CN112639091A - 新型脂肪酶和其用途 - Google Patents
新型脂肪酶和其用途 Download PDFInfo
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- CN112639091A CN112639091A CN202080003765.4A CN202080003765A CN112639091A CN 112639091 A CN112639091 A CN 112639091A CN 202080003765 A CN202080003765 A CN 202080003765A CN 112639091 A CN112639091 A CN 112639091A
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- Prior art keywords
- lipase
- amino acid
- enzyme
- ala
- acid sequence
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Abstract
本发明的课题在于发现适于油脂加工的新型脂肪酶并谋求其利用·应用。本发明提供由与序列号1或2的氨基酸序列具有90%以上的同源性的氨基酸序列构成的新型脂肪酶和以该新型脂肪酶作为有效成分的酶制剂。该酶制剂被用于油脂的物性的改性·改良等。
Description
技术领域
本发明涉及显示酯交换能力的新型脂肪酶和其用途。本申请主张基于2019年8月1日提出申请的日本专利申请第2019-142145号的优先权,该专利申请的全部内容通过参照而被援引。
背景技术
油脂的酯交换反应是改良油脂的物性(熔点、结晶性、耐热性等)有效的方法,大致分为化学酯交换和酶酯交换这2种(例如参照非专利文献1、2)。在化学酯交换中,环境负荷高、作业安全性差等课题较多。近年来,从反式脂肪酸对健康风险的顾虑出发,作为成为反式脂肪酸的原因的部分氢化的替代,酯交换油脂的制造备受关注。酶酯交换可以利用来自假丝酵母属(Candida)、产碱杆菌属(Alcaligenes)、假单胞菌属(Pseudomonas)、嗜热真菌属、伯克氏菌属等的脂肪酶(例如参照专利文献1~4、非专利文献3、4)。
现有技术文献
专利文献
专利文献1:国际公开第2006/059592号小册子
专利文献2:日本专利第3791943号公报
专利文献3:日本特开2008-194011号公报
专利文献4:日本特表平2-504342号公报
非专利文献
非专利文献1:油化学会志,48,1151-1159(1999)
非专利文献2:Oleoscience,6,145-151(2006)
非专利文献3:J.Am.Oil Chem.Soc.,60,291-294(1983)
非专利文献4:J.Am.Oil Chem.Soc.,75,953-959(1998)
发明内容
在油脂加工中酶酯交换的需求高。然而,酯交换能力高且在反应中不易产生作为副反应的水解反应的酶尚未实用化。在酯交换反应中产生水解使油脂即甘油三酯的收率降低,生成对油脂的品质造成不良影响的部分甘油酯(甘油二酯、甘油单酯)和脂肪酸。本发明为了打破该现状,其课题在于发现在酯交换反应中不易产生作为副反应的水解反应的酶(酯交换脂肪酶),并谋求其利用·应用。
本发明人等为了发现适于油脂加工的酯交换脂肪酶,以各种来自微生物的脂肪酶作为对象进行了探索。其结果,发现酯交换能力优异的新型脂肪酶(本脂肪酶)。即,成功地取得·鉴定了产业上的有用性高的酯交换脂肪酶。进而,也成功地鉴定了该酶的氨基酸序列和编码该酶的基因的碱基序列。
基于上述的成果和考察,可提供以下的发明。
[1]一种脂肪酶,由与序列号1或2的氨基酸序列具有90%以上的同源性的氨基酸序列构成。
[2]根据[1]所述的脂肪酶,其中,所述脂肪酶的氨基酸序列为与序列号1或2所示的氨基酸序列显示93%以上的同源性的氨基酸序列。
[3]根据[1]所述的脂肪酶,其中,所述脂肪酶的氨基酸序列为与序列号1或2所示的氨基酸序列显示95%以上的同源性的氨基酸序列。
[4]一种酶制剂,以[1]~[3]中任一项所述的脂肪酶作为有效成分。
[5]一种脂肪酶的制造方法,包括以下的步骤(1)和(2):
(1)对生产[1]~[3]中任一项所述的脂肪酶的微生物进行培养的步骤;
(2)从培养后的培养液和/或菌体回收脂肪酶的步骤。
[6]一种油脂的制造方法,包括在受体底物和供体底物的存在下利用[1]~[3]中任一项所述的脂肪酶或[4]所述的酶制剂进行的酶反应。
[7]根据[6]所述的制造方法,其中,受体底物为油脂、甘油脂肪酸酯或甘油,
供体底物为脂肪酸、酯化合物或油脂。
附图说明
图1是酯交换活性的研究。将本脂肪酶的酯交换活性与各种脂肪酶(来自米根霉(Rhizopus oryzae)的脂肪酶(脂肪酶DF)、来自荧光假单胞菌(Pseudomonas fluorescens)的脂肪酶(脂肪酶AK)、来自洋葱伯克氏菌(Burkholderia cepacia)的脂肪酶(脂肪酶PS)、来自卡门柏青霉(Penicillium camembertii)的脂肪酶(脂肪酶G))进行比较。
图2是本脂肪酶的最适pH。
图3是本脂肪酶的pH稳定性。
图4是本脂肪酶的最适温度。
图5是本脂肪酶的温度稳定性。
具体实施方式
1.酯交换脂肪酶剂和其有效成分(酯交换脂肪酶)
本发明涉及对油脂的酯交换有用的酶制剂(酯交换脂肪酶剂)。本发明的酶制剂(以下,也称为“本酶制剂”)含有酯交换脂肪酶(以下,也称为“本酶”)作为有效成分。有效成分的酯交换脂肪酶、即本酶由序列号1或2所示的氨基酸序列或与该氨基酸序列等效的氨基酸序列构成。在此的“等效的氨基酸序列”是指与基准的氨基酸序列(序列号1的氨基酸序列或序列号2的氨基酸序列)一部分不同,但该不同不会对蛋白质的功能(在此为酯交换能力)造成实质性影响的氨基酸序列。因此,具有等效的氨基酸序列的酶催化酯交换反应。活性的程度只要能够发挥作为酯交换脂肪酶的功能就没有特别限制。但是,优选与由作为基准的氨基酸序列构成的酶同等程度或比其高。
序列号1的氨基酸序列为包含酯交换脂肪酶的信号肽序列的氨基酸序列,序列号2为从序列号1除去信号肽序列而得的氨基酸序列(成熟体的氨基酸序列)。
“氨基酸序列的一部分不同”例如通过构成氨基酸序列的氨基酸的中的1个或多个氨基酸的缺失、取代、对氨基酸序列的1个或多个氨基酸的附加、插入或者它们的任意组合而产生。氨基酸序列的一部分不同只要可保持酯交换活性就被允许(也可以活性稍微变动)。只要满足该条件,则氨基酸序列不同的位置没有特别限定。另外,氨基酸序列的不同可以在多个部位(场所)产生。
带来氨基酸序列的一部分不同的氨基酸的数量是相当于构成氨基酸序列的全部氨基酸的例如小于约30%的数量,优选为相当于小于约20%的数量,更优选为相当于约15%的数量,进一步优选为相当于小于约10%的数量,更进一步优选为相当于小于约7%的数量,再更进一步优选为相当于小于约5%的数量,进一步优选为相当于小于约3%的数量,更进一步优选为相当于小于约2%的数量,最优选为相当于小于约1%的数量。因此,等效蛋白质与基准的氨基酸序列具有例如约70%以上、优选约80%以上、更优选约85%以上、进一步优选约90%以上、更进一步优选约93%以上、再更进一步优选约95%以上、进一步优选约97%以上、更进一步优选约98%以上、最优选约99%以上的同源性。
“氨基酸序列的一部分不同”的典型例之一是通过构成氨基酸序列的氨基酸中的1~40个(优选1~30个、更优选1~10个、进一步优选1~7个、更进一步优选1~5个、更进一步优选1~3个)的氨基酸的缺失、取代、对氨基酸序列的1~40个(优选1~30个、更优选1~10个、进一步优选1~7个、更进一步优选1~5个、更进一步优选1~3个)的氨基酸的附加、插入或者它们的组合而在氨基酸序列产生变异(变化)。
优选通过在对酯交换能力非必需的氨基酸残基中产生保守性氨基酸取代而得到等效的氨基酸序列。在此的“保守性氨基酸取代”是指将某个氨基酸残基取代成具有同样性质的侧链的氨基酸残基。氨基酸残基根据其侧链而被分类为碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、无电荷极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)、芳香族侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)之类的几个家族。保守性氨基酸取代优选为相同家族内的氨基酸残基间的取代。应予说明,本发明的脂肪酶(序列号2)的活性残基被推测为87位氨基酸丝氨酸、264位氨基酸天冬氨酸、286位氨基酸组氨酸,因此,优选在该氨基酸残基以外的氨基酸残基中产生取代。
然而,两个氨基酸序列的同源性(%)例如可以通过以下的步骤来确定。首先,以能够进行最佳的比较的方式将两个序列进行排列(例如,可以在第一序列导入空位(gap)而使其与第二序列的比对最佳化)。第一序列的特定位置的分子(氨基酸残基)与第二序列的对应的位置的分子相同时,可以说该位置的分子相同。两个序列的同源性是该两个序列所共同的相同位置的数量的函数(即,同源性(%)=相同位置的数量/位置的总数×100),优选将比对的最佳化所需的空位的数量和尺寸也纳入考虑。
两个序列的比较和同源性的确定可以使用数学算法来实现。作为可用于序列比较的数学算法的具体例,有在Karlin和Altschul(1990)Proc.Natl.Acad.Sci.USA 87:2264-68中记载的、在Karlin和Altschul(1993)Proc.Natl.Acad.Sci.USA 90:5873-77中进行了改变的算法,但并不限定于此。这样的算法已被编入Altschul等(1990)J.Mol.Biol.215:403-10中记载的NBLAST程序和XBLAST程序(版本2.0)。为了得到与基准的氨基酸序列等效的氨基酸序列,例如只要在XBLAST程序中使score=50、wordlength=3进行BLAST多肽检索即可。为了得到用于比较的空位比对,可以利用Altschul等(1997)Amino Acids Research25(17):3389-3402中记载的Gapped BLAST。利用BLAST和Gapped BLAST时,可以使用对应的程序(例如XBLAST和NBLAST)的默认参数。详细而言,请参照http://www.ncbi.nlm.nih.gov。作为可用于序列比较的其它数学算法的例子,有Myers和Miller(1988)Comput Appl Biosci.4:11-17中记载的算法。这样的算法例如已被编入可在GENESTREAM网络服务器(IGH Montpellier,法国)或ISREC服务器中利用的ALIGN程序。在氨基酸序列的比较中利用ALIGN程序时,例如可以使用PAM120残基质量表,使空位长罚分=12,空位罚分=4。
可以使用GCG软件包的GAP程序,使用Blossom 62矩阵或PAM250矩阵,使空位权重=12、10、8、6或4,空位长权重=2、3或4来确定两个氨基酸序列的同源性。
作为本酶制剂的有效成分的酯交换脂肪酶、即本酶,可以为更大的蛋白质(例如融合蛋白质)的一部分。作为在融合蛋白质中附加的序列,例如可举出多重组氨酸残基这样的对纯化有用的序列、确保重组生产时的稳定性的附加序列等。
本酶可以通过对产生该酯交换脂肪酶的微生物(酯交换脂肪酶产生株)进行培养而得到。酯交换脂肪酶产生株可以为野生株,也可以为变异株(例如可通过紫外线照射而得到变异株)。酯交换脂肪酶产生株的具体例为乌汶伯克霍尔德氏菌DSM 17311株(本酶生产株)。该菌株为保存于莱布尼茨研究所DSMZ(German Collection Microorganisms andCell Cultures,德国微生物及细胞培养物)(Inhoffenstr.7B,D-38124Braunschweig,Germany)的菌株,可以通过经由规定的手续而获得。变异株可以通过紫外线、X射线、γ射线等的照射、利用亚硝酸、羟基胺、N-甲基-N’-硝基-N-亚硝基胍等的处理等而得到。变异株只要产生本酶就没有限定。作为变异株,可举出本酶的生产率得到提高的菌株、夹杂物的生产率得到降低的菌株、培养变得容易的菌株、从培养液的回收变得容易的菌株等。
根据本发明人等的研究,如下所述确定了本发明的脂肪酶的各种性质(详细情况参照后述的实施例)。因此,本酶除其来源以外,还可以通过以下的酶化学性质来特定。应予说明,对各酶化学性质进行评价时的脂肪酶活性的测定只要使用市售的试剂盒即可(详细的测定条件、测定步骤示于后述的实施例)。
(1)作用
本酶为脂肪酶,催化酯交换反应。
(2)最适pH
本酶的最适pH约为8。最适pH例如可基于对于pH3~8的pH区域而言在麦吉尔文缓冲液(McIlvaine buffer)中测定、对于pH9~10的pH区域而言在0.1M Tris-盐酸缓冲液(Tris-HCl)中测定而得到的结果进行判断。
(3)pH稳定性
本酶在pH3~10的pH区域中显示稳定的活性。例如,如果供于处理的酶溶液的pH为该范围内,则在30℃、1小时的处理后,显示最大活性的90%以上的活性。pH稳定性例如可基于对于pH3~8的pH区域而言在麦吉尔文缓冲液(McIlvaine buffer)中测定、对于pH9~10的pH区域而言在0.1M Tris-盐酸缓冲液(Tris-HCl)中测定而得到的结果进行判断。
(4)最适温度
本酶的最适温度为40℃~70℃。
(5)温度稳定性
本酶即使在纯化水中、在20℃~70℃的条件下进行1小时处理,也维持90%以上的活性。
作为本酶制剂的有效成分的本酶催化酯交换反应。本发明中的酯交换反应是指接近于油脂中所含的脂肪酸部位非特异性结合于甘油的1位、2位、3位的羟基的状态的酯交换反应,也被称为部位非特异性酯交换反应。另一方面,将对1位和3位的羟基特异性的酯交换反应称为1位、3位特异性酯交换反应。酯交换活性的测定或评价可以通过后述的实施例所示的方法(以纯化棕榈油作为底物的酯交换活性测定方法、以可可油(可可脂)作为底物的酯交换活性评价方法)进行。在以纯化棕榈油(脂肪酸组成PPP:约8%、POP+PPO:约41%、POO:约41%、OOO:约10%)作为底物的评价方法中,纯化棕榈油含有油酸(O)和棕榈酸(P)作为构成脂肪酸,甘油三酯的脂肪酸组成的比率大致是PPP:约8%、POP+PPO:约41%、POO:约41%、OOO:约10%,POP+PPO中约90%为POP,因此,三棕榈酸甘油酯(PPP)主要通过酯交换反应而增加。因此,可以以三棕榈酸甘油酯增加量作为指标来评价酯交换活性。另一方面,可可油含有油酸、棕榈酸、硬脂酸等作为构成脂肪酸,主要以在2位结合有油酸的甘油三酯作为主成分。因此,棕榈酸结合于1位、2位、3位全部的三棕榈酸甘油酯(PPP)仅通过酯交换反应(部位非特异性酯交换反应)而产生(在需要将1位或3位的棕榈酸与2位的油酸进行替换,用1位、3位特异性的脂肪酶进行反应时,不产生在2位插入有棕榈酸的甘油三酯)。因此,在以可可油作为底物的评价方法中,可以以三棕榈酸甘油酯生成量作为指标来评价酯交换活性。酯交换能力的评价也可以以单位重量的酯交换活性计算而进行评价,但通过计算单位酶活性的酯交换活性,能够适当地评价酯交换能力(部位非特异性酯交换反应性)的优劣。
如后述的实施例所示,作为本酶制剂的有效成分的本酶的代表例(由序列号1或2的氨基酸序列构成的酯交换脂肪酶),酯交换反应快且反应效率也优异。进而,与油脂反应时的甘油二酯的生成少,酯交换反应优先于水解反应。因此,如果将本酶制剂用于油脂、油脂加工品等的物性的改性·改良(详细内容后述),则可实现处理时间的缩短化,此外,通过部位非特异性的酯交换,可期待由变为与处理前不同的脂肪酸组成(特别是2位的脂肪酸不同的组成)的油脂所带来的品质的提高(期望物性的获得)以及甘油三酯的收率提高、对油脂的品质造成不良影响的甘油二酯、脂肪酸的生成的抑制。
本酶制剂中的有效成分(本酶)的含量没有特别限定,例如可以以每1g本酶制剂的酶活性成为1U~1000U、优选10U~300U的方式设定或调整有效成分的含量。本发明的酶制剂通常以固体状(例如,颗粒、粉体或者在二氧化硅、硅藻土以及多孔质聚合物等可于载体表面或内部固定酶的原材料中固定化有该酶的固定化酶)或液体状提供。本酶制剂除有效成分(本酶)以外,还可以含有赋形剂、缓冲剂、悬浮剂、稳定剂、保存剂、防腐剂、生理盐水等。作为赋形剂,可以使用乳糖、山梨糖醇、D-甘露醇、麦芽糊精、白糖等。作为缓冲剂,可以使用磷酸盐、柠檬酸盐、乙酸盐等。作为稳定剂,可以使用丙二醇、抗坏血酸等。作为保存剂,可以使用苯酚、苯扎氯铵、苄醇、氯丁醇、对羟基苯甲酸甲酯等。作为防腐剂,可以使用苯扎氯铵、对羟基苯甲酸、氯丁醇等。
2.本酶的制造方法
本发明的另一方面提供本酶的制造方法。在本发明的制造方法中,进行对产生本酶的微生物进行培养的步骤(步骤(1))以及从培养后的培养液和/或菌体回收脂肪酶的步骤(步骤(2))。
培养条件、培养法只要可生产本酶就没有特别限定。即,可以以生产本酶作为条件来适当设定适于使用的微生物的培养的方法、培养条件。作为培养法,可以为液体培养、固体培养中的任一者,优选利用液体培养。以液体培养为例对其培养条件进行说明。
作为培养基,只要是使用的微生物可生长的培养基就没有特别限定。例如可以使用添加有葡萄糖、蔗糖、龙胆二糖、可溶性淀粉、甘油、糊精、糖蜜、有机酸等碳源,进一步添加有硫酸铵、碳酸铵、磷酸铵、乙酸铵、或者蛋白胨、酵母提取物、玉米浆、酪蛋白水解物、麦糠、肉提取物等氮源,进一步添加有钾盐、镁盐、钠盐、磷酸盐、锰盐、铁盐、锌盐等无机盐的培养基。为了促进使用的微生物的生长,可以在培养基中添加维生素、氨基酸等。培养基的pH例如调整为约3~8,优选调整为约4~7左右,培养温度通常为约20~40℃,优选为约25~35℃左右,在需氧条件下培养1~20天、优选3~10天左右。作为培养方法,例如可利用振荡培养法、利用发酵罐的需氧深层培养法。
在以上的条件下培养后,从培养液或菌体回收目标酶(步骤(2))。从培养液回收时,例如通过对培养上清液进行过滤、离心处理等而除去不溶物后,适当地组合利用超滤膜的浓缩、硫酸铵沉淀等盐析、透析、离子交换树脂等各种层析等而进行分离、纯化,由此能够得到本酶。另一方面,从菌体内回收时,例如通过加压处理、超声波处理等将菌体破碎后,与上述同样地进行分离、纯化,由此能够得到本酶。应予说明,也可以通过过滤、离心处理等预先从培养液中回收菌体后,进行上述一系列的工序(菌体的破碎、分离、纯化)。
本酶也可以通过基因工程学方法而容易地制备。例如可以通过用编码本酶的DNA(作为一个例子,将编码由序列号1的氨基酸序列构成的本酶的DNA序列示于序列号3,将编码由序列号2的氨基酸序列构成的本酶的DNA序列示于序列号4)转化适当的宿主细胞(例如大肠杆菌),回收转化体内表达的蛋白质而制备。所回收的蛋白质可以根据目的而适当纯化。如果如此以重组蛋白质的形式得到本酶,则可以进行各种修饰。例如,如果将编码本酶的DNA和其它适当的DNA插入到相同的载体并使用该载体进行重组蛋白质的生产,则能够得到由连接有任意的肽或蛋白质的重组蛋白质构成的本酶。另外,可以实施糖链和/或脂质的附加、或者产生N末端或C末端的处理这样的修饰。通过如上所述的修饰,能够简化重组蛋白质的提取、纯化,或者附加生物学功能等。
通常,如上所述利用适当的宿主-载体系统而进行基因的表达~表达产物(本酶)的回收,但也可以利用无细胞合成系统。在此,“无细胞合成系统(无细胞转录系统、无细胞转录/翻译系统)”是指不使用活细胞,而是使用来自活细胞的(或者通过基因工程学方法得到的)核糖体、转录·翻译因子等,从作为模板的核酸(DNA、mRNA)体外合成其所编码的mRNA、蛋白质。在无细胞合成系统中,一般使用将细胞破碎液根据需要进行纯化而得到的细胞提取液。在细胞提取液中一般含有蛋白质合成所需的核糖体、起始因子等各种因子、tRNA等各种酶。在进行蛋白质的合成时,在该细胞提取液中添加各种氨基酸、ATP、GTP等能量源、肌酸磷酸等蛋白质的合成所需的其它物质。当然,在合成蛋白质时,也可以根据需要补充另行准备的核糖体、各种因子和/或各种酶等。
也报告了重构蛋白质合成所需的各分子(因子)的转录/翻译系统的开发(Shimizu,Y.et al.:Nature Biotech.,19,751-755,2001)。在该合成系统中,将由构成细菌的蛋白质合成系统的3种起始因子、3种伸长因子、参与终止的4种因子、使各氨基酸与tRNA结合的20种氨基酸酰基tRNA合成酶和甲硫氨酰tRNA甲酰转移酶组成的31种因子的基因从大肠菌基因组进行扩增,使用它们在体外重构蛋白质合成系统。在本发明中也可以利用这样的重构的合成系统。
术语“无细胞转录/翻译系统”可与无细胞蛋白质合成系统、体外翻译系统或体外转录/翻译系统交换使用。在体外翻译系统中,将RNA作为模板使用而合成蛋白质。作为模板RNA,可使用全RNA、mRNA、体外转录产物等。在其它的体外转录/翻译系统中,使用DNA作为模板。模板DNA应含有核糖体结合区域,另外优选含有适当的终止子序列。应予说明,在体外转录/翻译系统中,可设定添加各反应所需的因子以使得转录反应和翻译反应连续进行的条件。
也可以将如上所述得到的纯化酶通过例如冷冻干燥、真空干燥或者喷雾干燥等制成粉末而提供。此时,可以预先将纯化酶溶解于乙酸缓冲液、磷酸缓冲液、三乙醇胺缓冲液、tris-盐酸缓冲液、GOOD的缓冲液。优选可以使用乙酸缓冲液、磷酸缓冲液、三乙醇胺缓冲液。应予说明,在此,作为GOOD的缓冲液,可举出PIPES、MES或MOPS。
酶的纯化度没有特别限定,例如可以纯化至水解活性的比活性为1~100000(U/μg)、优选比活性为10~20000(U/μg)的状态。另外,最终的形态可以为液体状,也可以为固体状(包含粉体状)。
3.本酶制剂/本酶的用途(油脂的酯交换法)
本发明的再一方面涉及本酶制剂/本酶的用途,提供使用本酶制剂或本酶的油脂的酯交换法。在本发明的酯交换法中,进行使本酶制剂或本酶作用于油脂的步骤,通过该步骤进行油脂的酯交换,即,使油脂中的三酰化甘油(简称为TG或TAG)的构成脂肪酸重组(重排)。
作为可利用本发明的酯交换法处理的油脂,可例示大豆油、菜籽油、米油、玉米油、葵花籽油、棉籽油、花生油、红花油、棕榈油、软质棕榈油、棕榈分馏油、棕榈核油、椰子油、可可脂等植物油脂、鱼油、猪油、牛油、乳脂等动物油脂和它们的分馏油、氢化油、甘油三月桂酸酯、甘油三油酸酯、三棕榈酸甘油酯等合成油脂。本发明的酯交换法除油脂彼此的酯交换以外,还可以用于油脂与脂肪酸或脂肪酸酯之间的酯交换。脂肪酸的例子为硬脂酸、棕榈酸、月桂酸、花生酸、山嵛酸、油酸、亚油酸,脂肪酸酯的例子为硬脂酸乙酯、棕榈酸乙酯、油酸乙酯、亚油酸乙酯。
在本发明的酯交换法中,将本酶制剂或本酶添加于油脂,例如在30~100℃,优选35~80℃的条件下反应规定时间(例如1小时~48小时)。为了促进反应,反应中可以搅拌。
也可以将本酶制剂或本酶供于固定化处理,进行利用固定化酶的反应。在利用固定化酶的反应中,可利用间歇式搅拌槽型反应器、流通式搅拌槽型反应器、填充层型反应器、流动层型反应器等。
本发明的酯交换法对油脂或油脂加工品(例如起酥油、人造黄油)的物性的改性·改良有用。例如出于铺展性的提高、乳液稳定性的提高、固体脂肪含有值(SFC)的优化、固化性的提高、特定脂肪酸的选择性浓缩、低反式酸油脂或低反式酸油脂加工品的制造等目的,可以应用本发明的酯交换法。应用本发明的酯交换法而得到的油脂或含有其的油脂加工品与处理前相比,在物性上确认到改善,产业上的利用价值高。
如以上的说明可知,根据本发明的酯交换法,能够制造酯交换油脂。即本发明也提供酯交换油脂的制造方法。典型而言,在本发明的酯交换油脂的制造方法中,准备受体底物(油脂(甘油三酯)、甘油脂肪酸酯(甘油二酯、甘油单酯)、甘油)和供体底物(脂肪酸、酯化合物(脂肪酸酯等)或油脂(可以与受体底物相同)),使本酶制剂或本酶作用(即,进行在受体底物和供体底物的存在下的酶反应)。受体底物、供体底物中使用的油脂、脂肪酸、脂肪酸酯可以使用上述的油脂、脂肪酸、脂肪酸酯。酶反应的条件可以采用上述的条件(例如在30~100℃,优选35~80℃的条件下反应规定时间(例如1小时~48小时))。
实施例
1.本脂肪酶的制备
在杀菌后的预培养用培养基中接种本酶生产株,在30℃进行3天培养,得到预培养液。
<预培养用培养基>
酵母提取物(Becton·Dickinson株式会社制)0.5%
细菌蛋白胨(Becton·Dickinson株式会社制)1.0%
氯化钠(富士胶片和光纯药株式会社制)0.5%
pH 7.2
在蒸气杀菌后的主培养用培养基中添加预培养液,在30℃进行3天振荡培养,取得培养液。
<主培养用培养基>
大豆油2.0%
蛋白胨0.5%(Difco公司制)
肉提取物0.3%(Difco公司制)
磷酸二氢钾0.1%
硫酸镁7水合物0.02%
硫酸铁(II)7水合物0.001%
Adekanol LG-126(株式会社ADEKA制)0.2%
将得到的培养液进行离心分离,回收培养上清液。将培养上清液供于硅藻土过滤,得到澄清液。将澄清液通过超滤进行浓缩脱盐,将得到的脱盐液进行冷冻干燥,由此制成粗制酶粉末(本脂肪酶)。利用SDS-PAGE确认本脂肪酶的分子量,结果可知为约33kDa。
来自米根霉(Rhizopus oryzae)的脂肪酶(脂肪酶DF)、来自荧光假单胞菌(Pseudomonas fluorescens)的脂肪酶(脂肪酶AK)、来自洋葱伯克氏菌(Burkholderiacepacia)的脂肪酶(脂肪酶PS)、来自卡门柏青霉(Penicillium camembertii)的脂肪酶(脂肪酶G)分别从天野酶株式会社获得。
2.单位水解活性的酯交换活性的研究
(1)评价方法
即将使用前将纯化棕榈油(有限会社imagine公司,脂肪酸组成PPP:约8%,POP:约41%,POO:约41%,OOO:约10%)在60℃下溶解。在带螺旋盖的100mL容积三角烧瓶中称量使用的酶0.2g。加入溶解完毕的纯化棕榈油20mL。将加入的时刻设为t=0。在60℃下以160rpm振荡。在开始30分钟后、60分钟后、120分钟后进行取样(10μL),溶解于1mL己烷中。将通过了0.45μm过滤器的样品供于气相色谱(GC)分析。测定三棕榈酸甘油酯(PPP)的生成量。计算PPP量(PPP)相对于纯化棕榈油的三酰化甘油的总量(Total TAG)的比率(%),将反应前与反应后的差设为PPP增加量(%)。通过用将脂肪酶BU的水解活性设为1时的各酶的水解活性的比率乘以各酶的PPP增加量而得的数值进行比较,从而评价单位水解活性的酯交换活性。水解活性使用脂肪酶试剂盒S测定。在显色原液250μL中加入试剂盒附带的缓冲液250μL和纯化水2000μL而制成显色液。在试管中加入显色液1mL、将5mg/mL酶液稀释成适当的浓度的稀释液50μL和酯酶抑制剂20μL,在30℃保温5分钟后,添加底物液100μL,进一步在30℃反应30分后,放入反应停止液2mL。将反应停止后的样品全部移至15mL管中,以8000g离心分离1分钟后,回收上清液,在412nm处测定吸光度。将反应停止液添加后放入酶液、酯酶抑制剂而得的液体作为空白,由以下的计算式计算。
U/mg=(A412样品-A412空白)×1/0.05×1/5×n)
(其中,A412样品为样品的412nm的吸光度,A412空白为空白的412nm的吸光度,0.05为样品添加量(mL),5为酶液的浓度(5mg/mL),n为酶液的稀释倍数)
GC分析条件
柱:DB-1ht(5m)
检测器:FID
炉温:初期120℃保持1分钟→升温25℃/min(到150℃为止)→升温30℃/min(到260℃为止)→升温20℃/min(到370℃为止)→在370℃保持3分钟
气化室温度:370℃
检测器温度:370℃
注入量:1μL
柱入口压:100kpa(仅注射时)
载气:He
分流比:1:50
(2)结果
可知本脂肪酶与其它脂肪酶相比,单位水解活性的酯交换活性高(图1、表1)。
[表1]
评价结果(PPP增加量(%)/水解活性的比较)的汇总
3.单位酶活性的酯交换活性的研究
(1)酶活性评价方法(GC)
将甘油三油酸酯(东京化成工业)2.5mL添加于螺口瓶中。测量并添加一定量的各脂肪酶粉末,在60℃油浴中一边搅拌一边预热。添加棕榈酸甲酯(东京化成工业)3.0mL,在60℃开始反应。经时实施取样(30μL样品/1.0mL己烷),通过气相色谱(GC)测定所生成的油酸甲酯的量。将在本反应条件下在1分钟内生成1μmol的油酸甲酯的酶量设为1U,通过以下的计算式计算。
酶活性(U/g)=A/a×33×(1/T)×(1/G)×5.5
(A:油酸甲酯的区域面积,a:标准曲线的斜率,1/T:换算为反应时间1min、1/G:换算为试样1g)
GC分析条件
柱:DB-1HT(长度:5m、膜厚:0.1μm、内径:0.25mm)
检测器:FID
炉温:初期50℃、升温40℃/min、最终370℃
气化室温度:370℃
检测器温度:370℃
注入量:1μL
柱入口压:100kPa
载气:He
分流比:1∶50
(2)酯交换活性评价方法(GC)
将酶活性3U的酶液冷冻干燥,添加可可油3mL和超纯水6μL,一边搅拌一边在60℃下开始反应。在反应1天后和4天后实施取样,通过GC测定作为指标的三棕榈酸甘油酯(PPP)的比例。
GC分析条件
柱:DB-1HT(长度:5m、膜厚:0.1μm、内径:0.25mm)
检测器:FID
柱温:在120℃下4分钟→升温至150℃(20℃/分钟)→升温至315℃(30℃/分钟)→在315℃下3分钟→升温至325℃(1.5℃/分钟)→升温至370℃(30℃/分钟)→在370℃下2分钟
载气:He
分流比:50∶1
分析:由峰总面积计算PPP的比例
(3)结果
可知本脂肪酶与其它脂肪酶相比,单位酶活性的酯交换活性高(表2)。
[表2]
各样品的酶添加量和PPP上升量
4.油脂组成变化的研究
(1)评价方法
在即将使用前将纯化棕榈油(有限会社imagine公司,脂肪酸组成PPP:8%、POP:41%、POO:41%、OOO:10%)在60℃下溶解。在带螺旋盖的100mL容积三角烧瓶中称量使用的酶1g。加入溶解完毕的纯化棕榈油20mL。将加入的时刻设为t=0。在60℃下以160rpm振荡。在开始120分钟后取样(10μL),溶解于1mL己烷中。将通过了0.45μm过滤器的样品供于气相色谱(GC)分析,测定甘油三酯(TG)、甘油二酯(DG)、甘油单酯(MG)、游离脂肪酸(FFA)的比率(%),确认油脂组成的变化。应予说明,在该评价中,使用依据常规方法将本脂肪酶固定化于硅藻土的本脂肪酶(固定化),与市售的来自疏绵状嗜热丝孢菌的固定化脂肪酶(比较品1)进行比较。
GC分析条件
柱:DB-1ht(5m)
检测器:FID
炉温:初期在120℃下保持1分钟→升温25℃/min(到150℃为止)→升温30℃/min(到260℃为止)→升温20℃/min(到370℃为止)→在370℃下保持3分钟
气化室温度:370℃
检测器温度:370℃
注入量:1μL
柱入口压:100kpa(仅注射时)
载气:He
分流比:1:50
(2)结果
可知本脂肪酶(固定化)与比较品1相比,甘油三酯的比率高,作为副产物的部分甘油酯(甘油二酯、甘油单酯)、游离脂肪酸的生成少,产业上有用(表3)。
[表3]
酶 | TG | DG | MG | FFA |
比较品1 | 86 | 9 | 1 | 4 |
本脂肪酶(固定化) | 93 | 6 | 0 | 1 |
油脂组成(甘油三酯(TG)、甘油二酯(DG)、
甘油单酯(MG)、游离脂肪酸(FFA)的比率(%))的比较
作为进一步的研究,使用本脂肪酶(固定化)以及比较对象的固定化酶2种,在油脂中进行反应。比较对象使用市售的来自疏绵状嗜热丝孢菌的固定化脂肪酶(比较品1)和市售的来自米黑根毛霉的固定化脂肪酶(比较品2)。反应体系依据上述(1)中记载的方法,设置对纯化棕榈油20mL添加各酶2.0g的试验区和未添加的试验区。在反应开始1小时的时刻分取样品,在上述GC分析条件中记载的条件下进行分析。
分析的结果,本脂肪酶与比较对象品相比,由于DG、FFA的生成能力低,因此,推测与水解反应相比主要发生酯交换反应。进而,本脂肪酶与比较对象的酶相比,TG中的三棕榈酸甘油酯(PPP)比率大幅增加,因此,可以说将POP、POO、OOO转换为PPP的能力优异,产业上有用(表4)。
[表4]
油脂组成(%):将甘油三酯(TG)、甘油二酯(DG)、甘油
单酯(MG)、游离脂肪酸(FFA)的总量设为100%时的TG、DG、
MG、FFA、PPP的存在比率(%)
TG中的PPP比率的增加量(%):甘油三酯(TG)中的三棕
榈酸甘油酯(PPP)的比率(%)相对于不添加酶的增加量((酶
添加时的TG中的PPP比率)-(不添加酶时的TG中的PPP比率))
5.本脂肪酶的酶学性质的确认
5-1.最适pH
(1)方法
制备麦吉尔文缓冲液(pH3、4、5、6、7、8)和0.1mol/L Tris-HCl(p9、9.5)。使用脂肪酶试剂盒S分别测定各pH下的活性。在显色原液250μL中加入使用的缓冲液2250μL而制成显色液。在试管中添加显色液1mL、稀释成适当的浓度的酶液50μL和酯酶抑制剂20μL,在30℃下保温5分钟后,添加底物液100μL,进一步在30℃下反应30分钟后,放入反应停止液2mL。将反应停止后的样品全部移至15mL管中,以8000g进行1分钟离心分离后,回收上清液,在412nm处测定吸光度。以将反应液的pH实测值为pH 7.9(使用麦吉尔文缓冲液pH8.0的样品)的样品的活性设为100%时的相对活性进行评价。应予说明,使用在反应停止液添加后放入酶液、酯酶抑制剂而得的液体作为空白。
(2)结果
可知本脂肪酶的最适pH为约8(图2)。
5-2.pH稳定性
(1)方法
制备麦吉尔文缓冲液(pH3、4、5、6、7、8)和0.1mol/L Tris-HCl(pH9、10)。使用上述缓冲液制备15mg/mL酶液,在30℃下进行1小时处理后,加入等量的脂肪酶试剂盒S附带的缓冲液进行混合,制成处理样品。使用脂肪酶试剂盒S测定处理样品的活性。在试管中添加显色液1mL、稀释成适当的浓度的处理样品50μL和酯酶抑制剂20μL,在30℃下保温5分钟后,添加底物液100μL,进一步在30℃下反应30分钟后,放入反应停止液2mL。将反应停止后的样品全部移至15mL管中,以8000g进行1分钟离心分离后,回收上清液,在412nm处测定吸光度。以将在30℃下进行1小时处理的酶液的pH实测值为pH 3.1(使用麦吉尔文缓冲液pH 3.0的样品)的样品的活性设为100%时的相对活性进行评价。应予说明,使用在反应停止液添加后放入酶液、酯酶抑制剂而得的液体作为空白。
(2)结果
可知本脂肪酶在pH3~10下稳定(图3)。
5-3.最适温度
(1)方法
使用脂肪酶试剂盒S测定各温度下的活性。在试管中添加显色液1mL、稀释成适当的浓度的酶液50μL和酯酶抑制剂20μL,在各温度(20、30、40、50、60、70℃)下保温5分钟后,添加底物液100μL,进一步反应30分钟后,放入反应停止液2mL。将反应停止后的样品全部移至15mL管中,以8000g进行1分钟离心分离后,回收上清液,在412nm处测定吸光度。以将40℃下的活性设为100%时的相对活性进行评价。应予说明,使用在反应停止液添加后放入酶液、酯酶抑制剂而得的液体作为空白。
(2)结果
可知本脂肪酶的最适温度为40~70℃(图4)。
5-4.温度稳定性
(1)方法
将酶粉末以成为5mg/mL的方式溶解于纯化水后,在各温度下进行1小时处理。使用脂肪酶试剂盒S测定处理样品的活性。在试管中添加显色液1mL、稀释成适当的浓度的处理样品50μL和酯酶抑制剂20μL,在30℃下保温5分钟后,添加底物液100μL,进一步在30℃下反应30分钟后,放入反应停止液2mL。将反应停止后的样品全部移至15mL管,以8000g进行1分钟离心分离后,回收上清液,在412nm处测定吸光度。以将在30℃下进行1小时处理的样品的活性设为100%时的相对活性进行评价。应予说明,使用在反应停止液添加后放入酶液、酯酶抑制剂而得的液体作为空白。
(2)结果
可知本脂肪酶在20~70℃下稳定(图5)。
6.序列信息的确认
将通过基因组解析而提取的本脂肪酶的氨基酸序列和编码其的碱基序列示于序列表。应予说明,序列号与各序列的对应关系如下。
序列号1:氨基酸序列(包含信号肽)
序列号2:氨基酸序列(成熟体)
序列号3:碱基序列(包含信号序列)
序列号4:碱基序列(成熟体)
产业上的可利用性
本发明的酶制剂特别适于食品用途中的使用。例如,被用于人造黄油、起酥油等食用油脂的改性。另外,也可期待用于各种酯交换油脂的制造。
本发明不受上述发明的实施方式和实施例的说明任何限定。在不脱离权利要求书的记载的情况下,在本领域技术人员能够容易地想到的范围内进行的各种变形方式也包含在本发明中。本说明书中明示的论文、公开专利公报和专利公报等的内容通过援引而引用其全部内容。
序列表
<110> 天野酶制品株式会社
<120> 新型脂肪酶和其用途
<130> 20069WO
<150> JP P2019-142145
<151> 2019-08-01
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 364
<212> PRT
<213> 乌汶伯克霍尔德氏菌
<400> 1
Met Ala Arg Ser Met Arg Ser Arg Val Val Ala Gly Ala Val Ala Cys
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Ala Met Ser Ala Ala Pro Phe Ala Gly Met Thr Ala Leu Ala Thr Val
20 25 30
Ala Thr Thr Arg Ala Ala Val Ala Ala Thr Ala Pro Ala Asp Asp Tyr
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Ala Thr Thr Arg Tyr Pro Ile Ile Leu Val His Gly Leu Thr Gly Thr
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Asp Lys Tyr Ala Gly Val Leu Asp Tyr Phe Tyr Gly Ile Gln Gln Asp
65 70 75 80
Leu Gln Gln His Gly Ala Thr Val Tyr Val Ala Asn Leu Ser Gly Tyr
85 90 95
Gln Ser Asp Asp Gly Pro Asn Gly Arg Gly Glu Gln Leu Leu Ala Gln
100 105 110
Val Lys Gln Val Leu Ala Gln Thr Gly Ala Ala Lys Val Asn Leu Ile
115 120 125
Gly His Ser Gln Gly Gly Leu Ser Ser Arg Tyr Val Ala Ala Val Ala
130 135 140
Pro Glu Leu Val Ala Ser Val Thr Thr Ile Gly Thr Pro His Arg Gly
145 150 155 160
Ser Glu Phe Ala Asp Phe Val Gln Gly Val Leu Ala Tyr Asp Pro Thr
165 170 175
Gly Leu Ser Ser Thr Val Ile Ala Ala Phe Val Asn Val Phe Gly Met
180 185 190
Leu Thr Ser Ser Thr His Asn Thr Asn Gln Asp Ala Leu Ala Ala Leu
195 200 205
Gln Thr Leu Thr Thr Ala Arg Ala Ala Thr Tyr Asn Gln Asn Phe Pro
210 215 220
Ser Ala Gly Leu Gly Ala Pro Gly Ser Cys Gln Ser Gly Ala Pro Thr
225 230 235 240
Glu Thr Val Gly Gly Asn Thr His Leu Leu Tyr Ser Trp Ala Gly Thr
245 250 255
Ala Ile Gln Pro Thr Phe Ser Ala Leu Gly Val Thr Gly Ala Lys Asp
260 265 270
Thr Ser Thr Ile Pro Val Val Asp Pro Ala Asn Ala Leu Asp Ala Ser
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Thr Leu Ala Leu Leu Gly Ser Gly Thr Val Met Ile Asn Arg Gly Ser
290 295 300
Gly Glu Asn Asp Gly Val Val Ser Lys Cys Ser Ala Leu Phe Gly Gln
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Val Leu Ser Thr Ser Tyr Lys Trp Asn His Val Asp Glu Ile Asn Gln
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Leu Leu Gly Val Arg Gly Ala Tyr Ala Glu Asp Pro Val Ala Val Ile
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Ala Asp Asp Tyr Ala Thr Thr Arg Tyr Pro Ile Ile Leu Val His Gly
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Ile Gln Gln Asp Leu Gln Gln His Gly Ala Thr Val Tyr Val Ala Asn
35 40 45
Leu Ser Gly Tyr Gln Ser Asp Asp Gly Pro Asn Gly Arg Gly Glu Gln
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Leu Leu Ala Gln Val Lys Gln Val Leu Ala Gln Thr Gly Ala Ala Lys
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Val Asn Leu Ile Gly His Ser Gln Gly Gly Leu Ser Ser Arg Tyr Val
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Ala Ala Val Ala Pro Glu Leu Val Ala Ser Val Thr Thr Ile Gly Thr
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Pro His Arg Gly Ser Glu Phe Ala Asp Phe Val Gln Gly Val Leu Ala
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Tyr Asp Pro Thr Gly Leu Ser Ser Thr Val Ile Ala Ala Phe Val Asn
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Val Phe Gly Met Leu Thr Ser Ser Thr His Asn Thr Asn Gln Asp Ala
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Leu Ala Ala Leu Gln Thr Leu Thr Thr Ala Arg Ala Ala Thr Tyr Asn
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Gln Asn Phe Pro Ser Ala Gly Leu Gly Ala Pro Gly Ser Cys Gln Ser
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<213> 乌汶伯克霍尔德氏菌
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atggccagat caatgcgttc cagggtggtg gcaggggcag tggcatgcgc gatgagcgcc 60
gcgccgttcg cgggcatgac cgcgctcgcg acggttgcga cgacgcgcgc ggcggtggcg 120
gcaaccgcac cggccgacga ttacgcgacg acgcgttatc cgatcattct cgtgcacggg 180
ctgacgggca ccgacaagta tgcgggcgtg ctcgattact tttacggcat ccagcaggac 240
ctgcagcagc atggcgcgac cgtgtacgtc gcgaacctgt ccggctacca gagcgacgac 300
ggcccgaacg ggcgcggcga gcaattgctc gcgcaagtga agcaggtgct tgcgcagacc 360
ggcgcggcca aggtcaacct gatcggccac agccagggcg gcctgtcgtc gcgctatgtc 420
gccgccgtcg cgccggagct ggtcgcgtcg gtgacgacga tcggcacgcc gcaccgcggc 480
tcggaattcg cggacttcgt gcagggcgtg ctcgcgtacg acccgaccgg tctttcgtcg 540
acagtgatcg cggcgttcgt caatgtgttc ggcatgctga cgagcagcac ccacaacacc 600
aaccaggacg cgctcgccgc gctgcagacg ctgaccacgg cgcgggccgc gacctataac 660
cagaacttcc cgagcgcggg gctcggcgcg cccggctcgt gccagagcgg cgcgccgacg 720
gagacggtcg gcggcaacac gcacctgctg tactcgtggg ccggcaccgc gatccagccg 780
acgttctccg cgctgggcgt gacgggcgcg aaggatacga gcacgatccc ggtcgtcgat 840
ccggcgaatg cgctcgacgc gtcgacgctc gcgctgctcg gcagcggcac ggtgatgatc 900
aaccgcggct cgggcgaaaa cgacggcgtc gtgtcgaaat gcagcgcgct gttcgggcag 960
gtgctgagca cgagctacaa gtggaaccac gtcgacgaga tcaaccagct gctgggcgtg 1020
cgcggcgcgt atgcggaaga cccggtcgcg gtgatccgca cgcacgcgaa ccggctgaag 1080
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<210> 4
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<212> DNA
<213> 乌汶伯克霍尔德氏菌
<400> 4
gccgacgatt acgcgacgac gcgttatccg atcattctcg tgcacgggct gacgggcacc 60
gacaagtatg cgggcgtgct cgattacttt tacggcatcc agcaggacct gcagcagcat 120
ggcgcgaccg tgtacgtcgc gaacctgtcc ggctaccaga gcgacgacgg cccgaacggg 180
cgcggcgagc aattgctcgc gcaagtgaag caggtgcttg cgcagaccgg cgcggccaag 240
gtcaacctga tcggccacag ccagggcggc ctgtcgtcgc gctatgtcgc cgccgtcgcg 300
ccggagctgg tcgcgtcggt gacgacgatc ggcacgccgc accgcggctc ggaattcgcg 360
gacttcgtgc agggcgtgct cgcgtacgac ccgaccggtc tttcgtcgac agtgatcgcg 420
gcgttcgtca atgtgttcgg catgctgacg agcagcaccc acaacaccaa ccaggacgcg 480
ctcgccgcgc tgcagacgct gaccacggcg cgggccgcga cctataacca gaacttcccg 540
agcgcggggc tcggcgcgcc cggctcgtgc cagagcggcg cgccgacgga gacggtcggc 600
ggcaacacgc acctgctgta ctcgtgggcc ggcaccgcga tccagccgac gttctccgcg 660
ctgggcgtga cgggcgcgaa ggatacgagc acgatcccgg tcgtcgatcc ggcgaatgcg 720
ctcgacgcgt cgacgctcgc gctgctcggc agcggcacgg tgatgatcaa ccgcggctcg 780
ggcgaaaacg acggcgtcgt gtcgaaatgc agcgcgctgt tcgggcaggt gctgagcacg 840
agctacaagt ggaaccacgt cgacgagatc aaccagctgc tgggcgtgcg cggcgcgtat 900
gcggaagacc cggtcgcggt gatccgcacg cacgcgaacc ggctgaagct cgcgggcgtg 960
taa 963
Claims (7)
1.一种脂肪酶,由与序列号1或2的氨基酸序列具有90%以上的同源性的氨基酸序列构成。
2.根据权利要求1所述的脂肪酶,其中,所述脂肪酶的氨基酸序列为与序列号1或2所示的氨基酸序列显示93%以上的同源性的氨基酸序列。
3.根据权利要求1所述的脂肪酶,其中,所述脂肪酶的氨基酸序列为与序列号1或2所示的氨基酸序列显示95%以上的同源性的氨基酸序列。
4.一种酶制剂,以权利要求1~3中任一项所述的脂肪酶作为有效成分。
5.一种脂肪酶的制造方法,包括以下的步骤(1)和(2):
(1)对生产权利要求1~3中任一项所述的脂肪酶的微生物进行培养的步骤;
(2)从培养后的培养液和/或菌体回收脂肪酶的步骤。
6.一种油脂的制造方法,包括在受体底物和供体底物的存在下利用权利要求1~3中任一项所述的脂肪酶或权利要求4所述的酶制剂进行的酶反应。
7.根据权利要求6所述的制造方法,其中,受体底物为油脂、甘油脂肪酸酯或甘油,
供体底物为脂肪酸、酯化合物或油脂。
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EP3805393A4 (en) | 2022-02-23 |
JP2021090440A (ja) | 2021-06-17 |
US20210214696A1 (en) | 2021-07-15 |
EP3805393A1 (en) | 2021-04-14 |
JP6846577B1 (ja) | 2021-03-24 |
WO2021020458A1 (ja) | 2021-02-04 |
US11718837B2 (en) | 2023-08-08 |
JP2023171429A (ja) | 2023-12-01 |
BR112021002182A2 (pt) | 2022-02-15 |
JP7358592B2 (ja) | 2023-10-10 |
JP7148659B2 (ja) | 2022-10-05 |
JPWO2021020458A1 (ja) | 2021-09-13 |
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