
         45 . The method of  claim 44 , wherein the activity of said ALDC enzyme is in the range of 1000 to 2500 Units per mg of protein. 
     
     
         46 . The method of  claim 45 , wherein the ALDC enzyme is from  Lactobacillus casei, Brevibacterium acetylicum, Lactococcus lactis, Leuconostoc lactis, Enterobacter aerogenes, Bacillus subtilis, Bacillus brevis, Lactococcus lactis  DX, or  Bacillus licheniformis.    
     
     
         47 . The method of  claim 45 , wherein the ALDC enzyme is from  Bacillus brevis  or  Bacillus licheniformis.    
     
     
         48 . The method of  claim 45 , wherein said ALDC enzyme has an amino acid sequence having at least 80% identity with any one selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8 or any functional fragment thereof. 
     
     
         49 . A method for increasing the activity and/or stability of an ALDC enzyme in a composition comprising ALDC wherein said method comprises the step of adding a metal ion to the composition at a concentration of about 1 μM to about 200 mM, preferably about 100 μM to about 200 mM. 
     
     
         50 . The method of  claim 49 , wherein the atomic radius for the metal ion is about 140 pm to about 165 pm. 
     
     
         51 . The method of  claim 50 , wherein the atomic radius for the metal ion is about 140 pm to about 150 pm. 
     
     
         52 . The method of  claim 51 , wherein the atomic radius for the metal ion is about 142 pm to about 146 pm. 
     
     
         53 . The method of  claim 49 , wherein the metal ion is selected from the group consisting of Mg 2+ , Mn 2+ , Co 2+ , Cu 2+ , Ca 2+ , Ba 2+ , and Fe 2+  and combinations thereof. 
     
     
         54 . The method of  claim 53 , wherein the metal ion is selected from the group consisting of Zn 2+ , Mn 2+ , and Co 2+ . 
     
     
         55 . The method of  claim 54 , wherein the metal ion is Zn 2+ . 
     
     
         56 . The method of  claim 55 , wherein the ALDC enzyme is from  Lactobacillus casei, Brevibacterium acetylicum, Lactococcus lactis, Leuconostoc lactis, Enterobacter aerogenes, Bacillus subtilis, Bacillus brevis, Lactococcus lactis  DX, or  Bacillus licheniformis.    
     
     
         57 . The method of  claim 55 , wherein the ALDC enzyme is from  Bacillus brevis  or  Bacillus licheniformis.    
     
     
         58 . The method of  claim 55 , wherein said ALDC enzyme has an amino acid sequence having at least 80% identity with any one selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8 or any functional fragment thereof. 
     
     
         59 . A composition comprising an acetolactate decarboxylase (ALDC) enzyme and a metal ion, wherein said metal ion is present at a concentration of about 1 μM to about 200 mM, preferably about 100 μM to about 200 mM. 
     
     
         60 . The composition of  claim 59 , wherein the atomic radius for the metal ion is about 140 pm to about 165 pm. 
     
     
         61 . The composition of  claim 59 , wherein the atomic radius for the metal ion is about 140 pm to about 150 pm. 
     
     
         62 . The composition of  claim 61 , wherein the atomic radius for the metal ion is about 142 pm to about 146 pm. 
     
     
         63 . The composition of  claim 59 , wherein the metal ion is selected from the group consisting of Mg 2+ , Mn 2+ , Co 2+ , Cu 2+ , Ca 2+ , Ba 2+ , and Fe 2+  and and combinations thereof. 
     
     
         64 . The composition of  claim 63 , wherein the metal ion is selected from the group consisting of Zn 2+ , Mn 2+ , and Co 2+ . 
     
     
         65 . The composition of  claim 64 , wherein the metal ion is Zn 2+ . 
     
     
         66 . A method for decomposing acetolactate comprising the step of treating a substrate with a composition comprising an ALDC enzyme and a metal ion, wherein the metal ion is present at a concentration of about 1 μM to about 200 mM in said composition; preferably said substrate is a carbohydrate containing substrate such as a wort or a fruit juice; preferably said substrate is a fermentation and/or maturation media. 
     
     
         67 . The method of  claim 66 , where the metal ion is present in said composition at a concentration of about 10 μM to about 150 mM, or about 20 μM to about 120 mM, or about 25 μM to about 100 mM, or about 25 μM to about 50 mM, or about 25 μM to about 20 mM, or about 100 μM to about 20 mM, or about 250 μM to about 20 mM, or about 1 mM to about 20 mM, or about 1 mM to about 5 mM. 
     
     
         68 . The method of  claim 66 , wherein the metal ion is present in said substrate at a concentration of:
 (i) about 1 μM to about 500 μM, or about 1 μM to about 300 μM, such as about 6 μM to about 300 μM, or   (ii) about 1 μM to about 100 μM or about 1 μM to about 50 μM, or   (iii) about 1 μM to about 25 μM, or about 6 μM to about 50 μM, or about 6 μM to about 25 μM, or about 25 μM to about 50 μM.   
     
     
         69 . The method of  claim 66 , wherein the metal ion and ALDC is added in a composition, and wherein
 (i) said metal ion is present in the composition at a concentration of about 1 mM to 5 mM; or   (ii) the molar ratio of said metal ion to ALDC enzyme in the composition is higher than 1; or 2:1 or higher; or 10:1 or higher; or 20:1 or higher; or 30:1 or higher; or 60:1 or higher.   
     
     
         70 . The method of  claim 69 , wherein the metal ion is selected from the group consisting of Mg 2+ , Mn 2+ , Co 2+ , Cu 2+ , Ca 2+ , Ba 2+ , and Fe 2+  and combinations thereof. 
     
     
         71 . The method of  claim 70 , wherein the metal ion is selected from the group consisting of Zn 2+ , Mn 2+ , and Co 2+ . 
     
     
         72 . The method of  claim 71 , wherein the metal ion is Zn 2+ . 
     
     
         73 . The method of  claim 72 , wherein the substrate is treated during a beer and/or wine and/or cider and/or perry and/or sake fermentation or maturation process. 
     
     
         74 . The method of  claim 72 , wherein the ALDC enzyme is from  Lactobacillus casei, Brevibacterium acetylicum, Lactococcus lactis, Leuconostoc lactis, Enterobacter aerogenes, Bacillus subtilis, Bacillus brevis, Lactococcus lactis  DX, or  Bacillus licheniformis.    
     
     
         75 . The method of  claim 72 , wherein the ALDC enzyme is from  Bacillus brevis  or  Bacillus licheniformis.    
     
     
         76 . The method of  claim 72 , wherein said ALDC enzyme has an amino acid sequence having at least 80% identity with any one selected from SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 8 or any functional fragment thereof.