CN106459938B - 固定脂肪酶的方法 - Google Patents
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Abstract
公开了一种将脂肪酶固定到含有氨基官能团的支持物上的方法，所述方法包括在表面活性材料的存在下使所述脂肪酶与所述支持物接触。
Description
技术领域
本发明涉及将脂肪酶固定到具有氨基官能团的支持物上的方法，采用所述固定的脂肪酶产生甘油三酯组合物的方法，以及脂肪酶在酯交换反应中的应用。
背景技术
脂肪酶(E.C.3.1.1.3)属于酶家族，特异性催化三-、二和单酰基甘油的酯键形成甘油和脂肪酸。它们还能催化诸如相互酯化、酯化、酸解、醇解和氨解的其他反应。脂肪酶的高成本导致酶促反应在经济上不具吸引力。脂肪酶的固定是提高脂肪酶工业敏感性的一种途径并且能够回收脂肪酶蛋白。采用各种途径预先处理支持物或者脂肪酶可以将脂肪酶固定在不同的支持物上。
Nevena等(NEVENA,Z.将来自皱褶念珠菌的脂肪酶固定到赛帕珠上：高碘酸氧化脂肪酶的作用(Immobilization of lipase from Candida rugosa on Sepabeads:theeffect of lipase oxidation by periodates).Bioprocess Biosyst Eng.2011,第34篇,第803-810页)描述了使用某些具有氨基官能团或者环氧基团的
作为固定来自皱褶念珠菌的非特异性脂肪酶的合适的支持物。具有氨基官能团的
需要用戊二醛或高碘酸活化以显示其改善的活性。
Palomo等(PALOMO,Jose M等，脂肪酶在高度疏水的支持物(十八烷基-赛帕珠)上的界面吸附：脂肪酶的开放形式的固定、过度活化和稳定，Journal of MolecularCatalysis B:Enzymatic.2002,第19期，第20篇，第279-286页)测试了各种脂肪酶在高度疏水支持物(例如十八烷基-
上的固定。
酶活性易受到固定试剂如戊二醛或固定支持物的影响。为了确保酶固定后的稳定性和活性，常常加入非脂肪酶蛋白如鸡蛋白蛋白或牛血清白蛋白。然而，已知这些动物蛋白质可导致过敏反应。
仍然需要简化的固定方法而不需要预先活化支持物，并且需要正确选择保留脂肪酶活性和稳定性以利于商业相关甘油三酯组合物的制备的支持物。
发明内容
本发明的目的是提供一种固定方法，其中可以避免支持物的预先活化，并且如果确实这样的话，疏水支持物如具有十八烷基的
(EC-OD)能够进行酯交换反应以获得商业上重要的产物。本发明的另一个目的是提供一种固定方法，所述方法避免用非脂肪酶蛋白如动物来源的白蛋白的处理以确保脂肪酶的活性和稳定性而制备甘油三酯组合物。
根据本发明，提供了一种在表面活性材料的存在下将脂肪酶固定到具有氨基官能团的支持物上的方法。术语氨基官能团指致力于与脂肪酶相互作用或结合的氨基基团。
本发明的另一方面是采用固定到具有氨基官能团的支持物上的脂肪酶，通过酶促酯交换制备甘油三酯的方法。
本发明的另一方面还提供了固定的脂肪酶在制备包含至少15％OPO的甘油三酯脂肪组合物中的应用。
具有氨基官能团的支持物可以是具有氨基官能团如氨基-环氧、或具有C1-C24，优选的是C2-C10碳链的烷基氨基的支持物。所述支持物包含甲基丙烯酸类聚合物。优选的是所述聚合物形成基质。
本发明优选的支持物含有烷基氨基官能团，如乙基氨基或己基氨基。
支持物与脂肪酶之间的作用机制或者是通过离子相互作用或者是化学结合，优选离子相互作用。
表面活性剂可以由糖(单糖、双糖或多糖)，多元醇(例如去水山梨糖醇和山梨醇)或者分子量为350到35000的聚乙二醇如PEG600、1500、4000形成。尤其合适的非离子表面活性剂是聚氧乙烯去水山梨糖醇C8-C24脂肪酸酯，具体是源自月桂酸的那些，例如吐温
或者源自油酸的那些，例如吐温
水性溶液中表面活性剂的浓度应足以确保支持物被酶有效负载。对水性溶液施用至少0.01重量％、优选0.01-10重量％、最优选0.1-5重量％的表面活性剂浓度可得到非常好的结果。
支持物(克)上脂肪酶的理想载量(克)为1-20重量％，优选5-15重量％。
施用的接触时间可以在宽范围内变化。然而，适当的接触时间为1-72小时。
水性脂肪酶溶液优选的浓度为1-20克/升。
虽然脂肪酶可以是任意现有技术的脂肪酶，但优选是选自以下的脂肪酶：1)来自米赫根毛霉(Rhizomucor miehei)、稻根霉菌(Rhizopus oryzae)和嗜热真菌(ThermomycesLanuginosus)的1,3-特异性脂肪酶，2)来自卡门柏青霉(Penicillium Camembertii)、对部分甘油三酯水解特异性的脂肪酶，如Amano G，和3)酯或甘油三酯水解特异性的脂肪酶，优选来自皱褶念珠菌(Candida rugosa)的脂肪酶。尤其优选来自稻根霉菌(Rhizopusoryzae)的1,3-特异性脂肪酶如来自Amano的脂肪酶D。
脂肪酶的固定可以以许多不同的方式进行。适当地，支持物、脂肪酶和/或表面活性剂之间的接触作为批量工艺，作为固定床中连续工艺，作为在流化床中或者在连续搅拌罐中连续工艺进行，用脂肪酶溶液的连续移动实现所述接触。
本发明固定的脂肪酶可以以任意酶转化工艺施加，例如甘油三酯、甘油二酯或酯的水解，以及脂肪酸或甘油二酯或甘油三酯的酯化或酯交换。这些工艺也是本发明的一部分，前提是工艺中使用本发明固定的脂肪酶。
制备甘油三酯的优选工艺是制备包含通式为ABA的对称甘油三酯例如OPO或SOS的甘油三酯组合物，其中O是油酸，P是棕榈酸，S是选自棕榈酸和硬脂酸的饱和脂肪酸。本发明尤其优选的甘油三酯组合物包含至少15％OPO。
甘油三酯脂肪和油是重要的商业产品，在食品工业等利于广泛使用。一些甘油三酯具有重要营养，甘油三酯1,3-二油酰-2-棕榈酰甘油酯(OPO)是母乳脂肪的重要组分。
实施例
以下非限制性实施例阐述了本发明，并不以任意方式限制本发明的范围。除非另有说明，在实施例和通篇说明书中，所有百分比、份数和比例是基于重量的。
实施例1：具有水性脂肪酶D的
的制备
制备脂肪酶溶液：根据表1制备七种脂肪酶溶液。样品N° 7是对照样品。所有试剂在室温下以150转/分钟混合3-24小时，然后离心收集固定的脂肪酶小丸。
表1
实施例2(比较)
采用以下酸解试剂，所有七种脂肪酶制剂在60℃进行酸解反应：
1g固定的酶(使用离心后的团块)
35g棕榈油硬脂馏分(原料)
49g油酸
0.126g H2O
组成原料参见表2
表2
| 原料 | |
| 碳数目 | |
| C48 | 62.1 |
| C50 | 24.3 |
| C52 | 8.6 |
| C54 | 1.9 |
| C56 | 0.0 |
根据AOCS Ce 5.86，通过GC确定碳数目。
表3提供了24小时后各种原料的酸解反应的结果。
表3
| HA | OD | BU | HFA | EA | EP | 对照 | |
| 碳数目 | |||||||
| C48 | 59.9 | 60.1 | 60.0 | 57.4 | 60.3 | 60.1 | 60.3 |
| C50 | 25.4 | 25.3 | 25.3 | 26.8 | 25.1 | 25.3 | 25.2 |
| C52 | 9.2 | 9.2 | 9.2 | 10.2 | 9.1 | 9.2 | 9.1 |
| C54 | 2.1 | 2.1 | 2.2 | 2.5 | 2.1 | 2.1 | 2.1 |
| C56 | 0.2 | 0.1 | 0.2 | 0.2 | 0.2 | 0.2 | 0.2 |
24小时后，所有脂肪酶制剂几乎没有产生产物OPO或OOP(C52)。
实施例3
将70ml实施例1的脂肪酶制剂与2.4g鸡蛋白蛋白、0.65g吐温
和1.5g各自的支持物混合。根据实施例2进行酸解反应。
表4显示了在鸡蛋白蛋白和吐温20的存在下，采用各种赛帕珠和水性脂肪酶D溶液的酸解结果(24小时)。
表4
| HA | OD | BU | HFA | EA | EP | |
| 碳数目 | ||||||
| C48 | 7.2 | 9.5 | 61.2 | 6.9 | 7.2 | 61.4 |
| C50 | 29.3 | 31.3 | 24.8 | 29.0 | 25.0 | 24.7 |
| C52 | 41.5 | 41.5 | 8.8 | 42.8 | 42.3 | 8.8 |
| C54 | 20.4 | 16.9 | 2.0 | 20.4 | 20.7 | 1.9 |
| C56 | 0.5 | 0.4 | 0.0 | 0.5 | 0.4 | 0.0 |
实施例4
将70ml实施例1的脂肪酶制剂与250mgPEG 1500、0.65g吐温
和1.5g各自的支持物混合。根据实施例2进行酸解反应。在相同的反应条件下在聚丙烯(Accurel)上进行固定作为比较。
表5显示了在PEG 1500和吐温
的存在下，采用各种赛帕珠和水性脂肪酶D溶液的酸解结果(24小时)。
表5
| HA | OD | HFA | EA | Accurel | ||
| 碳数目 | ||||||
| C48 | 6.7 | 61.9 | 6.8 | 6.8 | 58.2 | |
| C50 | 27.3 | 24.4 | 27.6 | 27.3 | 25.2 | |
| C52 | 42.4 | 8.5 | 42.5 | 42.4 | 10.0 | |
| C54 | 22.5 | 1.9 | 22.0 | 22.4 | 2.3 | |
| C56 | 0.2 | 0.0 | 0.5 | 0.4 | 4.1 |
实施例5
支持物EC-HA上固定脂肪酶D的多重用途
将70ml实施例1的脂肪酶制剂与30mg PEG 600、0.65g吐温
和1.5g支持物EC-HA混合。根据实施例2进行酸解反应。3.5小时之后，终止酸解反应，通过过滤从反应混合物分离固定的脂肪酶。收集固定的脂肪酶并用于第二轮的酸解分析。重复酸解八次。每一轮中，在时间3.5小时时取样(～2ml)进行碳数目分析。
表6显示了在连续的8轮中重复使用在EC-HA支持物上的固定的脂肪酶D的酸解结果。
表6
| 第1轮 | 第2轮 | 第3轮 | 第4轮 | 第5轮 | 第6轮 | 第7轮 | 第8轮 | |
| C46 | 0.8 | 1.2 | 1.3 | 1.5 | 1.4 | 1.5 | 1.5 | 1.6 |
| C48 | 11.2 | 19.6 | 22.7 | 25.3 | 24.7 | 26 | 27.7 | 28.5 |
| C50 | 34.1 | 35.9 | 36.4 | 35.9 | 35.8 | 35.6 | 35.3 | 35.2 |
| C52 | 42.4 | 34.2 | 31.3 | 29.4 | 30.1 | 29.1 | 28 | 27.4 |
| C54 | 11.2 | 9.1 | 8.2 | 7.8 | 8 | 7.8 | 7.6 | 7.3 |
实施例6
脂肪酶D(0.9g/77ml)溶液与表7所示用量的各种吐温混合，搅拌15分钟。在每种制剂中加入1.5g赛帕珠EC-HA，混合物搅拌24小时。然后，过滤去除固定的酶，根据实施例2所述在酸解反应中进行测试。表7显示了3.5小时后酸解后5种不同的吐温的结果。
表7
| 碳数目 | 吐温20 | 吐温40 | 吐温60 | 吐温80 | 吐温85 |
| 数量(克) | 0.650 | 0.676 | 0.693 | 0.694 | 0.974 |
| C46 | 1.5 | 2.14 | 2.79 | 1.34 | 2.4 |
| C48 | 16.6 | 41.19 | 54.37 | 18.02 | 46.5 |
| C50 | 34.2 | 31.4 | 27.49 | 33.26 | 30.5 |
| C52 | 36.9 | 18.73 | 11.85 | 36.11 | 15.8 |
| C54 | 10.6 | 5.78 | 3.48 | 10.77 | 4.5 |
| C56 | 0.3 | 0.53 | 0 | 0.38 | 0.3 |
实施例7
用吐温
重复实施例6，只是省去脂肪酶溶液与吐温
的预混。将脂肪酶溶液、吐温
和支持物材料混合，混合物搅拌24小时。然后，过滤除去固定的酶，根据实施例2所述在酸解反应中进行测试。表8显示了3.5小时后酸解后的结果。
表8
| 碳数目 | 预混 | 不预混 |
| C46 | 1.3 | 1.51 |
| C48 | 13.8 | 16.38 |
| C50 | 33 | 32.32 |
| C52 | 40.2 | 38.34 |
| C54 | 11.4 | 11.14 |
| C56 | 0.3 | 0.3 |
结果表明吐温
与脂肪酶溶液的预混并非必要。
Claims (4)
1.一种将1,3-特异性脂肪酶固定到含有氨基官能团的支持物上的方法，所述方法包括在水性溶液中的表面活性材料的存在下使所述脂肪酶与所述支持物接触，其中，所述表面活性材料是非离子表面活性剂，所述氨基官能团是具有C1-C22碳原子的烷基氨基官能团。
2.如权利要求1所述的方法，其特征在于，所述脂肪酶源自稻根霉菌。
3.如权利要求1或2所述的方法，其特征在于，所述表面活性材料选自：聚乙二醇、甲氧基聚乙二醇、聚山梨酯以及它们的混合物。
4.如前述权利要求中任一项所述的方法，其特征在于，所述水性溶液中表面活性材料的浓度为至少0.01重量％。
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| US5108916A (en) * | 1989-06-05 | 1992-04-28 | Rhone-Poulenc Rorer, S.A. | Process for stereoselectively hydrolyzing, transesterifying or esterifying with immobilized isozyme of lipase from candida rugosa |
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| CN101278047B (zh) * | 2005-09-30 | 2012-12-12 | 诺维信公司 | 酶的固定化 |
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| EP4242305A2 (en) | 2023-09-13 |
| DK3146044T3 (da) | 2019-09-02 |
| EP3502247A1 (en) | 2019-06-26 |
| WO2015177042A1 (en) | 2015-11-26 |
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