CN110621162B - 脂肪酶 - Google Patents
脂肪酶 Download PDFInfo
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- CN110621162B CN110621162B CN201880031054.0A CN201880031054A CN110621162B CN 110621162 B CN110621162 B CN 110621162B CN 201880031054 A CN201880031054 A CN 201880031054A CN 110621162 B CN110621162 B CN 110621162B
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- variant
- lipase
- variant polypeptide
- dough
- enzyme
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- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
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Abstract
脂肪酶,制备脂肪酶的方法,在食品、饲料、个人护理、洗涤剂、谷物加工、纸浆和纸加工、生物燃料、乙醇生产、纺织品、乳品加工、可可脂加工、可可提取、膳食补充剂、咖啡加工、涂料、水处理和油加工中使用脂肪酶的方法。
Description
序列表
根据“专利合作条约(PCT)下国际专利申请中核苷酸和氨基酸序列表的呈现标准”ST.25,本申请包括计算机可读形式(CRF)的以ASC II文本(.txt)形式的核苷酸和氨基酸序列表。序列表在下文中被标识,并出于所有目的通过引用整体并入本申请的说明书中。
技术领域
数千年来,面包一直是人类营养的主食。面包通常这样来制作:将面粉,水,盐,酵母和/或其他食品添加剂组合以制成面团(dough)或糊状物(paste);然后将面团烘烤以制备面包。已知酶在烘焙中是有用的,因为酶对烘焙过程的影响可以与化学替代物相似或更好,酶可用于保鲜,并增加面包体积。几种不同的酶可用于制作面包,例如已知脂肪酶能改善面包的稳定性和体积;然而,工业上仍然需要能改善体积、稳定性、耐受性,减少或消除添加剂二乙酰酒石酸单甘油酯(DATEM)的脂肪酶。本公开涉及满足或超过这些工业要求的变体脂肪酶。
发明概要
变体多肽,包含与SEQ ID NO:1的氨基酸序列至少80%同一的氨基酸序列,并且该变体多肽具有脂肪酶活性。
变体多肽,相对于SEQ ID NO:1的氨基酸序列包含氨基酸残基***、缺失或取代,且该变体多肽具有脂肪酶活性。
变体多肽,相对于SEQ ID NO:1的氨基酸序列,包含在氨基酸残基位置编号23、33,82、83、84、85、160、199、254、255、258、263、264、265、268、308、311或其任何组合处的氨基酸残基***、缺失或取代,且所述变体多肽具有脂肪酶活性。
包含氨基酸取代的变体多肽,所述氨基酸取代选自,相对于SEQ ID NO:1的氨基酸序列,Y23A、K33N、S82T、S83D、S83H、S83I、S83N、S83R、S83T、S83Y、S84S、S84N、84′Y、84′L、84′S、I85A、I85C、I85F、I85H、I85L、I85M、I85P、I85S、I85T、I85V、I85Y、K160N、P199I、P199V、I254A、I254C、I254E、I254F、I254G、I254L、I254M、I254N、I254R、I254S、I2454V、I254W、I254Y、I255A、I255L、A256D、L258A、L258D;L258E、L258G、L258H、L258N、L258Q、L258R、L258S、L258T、L258V、D263G、D263K、D263P、D263R、D263S;T264A、T264D、T264G、T264I、T264L、T264N、T264S、D265A、D265G、D265K、D265L、D265N、D265S、D265T、T268A、T268G、T268K、T268L、T268N、T268S、D308A和Y311E,或其任意组合,且这些变体多肽具有脂肪酶活性。
变体多肽,其包含与SEQ ID NO:1所示的氨基酸序列至少80%同一的氨基酸序列,其中所述变体多肽相对于SEQ ID NO:1的氨基酸序列具有至少一个单氨基酸取代,所述一个单氨基酸取代选自:相对于SEQ ID NO:1的氨基酸序列,Y23A、K33N、S82T、S83D、S83H、S83I、S83N、S83R、S83T、S83Y、S84S、S84N、84′Y、84′L、84′S、I85 A、I85C、I85F、I85H、I85L、I85M、I85P、I85S、I85T、I85V、I85Y、K160N、P199I、P199V、I254A、I254C、I254E、I254F、I254G、I254L、I254M、I254N、I254R、I254S、I2454V、I254W、I254Y、I255A、I255L、A256D、L258A、L258D;L258E、L258G、L258H、L258N、L258Q、L258R、L258S、L258T、L258V、D263G、D263K、D263P、D263R、D263S;T264A、T264D、T264G、T264I、T264L、T264N、T264S、D265A、D265G、D265K、D265L、D265N、D265S、D265T、T268A、T268G、T268K、T268L、T268N、T268S、D308A和Y311E;其中所述变体多肽具有脂肪酶活性。
变体多肽,其包含与SEQ ID NO:1所示的氨基酸序列至少80%同一的氨基酸序列,其中所述变体多肽具有表:1所示的修饰,并且所述变体多肽具有脂肪酶活性。
变体多肽,其中所述变体多肽由与SEQ ID NO:2所示核酸序列至少80%同一的核酸序列编码,并且所述变体多肽具有脂肪酶活性。
SEQ ID NO:2所示核酸序列的变体核苷酸,其中所述变体核苷酸是与SEQ ID NO:2所示的核酸序列至少80%同一的核酸序列,其中变体核苷酸编码具有脂肪酶活性的多肽。
变体多肽,其包含SEQ ID NO:1的全长氨基酸序列的片段,该片段是具有脂肪酶活性的变体多肽。
变体多肽,其包含本文公开的至少一种变体多肽和具有脂肪酶活性的第二多肽的杂合体,其中所述杂合体具有脂肪酶活性。
组合物,其包含本文公开的变体多肽。
组合物,其包含本文公开的变体多肽,和至少第二种酶。所述组合物进一步包含的第二种酶选自:第二脂肪酶,淀粉酶,木聚糖酶,蛋白酶,纤维素酶,葡糖淀粉酶,氧化还原酶,磷脂酶和环糊精葡聚糖转移酶。
组合物,包含本文公开的变体多肽并且还进一步包含载体、稳定剂、缓冲剂、防腐剂或其任何组合。包含本文公开的变体多肽的组合物,其中载体是小麦粉。包含本文公开的变体多肽的组合物,其中稳定剂是乙酸钙、氯化钙、氯化镁、氯化钠、硫酸钠、瓜尔胶或其任何组合。包含本文公开的变体多肽的组合物,其中缓冲剂是乙酸钙、乙酸钠、柠檬酸钠、磷酸钠、磷酸钾或其任何组合。包含本文公开的变体多肽的组合物,其中防腐剂是乙酸钙、乙酸钠、丙酸钠、丙酸钙、丙酸、山梨酸钾、山梨酸、苯甲酸钠、苯甲酸、乙酸或任何组合。组合物,包含本文公开的变体多肽,其中组合物。组合物,包含本文公开的变体多肽和一种或多种选自以下的组分:糖类如蔗糖、海藻糖、乳糖;奶粉、面筋、颗粒化脂肪、氨基酸、盐、氧化剂如抗坏血酸、溴酸盐和偶氮二甲酰胺,还原剂如L-半胱氨酸、乳化剂如单甘油酯、甘油二酯、甘油单硬脂酸酯、硬脂酰乳酸钠、硬脂酰乳酸钙、脂肪酸聚甘油酯和甘油单酯和二酯的二乙酰酒石酸酯,树胶如瓜尔胶和黄原胶、香料、酸如柠檬酸和丙酸、淀粉、改性淀粉、保湿剂如甘油和防腐剂。
制备变体多肽的方法,包括:提供SEQ ID NO:2的或本文公开的模板核酸序列,将模板核酸序列转化到表达宿主中,培养表达宿主以产生变体多肽,并纯化变体多肽。方法,其进一步包含选自以下的表达宿主:细菌表达***,酵母表达***,真菌表达***和合成表达***。方法,其中细菌表达***选自大肠杆菌,芽孢杆菌属(Bacillus),假单胞菌属(Pseudomonas)和链霉菌属(Streptomyces)。方法,其中酵母表达***选自念珠菌属(Candida),毕赤酵母属(Pichia),酵母属(Saccharomyces),裂殖酵母属(Schizosaccharomyces)。方法,其中真菌表达***选自青霉属(Penicillium),曲霉属(Aspergillus),镰刀菌属(Fusarium),毁丝霉属(Myceliopthora),根毛菌属(Rhizomucor),根霉菌属(Rhizopus),嗜热真菌属(Thermomyces)和木霉属(Trichoderma)。
一种不添加乳化剂的,制备面团或由面团制备的烘焙产品的方法,该方法包括将如本文公开的一种变体多肽添加到面团中并烘焙。方法,其中所述乳化剂选自:硬脂酰乳酸钙(CSL),单甘油二乙酰酒石酸酯(DATEM),乙氧基化甘油单酯和甘油二酯(EMG),聚山梨醇酯(PS),硬脂酰乳酸钠(SSL)和琥珀酰化甘油单酯(SMG)。
用于制备面团或由面团制备的烘焙产品的预混物,其包含至少一种本文公开的变体多肽。
附图简述
图1A和图1B显示了烘焙试验的结果。
发明详述
面包包括但不限于面包卷、小奶油面包、糕点、蛋糕、小面包干、比萨面包、比塔面包、薄饼、派皮、lavish、pitta、佛卡恰、发酵面团、面条、曲奇饼、玉米粉圆饼、薄煎饼、小薄煎饼、烤碎面包片和饼干。烘焙面包通常涉及混合成分形成面团、揉面、发面、成型、烘焙、冷却和保存。用于制作面团的成分通常包括面粉、水、盐、酵母和其他食品添加剂。
面粉通常由小麦制成,可以为不同目的而研磨,如制作面包、糕点、蛋糕、饼干面团和面条。小麦粉的替代品包括但不限于杏仁粉、椰粉、芡欧鼠尾草粉、玉米粉、大麦粉、二粒小麦粉、大豆粉、***粉、马铃薯粉、昆诺阿藜、埃塞俄比亚画眉草粉、黑麦粉、苋菜粉、竹芋根粉、鹰嘴豆粉、腰果粉、亚麻籽饼粉、夏威夷果粉、稷粉、高粱粉、米粉、木薯粉及其任意组合。已知全世界范围内面粉类型在不同区域和不同国家间不同。
酵母将糖分解为二氧化碳和水。多种面包酵母(通常源自酿酒酵母(Saccharomyces cerevisiae))为本领域已知,包括但不限于酵母浆、压缩酵母、压块酵母、活性干酵母、即发酵母、耐渗压酵母、速发酵母、灭活酵母。其他类型的酵母包括营养酵母、啤酒酵母、酒糟和酒酵母。
甜味剂包括但不限于液态糖、糖浆、白(砂)糖、棕(粗)糖、蜂蜜、果糖、右旋糖、葡萄糖、高果糖玉米糖浆、糖蜜和人工甜味剂。
乳化剂包括但不限于二乙酰酒石酸单甘油酯(DATEM)、硬脂酰乳酸钠(SSL)、硬脂酰乳酸钙(CSL)、乙氧基化单酰和二酰甘油(EMG)、聚山梨酯(PS)和琥珀酰化单酰甘油(SMG)。
其他可用于本公开的方法的食品添加剂包括:脂质、油、黄油、人造黄油、起酥油、乳脂、甘油、蛋、膳食、非膳食替代品、增稠剂、防腐剂、着色剂和酶。
用于烘焙的成分或添加剂可以在烘焙过程中单独添加。成分或添加剂也可以与一种以上的成分或添加剂组合以形成预混物,然后在烘焙过程中添加预混物。此外,酶可以在烘焙过程之前直接添加到面粉中。
酶是包含氨基酸序列的生物分子,其中酶可以催化反应。根据国际生物化学和分子生物学(IUBMB)联合会命名委员会的建议,酶名称为本领域技术人员已知。酶名称包括:EC(酶学委员会)号、建议名称、别名(若有)、催化活性和其他因子。酶也称为多肽、蛋白质、肽、氨基酸序列,或通过SEQ ID NO标识。在本公开中,酶的别名可互换使用。
已知不同种类的酶可用于烘焙,包括:脂肪酶E.C.3.1.3;α-淀粉酶(E.C.3.2.1.1);β-淀粉酶(E.C.3.2.1.2);葡聚糖1,4-α-麦芽四糖水解酶(E.C.3.2.1.60),也称为外切-麦芽四糖水解酶、G4-淀粉酶;葡聚糖1,4-α-麦芽糖水解酶(E.C.3.2.1.133),也称为产麦芽糖α-淀粉酶;内切-1,4-β-木聚糖酶(E.C.3.2.1.8);氧化还原酶;磷脂酶A1(E.C.3.1.1.32);磷脂酶A2(E.C.3.1.1.4);磷脂酶C(E.C.3.1.4.3);磷脂酶D(E.C.3.1.4.4);半乳糖脂酶(E.C.3.1.1.26);纤维素酶(EC 3.2.1.4)、转谷酰胺酶(EC2.3.2.13)、植酸酶(EC 3.1.3.8;3.1.3.26;和3.1.1.72)和蛋白酶。酶可用作食品成分、食品添加剂和/加工助剂。
脂肪酶(E.C.3.1.1.3)是已知切割脂质中的酯键的水解酶。脂肪酶包括磷脂酶、三酰甘油脂酶和半乳糖脂酶。已从植物、哺乳动物及包括但不限于以下的微生物鉴定了脂肪酶:假单胞菌属(Pseudomonas)、弧菌属(Vibrio)、不动杆菌属(Acinetobacter)、伯克氏菌属(Burkholderia)、色杆菌属(Chromobacterium),来自腐皮镰刀菌(Fusarium solani)的角质酶(FSC),南极假丝酵母(Candida antarctica)A(CalA)、米根霉(Rhizopus oryzae)(ROL)、疏绵状嗜热丝孢菌(Thermomyces lanuginosus)(TLL)、米黑根毛霉(Rhizomucormiehei)(RML)、黑曲霉(Aspergillus Niger)、异孢镰刀菌(Fusarium heterosporum)、尖孢镰刀菌(Fusarium oxysporum)、大刀镰刀菌(Fusarium culmorum)脂肪酶。
此外,专利和公开的专利申请中已公开了许多脂肪酶、磷脂酶和半乳糖脂酶,包括但不限于WO1993/000924、WO2003/035878、WO2003/089620、WO2005/032496、WO2005/086900、WO2006/031699、WO2008/036863和WO2011/046812。
用于食品加工和烘焙的市售脂肪酶包括但不限于:LIPOPANTM、NOOPAZYME、LIPOPANMAX、LIPOPAN Xtra(可获自Novozymes);PANAMORE、CAKEZYME和BAKEZYME(可获自DSM);及GRINDAMYL EXEL 16、GRINDAMYL POWERBAKE和TS-E 861(可获自Dupont/Danisco)。
(亲本蛋白质或酶,也称为“亲本酶”的)“亲本”序列是用于向序列中引入改变(例如通过引入一个或多个氨基酸取代、***、缺失或其组合)而得到亲本序列的“变体”的起始序列。术语亲本酶(或亲本序列)包括
1.野生型酶(序列)和
2.合成产生的序列(酶),其用作引入(进一步)改变的起始序列。
“酶变体”或“序列变体”或“变体酶”是指与其亲本酶在其氨基酸序列上一定程度上不同的酶。如果没有另外说明,变体酶“具有酶活性”是指该变体酶具有与各自亲本酶相同类型的酶活性。
在一个实施方案中,具有氨基酸取代的变体多肽可以是保守氨基酸取代。“保守氨基酸取代”是指,与亲本氨基酸序列相比,在同一位置用具有相似特性的不同氨基酸残基替换氨基酸序列中的一个氨基酸残基。保守氨基酸取代的一些实例包括但不限于用不同的带正电荷氨基酸残基替换带正电荷的氨基酸残基;用不同的极性氨基酸残基替换极性氨基酸残基;用不同的非极性氨基酸残基替换非极性氨基酸残基,用不同的碱性氨基酸残基替换碱性氨基酸残基,或用不同的芳香族氨基酸残基替换芳香族氨基酸残基。
WIPO标准ST.25(1998)规定,氨基酸残基应在序列表中使用以下三个字母的代码表示,第一个字母为大写字母。下表提供了氨基酸识别符的概述,以及相应编码使用标准遗传标准的编码氨基酸的DNA密码子。编码氨基酸残基的DNA密码子可以根据使用的生物体而不同,并且可以应用稍微不同的遗传密码子翻译表。这种非标准代码翻译表的汇编在NCBI维护。参见例如
https://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi,供参考。
在另一实施方案中,具有脂肪酶活性的变体多肽是“成熟多肽”。成熟多肽指处于其最终形式的酶,包括任何翻译后修饰、糖基化、磷酸化、截短、N端修饰、C端修饰、信号序列删除。成熟多肽可取决于表达***、载体、启动子和/或产生方法而不同。
在另一个实施方案中,脂肪酶在宽泛pH下,在约pH4.0到约pH12.0范围的任何单一点上具有活性。在一个实施方案中,脂肪酶在pH4.0至pH11.0,pH4.0至pH10.0,pH4.0至pH9.0,pH4.0至pH8.0,pH4.0至pH7.0,pH4.0至pH6.0或pH4.0至pH 5.0范围内具有活性。在另一个实施方案中,脂肪酶在pH 4.0、pH 4.1、pH 4.2、pH 4.3、pH 4.4、pH 4.5、pH 4.6、pH4.7、pH 4.8、pH 4.9、pH 5.0、pH 5.1、pH 5.2、pH 5.3、pH 5.4、pH 5.5、pH 5.6、pH 5.7、pH5.8、pH 5.9、pH 6.0、pH 6.1、pH 6.2、pH 6.3、pH 6.4、pH 6.5、pH 6.6、pH 6.7、pH 6.8、pH6.9、pH 7.0、pH 7.1、pH 7.2、pH 7.3、pH 7.4、pH 7.5、pH 7.6、pH 7.7、pH 7.8、pH 7.9、pH8.0、pH 8.1、pH 8.2、pH 8.3、pH 8.4、pH 8.5、pH 8.6pH 8.7、pH 8.8pH 8.9、pH 9.0、pH9.1、pH 9.2、pH 9.3、pH 9.4、pH 9.5、pH 9.6、pH 9.7、pH 9.8、pH 9.9、pH 10.0、pH 10.1、pH 10.2、pH 10.3、pH 10.4、pH 10.5、pH 10.6、pH 10.7、pH 10.8、pH 10.9、pH 11.0、pH11.1、pH 11.2、pH 11.3、pH 11.4、pH 11.5、pH 11.6、pH 11.7、pH 11.8、pH 11.9、pH 12.0、pH 12.1、pH 12.2、pH 12.3、pH 12.4、and pH 12.5、pH 12.6、pH 12.7、pH 12.8、pH 12.9和更高pH值下具有活性。
在另一实施方案中,脂肪酶在烘焙过程中任意时间所用的宽温度范围内具有活性,其中温度是约20℃至约60℃范围内的任意点。在另一实施方案中,脂肪酶在20℃至55℃、20℃至50℃、20℃至45℃、20℃至40℃、20℃至35℃、20℃至30℃或20℃至25℃温度范围内具有活性。在另一实施方案中,脂肪酶在至少19℃、20℃、21℃、22℃、23℃、24℃、25℃、26℃、27℃、28℃、29℃、30℃、31℃、32℃、33℃、34℃、35℃、36℃、37℃、38℃、39℃、40℃、41℃、42℃、43℃、44℃、45℃、46℃、47℃、48℃、49℃、50℃、51℃、52℃、53℃、54℃、55℃、56℃、57℃、58℃、59℃、60℃、61℃、62℃的温度或更高温度具有活性。
“序列同一性”、“%序列同一性”、“%同一性”或“序列比对”指第一氨基酸序列与第二种氨基酸序列的比较,或第一核酸序列与第二种核酸序列的比较,且基于该比较计算为百分比。该计算的结果可以描述为“百分比相同”或“百分比ID”
通常,可以用序列比对通过两种不同方法之一计算序列同一性。在第一种方法中,在最终序列同一性计算中将单个位置的错配和单个位置的缺口都计数为非同一位置。在第二种种方法中,在最终序列同一性计算中将单个位置的错配计数为非同一位置;但是,在最终序列同一性计算中不将单个位置的缺口计数(忽略)为非同一位置。换言之,在第二种种方法中,在最终序列同一性计算中忽略缺口。这两种方法之间的差异(在单个位置将缺口计数为非同一位置对忽略缺口)可导致两个序列间序列同一性值的变异。
在本公开的一个实施方案中,通过程序测定序列同一性,该程序产生比对,并在最终序列同一性计算中将单个位置的错配和单个位置的缺口都计数为非同一位置来计算同一性。例如程序(EMBOS),其执行Needleman和Wunsch(Needleman和Wunsch,1970,J.Mol.Biol.48:443-453)的算法,其这样计算序列同一性:首先产生第一序列和第二种序列之间的比对,然后计数比对长度内相同位置的数目,然后将相同残基的数目除以比对长度,然后将此数乘以100来产生%序列同一性[%序列同一性=(相同残基数/比对长度)x100]。
在本公开的另一实施方案中,可以从配对比对计算序列同一性,该配对比对在全长范围内显示两个序列,所以以其全长显示第一序列和第二种序列(“总体序列同一性”)。例如,程序Needle(EMBOSS)产生这类比对;%序列同一性=(相同残基数/比对长度)x100。
在本公开的另一实施方案中,可以从仅显示第一序列或第二种序列的局部区域的配对比对计算序列同一性(“局部同一性”)。例如,程序Blast(NCBI)产生这类比对;%序列同一性=(相同残基数/比对长度)x100。
在另一个实施方案中,序列比对是这样计算的:单个位置的错配在最终序列同一性计算中被计为非相同位置;但,单个位置的空位不被计作(忽略)最终序列同一性计算中的非相同位置。
在一个优选的实施方案中,使用Needleman和Wunsch的算法(J.Mol.Biol.(1979)48,p.443-453)生成序列比对。优选地,程序“NEEDLE”(欧洲分子生物学开放软件套件(EMBOSS))被用于本发明的目的,使用程序默认参数(空位开放=10.0,空位延伸=0.5和矩阵=EBLOSUM62)。序列同一性可以根据两个序列相对全长序列的比对计算,从而以其全长显示第一序列和第二序列(“全局序列同一性”)。例如,%序列同一性=(#相同残基的#/比对长度)×100)]。
在另一个优选实施方案中,优选的比对程序是“NEEDLE”,使用程序默认参数(空位开放=10.0,空位延伸=0.5和矩阵=EDNAFULL)。
根据本发明,酶变体可以描述为与相应亲本酶的氨基酸序列至少n%同一的氨基酸序列,其中“n”是10-100的整数。在一个实施方案中,变体酶与亲本酶的全长氨基酸序列相比,有至少10%,至少15%、至少20%、至少25%、至少30%、至少35%、至少40%、至少45%、至少50%、至少55%、至少60%、至少65%、至少70%、至少75%、至少80%、至少81%、至少82%、至少83%、至少84%、至少85%、至少86%、至少87%、至少88%、至少89%、至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%同一,其中酶变体具有酶活性。
本发明进一步涉及编码本发明的变体多肽的多核苷酸。术语“多核苷酸”、“核酸序列”、“核苷酸序列”、“核酸”、“核酸分子”在本文中可互换使用,并且指任何长度的聚合无分支形式的核苷酸,核糖核苷酸或脱氧核糖核苷酸或两者的组合。“基因”是携带特定遗传信息的DNA片段。
“亲本”或“模板核酸序列”是多核苷酸酸序列,是用于向序列中引入突变的起始序列,并产生所述亲本多核苷酸序列的“变体”。“变体多核苷酸”是指与亲本多核苷酸编码相同酶的多核苷酸。在这种情况下,变体多核苷酸与其亲本多核苷酸的核酸序列不同,但编码的多肽保持不变。
在本公开的一个实施方案中,脂肪酶可以与至少一种其他酶组合使用。其他酶可以来自相同类别的酶,例如,组合物包含第一脂肪酶和第二脂肪酶。其他酶也可以来自不同类别的酶,例如,组合物包含脂肪酶和淀粉酶。具有至少一种其他酶的组合可以是包含至少三种酶的组合物。这三种酶可以具有来自相同类的酶,例如第一脂肪酶,第二脂肪酶和第三脂肪酶,或者酶可以来自不同类别的酶,例如脂肪酶,淀粉酶和木聚糖酶。在另一个实施方案中,第二种酶包含或选自:α-淀粉酶;β-淀粉酶,葡聚糖1,4-α-麦芽四糖水解酶,也称为外切-麦芽四糖水解酶、G4-淀粉酶;葡聚糖1,4-α-麦芽糖水解酶,也称为产麦芽糖α-淀粉酶、环糊精葡聚糖转移酶、葡糖淀粉酶;内切-1,4-β-木聚糖酶;木聚糖酶、纤维素酶、氧化还原酶;磷脂酶A1、磷脂酶A2、磷脂酶C、磷脂酶D;半乳糖脂酶、三酰甘油脂酶、***呋喃糖苷酶、转谷氨酰胺酶、果胶酶、果胶酸裂合酶、蛋白酶或其任意组合。在另一实施方案中,该酶组合是本文公开的脂肪酶和产麦芽糖α-淀粉酶,或者该酶组合是本文公开的脂肪酶、产麦芽糖α-淀粉酶和木聚糖酶。
在本公开的另一实施方案中,该脂肪酶可以是一种以上脂肪酶的杂合体。“杂合体”或“嵌合”或“融合蛋白质”指将本公开的第一脂肪酶的结构域与第二种脂肪酶的结构域组合形成杂合脂肪酶,且该杂合体具有脂肪酶活性。在一个实施方案中,将本公开的脂肪酶的结构域与市售脂肪酶如LIPOPAN(可获自Novozymes)或PANAMORE(可获自DSM)的结构域组合形成杂合脂肪酶,且该杂合脂肪酶具有脂肪酶活性。
工业酶通常是用细菌、真菌或酵母表达***产生的重组蛋白质。“表达***”还指宿主微生物、表达宿主、宿主细胞、产生生物或产生菌株,对于本公开,这些术语中的每一个可互换使用。表达***的实例包括但不限于:黑曲霉、米曲霉(Aspergillus oryzae)、多形汉逊酵母(Hansenula polymorpha)、疏绵状嗜热丝孢菌、尖孢镰刀菌、异孢镰刀菌、大肠杆菌(Escherichia coli),芽孢杆菌属(Bacillus),优选枯草芽孢杆菌(Bacillus subtilis)或地衣芽孢杆菌(Bacillus licheniformis),假单胞杆菌属(Pseudomonas),优选荧光假单胞菌(Pseudomonas fluorescens),巴斯德毕赤酵母(Pichia pastoris)(也称为Komagataella phaffii),Thermothelomyces thermophila(也已知为Myceliopthorathermophile(C1)),嗜热毁丝霉(Myceliopthora thermophile)(C1),粟酒裂殖酵母(Schizosaccharomyces pombe),木霉属(Trichoderma),优选里氏木霉(Trichodermareesei)和酵母属(Saccharomyces),优选地酿酒酵母(Saccharomyces cerevisiae)。在一个实施方案中,用上文所列表达***产生本公开的脂肪酶。
已知脂肪酶可用于其他工业应用。在本公开的一个实施方案中,该脂肪酶用于去垢剂。在本公开的一个实施方案中,该脂肪酶用于个人护理产品,如隐形眼镜溶液。在另一实施方案中,本公开的脂肪酶用于织物加工,如制革。在另一实施方案中,本公开的脂肪酶可用于纸浆和纸张加工。在另一实施方案中,该纸浆和纸张加工是树脂控制(pitchcontrol)或脱墨。在另一个实施方案中,本公开的脂肪酶可用于制造生物柴油。在另一实施方案中,本公开的脂肪酶可用于干酪成熟。在另一实施方案中,本公开的脂肪酶可用于制备肉味和/或香气。在另一实施方案中,本公开的脂肪酶可用于改性油和脂肪。在另一实施方案中,本公开的脂肪酶可用于酶促油脱胶。在另一实施方案中,本公开的脂肪酶可用于产生乙醇。
本文所用的术语“烘焙产品”包括诸如面包、条状面包(loaf bread)、盘式面包(pan bread)、脆皮卷、三明治面包、小奶油面包、长棍面包、夏巴塔、羊角面包、面条,以及精细烘焙食品,如炸面圈、奶油鸡蛋卷、葡萄干甜面包、蛋糕、小松饼等。
本文所用的术语“面团”定义为面粉、盐、酵母和水的混合物,其可在烘焙前揉和、模制、成型或碾压。此外,也可以使用其他成分,如糖、人造黄油、蛋、乳等。该术语包括用于制备烘焙商品如面包、面包卷、三明治面包、长棍面包、夏巴塔、羊角面包、甜酵母面团等的面团。
本文所用的术语“面包体积”是通过用激光扫描仪(例如Volscan Profiler exMicro Stable System)测量体积以及比体积而测定的烘焙商品的体积。该术语还包括通过测量某些烘焙商品的长度、宽度和高度而测定的体积。
本文所用的术语“包含”与“包括”、“含有”或“表征为”同义,且是包容性的或开放性的,不排除其他未引用的要素或方法步骤。
应理解,本文所述的本发明的方面和实施方案包括“由方面和实施方案组成”和/或“基本由方面和实施方案组成”。
本公开通篇中,多种方面以范围型式呈现。应理解,范围型式的描述仅仅是为了方便和简洁,不应解释为对本公开的范围的僵化限制。因此,范围的描述应视为已明确公开了所有可能的子范围以及该范围内的单个数值。例如,如1至6的范围描述应视为已明确公开了如1至3、1至4、1至5、2至4、2至6、3至6等的子范围,以及该范围内的单个数字,例如1、2、3、4、5和6。无论范围的宽度如何,这都适用。
本公开的其他目的、优势和特征将从以下说明书结合附图变得显而易见。
在以下描述中,给出许多具体细节以提供对本公开的更充分理解。但是,对本领域技术人员而言,显而易见的是,可以在没有一个或多个这些具体细节的情况下实施本公开的方法。在其他情况下,为了避免模糊本公开,未描述本领域技术人员公知的公知特征和方法。
实施例1:变体脂肪酶
使用合理设计的单点诱变和多位点诱变在实验室中产生非天然存在的变体脂肪酶。变体脂肪酶包括在亲本酶(LIP062,其是SEQ ID NO:1的氨基酸序列,并且由SEQ ID NO:2的核酸序列编码)的下述18种不同氨基酸残基位置的单位点氨基酸修饰、***或缺失:23、33、82、83、84、85、160、199、254、255、256、258、263、264、265、268、308、311或其任意组合,其中变体脂肪酶具有脂肪酶活性。
变体脂肪酶也通过亲本酶(LIP062)的单点修饰的各种组合产生,其中变体脂肪酶具有脂肪酶活性。例如,单点修饰和单点修饰的各种组合列于表:1中。
该表显示了LIP096的变体脂肪酶,其是具有LIP062的氨基酸序列和一个氨基酸取代A256D的变体多肽,其中所述变体多肽具有脂肪酶活性。该表还显示了LIP182的变体脂肪酶,其是具有LIP062的氨基酸序列和S83H,I85S,I255A,T264A和D265T的氨基酸取代组合的变体多肽,其中所述变体多肽具有脂肪酶活性。表1还显示了亲本脂肪酶的脂肪酶变体,其中变体包括氨基酸残基的***。氨基酸残基的***显示为(‘),例如(84′)
表:1
表:1
表:1
表:1
表:1
实施例2:脂肪酶的表达和纯化
表达
通过构建含有编码多核苷酸序列的表达质粒、转化质粒入巴斯德毕赤酵母(Komagataella phaffii)并按以下方式培养所得到的表达菌株来获得变体脂肪酶。通过将序列经确认的菌株的甘油原种涂布在含Zeocin的酵母提取物蛋白胨葡萄糖(YPD)琼脂平板上来获得表达菌株的新鲜巴斯德毕赤酵母细胞。2天后,用来自这些平板的细胞将产生菌株的起子种子培养物接种入100mL缓冲甘油复合培养基(BMGY),并在30℃、225-250转/分钟培养20-24小时。通过适量转入折流反应器中的2-4L BMMY培养基来放大种子培养物。在30℃和平桨叶搅拌桨提供的1100转/分钟搅拌下进行发酵48-72小时。发酵的起始批次期之后,无论何时培养物中溶解氧水平下降至30%以下,加入经除菌过滤的甲醇作为补料。备选地,每3小时按起始批次培养物的0.5%v/v加入补料。最终发酵液在4℃7000xg离心30分钟以获得无细胞上清。
变体脂肪酶的表达水平示于表2中,通过以下方法测定:通过SDS-PAGE或毛细管电泳或通过使用PNP-辛酸盐作为底物的酶活性测定上清液的目的蛋白质的表达。结果如下表2所示,数据显示为与亲本(LIP062)表达相比的百分比。对于一些变体脂肪酶,“n.d.”表示表达水平未确定;但是,生成足够的原料以将变体脂肪酶移送到实施例3的脂肪酶活性测试中,并进行如上文实施例1表1中所描述的送氨基酸序列鉴定。
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回收
过滤通过纱布(cheese-cloth)后,用截留分子量5kD的实验室规模切向流过滤(TFF)***(SpectrumLabs)超滤无细胞上清。样品首先浓缩10-20X,然后缓冲液交换5X入50mM HEPES pH 7.5。得到的截留物27000xg离心1小时,然后通过0.2μm滤器除菌过滤,以去除任何产生微生物或颗粒物质。用Braford测定法测定最终样品的总蛋白质含量。脂肪酶冷冻干燥形成粉末。
实施例3-脂肪酶活性
使用溶液中的天然底物测定变体脂肪酶的活性。通过超声处理在0.25%脱氧胆酸钠中以5mM终浓度制备天然脂质底物。将底物(15μL)与30μL荧光素(0.25μg/mL,在10mMCaCl2中)和10μL在5mM Hepes pH 7.5中预稀释的回收的脂肪酶(~1-2μg/mL)混合。通过由于pH变化引起的荧光下降(激发/发射为485nm/525nm)监测脂质水解产物,在26℃下每30秒动态记录10分钟。对数刻度的活性与每分钟的荧光变化成比例。结果如下表3所示,数据表示为相同蛋白质浓度下亲本酶(LIP062)荧光变化的百分比。对于在一些底物上的一些变体脂肪酶,变体脂肪酶活性未确定,用“n.d.”表示;但是,如实施例2中所述,产生足够的材料,并送氨基酸序列鉴定,如上文实施例1表1中所述。
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实施例4:HPLC评估的面团中脂解酶活性
用几种浓度的变体脂肪酶处理简化面团,以确定它们对面粉脂质的相对比活性。面团由1份面粉和2份含有34mg/ml氯化钠和6种浓度的酶的水制备,酶的6种浓度为:0.02、0.04、0.2、0.4、2.0、4.0μg酶/500μl面团。将面团以3000rpm混合5分钟,然后在30℃的湿度控制室中温育总共60分钟。对于脂质分析,向每个样品中加入500μl 1-丁醇,并通过在3000rpm下涡旋10分钟使面团均质化。然后室温下4000×g离心5分钟分离固体。有机相被移除并直接注入用于脂质分析。通过HPLC(Agilent 1100系列)用硅胶柱(ChromolithPerformance Si 100-4.6mm,Merck)分离脂质,并通过ELSD(Agilent 1260 Infinity)分析。脂质分离的色谱方法来自Gerits,等人“Single run HPLC separation coupled toevaporative light scattering detection unravels wheat flour endogenous lipidredistribution during bread dough making”LWT-Food Science and Technology,53(2013)426-433。六种酶剂量和阴性对照用于确定由于酶处理的各个脂质类别(表4)是否增加,减少或显示没有变化。
表4:脂质类别
表5显示了相对于亲本酶的脂质类测量的变化结果。表5的酶列列出了亲本脂肪酶(LIP062);和70种不同的变体脂肪酶,其中变体脂肪酶与亲本酶相比具有至少一个氨基酸修饰。表5中,在底物TAG,MGDG,DGDG和NAPE上的脂肪酶变体活性以相对于亲本脂肪酶(LIP062)活性的%列出。表5还显示了相对于亲本脂肪酶(LIP062)作为%列出的变体脂肪酶的产物(FFA,MAG,MGMG和DGMG)的累积。
表5:HPLC分析面团中的酶活性
表5:HPLC分析面团中的酶活性
实施例5:在各种pH值下的脂肪酶比活性
将变体脂肪酶在5mM Hepes pH 7.5中以适当浓度稀释,然后进一步稀释16倍,至在pH 6.5至pH 12.0的宽范围缓冲液中制备的0.4mM PNP-辛酸盐中。宽范围缓冲液含有:25mM磷酸,25mM柠檬酸,25mM硼酸,25mM CAPS和50mM NaCl。对于pH 8.0,缓冲液补充有10mMTris pH 8.0。在26℃下每40秒记录405nm处的吸光度15分钟测量活性。针对相同条件各pH下的背景(无酶)和PNP吸光度来校正活性。结果列于表6中,数据显示为微克PNP/min/mg酶。
实施例6:烘焙试验
在快速直接面团***Pistolet测试中测试变体脂肪酶的烘焙性能。使用2000g的550型面粉(Vogtmühlen Illertissen)、120g压缩酵母、40g盐、30g葡萄糖、22g小麦淀粉、120ppm抗坏血酸、5ppm Nutrilife AM 100(真菌α-淀粉酶)、200ppm Nutrilife CS 30(真菌木聚糖酶、纤维素酶、真菌α-淀粉酶)和1180g水的成分在Kemper SP 15螺旋式混合器中按速度1混合5.5分钟并按速度2混合0.5分钟,最终面团温度28℃。静置12分钟后,将面团按比例变大(scale)至1500g的团块,滚圆,并再醒发12分钟。然后,通过使用自动面团分块机和滚圆机来将面团分块和滚圆为每个50g的30个团块。然后将面团团块在85%相对湿度下在35℃醒发35分钟(正常醒发)和45分钟(延长醒发)。12分钟醒发时间后,在面团团块中央压出凹槽。在柜式烤炉中240℃烘焙经醒发的面团团块12分钟,蒸汽注射15秒。
变体脂肪酶的每种变体测试多达六次重复,结果如图1所示。这些结果报告为所测试的重复的平均值。在烘焙试验之前,测试每种酶的活性,其在不同的酶之间有变化,然后测试每种酶以确定该酶的最佳剂量,并且最后以最佳剂量添加酶。对于对照,已经使用10-28个重复来计算平均值。Panamore Golden 2.2(PG2.2)(DSM)的剂量基于制造商推荐的0.68mg脂肪酶/kg面粉。DATEM,Lametop LT 552(BASF)按照制造商的建议,剂量为0.4%。LIP062是脂肪酶变体的亲本脂肪酶,以1mg脂肪酶/kg面粉的最佳剂量使用。
变体脂肪酶对面团特性和最终烘焙食品的影响与亲本脂肪酶(LIP062),阴性对照(无DATEM)和含有0.4%(基于面粉)的DATEM(Lametop LT 552)参照进行比较。通过涉及重量的15个面包卷的长度、宽度和高度的测量,来测定体积效应。阴性对照定义为0%。由熟练的烘焙师评估面团特性,并与阴性对照比较进行描述。
Claims (17)
1.亲本脂肪酶的变体多肽,其中所述氨基酸取代组合为S83T、I85V和D265T,或者S83T、I85V、I255A和D265S,其中所述亲本脂肪酶由SEQ ID NO:1的氨基酸序列组成,或所述亲本脂肪酶由SEQ ID NO:2的核苷酸序列编码,并且其中变体多肽具有脂肪酶活性。
2.组合物,其包含权利要求1所述的变体多肽。
3.组合物,其包含权利要求1所述的变体多肽和至少第二种酶。
4.权利要求3所述的组合物,其中所述第二种酶选自:第二脂肪酶、淀粉酶、木聚糖酶、蛋白酶、纤维素酶、葡糖淀粉酶、氧化还原酶、磷脂酶和环糊精葡聚糖转移酶。
5.权利要求3所述的组合物,其中所述第二种酶为β-淀粉酶。
6.权利要求2-5中任一项所述的组合物,进一步包含载体、稳定剂、缓冲剂、防腐剂或其任何组合。
7.变体多核苷酸,其编码权利要求1所述的变体多肽。
8.权利要求7所述的变体多核苷酸,其中所述变体多核苷酸是与SEQ ID NO:2所示的核酸序列至少80%同一的核酸序列。
9.制备变体多肽的方法,其包括:提供权利要求7或8所述的变体多核苷酸,将模板核酸序列转化到表达宿主中,培养表达宿主以产生变体多肽,并纯化所述变体多肽。
10.权利要求9所述的方法,其中所述表达宿主选自:细菌表达***、真菌表达***和合成表达***。
11.权利要求9所述的方法,其中所述表达宿主为酵母表达***。
12.权利要求10所述的方法,其中所述细菌表达***选自大肠杆菌、芽孢杆菌属(Bacillus)、假单胞菌属(Pseudomonas)和链霉菌属(Streptomyces)。
13.权利要求11所述的方法,其中所述酵母表达***选自念珠菌属(Candida)、毕赤酵母属(Pichia)、酵母属(Saccharomyces)和裂殖酵母属(Schizosaccharomyces)。
14.权利要求10所述的方法,其中所述真菌表达***选自青霉属(Penicillium)、曲霉属(Aspergillus)、镰刀菌属(Fusarium)、毁丝霉属(Myceliopthora)、根毛菌属(Rhizomucor)、根霉菌属(Rhizopus)、嗜热真菌属(Thermomyces)和木霉属(Trichoderma)。
15.一种不添加乳化剂的制备面团或由面团制备的烘焙产品的方法,所述方法包括将一种如权利要求1所述的变体多肽或权利要求2-5中任一项所述的组合物添加到面团中并烘焙所述面团。
16.权利要求15所述的方法,其中所述乳化剂选自:硬脂酰乳酸钙(CSL)、单甘油二乙酰酒石酸酯(DATEM)、乙氧基化甘油单酯和甘油二酯(EMG)、聚山梨醇酯(PS)、硬脂酰乳酸钠(SSL)和琥珀酰化甘油单酯(SMG)。
17.用于面团或由面团制备的烘焙产品的预混物,其包含至少一种权利要求1所述的变体多肽或权利要求2-5中任一项所述的组合物。
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- 2018-05-10 EP EP18797619.6A patent/EP3621984A4/en active Pending
- 2018-05-10 CN CN201880046631.3A patent/CN110914292A/zh active Pending
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- 2018-05-10 CA CA3061178A patent/CA3061178A1/en active Pending
- 2018-05-10 EP EP18798843.1A patent/EP3621443A4/en active Pending
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CA3061178A1 (en) | 2018-11-15 |
US11214777B2 (en) | 2022-01-04 |
CN110621162A (zh) | 2019-12-27 |
EP3621443A4 (en) | 2021-03-17 |
EP3621443A1 (en) | 2020-03-18 |
US20200208082A1 (en) | 2020-07-02 |
AU2018265136A1 (en) | 2019-11-21 |
RU2019140871A3 (zh) | 2021-09-09 |
RU2019140871A (ru) | 2021-06-16 |
WO2018209018A1 (en) | 2018-11-15 |
US20200205423A1 (en) | 2020-07-02 |
WO2018209026A1 (en) | 2018-11-15 |
MX2019013532A (es) | 2020-08-03 |
US11162086B2 (en) | 2021-11-02 |
CN110914292A (zh) | 2020-03-24 |
JP2020519288A (ja) | 2020-07-02 |
EP3621984A1 (en) | 2020-03-18 |
BR112019023695A2 (pt) | 2020-06-09 |
EP3621984A4 (en) | 2021-03-03 |
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