CN1059168A - 乙酰乳酸脱羧酶衍生物及其应用 - Google Patents

乙酰乳酸脱羧酶衍生物及其应用 Download PDF

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CN1059168A
CN1059168A CN91105715A CN91105715A CN1059168A CN 1059168 A CN1059168 A CN 1059168A CN 91105715 A CN91105715 A CN 91105715A CN 91105715 A CN91105715 A CN 91105715A CN 1059168 A CN1059168 A CN 1059168A
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斯文·佩德森
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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Abstract

用限定比例的戊二醛处理含水介质中的所产生 的、且可在继后固相化的乙酰乳酸脱羧酶乙酰乳酸脱 羧酶衍生物。该用于啤酒发酵中的乙酰乳酸脱羧酶 衍生物可在低pH条件下显示出令人满意的稳定 性。

Description

本发明涉及乙酰乳酸脱羧酶（ALDC）衍生物及其应用。
在发酵含碳水化合物物质如麦芽汁或葡萄汁的过程中，可经过生醇发酵之外的途径生成多种付产物。其中一种不需要的付产物便是双乙酰。
欧洲专利EP46066号中述及，可用ALDC来防止双乙酰的生成。然而，天然存在之ALDC的最适pH均在pH6左右，而且在发酵麦芽汁的pH3.8-4.7，特别是以低麦芽含量发酵麦芽汁时低于4的常用pH条件下，该酶只能产生很低的活性，而不适于生产实践之目的。为此，这一现有技术并没有找到一种适于在任何大规模工业生产实践中防止发酵生产啤酒或葡萄酒时产生双乙酰的有效方法。欧洲专利46066号述及可以制备固相化ALDC。该专利还提到可参照Biochem.，Vol.11，No.22.PP.4022-4084（1972）中所述方法，对ALDC进行化学修饰，以使之在较低pH值时发挥最佳活性。它所引用的上述参考文献中提到的修饰方法包括在胰凝乳蛋白酶分子上加入聚（鸟氨酰）侧链;故该文献并没有提出修饰ALDC的方法。该现有技术提出的使酶在低pH值条件下产生最佳活性的修饰方法不适于工业应用，也不知道这种用于修饰胰凝乳蛋白酶的聚（鸟氨酰）方法是否适于ALDC。最后，上述方法也没有说明按现有技术修饰的凝乳蛋白酶是否在低pH条件下仍具有令人满意的稳定性。
因此，本发明的目的是提供一种可适用于工业生产，并可在低pH下表现有令人满意之稳定性的ALDC衍生物。
已令人惊异地发现，用戊二醛处理ALDC，所得修饰产物首先表现有期望的pH曲线，第二是易于制造且成本低，第三是有令人满意的稳定性。另外，如下文将进一步说明的，本发明的ALDC衍生物可以可溶性和固相化衍生物的形式存在。
因此，本发明的ALDC衍生物之特征在于是用浓度相当于每克纯ALDC蛋白质约0.1至5克、较好是相当于每克纯ALDC蛋白质0.25至2克的戊二醛处理含水介质中的ALDC。已经发现，如果不作进一步处理，这种ALDC衍生物是可溶性的，而且能够以高活性收率制得。
Biotechnology  Letters，Vol.10.N0.5，PP.325-330（1988）中描述了β-葡糖苷酶与戊二醛交联可提供一种改善了热稳定性的衍生物。Rolf  D.Schmid（Adv.Biochem.Eng.12，PP.41-118.1979）述及可交联木瓜蛋白酶、葡萄糖氧化酶、过氧化氢酶和尿酸酶等不同的酶，以提供具有更好热稳定性的酶衍生物，但其中没有交联ALDC。因此，现有技术并没有涉及用戊二醛作交联剂实现本发明的目的。
应明确的是，所有ALDC酶，即所有由微生物产生的ALDC，更好是由短芽孢杆菌（Bacillus  brevis）和地衣形芽孢杆菌（S.licheniformis）产生的ALDC均可用于本发明。
在一优选实施例中，本发明的ALDC衍生物特征在于上文中指出的ALDC衍生物是被固相化的。可按任何一种已知方法，如吸附法进行固相化，其优点包括可得到不含ALDC的啤酒。
欧洲专利EP131125号介绍了一种借助吸附或与戊二醛交联来固定酶（如ALDC）的特定无机载体。但该专利并没有描述改变pH即可导致ALDC之pH-活性曲线的问题，而这一点正是本发明的一个主要特征。该EP专利中没有述及的本发明的另一个特征是，先用有限的小浓度戊二醛处理ALDC，然后实现最佳固相化。
另外，本发明还涉及本发明的ALDC在啤酒发酵中的应用。
本发明的一个优选使用方案是将可溶ALDC衍生物与普通酵母一起用于批量发酵。这是使用本发明ALDC衍生物的一种最简单的方式。
本发明的另一个优选使用方案是将固相化ALDC衍生物与普通酵母一起用于批量发酵。其优点是终产品不存在ALDC。
本发明的再一个优选使用方案是将固相化ALDC衍生物与固相化酵母一起用于连续发酵中。其优点包括直接除去双乙酰前体物α-乙酰乳酸。
下面借助实施例进一步阐述本发明。这些实施例中使用的是培养枯草芽孢杆菌重组体菌株所产生的ALDC制剂;所说的菌株含有编码并表达短芽孢杆菌ALDC、具有DK149335B中所述性质的基因。
实施例1
使100mlALDC溶液（批号EDF212，其为发酵液离心物，ALDC活性为1700ADU/ml）与2.5ml2%戊二醛溶液（戊二醛终浓度0.05%（W/W，相当于每克ALDC加0.5g戊二醛）混合。用冰将反应混合物冷却3小时。pH被调至7.5并维持之。
将底物中pH值调到3.6至6.0，用Novo分析法（AF278/1-GB，有关资料可从Novo  Nordisk  A/S，Novo  Alle，DK-2880  Bagsvaerd得到）测定ALDC活性（ADU/ml）。所得pH-活性曲线如图1所示。
检测ALDC和其衍生样品在普通的、灭菌之丹麦啤酒（HOF）中的稳定性。向pH分别调到4.0和6.0的每毫升啤酒中加入20ADU。每天取啤酒样品检测pH6.0时残留的ALDC活性。结果示于图2和3中。
结论是用戊二醛进行处理可改善在pH4.0条件下ALDC的活性和稳定性。
实施例2
在传统的分批发酵中检测衍生的ALDC
使用ALDC（批号EDF212，1700ADU/ml）（制剂1）和按实施例1中所述方法制备的戊二醛处理的ALDC（1100ADU/ml）（制剂2）。
用啤酒厂麦芽汁（FAXE）和加入不同量（10、20、30和40%W/W）辅料（蔗糖）的同样麦芽汁进行发酵。每升麦芽汁加入2.5g酵母。ALDC连同酵母一起加入（每升46ADU）。发酵于12℃下进行7天。每天检测pH值（图4）、ALDC残留活性（图5）和总双乙酰（α-乙酰乳酸十双乙酰）（图6-9）。
结果表明，当使用衍生的ALDC（制剂2）时，总双乙酰的降低量比使用未处理的ALDC时更大，而且衍生的制剂更为稳定。
实施例3
将3ml按实施例1制得的戊二醛处理的ALDC（1100ADU/ml）加到1g载体（得自Grace的硅载体，Grade500MP，详见Biocatalyst  Supports  SG  SC  IE/June1987所述）。将pH调到4.5，并于5℃下将载体和ALDC溶液缓慢旋转搅拌20小时，然后真空过滤。向吸附了ALDC的颗粒内加入0.5ml聚氮杂环丁烷（Kymene，Hercules  BV）的3%溶液，并真空干燥所得混合物。pH6.0条件下固相化ALDC的活性为589ADU/g，pH3.8条件下为331ADU/g。

Claims (6)

1、ALDC衍生物，其中用浓度相当于每克纯ALDC蛋白质为0.1至5克、更好是相当于每克纯ALDC蛋白质为0.25至2克的戊二醛处理含水介质中的ALDC。
2、根据权利要求1的ALDC衍生物，其中戊二醛处理的ALDC衍生物是固相化的。
3、根据权利要求1至2的ALDC衍生物在啤酒发酵中的应用。
4、根据权利要求3的应用，其中将根据权利要求1的ALDC衍生物与普通酵母一同用于分批发酵中。
5、根据权利要求3的应用，其中将根据权利要求2的ALDC衍生物与普通酵母一起用于分批发酵中。
6、根据权利要求3的应用，其中根据权利要求2的ALDC衍生物与固相化酵母一起用于连续发酵中。
CN91105715A 1990-08-16 1991-08-16 乙酰乳酸脱羧酶衍生物及其应用 Expired - Fee Related CN1054638C (zh)

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CN107636151A (zh) * 2015-05-22 2018-01-26 杜邦营养生物科学有限公司 乙酰乳酸脱羧酶

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ES2596402T3 (es) 2010-01-19 2017-01-09 Basf Corporation Proteasas estabilizadas para su uso en el cuidado de la piel
JP5917166B2 (ja) * 2012-01-25 2016-05-11 アサヒビール株式会社 発酵麦芽飲料の製造方法
US20170121741A1 (en) 2014-04-01 2017-05-04 Dupont Nutrition Biosciences Aps Method for increasing crude palm oil yields
WO2016097266A1 (en) 2014-12-19 2016-06-23 Dupont Nutrition Biosciences Aps Method of improving palm oil yields from palm fruit or palm fruit liquid

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US4241185A (en) * 1979-02-23 1980-12-23 The Great Western Sugar Company Method of stabilizing α-galactosidase
DK145502C (da) * 1980-08-07 1983-05-02 Forenede Bryggerier As Fremgangsmaade til fremstilling af fermenterede alkoholiske produkter
DK252483D0 (da) * 1983-06-03 1983-06-03 Novo Industri As Alpha-acetolactate decarboxylaseemzymprodukt og fremstilling deraf
DK317483D0 (da) * 1983-07-08 1983-07-08 Superfos As Immobiliseret enzympraeparat og fremgangsmade til fremstilling deraf
JPH0614865B2 (ja) * 1985-12-13 1994-03-02 麒麟麦酒株式会社 α―アセト乳酸脱炭酸酵素をコードするＤＮＡ鎖およびこのＤＮＡ鎖により形質転換された酵母
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CN107636151A (zh) * 2015-05-22 2018-01-26 杜邦营养生物科学有限公司 乙酰乳酸脱羧酶
CN107636151B (zh) * 2015-05-22 2023-04-04 杜邦营养生物科学有限公司 乙酰乳酸脱羧酶

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FI930654A (fi) 1993-02-15
JP3126730B2 (ja) 2001-01-22
CN1113093C (zh) 2003-07-02
CN1054638C (zh) 2000-07-19
DK194990D0 (da) 1990-08-16
CA2089498A1 (en) 1992-02-17
FI930654A0 (fi) 1993-02-15
BR9106762A (pt) 1993-07-20
FI105696B (fi) 2000-09-29
DK0575323T3 (da) 1995-05-01
DE69106896T2 (de) 1995-06-14
ATE117368T1 (de) 1995-02-15
EP0575323B1 (en) 1995-01-18
CN1257913A (zh) 2000-06-28
JPH06500013A (ja) 1994-01-06
AU645343B2 (en) 1994-01-13
CA2089498C (en) 2002-07-16
GR3014933T3 (en) 1995-05-31
DE69106896D1 (de) 1995-03-02
MX9100626A (es) 1992-04-01
US5306634A (en) 1994-04-26
WO1992003543A1 (en) 1992-03-05
AU8390891A (en) 1992-03-17

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