CN116828989A - 天然干酪的制造方法 - Google Patents
天然干酪的制造方法 Download PDFInfo
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Abstract
本发明的目的在于，提供一种在使用来自微生物的脂肪酶的天然干酪的制造技术中促进短链脂肪酸游离的技术。通过天然干酪的制造方法能够促进短链脂肪酸的游离，所述制造方法包括使来自微生物的脂肪酶和来自丝状菌的蛋白酶作用于乳汁的工序。
Description
技术领域
本发明涉及天然干酪的制造方法。
背景技术
脂肪酶是催化脂质基质中的酯键水解的酶，带来脂肪酸的游离。在乳业用途中，脂肪酶被用于乳制品的风味生成和/或风味增强，特别是在干酪的制造中发挥重要的作用。
作为在干酪的制造中使用的脂肪酶，传统上使用来自仔山羊、仔牛或仔羊等反刍动物的脂肪酶。来自反刍动物的脂肪酶从乳脂肪游离短链脂肪酸(特别是碳原子数为4的丁酸)的特性优异，该优异的特性长期用于干酪的风味生成和/或风味增强。
另一方面，为了对食品加工中利用的酶制剂的洁食品质或清真品质的要求，对于来自动物的脂肪酶的替代品有强烈的产业要求。为了应对该要求，提出了来自微生物的酶的利用(例如专利文献1)和重组酶的利用(例如专利文献2)等，另外，为了应用于特定用途，还有在基因工程学上改变脂肪酶的尝试(例如专利文献3～5)，还提出了对短链脂肪酸的游离能力低的特定的来自微生物的脂肪酶，导入通过提高短链脂肪酸的游离能力而赋予短链脂肪酸选择性的突变的脂肪酶(专利文献6和7)。
现有技术文献
专利文献
专利文献1：日本特开昭61-135541号公报
专利文献2：美国专利申请公开第2004/0001819号说明书
专利文献3：日本特表2011-512809号公报
专利文献4：日本特表2003-524386号公报
专利文献5：日本特表2004-517639号公报
专利文献6：国际公开第2015/087833号
专利文献7：国际公开第2017/211930号
发明内容
发明所要解决的技术问题
本发明人认为能够将在天然干酪的制造中传统使用的来自反刍动物的脂肪酶替换为具有短链脂肪酸选择性的来自微生物的脂肪酶。然而，即使在天然干酪的制造中使用该来自微生物的脂肪酶，也与预测相反，面临着无法充分发挥短链脂肪酸的游离能力这样的新课题。
因此，本发明的目的在于，提供在使用来自微生物的脂肪酶的天然干酪的制造技术中促进短链脂肪酸的游离的技术。
用于解决技术问题的技术方案
本发明人认为，即使在天然干酪的制造中使用来自微生物的脂肪酶，也无法发挥短链脂肪酸的游离能力的原因在于脂肪酶处于无法与脂肪球反应的环境。脂肪球的破坏通常使用乳化剂，但在天然干酪的制造中伴随不使用乳化剂的制约，因此无法使用乳化剂。因此，本发明人进行了深入研究，结果发现：通过与脂肪酶一起并用来自丝状菌的蛋白酶，脂肪酶变得容易作用，促进短链脂肪酸的游离。
即，本发明提供下述揭示的方式的发明。
项1.一种天然干酪的制造方法，其包括使来自微生物的脂肪酶和来自丝状菌的蛋白酶作用于乳汁的工序。
项2.根据项1所述的制造方法，其中，所述来自微生物的脂肪酶包含以下(1)～(3)所示的至少任一种多肽：
(1)多肽，其包含序列号1～15中任一者所示的氨基酸序列、或从序列号1～15所示的氨基酸序列中分别去除第1～15位的氨基酸序列而成的氨基酸序列；
(2)多肽，其是在序列号1～15中任一者所示的氨基酸序列、或从序列号1～15所示的氨基酸序列中分别去除第1～15位的氨基酸序列而成的氨基酸序列中，1个或多个氨基酸残基被替换、添加、插入或缺失而成的，且具有短链脂肪酸选择性；以及
(3)多肽，其相对于序列号1～15中任一者所示的氨基酸序列、或从序列号1～15所示的氨基酸序列中分别去除第1～15位的氨基酸序列而成的氨基酸序列的序列一致性为70％以上，且具有短链脂肪酸选择性。
项3.根据项1或2所述的制造方法，其中，所述来自微生物的脂肪酶的使用量相对于上述乳汁中的1g乳脂肪为1LU以上。
项4.根据项1～3中任一项所述的制造方法，其中，所述来自丝状菌的蛋白酶的使用量相对于所述来自微生物的脂肪酶1LU为0.02U以上。
项5.根据项1～4中任一项所述的制造方法，其中，所述来自丝状菌的蛋白酶的使用量相对于所述乳汁中的1g乳蛋白为0.3U以上。
项6.根据项5所述的制造方法，其中，所述来自丝状菌的蛋白酶的使用量相对于所述乳汁中的1g乳蛋白为20U以下。
项7.根据项1～6中任一项所述的制造方法，其中，所述来自丝状菌的蛋白酶为来自曲霉属的蛋白酶。
项8.一种天然干酪，其包含来自微生物的脂肪酶和来自丝状菌的蛋白酶。
项9.一种使用来自微生物的脂肪酶的天然干酪制造中的短链脂肪酸游离促进剂，其包含来自丝状菌的蛋白酶。
项10.一种干酪加工品的制造方法，其包括通过项1～7中任一项所述的制造方法制造天然干酪的工序；以及加工所述天然干酪的工序。
项11.一种干酪加工品，其通过项10所述的制造方法制造。
发明效果
根据本发明，提供一种在使用来自微生物的脂肪酶的天然干酪的制造技术中，促进短链脂肪酸的游离的技术。
附图说明
图1表示试验例1中得到的天然干酪中的游离脂肪酸组成。表示各脂肪酸游离量的6根柱状图，从左起依次表示比较例1、参考例1、比较例2、实施例1、实施例2和比较例3的游离量。
图2表示试验例2中得到的天然干酪中的游离脂肪酸组成。
具体实施方式
以下，详细地说明本发明。应予说明，在序列表以外，氨基酸序列中的20种氨基酸残基有时用单字符缩写来表示。即，甘氨酸(Gly)为G，丙氨酸(Ala)为A，缬氨酸(Val)为V，亮氨酸(Leu)为L，异亮氨酸(Ile)为I，苯丙氨酸(Phe)为F，酪氨酸(Tyr)为Y，色氨酸(Trp)为W，丝氨酸(Ser)为S，苏氨酸(Thr)为T，半胱氨酸(Cys)为C，蛋氨酸(Met)为M，天冬氨酸(Asp)为D，谷氨酸(Glu)为E，天冬酰胺(Asn)为N，谷氨酰胺(Gln)为Q，赖氨酸(Lys)为K，精氨酸(Arg)为R，组氨酸(His)为H，脯氨酸(Pro)为P。
本说明书中，所示的氨基酸序列的左端为N末端，右端为C末端。
在本说明书中，“非极性氨基酸”包含丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、蛋氨酸、苯丙氨酸和色氨酸。“非电荷氨基酸”包含甘氨酸、丝氨酸、苏氨酸、半胱氨酸、酪氨酸、天冬酰胺和谷氨酰胺。“酸性氨基酸”包含天冬氨酸和谷氨酸。“碱性氨基酸”包含赖氨酸、精氨酸和组氨酸。
本说明书中，“替换”不仅包括人为地导入氨基酸残基替换的情况，还包括自然地导入氨基酸残基替换的情况，即还包含原本氨基酸残基就不同的情况。本说明书中，氨基酸残基的替换可以为人为的替换，也可以为自然的替换，优选人为的替换。
1.天然干酪的制造方法
本发明的天然干酪的制造方法的特征在于，包括使来自微生物的脂肪酶和来自丝状菌的蛋白酶作用于乳汁的工序。以下，对本发明的天然干酪的制造方法进行详述。
1-1.天然干酪
在本发明中，天然干酪属于使乳汁的凝乳熟化而成的干酪类，且不包含乳化剂。天然干酪可以是软质干酪、半硬质干酪(半硬干酪)、硬质/超硬质干酪(硬质干酪)中的任一种。
1-2.乳汁
作为乳汁的种类，可没有特别限定地使用通常的干酪的制造中使用的乳汁。作为具体的乳汁，可举出牛奶、山羊奶、水牛奶、羊奶等。这些乳汁可以单独使用1种，也可以组合使用多种。这些乳汁中，可优选举出牛奶。
1-3.来自微生物的脂肪酶
本发明中使用的来自微生物的脂肪酶中，只要具有短链脂肪酸选择性，就包含微生物中发现的天然脂肪酶和将上述天然脂肪酶的序列作为基本骨架而人为改变得到的脂肪酶中的任一种。
在本发明中，“短链脂肪酸选择性”是指，从脂质游离的脂肪酸中，与碳原子数为5以上的脂肪酸分别相比，提高碳原子数为4的丁酸的游离量(以摩尔基准进行对比)的特性。具体而言，“短链脂肪酸选择性”可以通过以下方式确认：以乳脂肪(破坏脂肪球，使乳脂肪露出的状态的乳脂肪)2g为底物，将多肽(脂肪酶)供于以每1g乳脂肪3LU的量在40℃下作用30分钟的条件后，测定C4、C6、C8、C10、C12、C14、C16、C18和C18：1的脂肪酸的游离量时，C4的脂肪酸(丁酸)的游离量大于C6、C8、C10、C12、C14、C16、C18和C18：1的脂肪酸各自的游离量(以摩尔基准进行对比)。
在本发明的制造方法中，上述的来自微生物的脂肪酶其自身具有提高短链脂肪酸的游离能力的特性，但即使在天然干酪的制造中仅替换来自反刍动物的脂肪酶无法发挥该特性，通过与来自丝状菌的蛋白酶并用，才能够发挥该特性。
作为本发明中使用的来自微生物的脂肪酶的具体例，可举出包含以下(1)～(3)所示的至少任一种多肽的脂肪酶。
(1)多肽，其中，包含序列号1～15中任一者所示的氨基酸序列、或从序列号1～15所示的氨基酸序列中分别去除第1～15位的氨基酸序列而成的氨基酸序列；
(2)多肽，其中，在序列号1～15中任一者所示的氨基酸序列、或从序列号1～15所示的氨基酸序列中分别去除第1～15位的氨基酸序列而成的氨基酸序列中，1个或多个氨基酸残基被替换、添加、插入或缺失而成，且具有短链脂肪酸选择性；以及
(3)多肽，其中，相对于序列号1～15中任一者所示的氨基酸序列、或从序列号1～15所示的氨基酸序列中分别去除第1～15位的氨基酸序列而成的氨基酸序列的序列一致性为70％以上，且具有短链脂肪酸选择性。
上述(1)～(3)所示的多肽具有脂肪酶活性和短链脂肪酸选择性。
上述(1)的多肽中，包含序列号1所示的氨基酸序列的多肽是以来自柱状假丝酵母(Candida cylindracea)的野生型脂肪酶LIP1(包含信号肽)为基本骨架，通过将LIP1的由549个氨基酸残基构成的氨基酸序列的第261位氨基酸残基即P替换为A而以具备短链脂肪酸选择性的方式突变得到的公知的脂肪酶。
上述(1)的多肽中，包含序列号2所示的氨基酸序列的多肽是以上述脂肪酶LIP1(包含信号肽)为基本骨架，通过将LIP1的由549个氨基酸残基构成的氨基酸序列的第261位氨基酸残基即P替换为F而以具备短链脂肪酸选择性的方式突变得到的公知的脂肪酶。
上述(1)的多肽中，包含序列号3所示的氨基酸序列的多肽是以上述脂肪酶LIP1(包含信号肽)为基本骨架，通过将LIP1的由549个氨基酸残基构成的氨基酸序列的第261位氨基酸残基即P替换为L而以具备短链脂肪酸选择性的方式突变得到的公知的脂肪酶。
上述(1)的多肽中，包括序列号4所示的氨基酸序列的多肽是以上述脂肪酶LIP1(包含信号肽)为基本骨架，通过将LIP1的由549个氨基酸残基构成的氨基酸序列的第261位氨基酸残基即P替换为S而以具备短链脂肪酸选择性的方式突变得到的公知的脂肪酶。
上述(1)的多肽中，包含序列号5所示的氨基酸序列的多肽是以上述脂肪酶LIP1(包含信号肽)为基本骨架，通过将LIP1的由549个氨基酸残基构成的氨基酸序列的第322位氨基酸残基即L替换为W而以具备短链脂肪酸选择性的方式突变得到的公知的脂肪酶。
上述(1)的多肽中，包含序列号6所示的氨基酸序列的多肽是以上述脂肪酶LIP1(包含信号肽)为基本骨架，通过将LIP1的由549个氨基酸残基构成的氨基酸序列的第360位氨基酸残基即F替换为L而以具备短链脂肪酸选择性的方式突变得到的公知的脂肪酶。
上述(1)的多肽中，包含序列号7所示的氨基酸序列的多肽是以上述脂肪酶LIP1(包含信号肽)为基本骨架，通过将LIP1的由549个氨基酸残基构成的氨基酸序列的第380位氨基酸残基即S替换为I而以具备短链脂肪酸选择性的方式突变得到的公知的脂肪酶。
上述(1)的多肽中，包含序列号8所示的氨基酸序列的多肽是以上述脂肪酶LIP1(包含信号肽)为基本骨架，通过将LIP1的由549个氨基酸残基构成的氨基酸序列的第380位氨基酸残基即S替换为L而以具备短链脂肪酸选择性的方式突变得到的公知的脂肪酶。
上述(1)的多肽中，包含序列号9所示的氨基酸序列的多肽是以上述脂肪酶LIP1(包含信号肽)为基本骨架，通过将LIP1的由549个氨基酸残基构成的氨基酸序列的第425位氨基酸残基即L替换为W而以具备短链脂肪酸选择性的方式突变得到的公知的脂肪酶。
上述(1)的多肽中，包含序列号10所示的氨基酸序列的多肽是以上述脂肪酶LIP1(包含信号肽)为基本骨架，通过将LIP1的由549个氨基酸残基构成的氨基酸序列的第428位氨基酸残基即L替换为F而以具备短链脂肪酸选择性的方式突变得到的公知的脂肪酶。
上述(1)的多肽中，包含序列号11所示的氨基酸序列的多肽是以上述脂肪酶LIP1(包含信号肽)为基本骨架，通过将LIP1的由549个氨基酸残基构成的氨基酸序列的第428位氨基酸残基即L替换为N而以具备短链脂肪酸选择性的方式突变得到的公知的脂肪酶。
上述(1)的多肽中，包含序列号12所示的氨基酸序列的多肽是以上述脂肪酶LIP1(包含信号肽)为基本骨架，通过将LIP1的由549个氨基酸残基构成的氨基酸序列的第429位氨基酸残基即G替换为F而以具备短链脂肪酸选择性的方式突变得到的公知的脂肪酶。
上述(1)的多肽中，包含序列号13所示的氨基酸序列的多肽是以上述脂肪酶LIP1(包含信号肽)为基本骨架，通过将LIP1的由549个氨基酸残基构成的氨基酸序列的第429位氨基酸残基即G替换M而以具备短链脂肪酸选择性的方式突变得到的公知的脂肪酶。
上述(1)的多肽中，包含序列号14所示的氨基酸序列的多肽是以上述脂肪酶LIP1(包含信号肽)为基本骨架，通过将LIP1的由549个氨基酸残基构成的氨基酸序列的第429位氨基酸残基即G替换为I而以具备短链脂肪酸选择性的方式突变得到的公知的脂肪酶。
上述(1)的多肽中，包含序列号15所示的氨基酸序列的多肽是以上述脂肪酶LIP1(包含信号肽)为基本骨架，通过将LIP1的由549个氨基酸残基构成的氨基酸序列的第549位氨基酸残基即V替换为F而以具备短链脂肪酸选择性的方式突变得到的公知的脂肪酶。
序列号1～15所示的氨基酸序列彼此的序列一致性极高。例如，相对于序列号13所示的氨基酸序列，序列号12和14的氨基酸序列仅被替换了1个氨基酸残基，序列号1～11和15的氨基酸序列仅被替换了2个氨基酸残基。序列号1～15所示的氨基酸序列均为1～15位的氨基酸序列形成信号肽序列，这些信号肽序列完全一致。
上述(1)的多肽中，包含从序列号1～15所示的氨基酸序列分别除去第1～15位的氨基酸序列(信号肽序列)的氨基酸序列的多肽分别为序列号1～15所示的氨基酸序列的成熟序列。
上述(2)和(3)的多肽包含以上述(1)的多肽的氨基酸序列为基本骨架的氨基酸序列。上述(2)和(3)的多肽与上述(1)的多肽相比，虽然氨基酸序列可见不同，但与上述(1)的多肽相同具有脂肪酶活性和短链脂肪酸选择性。
关于上述(2)和(3)的多肽的短链脂肪酸选择性，只要满足上述短链脂肪酸选择性就没有特别限定，作为短链脂肪酸选择性的程度的示例，可举出在测定将多肽供于上述的条件而得到的C4的脂肪酸的游离量(摩尔基准)与C6、C8、C10、C12、C14、C16、C18和C18：1的脂肪酸各自的游离量(摩尔基准)的总量的情况下，C4的脂肪酸的游离量相对于上述总量的比率为对应的上述(1)的多肽的上述比率的0.8～1.2倍的程度。应予说明，“对应的上述(1)的多肽”是指在上述(2)和(3)的多肽中包含作为基准的氨基酸序列的多肽。举出示例，在上述(2)和(3)的多肽分别为“在序列号13所示的氨基酸序列中，1个或数个氨基酸残基被替换、添加、插入或缺失而成，且具有短链脂肪酸选择性的多肽”和“相对于序列号13所示的氨基酸序列的序列一致性为70％以上且具有短链脂肪酸选择性的多肽”的情况下，与各个多肽“对应的上述(1)的多肽”为“包含序列号13所示的氨基酸序列的多肽”。
上述(2)的多肽中的氨基酸的差异可以仅包括替换、添加、插入和缺失中的1种差异(例如替换)，也可以包含2种以上差异(例如，替换和插入)。在上述(2)的多肽中，氨基酸的差异的数量可以为1个或数个，例如可举出1～50个，优选为1～20个、1～10个、1～8个、1～7个、
1～6个、1～5个、或1～4个，进一步优选为1～3个，特别优选为1或2个或者1个。
另外，在上述(3)的多肽中，相对于序列号1～15中任一者所示的氨基酸序列、或从序列号1～15所示的氨基酸序列分别去除第1～15位的氨基酸序列而得到的氨基酸序列的序列一致性只要为70％以上即可，可举出优选为80％以上、85％以上或90％以上，更优选为95％以上、96％以上、97％以上、或98％以上，进一步优选为99％以上、99.4％以上、99.6％以上、或99.8％以上。
在此，在上述(3)的多肽中，“序列一致性”表示通过BLASTPACKAGE[sgi32 bitedition，Version 2.0.12；available from National Center for BiotechnologyInformation(NCBI)]的bl2seq program(Tatiana A.Tatsusova，Thomas L.Madden，FEMSMicrobiol.Lett.，Vol.174，p247-250，1999)得到的氨基酸序列的一致性的值。参数设定为Gap insertion Cost value：11、Gap extension Cost value：1即可。
在上述(2)和(3)的多肽中，认为序列号1～15中任一者所示的氨基酸序列中的第224位(S)、第356位(E)、和第464位(H)的氨基酸残基有助于脂肪酶活性，因此优选在这些部位不导入替换或缺失。另外，认为序列号1、2、3、4的第261位、序列号5的第322位、序列号6的第360位、序列号7、8的第380位、序列号9的第425位、序列号10、11的第428位、序列号12、13、14的第429位、序列号15的第549位的氨基酸残基有助于底物特异性，因此优选在这些部位不导入替换或缺失。即，在上述(2)的多肽中，被替换、添加、插入或缺失的氨基酸的部位优选为被认为有助于上述脂肪酶活性的部位和被认为有助于底物特异性的部位以外的部位，在上述(3)的多肽中，上述序列一致性优选为除了被认为有助于上述脂肪酶活性的部位和被认为有助于底物特异性的部位以外的部分的序列一致性。
在上述(2)和(3)的多肽中导入有氨基酸替换的情况下，作为氨基酸替换的方式，可举出保守性替换。即，作为在(2)和(3)的多肽中导入的氨基酸替换，例如可举出：如果替换前的氨基酸为非极性氨基酸，则替换为其他非极性氨基酸；如果替换前的氨基酸为非带电性氨基酸，则替换为其他非带电性氨基酸；如果替换前的氨基酸为酸性氨基酸，则替换为其他酸性氨基酸；以及如果替换前的氨基酸为碱性氨基酸，则替换为其他碱性氨基酸。
应予说明，在上述(1)～(3)的多肽中，不仅包括人为替换而得到的多肽，还包括原本具有这样的氨基酸序列的多肽。
作为来自微生物的脂肪酶的使用量，只要能得到本发明期望的效果就没有特别限定，例如，可举出相对于材料中使用的乳汁中的1g乳脂肪为1LU以上。从更进一步提高短链脂肪酸的游离促进效果的观点出发，作为来自微生物的脂肪酶的使用量，可举出相对于乳汁中的1g乳脂肪，优选为1.4LU以上，更优选为2LU以上，进一步优选为2.5LU以上，进一步优选为3LU以上，更进一步优选为3.5LU以上，特别优选为4LU以上。作为来自微生物的脂肪酶的使用量范围的上限，没有特别限定，例如可举出相对于乳汁中的1g乳脂肪为20LU以下、10LU以下、8LU以下、5LU以下、或4.5LU以下。
应予说明，来自微生物的脂肪酶的脂肪酶活性是以三丁酸甘油酯为底物，通过脂肪酶活性试验法测定。即，在本发明中，将三丁酸甘油酯作为底物通过常规方法进行酶反应，将1分钟内带来1μmol丁酸的增加的酶量定义为1LU的脂肪酶活性。
1-4.来自丝状菌的蛋白酶
本发明中使用的来自丝状菌的蛋白酶与不并用来自丝状菌的蛋白酶而单独使用来自微生物的脂肪酶的情况相比，促进短链脂肪酸即丁酸的游离。作为能够得到这样的效果的机理之一，认为可能是来自丝状菌的蛋白酶破坏脂肪球，由此使来自微生物的脂肪酶容易作用于乳脂肪。
应予说明，本发明中使用的来自丝状菌的蛋白酶，在不具有凝乳活性的方面，与作为凝乳酶的示例的来自丝状菌的蛋白酶不同。即，从在本发明中使用的来自丝状菌的蛋白酶中除去凝乳酶。
作为来自丝状菌的蛋白酶的种类，在基于催化结构的分类中，可以是丝氨酸蛋白酶、金属蛋白酶、硫醇蛋白酶和天冬氨酸蛋白酶(酸性蛋白酶)，可优选举出金属蛋白酶。另外，作为来自丝状菌的蛋白酶的种类，在基于最适pH的分类中，可以是酸性蛋白酶、和中性蛋白酶、碱性蛋白酶，可优选举出中性蛋白酶。
来自丝状菌的蛋白酶只要能够得到本发明期望的效果且能够用于食品，就没有特别限定。作为来自丝状菌的蛋白酶的具体例，可举出来自曲霉(Aspergillus)属、毛霉(Mucor)属、脉孢菌(Neurospora)属、青霉(Penicillium)属、根毛霉(Rhizomucor)属、根霉(Rhizopus)属、和核盘菌(Sclerotinia)属等的蛋白酶。这些来自丝状菌的蛋白酶可以单独使用1种，也可以组合使用多种。在这些来自丝状菌的蛋白酶中，从进一步提高短链脂肪酸的游离促进效果的观点出发，可优选举出来自曲霉属的蛋白酶。
作为来自曲霉属的蛋白酶的具体例，可举出来自米曲霉(Aspergillus oryzae)、黑曲霉(Aspergillus niger)、蜂蜜曲霉(Aspergillus melleus)、日本曲霉(Aspergillusjaponicus)、泡盛曲霉(Aspergillus awamori)、白曲霉(Aspergillus kawachii)、酱油曲霉(Aspergillus sojae)、溜曲霉(Aspergillus tamarii)、臭曲霉(Aspergillusfoetidus)、烟曲霉(Aspergillus fumigatus)、构巢曲霉(Aspergillus nidulans)、棘孢曲霉(Aspergillus aculeatus)、亮白曲霉(Aspergillus candidus)、黄曲霉(Aspergillusflavus)、斋藤曲霉(Aspergillus saitoi)、乾氏曲霉(Aspergillus inuii)、灰绿曲霉(Aspergillus glaucus)、浅蓝灰曲霉(Aspergillus caesiellus)、棒曲霉(Aspergillusclavatus)、弯头曲霉(Aspergillus deflectus)、费雪式曲霉(Aspergillusfischerianus)、寄生曲霉(Aspergillus parasiticus)、青霉状曲霉(Aspergilluspenicilloides)、局限曲霉(Aspergillus restrictus)、聚多曲霉(Aspergillussydowii)、土曲霉(Aspergillus terreus)、焦曲霉(Aspergillus ustus)、杂色曲霉(Aspergillus versicolor)等的蛋白酶。这些来自曲霉属的蛋白酶可以单独使用1种，也可以组合使用多种。这些来自曲霉属的蛋白酶中，从更进一步提高短链脂肪酸的游离促进效果的观点出发，可优选举出来自米曲霉的蛋白酶。
来自丝状菌的蛋白酶可以通过公知的方法制备。作为示例，在制备来自曲霉属的蛋白酶的情况下，可以通过制曲曲霉属菌，使用公知的方法分离蛋白酶的方法、以及使用基因重组技术的方法等容易制备。另外，作为来自丝状菌的蛋白酶，可以使用市售品。作为市售品的来自丝状菌的蛋白酶，可举出天野酶株式会社制造的蛋白酶M“Amano”(来自米曲霉的酸性蛋白酶)、蛋白酶A“Amano”(来自米曲霉的中性蛋白酶)、蛋白酶A“Amano”2SD(来自米曲霉的中性蛋白酶)；新日本化学工业株式会社的Sumizyme MP(来自蜂蜜曲霉的碱性蛋白酶)、Sumizyme FL-G(来自米曲霉的碱性蛋白酶)等。
来自丝状菌的蛋白酶，从进一步提高短链脂肪酸的游离促进效果的观点出发，肽酶活性(外肽酶活性)相对于蛋白酶活性(全蛋白酶活性)的比率越多越好。
作为来自丝状菌的蛋白酶的使用量，只要能够得到本发明期望的效果就没有特别限定，例如，可举出相对于材料中使用的乳汁中的1g乳蛋白为0.3U以上。从更进一步提高短链脂肪酸的游离促进效果的观点出发，作为来自丝状菌的蛋白酶的使用量，可举出相对于乳汁中的1g乳蛋白，优选为0.6U以上，更优选为0.7U以上，进一步优选为0.8U以上，更进一步优选为0.9U以上、1.5U以上、2U以上、2.5U以上或3U以上。作为来自丝状菌的蛋白酶的使用量范围的上限，没有特别限定，例如，可举出相对于乳汁中的1g乳蛋白为20U以下，从提高得到的天然干酪的保形性的观点出发，相对于材料中使用的乳汁中的1g乳蛋白，优选为10U以下，更优选为5U以下，进一步优选为3U以下，进一步优选为2U以下，更进一步优选为1.5U以下，特别优选为1.2U以下。
关于来自丝状菌的蛋白酶的使用量相对于来自微生物的脂肪酶的使用量的比率，只要能得到本发明期望的效果就没有特别限定，从更进一步提高短链脂肪酸的游离促进效果的观点出发，作为相对于来自微生物的脂肪酶1LU的蛋白酶的使用量，例如可举出0.02U以上，优选为0.1U以上，更优选为0.15U以上，进一步优选为0.8U以上，进一步优选为2U以上，更进一步优选为5U以上、50U以上、或150U以上。作为来自丝状菌的蛋白酶的使用量相对于来自微生物的脂肪酶的使用量的比率的范围的上限，没有特别限定，例如可举出300U以下或200U以下。
应予说明，来自丝状菌的蛋白酶活性是以酪蛋白为底物，通过福林法进行测定。即，在本发明中，以酪蛋白为底物通过常规方法进行酶反应，将1分钟内带来相当于酪氨酸1μg的福林试液显色物质的增加的酶量定义为1U的来自丝状菌的蛋白酶活性。
1-5.凝乳酶
在本发明中，除了述酶以外，还可以使用凝乳酶。作为凝乳酶，可以没有特别限制地使用公知的凝乳酶，例如可举出：来自仔山羊、仔牛或仔羊等哺乳期的反刍动物的凝乳酶(rennet)；来自酵母、担子菌、丝状菌、细菌等微生物的凝乳酶(rennet)；基因重组凝乳酶(rennet或chymosin)；无花果的无花果蛋白酶、木瓜的木瓜蛋白酶、菠萝的菠萝蛋白酶等来自植物的凝乳酶(rennet)。
作为凝乳酶的使用量，本领域技术人员可以适当选择在天然干酪的制造工序中进行凝乳时使用的通常的量。
1-6.步骤和反应条件等、以及其他工序
本发明的天然干酪的制造方法中的具体的步骤只要设定为适时进行乳汁的乳酸发酵、利用凝乳酶的酪蛋白分子的凝固沉淀(凝乳化)、以及基于来自丝状菌的蛋白酶和来自微生物的脂肪酶的短链脂肪酸游离即可。
本发明中使用的来自丝状菌的蛋白酶和来自微生物的脂肪酶在乳汁的中添加时期只要能够得到本发明期望的效果就没有特别限定。例如，可举出乳汁的保温前或保温中；乳汁的乳酸发酵前、乳酸发酵中或乳酸发酵后；凝乳酶的添加前、添加时或添加后等。从提高乳汁中的乳脂肪与来自丝状菌的蛋白酶和来自微生物的脂肪酶的接触效率而提高天然干酪的制造效率的观点出发，来自丝状菌的蛋白酶和来自微生物的脂肪酶优选在凝乳酶添加前添加。
来自丝状菌的蛋白酶和来自微生物的脂肪酶可以同时添加，也可以阶段性添加。在阶段性添加来自丝状菌的蛋白酶和来自微生物的脂肪酶的情况下，来自丝状菌的蛋白酶和来自微生物的脂肪酶的添加顺序没有特别限定，作为示例，可以添加丝状菌蛋白酶，然后添加来自微生物的脂肪酶。
应予说明，用于乳酸发酵的乳酸菌的添加可以在任意的时机进行，优选可以在凝乳酶添加前的任意阶段添加。
来自丝状菌的蛋白酶、来自微生物的脂肪酶和凝乳酶添加时的温度只要能够得到本发明期望的效果就没有特别限定，例如可举出0～80℃，优选为10～60℃，进一步优选为25～40℃。另外，作为使来自丝状菌的蛋白酶和来自微生物的脂肪酶作用的时间(熟化期间)，可以根据天然干酪的种类来设定，从更进一步提高短链脂肪酸的游离量的观点出发，可举出优选为1个月以上，更优选为2个月以上，进一步优选为2.5个月以上。
在本发明的天然干酪的制造方法中，除了上述工序以外，还可以适当包括在通常的天然干酪的制造中进行的任意的工序。作为其他工序，可举出杀菌工序、加热工序、冷却工序、pH调整工序、热水中混炼工序、加盐工序、成型工序、在饱和食盐水中的浸渍工序、干燥工序和/或密封包装工序等。本领域技术人员可以根据天然干酪的种类等容易选择这些工序。
2.天然干酪
本发明的天然干酪包含制造时使用的上述来自微生物的脂肪酶和来自丝状菌的蛋白酶作为残留成分。本发明的天然干酪可以通过上述“1.天然干酪的制造方法”中记载的方法制造。
本发明的天然干酪与不使用上述来自丝状菌的蛋白酶以外同样制造的天然干酪相比，含有大量短链脂肪酸(丁酸)。对于短链脂肪酸(丁酸)的具体含量可以根据干酪的种类(水分量)、以及使用的来自微生物的脂肪酶和来自丝状菌的蛋白酶的量而不同，例如可举出0.8μmol/g以上，优选为4μmol/g以上。从进一步增强天然干酪的风味的观点出发，作为短链脂肪酸(丁酸)的具体含量，可举出优选为6μmol/g以上，更优选为8μmol/g以上，进一步优选为10μmol/g以上，更进一步优选为11μmol/g以上。
本发明的天然干酪其自身既可以作为天然干酪食用，也可以作为加工干酪的材料使用。加工干酪可以通过基于公知的方法适当进行天然干酪的加热熔融、乳化剂(柠檬酸盐、磷酸盐等)、添加物(油、防腐剂、调味料、增稠稳定剂、凝胶化剂、pH调节剂、香料等)的配合等来制造。进而，本发明的天然干酪也可以作为加工干酪以外的加工食品的材料使用。
3.天然干酪制造中的短链脂肪酸游离促进剂
如上所述，在使用来自微生物的脂肪酶的本发明的制造方法中，来自丝状菌的蛋白酶能够提高短链脂肪酸的游离促进效果。因此，本发明还提供一种使用来自生物的脂肪酶的天然干酪制造中的短链脂肪酸游离促进剂，其包含来自丝状菌的蛋白酶。
在本发明的短链脂肪酸游离促进剂中，关于各酶的详细情况和使用方法等，如上述“1.天然干酪的制造方法”中记载所示。
另外，本发明的短链脂肪酸游离促进剂，除各酶以外，还可以包含食品学上允许的其他成分。作为该其他成分，可举出赋形剂、崩解剂、防腐剂、保存材料、稳定剂、维生素、矿物质、甜味料、调味料等。
实施例
以下，举出实施例具体说明本发明，但本发明不被解释为限定于以下实施例。
使用酶
＜蛋白酶＞
·蛋白酶A“Amano”2SD(以下，记载为“PR-A2SD”)：来自丝状菌米曲霉(A.oryzae)的蛋白酶(天野酶株式会社制造)
·ProtinSD-NY10(以下，记载为“SD-NY10”)：来自细菌解淀粉芽孢杆菌(B.amyloliquefaciens)的蛋白酶(天野酶株式会社制造)
＜脂肪酶＞
·ItalaseC：来自动物的脂肪酶(DSM Food Specialties公司制造)
·G429M：属于包含从序列号13所示的氨基酸序列中除去了第1～15位的氨基酸序列的氨基酸序列的多肽的脂肪酶
活性值测定方法
蛋白酶和脂肪酶的活性值的测定方法如下所述。
＜蛋白酶活性＞
将0.6％(v/w)酪蛋白溶液(0.05mol/L磷酸氢钠，pH8.0[PRSD-NY10的情况]或pH6.0[PR-A2SD的情况])5mL在37℃下加热10分钟后，加入包含蛋白酶的试样溶液1mL并进行振荡混合。将该液体在37℃下放置10分钟后，加入三氯乙酸试液(1.8％三氯乙酸、1.8％乙酸钠和包含0.33mol/L乙酸的三氯乙酸[PRSD-NY10的情况]或0.44mol/L[PR-A2SD的情况])5mL并振荡混合，再次在37℃下放置30分钟，进行过滤。除去最初的滤液3mL，量取下一次滤液2mL，加入0.55mol/L碳酸钠试液5mL和福林试液(1→3)1mL并振荡混合，在37℃下放置30分钟。对于该液体(酶反应液)，以水作为对照，测定波长660nm处的吸光度AT。
另外，量取包含蛋白酶的试样溶液1mL，加入三氯乙酸试液(包含1.8％三氯乙酸、1.8％乙酸钠和0.33mol/L乙酸的三氯乙酸[PRSD-NY10的情况]或0.44mol/L[PR-A2SD的情况])5mL并振荡混合后，加入0.6％(v/w)酪蛋白溶液(0.05mol/L磷酸氢钠、pH8.0[PRSD-NY10的情况]或pH6.0[PR-A2SD的情况])5mL并振荡混合，对于除了在37℃下放置30分钟以外，与上述酶反应液相同进行操作的液体(空白)，测定吸光度AB。
将1分钟内带来相当于酪氨酸1μg的福林试液显色物质的增加的酶量设为1个单位(1U)。
量取1mg/mL酪氨酸标准原液(0.2mol/L盐酸中)1mL，2mL，3mL和4mL，分别加入0.2mol/L盐酸试液，制成100mL。量取各自的液体2mL，加入0.55mol/L碳酸钠试液5mL和福林试液(1→3)1mL并振荡混合，在37℃下放置30分钟。对于这些液体，将量取0.2mol/L盐酸试液2mL，与上述相同进行操作而得到的液体作为对照，测定波长660nm处的吸光度A1、A2、A3和A4。在纵轴取吸光度A1、A2、A3和A4，在横轴取各自的液体2mL中的酪氨酸量(μg)，制作标准曲线，求出相对于吸光度差1的酪氨酸量(μg)。
[数学式1]
蛋白酶活性(U/g，U/mL)＝(AT-AB)×F×11/2×1/10×1/M
AT：酶反应液的吸光度
AB：空白的吸光度
F：由酪氨酸标准曲线求出的吸光度差为1时的酪氨酸量(μg)
11/2：反应停止后的总液量的换算系数
1/10：反应时间每1分钟的换算系数
M：试样溶液1mL中的试样的量(g或mL)
＜脂肪酶活性＞
将0.6g/dL阿拉伯胶溶液(含有甘油)70mL、三丁酸甘油酯22.3mL和水329mL放入乳化器中，在14500±300min-1的条件下乳化15分钟(旋转：5分钟×3次，停止：5分钟×2次)，制成三丁酸甘油酯底物液。另一方面，量取适量的脂肪酶，使用冷水稀释，制成试样溶液。在平底试管中准确量取三丁酸甘油酯底物液30mL，在30±0.5℃下放置15分钟。放置后，浸渍pH电极，进行搅拌。(以后，在操作中连续搅拌。)将该液体使用0.05mol/L氢氧化钠液(定量用)调整至pH7.00±0.05。pH调整后，立即准确加入试样溶液2mL，开始反应。反应开始后，以保持pH7.00±0.05的方式立即开始0.05mol/L氢氧化钠液(定量用)的滴加，从反应开始准确地持续滴加5分钟的期间，每1分钟记录滴定量(V1～V5(mL))。
将1分钟内带来1.0μmol的丁酸的增加的酶量设为1个单位(1LU)。
[数学式2]
脂肪酶活性(LU/g)＝(V5-V1)/4×0.05×f×1000×D/2
V1：测定开始1分钟后的滴定量(mL)
V5：测定开始5分钟后的滴定量(mL)
0.05：0.05mol/L氢氧化钠液的当量浓度
f：0.05mol/L氢氧化钠液的系数
1000：单位换算系数(mmol→μmol)
D：稀释倍数(mL/g)
2：试样溶液添加量(mL)
短链脂肪酸选择性的确认
将乳脂肪(通过乳化剂破坏脂肪球，使乳脂肪露出的状态的脂肪)2g作为底物，将ItalaseC或G429M以每1g乳脂肪3LU的量供于在40℃下作用30分钟的条件后，测定C4、C6、C8、C10、C12、C14、C16、C18和C18：1的脂肪酸的游离量。其结果，确认到C4的脂肪酸(丁酸)的游离量大于C6、C8、C10、C12、C14、C16、C18和C18：1的脂肪酸各自的游离量(以摩尔基准的对比)。
试验例1
将牛奶(乳脂肪2.9重量％、乳蛋白3.3重量％)225kg(500lb)在73℃下加热杀菌19秒后用于材料。将杀菌后的牛奶冷却至34.4℃，添加乳酸菌(DSM CP-121)11.4g，搅拌约1小时直至pH降低至6.45。接着，添加14.3g的凝乳酶(rennet)(DSM Maxiren XDS BF；通过酵母表达来自牛的凝乳酶(chymosin)基因的重组凝乳酶(rennet))，停止搅拌并静置30分钟。确认凝乳的形成，将凝乳切成立方体状，边搅拌边加热至41℃后，分离乳清和凝乳。在凝乳20g中添加表1所示的酶并充分混合后，加热至65℃进行成型，移至容器，在8℃下熟化。对于熟化3个月后得到的天然干酪(半硬型的波罗夫洛干酪)，按照以下操作进行游离脂肪酸的分析。
将样品(天然干酪)5g和0.2M磷酸缓冲液(pH6.8)5mL充分混合而制成浆液，将浆液1g、2.5M硫酸0.05mL、0.01M壬酸(C9，内部标准)0.05mL、以及氯仿2.5mL以涡旋方式混合，提取游离脂肪酸。进行离心分离(7000rpm，10分钟)回收氯仿相，利用气相色谱(Agilent公司，柱：CP-Wax58)进行游离脂肪酸组成的分析。将结果示于表1和图1。
[表1]
如表1所示，在天然干酪的制造中使用来自动物的脂肪酶的情况下(参考例1)，短链脂肪酸(碳原子数为4的丁酸)充分游离，但在使用来自微生物的脂肪酶的情况下(比较例2)，只能确认到未使用脂肪酶的情况(比较例1)程度的短链脂肪酸。另一方面，在来自微生物的脂肪酶中并用来自丝状菌的蛋白酶的情况下(实施例1、2)，与比较例2相比，游离的短链脂肪酸量增加，其效果依赖于来自丝状菌的蛋白酶的添加量而增强。应予说明，在代替来自丝状菌的蛋白酶而使用来自细菌的蛋白酶的情况下，未确认到使短链脂肪酸量增加的效果(比较例3)。
试验例2
将市售牛奶(乳脂肪3.5重量％、乳蛋白3重量％)3.8L加热至34℃，添加中温性乳酸菌(Mesophilic Direct Set Cheese Culture(C101))1g和嗜热性乳酸菌(ThermophilicDirect Set Cheese Culture(C201))1g与表2所示的酶。搅拌30分钟后，添加凝乳酶(rennet)(DSM Maxiren XDS BF)0.24g，静置35分钟。确认凝乳的形成，将凝乳粗切，边搅拌边用30分钟加热至41℃。pH降低至5.9后，分离乳清和凝乳。将回收的凝乳在38℃下浸渍1小时后，将凝乳切成立方体状，进而确认到pH降低至5.2，与食盐11.8g充分混合。接着，将凝乳在70℃的热水中进行处理直至充分产生拉伸后，从热水中取出并成型，在冷水中浸渍30分钟。进而在10℃的饱和食盐水中浸渍一夜后，将取出的干酪在室温下干燥2小时，密封包装，在8℃下进行熟化。对于熟化3个月后得到的天然干酪(半硬型的波罗夫洛干酪)，与试验例1相同进行游离脂肪酸的分析。将结果示于表2和图2。
另外，对于得到的天然干酪的风味，基于以下基准进行感官评价。短链脂肪酸表示碳原子数为4的丁酸。将结果示于表2。
+++：强烈地感觉到来自短链脂肪酸的风味。
++：感觉到来自短链脂肪酸的风味
+：微弱地感觉到来自短链脂肪酸的风味
-：感觉不到来自短链脂肪酸的风味
此外，关于得到的天然干酪的保形性，将以熟化前的天然干酪的形状为基准的情况的熟化后的形状变化为何种程度作为保形性的指标，基于以下基准进行评价。将结果示于表2。
◎：形状没有变化(保持形状)
〇：形状的变化为稍微崩塌的程度(几乎保持了形状)
△：确认到形状的变化为稍微崩塌的程度(勉强保持了形状)
×：形状的变化剧烈(无法保持形状)
[表2]
由表2可知，在来自微生物的脂肪酶中并用来自丝状菌的蛋白酶的情况下(实施例3～实施例6)，能够感觉到来自短链脂肪酸的风味，特别是在实施例5、实施例6中，能够显著感觉到该风味。应予说明，虽然数据中未示出，但除不使用来自丝状菌的蛋白酶以外，进行与实施例3～6相同的工序而制造的天然干酪中，未感觉到来自短链脂肪酸的风味。另外，来自微生物的脂肪酶的添加量越多(相对于实施例3、实施例4的实施例5、实施例6)、以及来自丝状菌的蛋白酶的添加量越多(相对于实施例3的实施例4、相对于实施例5的实施例6)，短链脂肪酸的游离量越增加，但在来自丝状菌的蛋白酶的添加量被抑制的实施例5中，确认到显著优异的保形性。
序列表自由文本
序列号1是来自柱状假丝酵母的包含信号肽的脂肪酶LIP1的P261A突变体。
序列号2是来自柱状假丝酵母的包含信号肽的脂肪酶LIP1的P261F突变体。
序列号3是来自柱状假丝酵母的包含信号肽的脂肪酶LIP1的P261L突变体。
序列号4是来自柱状假丝酵母的包含信号肽的脂肪酶LIP1的P261S突变体。
序列号5是来自柱状假丝酵母的包含信号肽的脂肪酶LIP1的L322W突变体。
序列号6是来自柱状假丝酵母的包含信号肽的脂肪酶LIP1的F360L突变体。
序列号7是来自柱状假丝酵母的包含信号肽的脂肪酶LIP1的S380I突变体。
序列号8是来自柱状假丝酵母的包含信号肽的脂肪酶LIP1的S380L突变体。
序列号9是来自柱状假丝酵母的包含信号肽的脂肪酶LIP1的L425W突变体。
序列号10是来自柱状假丝酵母的包含信号肽的脂肪酶LIP1的L428F突变体。
序列号11是来自柱状假丝酵母的包含信号肽的脂肪酶LIP1的L428N突变体。
序列号12是来自柱状假丝酵母的包含信号肽的脂肪酶LIP1的G429F突变体。
序列号13是来自柱状假丝酵母的包含信号肽的脂肪酶LIP1的G429M突变体。
序列号14是来自柱状假丝酵母的包含信号肽的脂肪酶LIP1的G429I突变体。
序列号15是来自柱状假丝酵母的包含信号肽的脂肪酶LIP1的V549F突变体。
序列表
<110> 天野酶制品株式会社
<120> 天然干酪的制造方法
<130> 22004WO
<150> JP 2021-016920
<151> 2021-02-04
<160> 15
<170> PatentIn version 3.5
<210> 1
<211> 549
<212> PRT
<213> 人工序列
<220>
<223> 来自柱状假丝酵母（Candida cylindracea）的包含信号肽的脂肪酶LIP1的P261A突变体
<400> 1
Met Glu Leu Ala Leu Ala Leu Ser Leu Ile Ala Ser Val Ala Ala Ala
1 5 10 15
Pro Thr Ala Thr Leu Ala Asn Gly Asp Thr Ile Thr Gly Leu Asn Ala
20 25 30
Ile Ile Asn Glu Ala Phe Leu Gly Ile Pro Phe Ala Glu Pro Pro Val
35 40 45
Gly Asn Leu Arg Phe Lys Asp Pro Val Pro Tyr Ser Gly Ser Leu Asp
50 55 60
Gly Gln Lys Phe Thr Ser Tyr Gly Pro Ser Cys Met Gln Gln Asn Pro
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Glu Gly Thr Tyr Glu Glu Asn Leu Pro Lys Ala Ala Leu Asp Leu Val
85 90 95
Met Gln Ser Lys Val Phe Glu Ala Val Ser Pro Ser Ser Glu Asp Cys
100 105 110
Leu Thr Ile Asn Val Val Arg Pro Pro Gly Thr Lys Ala Gly Ala Asn
115 120 125
Leu Pro Val Met Leu Trp Ile Phe Gly Gly Gly Phe Glu Val Gly Gly
130 135 140
Thr Ser Thr Phe Pro Pro Ala Gln Met Ile Thr Lys Ser Ile Ala Met
145 150 155 160
Gly Lys Pro Ile Ile His Val Ser Val Asn Tyr Arg Val Ser Ser Trp
165 170 175
Gly Phe Leu Ala Gly Asp Glu Ile Lys Ala Glu Gly Ser Ala Asn Ala
180 185 190
Gly Leu Lys Asp Gln Arg Leu Gly Met Gln Trp Val Ala Asp Asn Ile
195 200 205
Ala Ala Phe Gly Gly Asp Pro Thr Lys Val Thr Ile Phe Gly Glu Ser
210 215 220
Ala Gly Ser Met Ser Val Met Cys His Ile Leu Trp Asn Asp Gly Asp
225 230 235 240
Asn Thr Tyr Lys Gly Lys Pro Leu Phe Arg Ala Gly Ile Met Gln Ser
245 250 255
Gly Ala Met Val Ala Ser Asp Ala Val Asp Gly Ile Tyr Gly Asn Glu
260 265 270
Ile Phe Asp Leu Leu Ala Ser Asn Ala Gly Cys Gly Ser Ala Ser Asp
275 280 285
Lys Leu Ala Cys Leu Arg Gly Val Ser Ser Asp Thr Leu Glu Asp Ala
290 295 300
Thr Asn Asn Thr Pro Gly Phe Leu Ala Tyr Ser Ser Leu Arg Leu Ser
305 310 315 320
Tyr Leu Pro Arg Pro Asp Gly Val Asn Ile Thr Asp Asp Met Tyr Ala
325 330 335
Leu Val Arg Glu Gly Lys Tyr Ala Asn Ile Pro Val Ile Ile Gly Asp
340 345 350
Gln Asn Asp Glu Gly Thr Phe Phe Gly Thr Ser Ser Leu Asn Val Thr
355 360 365
Thr Asp Ala Gln Ala Arg Glu Tyr Phe Lys Gln Ser Phe Val His Ala
370 375 380
Ser Asp Ala Glu Ile Asp Thr Leu Met Thr Ala Tyr Pro Gly Asp Ile
385 390 395 400
Thr Gln Gly Ser Pro Phe Asp Thr Gly Ile Leu Asn Ala Leu Thr Pro
405 410 415
Gln Phe Lys Arg Ile Ser Ala Val Leu Gly Asp Leu Gly Phe Thr Leu
420 425 430
Ala Arg Arg Tyr Phe Leu Asn His Tyr Thr Gly Gly Thr Lys Tyr Ser
435 440 445
Phe Leu Ser Lys Gln Leu Ser Gly Leu Pro Val Leu Gly Thr Phe His
450 455 460
Ser Asn Asp Ile Val Phe Gln Asp Tyr Leu Leu Gly Ser Gly Ser Leu
465 470 475 480
Ile Tyr Asn Asn Ala Phe Ile Ala Phe Ala Thr Asp Leu Asp Pro Asn
485 490 495
Thr Ala Gly Leu Leu Val Lys Trp Pro Glu Tyr Thr Ser Ser Ser Gln
500 505 510
Ser Gly Asn Asn Leu Met Met Ile Asn Ala Leu Gly Leu Tyr Thr Gly
515 520 525
Lys Asp Asn Phe Arg Thr Ala Gly Tyr Asp Ala Leu Phe Ser Asn Pro
530 535 540
Pro Ser Phe Phe Val
545
<210> 2
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<213> 人工序列
<220>
<223> 来自柱状假丝酵母（Candida cylindracea）的包含信号肽的脂肪酶LIP1的P261F突变体
<400> 2
Met Glu Leu Ala Leu Ala Leu Ser Leu Ile Ala Ser Val Ala Ala Ala
1 5 10 15
Pro Thr Ala Thr Leu Ala Asn Gly Asp Thr Ile Thr Gly Leu Asn Ala
20 25 30
Ile Ile Asn Glu Ala Phe Leu Gly Ile Pro Phe Ala Glu Pro Pro Val
35 40 45
Gly Asn Leu Arg Phe Lys Asp Pro Val Pro Tyr Ser Gly Ser Leu Asp
50 55 60
Gly Gln Lys Phe Thr Ser Tyr Gly Pro Ser Cys Met Gln Gln Asn Pro
65 70 75 80
Glu Gly Thr Tyr Glu Glu Asn Leu Pro Lys Ala Ala Leu Asp Leu Val
85 90 95
Met Gln Ser Lys Val Phe Glu Ala Val Ser Pro Ser Ser Glu Asp Cys
100 105 110
Leu Thr Ile Asn Val Val Arg Pro Pro Gly Thr Lys Ala Gly Ala Asn
115 120 125
Leu Pro Val Met Leu Trp Ile Phe Gly Gly Gly Phe Glu Val Gly Gly
130 135 140
Thr Ser Thr Phe Pro Pro Ala Gln Met Ile Thr Lys Ser Ile Ala Met
145 150 155 160
Gly Lys Pro Ile Ile His Val Ser Val Asn Tyr Arg Val Ser Ser Trp
165 170 175
Gly Phe Leu Ala Gly Asp Glu Ile Lys Ala Glu Gly Ser Ala Asn Ala
180 185 190
Gly Leu Lys Asp Gln Arg Leu Gly Met Gln Trp Val Ala Asp Asn Ile
195 200 205
Ala Ala Phe Gly Gly Asp Pro Thr Lys Val Thr Ile Phe Gly Glu Ser
210 215 220
Ala Gly Ser Met Ser Val Met Cys His Ile Leu Trp Asn Asp Gly Asp
225 230 235 240
Asn Thr Tyr Lys Gly Lys Pro Leu Phe Arg Ala Gly Ile Met Gln Ser
245 250 255
Gly Ala Met Val Phe Ser Asp Ala Val Asp Gly Ile Tyr Gly Asn Glu
260 265 270
Ile Phe Asp Leu Leu Ala Ser Asn Ala Gly Cys Gly Ser Ala Ser Asp
275 280 285
Lys Leu Ala Cys Leu Arg Gly Val Ser Ser Asp Thr Leu Glu Asp Ala
290 295 300
Thr Asn Asn Thr Pro Gly Phe Leu Ala Tyr Ser Ser Leu Arg Leu Ser
305 310 315 320
Tyr Leu Pro Arg Pro Asp Gly Val Asn Ile Thr Asp Asp Met Tyr Ala
325 330 335
Leu Val Arg Glu Gly Lys Tyr Ala Asn Ile Pro Val Ile Ile Gly Asp
340 345 350
Gln Asn Asp Glu Gly Thr Phe Phe Gly Thr Ser Ser Leu Asn Val Thr
355 360 365
Thr Asp Ala Gln Ala Arg Glu Tyr Phe Lys Gln Ser Phe Val His Ala
370 375 380
Ser Asp Ala Glu Ile Asp Thr Leu Met Thr Ala Tyr Pro Gly Asp Ile
385 390 395 400
Thr Gln Gly Ser Pro Phe Asp Thr Gly Ile Leu Asn Ala Leu Thr Pro
405 410 415
Gln Phe Lys Arg Ile Ser Ala Val Leu Gly Asp Leu Gly Phe Thr Leu
420 425 430
Ala Arg Arg Tyr Phe Leu Asn His Tyr Thr Gly Gly Thr Lys Tyr Ser
435 440 445
Phe Leu Ser Lys Gln Leu Ser Gly Leu Pro Val Leu Gly Thr Phe His
450 455 460
Ser Asn Asp Ile Val Phe Gln Asp Tyr Leu Leu Gly Ser Gly Ser Leu
465 470 475 480
Ile Tyr Asn Asn Ala Phe Ile Ala Phe Ala Thr Asp Leu Asp Pro Asn
485 490 495
Thr Ala Gly Leu Leu Val Lys Trp Pro Glu Tyr Thr Ser Ser Ser Gln
500 505 510
Ser Gly Asn Asn Leu Met Met Ile Asn Ala Leu Gly Leu Tyr Thr Gly
515 520 525
Lys Asp Asn Phe Arg Thr Ala Gly Tyr Asp Ala Leu Phe Ser Asn Pro
530 535 540
Pro Ser Phe Phe Val
545
<210> 3
<211> 549
<212> PRT
<213> 人工序列
<220>
<223> 来自柱状假丝酵母（Candida cylindracea）的包含信号肽的脂肪酶LIP1的P261L突变体
<400> 3
Met Glu Leu Ala Leu Ala Leu Ser Leu Ile Ala Ser Val Ala Ala Ala
1 5 10 15
Pro Thr Ala Thr Leu Ala Asn Gly Asp Thr Ile Thr Gly Leu Asn Ala
20 25 30
Ile Ile Asn Glu Ala Phe Leu Gly Ile Pro Phe Ala Glu Pro Pro Val
35 40 45
Gly Asn Leu Arg Phe Lys Asp Pro Val Pro Tyr Ser Gly Ser Leu Asp
50 55 60
Gly Gln Lys Phe Thr Ser Tyr Gly Pro Ser Cys Met Gln Gln Asn Pro
65 70 75 80
Glu Gly Thr Tyr Glu Glu Asn Leu Pro Lys Ala Ala Leu Asp Leu Val
85 90 95
Met Gln Ser Lys Val Phe Glu Ala Val Ser Pro Ser Ser Glu Asp Cys
100 105 110
Leu Thr Ile Asn Val Val Arg Pro Pro Gly Thr Lys Ala Gly Ala Asn
115 120 125
Leu Pro Val Met Leu Trp Ile Phe Gly Gly Gly Phe Glu Val Gly Gly
130 135 140
Thr Ser Thr Phe Pro Pro Ala Gln Met Ile Thr Lys Ser Ile Ala Met
145 150 155 160
Gly Lys Pro Ile Ile His Val Ser Val Asn Tyr Arg Val Ser Ser Trp
165 170 175
Gly Phe Leu Ala Gly Asp Glu Ile Lys Ala Glu Gly Ser Ala Asn Ala
180 185 190
Gly Leu Lys Asp Gln Arg Leu Gly Met Gln Trp Val Ala Asp Asn Ile
195 200 205
Ala Ala Phe Gly Gly Asp Pro Thr Lys Val Thr Ile Phe Gly Glu Ser
210 215 220
Ala Gly Ser Met Ser Val Met Cys His Ile Leu Trp Asn Asp Gly Asp
225 230 235 240
Asn Thr Tyr Lys Gly Lys Pro Leu Phe Arg Ala Gly Ile Met Gln Ser
245 250 255
Gly Ala Met Val Leu Ser Asp Ala Val Asp Gly Ile Tyr Gly Asn Glu
260 265 270
Ile Phe Asp Leu Leu Ala Ser Asn Ala Gly Cys Gly Ser Ala Ser Asp
275 280 285
Lys Leu Ala Cys Leu Arg Gly Val Ser Ser Asp Thr Leu Glu Asp Ala
290 295 300
Thr Asn Asn Thr Pro Gly Phe Leu Ala Tyr Ser Ser Leu Arg Leu Ser
305 310 315 320
Tyr Leu Pro Arg Pro Asp Gly Val Asn Ile Thr Asp Asp Met Tyr Ala
325 330 335
Leu Val Arg Glu Gly Lys Tyr Ala Asn Ile Pro Val Ile Ile Gly Asp
340 345 350
Gln Asn Asp Glu Gly Thr Phe Phe Gly Thr Ser Ser Leu Asn Val Thr
355 360 365
Thr Asp Ala Gln Ala Arg Glu Tyr Phe Lys Gln Ser Phe Val His Ala
370 375 380
Ser Asp Ala Glu Ile Asp Thr Leu Met Thr Ala Tyr Pro Gly Asp Ile
385 390 395 400
Thr Gln Gly Ser Pro Phe Asp Thr Gly Ile Leu Asn Ala Leu Thr Pro
405 410 415
Gln Phe Lys Arg Ile Ser Ala Val Leu Gly Asp Leu Gly Phe Thr Leu
420 425 430
Ala Arg Arg Tyr Phe Leu Asn His Tyr Thr Gly Gly Thr Lys Tyr Ser
435 440 445
Phe Leu Ser Lys Gln Leu Ser Gly Leu Pro Val Leu Gly Thr Phe His
450 455 460
Ser Asn Asp Ile Val Phe Gln Asp Tyr Leu Leu Gly Ser Gly Ser Leu
465 470 475 480
Ile Tyr Asn Asn Ala Phe Ile Ala Phe Ala Thr Asp Leu Asp Pro Asn
485 490 495
Thr Ala Gly Leu Leu Val Lys Trp Pro Glu Tyr Thr Ser Ser Ser Gln
500 505 510
Ser Gly Asn Asn Leu Met Met Ile Asn Ala Leu Gly Leu Tyr Thr Gly
515 520 525
Lys Asp Asn Phe Arg Thr Ala Gly Tyr Asp Ala Leu Phe Ser Asn Pro
530 535 540
Pro Ser Phe Phe Val
545
<210> 4
<211> 549
<212> PRT
<213> 人工序列
<220>
<223> 来自柱状假丝酵母（Candida cylindracea）的包含信号肽的脂肪酶LIP1的P261S突变体
<400> 4
Met Glu Leu Ala Leu Ala Leu Ser Leu Ile Ala Ser Val Ala Ala Ala
1 5 10 15
Pro Thr Ala Thr Leu Ala Asn Gly Asp Thr Ile Thr Gly Leu Asn Ala
20 25 30
Ile Ile Asn Glu Ala Phe Leu Gly Ile Pro Phe Ala Glu Pro Pro Val
35 40 45
Gly Asn Leu Arg Phe Lys Asp Pro Val Pro Tyr Ser Gly Ser Leu Asp
50 55 60
Gly Gln Lys Phe Thr Ser Tyr Gly Pro Ser Cys Met Gln Gln Asn Pro
65 70 75 80
Glu Gly Thr Tyr Glu Glu Asn Leu Pro Lys Ala Ala Leu Asp Leu Val
85 90 95
Met Gln Ser Lys Val Phe Glu Ala Val Ser Pro Ser Ser Glu Asp Cys
100 105 110
Leu Thr Ile Asn Val Val Arg Pro Pro Gly Thr Lys Ala Gly Ala Asn
115 120 125
Leu Pro Val Met Leu Trp Ile Phe Gly Gly Gly Phe Glu Val Gly Gly
130 135 140
Thr Ser Thr Phe Pro Pro Ala Gln Met Ile Thr Lys Ser Ile Ala Met
145 150 155 160
Gly Lys Pro Ile Ile His Val Ser Val Asn Tyr Arg Val Ser Ser Trp
165 170 175
Gly Phe Leu Ala Gly Asp Glu Ile Lys Ala Glu Gly Ser Ala Asn Ala
180 185 190
Gly Leu Lys Asp Gln Arg Leu Gly Met Gln Trp Val Ala Asp Asn Ile
195 200 205
Ala Ala Phe Gly Gly Asp Pro Thr Lys Val Thr Ile Phe Gly Glu Ser
210 215 220
Ala Gly Ser Met Ser Val Met Cys His Ile Leu Trp Asn Asp Gly Asp
225 230 235 240
Asn Thr Tyr Lys Gly Lys Pro Leu Phe Arg Ala Gly Ile Met Gln Ser
245 250 255
Gly Ala Met Val Ser Ser Asp Ala Val Asp Gly Ile Tyr Gly Asn Glu
260 265 270
Ile Phe Asp Leu Leu Ala Ser Asn Ala Gly Cys Gly Ser Ala Ser Asp
275 280 285
Lys Leu Ala Cys Leu Arg Gly Val Ser Ser Asp Thr Leu Glu Asp Ala
290 295 300
Thr Asn Asn Thr Pro Gly Phe Leu Ala Tyr Ser Ser Leu Arg Leu Ser
305 310 315 320
Tyr Leu Pro Arg Pro Asp Gly Val Asn Ile Thr Asp Asp Met Tyr Ala
325 330 335
Leu Val Arg Glu Gly Lys Tyr Ala Asn Ile Pro Val Ile Ile Gly Asp
340 345 350
Gln Asn Asp Glu Gly Thr Phe Phe Gly Thr Ser Ser Leu Asn Val Thr
355 360 365
Thr Asp Ala Gln Ala Arg Glu Tyr Phe Lys Gln Ser Phe Val His Ala
370 375 380
Ser Asp Ala Glu Ile Asp Thr Leu Met Thr Ala Tyr Pro Gly Asp Ile
385 390 395 400
Thr Gln Gly Ser Pro Phe Asp Thr Gly Ile Leu Asn Ala Leu Thr Pro
405 410 415
Gln Phe Lys Arg Ile Ser Ala Val Leu Gly Asp Leu Gly Phe Thr Leu
420 425 430
Ala Arg Arg Tyr Phe Leu Asn His Tyr Thr Gly Gly Thr Lys Tyr Ser
435 440 445
Phe Leu Ser Lys Gln Leu Ser Gly Leu Pro Val Leu Gly Thr Phe His
450 455 460
Ser Asn Asp Ile Val Phe Gln Asp Tyr Leu Leu Gly Ser Gly Ser Leu
465 470 475 480
Ile Tyr Asn Asn Ala Phe Ile Ala Phe Ala Thr Asp Leu Asp Pro Asn
485 490 495
Thr Ala Gly Leu Leu Val Lys Trp Pro Glu Tyr Thr Ser Ser Ser Gln
500 505 510
Ser Gly Asn Asn Leu Met Met Ile Asn Ala Leu Gly Leu Tyr Thr Gly
515 520 525
Lys Asp Asn Phe Arg Thr Ala Gly Tyr Asp Ala Leu Phe Ser Asn Pro
530 535 540
Pro Ser Phe Phe Val
545
<210> 5
<211> 549
<212> PRT
<213> 人工序列
<220>
<223> 来自柱状假丝酵母（Candida cylindracea）的包含信号肽的脂肪酶LIP1的L322W突变体
<400> 5
Met Glu Leu Ala Leu Ala Leu Ser Leu Ile Ala Ser Val Ala Ala Ala
1 5 10 15
Pro Thr Ala Thr Leu Ala Asn Gly Asp Thr Ile Thr Gly Leu Asn Ala
20 25 30
Ile Ile Asn Glu Ala Phe Leu Gly Ile Pro Phe Ala Glu Pro Pro Val
35 40 45
Gly Asn Leu Arg Phe Lys Asp Pro Val Pro Tyr Ser Gly Ser Leu Asp
50 55 60
Gly Gln Lys Phe Thr Ser Tyr Gly Pro Ser Cys Met Gln Gln Asn Pro
65 70 75 80
Glu Gly Thr Tyr Glu Glu Asn Leu Pro Lys Ala Ala Leu Asp Leu Val
85 90 95
Met Gln Ser Lys Val Phe Glu Ala Val Ser Pro Ser Ser Glu Asp Cys
100 105 110
Leu Thr Ile Asn Val Val Arg Pro Pro Gly Thr Lys Ala Gly Ala Asn
115 120 125
Leu Pro Val Met Leu Trp Ile Phe Gly Gly Gly Phe Glu Val Gly Gly
130 135 140
Thr Ser Thr Phe Pro Pro Ala Gln Met Ile Thr Lys Ser Ile Ala Met
145 150 155 160
Gly Lys Pro Ile Ile His Val Ser Val Asn Tyr Arg Val Ser Ser Trp
165 170 175
Gly Phe Leu Ala Gly Asp Glu Ile Lys Ala Glu Gly Ser Ala Asn Ala
180 185 190
Gly Leu Lys Asp Gln Arg Leu Gly Met Gln Trp Val Ala Asp Asn Ile
195 200 205
Ala Ala Phe Gly Gly Asp Pro Thr Lys Val Thr Ile Phe Gly Glu Ser
210 215 220
Ala Gly Ser Met Ser Val Met Cys His Ile Leu Trp Asn Asp Gly Asp
225 230 235 240
Asn Thr Tyr Lys Gly Lys Pro Leu Phe Arg Ala Gly Ile Met Gln Ser
245 250 255
Gly Ala Met Val Pro Ser Asp Ala Val Asp Gly Ile Tyr Gly Asn Glu
260 265 270
Ile Phe Asp Leu Leu Ala Ser Asn Ala Gly Cys Gly Ser Ala Ser Asp
275 280 285
Lys Leu Ala Cys Leu Arg Gly Val Ser Ser Asp Thr Leu Glu Asp Ala
290 295 300
Thr Asn Asn Thr Pro Gly Phe Leu Ala Tyr Ser Ser Leu Arg Leu Ser
305 310 315 320
Tyr Trp Pro Arg Pro Asp Gly Val Asn Ile Thr Asp Asp Met Tyr Ala
325 330 335
Leu Val Arg Glu Gly Lys Tyr Ala Asn Ile Pro Val Ile Ile Gly Asp
340 345 350
Gln Asn Asp Glu Gly Thr Phe Phe Gly Thr Ser Ser Leu Asn Val Thr
355 360 365
Thr Asp Ala Gln Ala Arg Glu Tyr Phe Lys Gln Ser Phe Val His Ala
370 375 380
Ser Asp Ala Glu Ile Asp Thr Leu Met Thr Ala Tyr Pro Gly Asp Ile
385 390 395 400
Thr Gln Gly Ser Pro Phe Asp Thr Gly Ile Leu Asn Ala Leu Thr Pro
405 410 415
Gln Phe Lys Arg Ile Ser Ala Val Leu Gly Asp Leu Gly Phe Thr Leu
420 425 430
Ala Arg Arg Tyr Phe Leu Asn His Tyr Thr Gly Gly Thr Lys Tyr Ser
435 440 445
Phe Leu Ser Lys Gln Leu Ser Gly Leu Pro Val Leu Gly Thr Phe His
450 455 460
Ser Asn Asp Ile Val Phe Gln Asp Tyr Leu Leu Gly Ser Gly Ser Leu
465 470 475 480
Ile Tyr Asn Asn Ala Phe Ile Ala Phe Ala Thr Asp Leu Asp Pro Asn
485 490 495
Thr Ala Gly Leu Leu Val Lys Trp Pro Glu Tyr Thr Ser Ser Ser Gln
500 505 510
Ser Gly Asn Asn Leu Met Met Ile Asn Ala Leu Gly Leu Tyr Thr Gly
515 520 525
Lys Asp Asn Phe Arg Thr Ala Gly Tyr Asp Ala Leu Phe Ser Asn Pro
530 535 540
Pro Ser Phe Phe Val
545
<210> 6
<211> 549
<212> PRT
<213> 人工序列
<220>
<223> 来自柱状假丝酵母（Candida cylindracea）的包含信号肽的脂肪酶LIP1的F360L突变体
<400> 6
Met Glu Leu Ala Leu Ala Leu Ser Leu Ile Ala Ser Val Ala Ala Ala
1 5 10 15
Pro Thr Ala Thr Leu Ala Asn Gly Asp Thr Ile Thr Gly Leu Asn Ala
20 25 30
Ile Ile Asn Glu Ala Phe Leu Gly Ile Pro Phe Ala Glu Pro Pro Val
35 40 45
Gly Asn Leu Arg Phe Lys Asp Pro Val Pro Tyr Ser Gly Ser Leu Asp
50 55 60
Gly Gln Lys Phe Thr Ser Tyr Gly Pro Ser Cys Met Gln Gln Asn Pro
65 70 75 80
Glu Gly Thr Tyr Glu Glu Asn Leu Pro Lys Ala Ala Leu Asp Leu Val
85 90 95
Met Gln Ser Lys Val Phe Glu Ala Val Ser Pro Ser Ser Glu Asp Cys
100 105 110
Leu Thr Ile Asn Val Val Arg Pro Pro Gly Thr Lys Ala Gly Ala Asn
115 120 125
Leu Pro Val Met Leu Trp Ile Phe Gly Gly Gly Phe Glu Val Gly Gly
130 135 140
Thr Ser Thr Phe Pro Pro Ala Gln Met Ile Thr Lys Ser Ile Ala Met
145 150 155 160
Gly Lys Pro Ile Ile His Val Ser Val Asn Tyr Arg Val Ser Ser Trp
165 170 175
Gly Phe Leu Ala Gly Asp Glu Ile Lys Ala Glu Gly Ser Ala Asn Ala
180 185 190
Gly Leu Lys Asp Gln Arg Leu Gly Met Gln Trp Val Ala Asp Asn Ile
195 200 205
Ala Ala Phe Gly Gly Asp Pro Thr Lys Val Thr Ile Phe Gly Glu Ser
210 215 220
Ala Gly Ser Met Ser Val Met Cys His Ile Leu Trp Asn Asp Gly Asp
225 230 235 240
Asn Thr Tyr Lys Gly Lys Pro Leu Phe Arg Ala Gly Ile Met Gln Ser
245 250 255
Gly Ala Met Val Pro Ser Asp Ala Val Asp Gly Ile Tyr Gly Asn Glu
260 265 270
Ile Phe Asp Leu Leu Ala Ser Asn Ala Gly Cys Gly Ser Ala Ser Asp
275 280 285
Lys Leu Ala Cys Leu Arg Gly Val Ser Ser Asp Thr Leu Glu Asp Ala
290 295 300
Thr Asn Asn Thr Pro Gly Phe Leu Ala Tyr Ser Ser Leu Arg Leu Ser
305 310 315 320
Tyr Leu Pro Arg Pro Asp Gly Val Asn Ile Thr Asp Asp Met Tyr Ala
325 330 335
Leu Val Arg Glu Gly Lys Tyr Ala Asn Ile Pro Val Ile Ile Gly Asp
340 345 350
Gln Asn Asp Glu Gly Thr Phe Leu Gly Thr Ser Ser Leu Asn Val Thr
355 360 365
Thr Asp Ala Gln Ala Arg Glu Tyr Phe Lys Gln Ser Phe Val His Ala
370 375 380
Ser Asp Ala Glu Ile Asp Thr Leu Met Thr Ala Tyr Pro Gly Asp Ile
385 390 395 400
Thr Gln Gly Ser Pro Phe Asp Thr Gly Ile Leu Asn Ala Leu Thr Pro
405 410 415
Gln Phe Lys Arg Ile Ser Ala Val Leu Gly Asp Leu Gly Phe Thr Leu
420 425 430
Ala Arg Arg Tyr Phe Leu Asn His Tyr Thr Gly Gly Thr Lys Tyr Ser
435 440 445
Phe Leu Ser Lys Gln Leu Ser Gly Leu Pro Val Leu Gly Thr Phe His
450 455 460
Ser Asn Asp Ile Val Phe Gln Asp Tyr Leu Leu Gly Ser Gly Ser Leu
465 470 475 480
Ile Tyr Asn Asn Ala Phe Ile Ala Phe Ala Thr Asp Leu Asp Pro Asn
485 490 495
Thr Ala Gly Leu Leu Val Lys Trp Pro Glu Tyr Thr Ser Ser Ser Gln
500 505 510
Ser Gly Asn Asn Leu Met Met Ile Asn Ala Leu Gly Leu Tyr Thr Gly
515 520 525
Lys Asp Asn Phe Arg Thr Ala Gly Tyr Asp Ala Leu Phe Ser Asn Pro
530 535 540
Pro Ser Phe Phe Val
545
<210> 7
<211> 549
<212> PRT
<213> 人工序列
<220>
<223> 来自柱状假丝酵母（Candida cylindracea）的包含信号肽的脂肪酶LIP1的S380I突变体
<400> 7
Met Glu Leu Ala Leu Ala Leu Ser Leu Ile Ala Ser Val Ala Ala Ala
1 5 10 15
Pro Thr Ala Thr Leu Ala Asn Gly Asp Thr Ile Thr Gly Leu Asn Ala
20 25 30
Ile Ile Asn Glu Ala Phe Leu Gly Ile Pro Phe Ala Glu Pro Pro Val
35 40 45
Gly Asn Leu Arg Phe Lys Asp Pro Val Pro Tyr Ser Gly Ser Leu Asp
50 55 60
Gly Gln Lys Phe Thr Ser Tyr Gly Pro Ser Cys Met Gln Gln Asn Pro
65 70 75 80
Glu Gly Thr Tyr Glu Glu Asn Leu Pro Lys Ala Ala Leu Asp Leu Val
85 90 95
Met Gln Ser Lys Val Phe Glu Ala Val Ser Pro Ser Ser Glu Asp Cys
100 105 110
Leu Thr Ile Asn Val Val Arg Pro Pro Gly Thr Lys Ala Gly Ala Asn
115 120 125
Leu Pro Val Met Leu Trp Ile Phe Gly Gly Gly Phe Glu Val Gly Gly
130 135 140
Thr Ser Thr Phe Pro Pro Ala Gln Met Ile Thr Lys Ser Ile Ala Met
145 150 155 160
Gly Lys Pro Ile Ile His Val Ser Val Asn Tyr Arg Val Ser Ser Trp
165 170 175
Gly Phe Leu Ala Gly Asp Glu Ile Lys Ala Glu Gly Ser Ala Asn Ala
180 185 190
Gly Leu Lys Asp Gln Arg Leu Gly Met Gln Trp Val Ala Asp Asn Ile
195 200 205
Ala Ala Phe Gly Gly Asp Pro Thr Lys Val Thr Ile Phe Gly Glu Ser
210 215 220
Ala Gly Ser Met Ser Val Met Cys His Ile Leu Trp Asn Asp Gly Asp
225 230 235 240
Asn Thr Tyr Lys Gly Lys Pro Leu Phe Arg Ala Gly Ile Met Gln Ser
245 250 255
Gly Ala Met Val Pro Ser Asp Ala Val Asp Gly Ile Tyr Gly Asn Glu
260 265 270
Ile Phe Asp Leu Leu Ala Ser Asn Ala Gly Cys Gly Ser Ala Ser Asp
275 280 285
Lys Leu Ala Cys Leu Arg Gly Val Ser Ser Asp Thr Leu Glu Asp Ala
290 295 300
Thr Asn Asn Thr Pro Gly Phe Leu Ala Tyr Ser Ser Leu Arg Leu Ser
305 310 315 320
Tyr Leu Pro Arg Pro Asp Gly Val Asn Ile Thr Asp Asp Met Tyr Ala
325 330 335
Leu Val Arg Glu Gly Lys Tyr Ala Asn Ile Pro Val Ile Ile Gly Asp
340 345 350
Gln Asn Asp Glu Gly Thr Phe Phe Gly Thr Ser Ser Leu Asn Val Thr
355 360 365
Thr Asp Ala Gln Ala Arg Glu Tyr Phe Lys Gln Ile Phe Val His Ala
370 375 380
Ser Asp Ala Glu Ile Asp Thr Leu Met Thr Ala Tyr Pro Gly Asp Ile
385 390 395 400
Thr Gln Gly Ser Pro Phe Asp Thr Gly Ile Leu Asn Ala Leu Thr Pro
405 410 415
Gln Phe Lys Arg Ile Ser Ala Val Leu Gly Asp Leu Gly Phe Thr Leu
420 425 430
Ala Arg Arg Tyr Phe Leu Asn His Tyr Thr Gly Gly Thr Lys Tyr Ser
435 440 445
Phe Leu Ser Lys Gln Leu Ser Gly Leu Pro Val Leu Gly Thr Phe His
450 455 460
Ser Asn Asp Ile Val Phe Gln Asp Tyr Leu Leu Gly Ser Gly Ser Leu
465 470 475 480
Ile Tyr Asn Asn Ala Phe Ile Ala Phe Ala Thr Asp Leu Asp Pro Asn
485 490 495
Thr Ala Gly Leu Leu Val Lys Trp Pro Glu Tyr Thr Ser Ser Ser Gln
500 505 510
Ser Gly Asn Asn Leu Met Met Ile Asn Ala Leu Gly Leu Tyr Thr Gly
515 520 525
Lys Asp Asn Phe Arg Thr Ala Gly Tyr Asp Ala Leu Phe Ser Asn Pro
530 535 540
Pro Ser Phe Phe Val
545
<210> 8
<211> 549
<212> PRT
<213> 人工序列
<220>
<223> 来自柱状假丝酵母（Candida cylindracea）的包含信号肽的脂肪酶LIP1的S380L突变体
<400> 8
Met Glu Leu Ala Leu Ala Leu Ser Leu Ile Ala Ser Val Ala Ala Ala
1 5 10 15
Pro Thr Ala Thr Leu Ala Asn Gly Asp Thr Ile Thr Gly Leu Asn Ala
20 25 30
Ile Ile Asn Glu Ala Phe Leu Gly Ile Pro Phe Ala Glu Pro Pro Val
35 40 45
Gly Asn Leu Arg Phe Lys Asp Pro Val Pro Tyr Ser Gly Ser Leu Asp
50 55 60
Gly Gln Lys Phe Thr Ser Tyr Gly Pro Ser Cys Met Gln Gln Asn Pro
65 70 75 80
Glu Gly Thr Tyr Glu Glu Asn Leu Pro Lys Ala Ala Leu Asp Leu Val
85 90 95
Met Gln Ser Lys Val Phe Glu Ala Val Ser Pro Ser Ser Glu Asp Cys
100 105 110
Leu Thr Ile Asn Val Val Arg Pro Pro Gly Thr Lys Ala Gly Ala Asn
115 120 125
Leu Pro Val Met Leu Trp Ile Phe Gly Gly Gly Phe Glu Val Gly Gly
130 135 140
Thr Ser Thr Phe Pro Pro Ala Gln Met Ile Thr Lys Ser Ile Ala Met
145 150 155 160
Gly Lys Pro Ile Ile His Val Ser Val Asn Tyr Arg Val Ser Ser Trp
165 170 175
Gly Phe Leu Ala Gly Asp Glu Ile Lys Ala Glu Gly Ser Ala Asn Ala
180 185 190
Gly Leu Lys Asp Gln Arg Leu Gly Met Gln Trp Val Ala Asp Asn Ile
195 200 205
Ala Ala Phe Gly Gly Asp Pro Thr Lys Val Thr Ile Phe Gly Glu Ser
210 215 220
Ala Gly Ser Met Ser Val Met Cys His Ile Leu Trp Asn Asp Gly Asp
225 230 235 240
Asn Thr Tyr Lys Gly Lys Pro Leu Phe Arg Ala Gly Ile Met Gln Ser
245 250 255
Gly Ala Met Val Pro Ser Asp Ala Val Asp Gly Ile Tyr Gly Asn Glu
260 265 270
Ile Phe Asp Leu Leu Ala Ser Asn Ala Gly Cys Gly Ser Ala Ser Asp
275 280 285
Lys Leu Ala Cys Leu Arg Gly Val Ser Ser Asp Thr Leu Glu Asp Ala
290 295 300
Thr Asn Asn Thr Pro Gly Phe Leu Ala Tyr Ser Ser Leu Arg Leu Ser
305 310 315 320
Tyr Leu Pro Arg Pro Asp Gly Val Asn Ile Thr Asp Asp Met Tyr Ala
325 330 335
Leu Val Arg Glu Gly Lys Tyr Ala Asn Ile Pro Val Ile Ile Gly Asp
340 345 350
Gln Asn Asp Glu Gly Thr Phe Phe Gly Thr Ser Ser Leu Asn Val Thr
355 360 365
Thr Asp Ala Gln Ala Arg Glu Tyr Phe Lys Gln Leu Phe Val His Ala
370 375 380
Ser Asp Ala Glu Ile Asp Thr Leu Met Thr Ala Tyr Pro Gly Asp Ile
385 390 395 400
Thr Gln Gly Ser Pro Phe Asp Thr Gly Ile Leu Asn Ala Leu Thr Pro
405 410 415
Gln Phe Lys Arg Ile Ser Ala Val Leu Gly Asp Leu Gly Phe Thr Leu
420 425 430
Ala Arg Arg Tyr Phe Leu Asn His Tyr Thr Gly Gly Thr Lys Tyr Ser
435 440 445
Phe Leu Ser Lys Gln Leu Ser Gly Leu Pro Val Leu Gly Thr Phe His
450 455 460
Ser Asn Asp Ile Val Phe Gln Asp Tyr Leu Leu Gly Ser Gly Ser Leu
465 470 475 480
Ile Tyr Asn Asn Ala Phe Ile Ala Phe Ala Thr Asp Leu Asp Pro Asn
485 490 495
Thr Ala Gly Leu Leu Val Lys Trp Pro Glu Tyr Thr Ser Ser Ser Gln
500 505 510
Ser Gly Asn Asn Leu Met Met Ile Asn Ala Leu Gly Leu Tyr Thr Gly
515 520 525
Lys Asp Asn Phe Arg Thr Ala Gly Tyr Asp Ala Leu Phe Ser Asn Pro
530 535 540
Pro Ser Phe Phe Val
545
<210> 9
<211> 549
<212> PRT
<213> 人工序列
<220>
<223> 来自柱状假丝酵母（Candida cylindracea）的包含信号肽的脂肪酶LIP1的L425W突变体
<400> 9
Met Glu Leu Ala Leu Ala Leu Ser Leu Ile Ala Ser Val Ala Ala Ala
1 5 10 15
Pro Thr Ala Thr Leu Ala Asn Gly Asp Thr Ile Thr Gly Leu Asn Ala
20 25 30
Ile Ile Asn Glu Ala Phe Leu Gly Ile Pro Phe Ala Glu Pro Pro Val
35 40 45
Gly Asn Leu Arg Phe Lys Asp Pro Val Pro Tyr Ser Gly Ser Leu Asp
50 55 60
Gly Gln Lys Phe Thr Ser Tyr Gly Pro Ser Cys Met Gln Gln Asn Pro
65 70 75 80
Glu Gly Thr Tyr Glu Glu Asn Leu Pro Lys Ala Ala Leu Asp Leu Val
85 90 95
Met Gln Ser Lys Val Phe Glu Ala Val Ser Pro Ser Ser Glu Asp Cys
100 105 110
Leu Thr Ile Asn Val Val Arg Pro Pro Gly Thr Lys Ala Gly Ala Asn
115 120 125
Leu Pro Val Met Leu Trp Ile Phe Gly Gly Gly Phe Glu Val Gly Gly
130 135 140
Thr Ser Thr Phe Pro Pro Ala Gln Met Ile Thr Lys Ser Ile Ala Met
145 150 155 160
Gly Lys Pro Ile Ile His Val Ser Val Asn Tyr Arg Val Ser Ser Trp
165 170 175
Gly Phe Leu Ala Gly Asp Glu Ile Lys Ala Glu Gly Ser Ala Asn Ala
180 185 190
Gly Leu Lys Asp Gln Arg Leu Gly Met Gln Trp Val Ala Asp Asn Ile
195 200 205
Ala Ala Phe Gly Gly Asp Pro Thr Lys Val Thr Ile Phe Gly Glu Ser
210 215 220
Ala Gly Ser Met Ser Val Met Cys His Ile Leu Trp Asn Asp Gly Asp
225 230 235 240
Asn Thr Tyr Lys Gly Lys Pro Leu Phe Arg Ala Gly Ile Met Gln Ser
245 250 255
Gly Ala Met Val Pro Ser Asp Ala Val Asp Gly Ile Tyr Gly Asn Glu
260 265 270
Ile Phe Asp Leu Leu Ala Ser Asn Ala Gly Cys Gly Ser Ala Ser Asp
275 280 285
Lys Leu Ala Cys Leu Arg Gly Val Ser Ser Asp Thr Leu Glu Asp Ala
290 295 300
Thr Asn Asn Thr Pro Gly Phe Leu Ala Tyr Ser Ser Leu Arg Leu Ser
305 310 315 320
Tyr Leu Pro Arg Pro Asp Gly Val Asn Ile Thr Asp Asp Met Tyr Ala
325 330 335
Leu Val Arg Glu Gly Lys Tyr Ala Asn Ile Pro Val Ile Ile Gly Asp
340 345 350
Gln Asn Asp Glu Gly Thr Phe Phe Gly Thr Ser Ser Leu Asn Val Thr
355 360 365
Thr Asp Ala Gln Ala Arg Glu Tyr Phe Lys Gln Ser Phe Val His Ala
370 375 380
Ser Asp Ala Glu Ile Asp Thr Leu Met Thr Ala Tyr Pro Gly Asp Ile
385 390 395 400
Thr Gln Gly Ser Pro Phe Asp Thr Gly Ile Leu Asn Ala Leu Thr Pro
405 410 415
Gln Phe Lys Arg Ile Ser Ala Val Trp Gly Asp Leu Gly Phe Thr Leu
420 425 430
Ala Arg Arg Tyr Phe Leu Asn His Tyr Thr Gly Gly Thr Lys Tyr Ser
435 440 445
Phe Leu Ser Lys Gln Leu Ser Gly Leu Pro Val Leu Gly Thr Phe His
450 455 460
Ser Asn Asp Ile Val Phe Gln Asp Tyr Leu Leu Gly Ser Gly Ser Leu
465 470 475 480
Ile Tyr Asn Asn Ala Phe Ile Ala Phe Ala Thr Asp Leu Asp Pro Asn
485 490 495
Thr Ala Gly Leu Leu Val Lys Trp Pro Glu Tyr Thr Ser Ser Ser Gln
500 505 510
Ser Gly Asn Asn Leu Met Met Ile Asn Ala Leu Gly Leu Tyr Thr Gly
515 520 525
Lys Asp Asn Phe Arg Thr Ala Gly Tyr Asp Ala Leu Phe Ser Asn Pro
530 535 540
Pro Ser Phe Phe Val
545
<210> 10
<211> 549
<212> PRT
<213> 人工序列
<220>
<223> 来自柱状假丝酵母（Candida cylindracea）的包含信号肽的脂肪酶LIP1的L428F突变体
<400> 10
Met Glu Leu Ala Leu Ala Leu Ser Leu Ile Ala Ser Val Ala Ala Ala
1 5 10 15
Pro Thr Ala Thr Leu Ala Asn Gly Asp Thr Ile Thr Gly Leu Asn Ala
20 25 30
Ile Ile Asn Glu Ala Phe Leu Gly Ile Pro Phe Ala Glu Pro Pro Val
35 40 45
Gly Asn Leu Arg Phe Lys Asp Pro Val Pro Tyr Ser Gly Ser Leu Asp
50 55 60
Gly Gln Lys Phe Thr Ser Tyr Gly Pro Ser Cys Met Gln Gln Asn Pro
65 70 75 80
Glu Gly Thr Tyr Glu Glu Asn Leu Pro Lys Ala Ala Leu Asp Leu Val
85 90 95
Met Gln Ser Lys Val Phe Glu Ala Val Ser Pro Ser Ser Glu Asp Cys
100 105 110
Leu Thr Ile Asn Val Val Arg Pro Pro Gly Thr Lys Ala Gly Ala Asn
115 120 125
Leu Pro Val Met Leu Trp Ile Phe Gly Gly Gly Phe Glu Val Gly Gly
130 135 140
Thr Ser Thr Phe Pro Pro Ala Gln Met Ile Thr Lys Ser Ile Ala Met
145 150 155 160
Gly Lys Pro Ile Ile His Val Ser Val Asn Tyr Arg Val Ser Ser Trp
165 170 175
Gly Phe Leu Ala Gly Asp Glu Ile Lys Ala Glu Gly Ser Ala Asn Ala
180 185 190
Gly Leu Lys Asp Gln Arg Leu Gly Met Gln Trp Val Ala Asp Asn Ile
195 200 205
Ala Ala Phe Gly Gly Asp Pro Thr Lys Val Thr Ile Phe Gly Glu Ser
210 215 220
Ala Gly Ser Met Ser Val Met Cys His Ile Leu Trp Asn Asp Gly Asp
225 230 235 240
Asn Thr Tyr Lys Gly Lys Pro Leu Phe Arg Ala Gly Ile Met Gln Ser
245 250 255
Gly Ala Met Val Pro Ser Asp Ala Val Asp Gly Ile Tyr Gly Asn Glu
260 265 270
Ile Phe Asp Leu Leu Ala Ser Asn Ala Gly Cys Gly Ser Ala Ser Asp
275 280 285
Lys Leu Ala Cys Leu Arg Gly Val Ser Ser Asp Thr Leu Glu Asp Ala
290 295 300
Thr Asn Asn Thr Pro Gly Phe Leu Ala Tyr Ser Ser Leu Arg Leu Ser
305 310 315 320
Tyr Leu Pro Arg Pro Asp Gly Val Asn Ile Thr Asp Asp Met Tyr Ala
325 330 335
Leu Val Arg Glu Gly Lys Tyr Ala Asn Ile Pro Val Ile Ile Gly Asp
340 345 350
Gln Asn Asp Glu Gly Thr Phe Phe Gly Thr Ser Ser Leu Asn Val Thr
355 360 365
Thr Asp Ala Gln Ala Arg Glu Tyr Phe Lys Gln Ser Phe Val His Ala
370 375 380
Ser Asp Ala Glu Ile Asp Thr Leu Met Thr Ala Tyr Pro Gly Asp Ile
385 390 395 400
Thr Gln Gly Ser Pro Phe Asp Thr Gly Ile Leu Asn Ala Leu Thr Pro
405 410 415
Gln Phe Lys Arg Ile Ser Ala Val Leu Gly Asp Phe Gly Phe Thr Leu
420 425 430
Ala Arg Arg Tyr Phe Leu Asn His Tyr Thr Gly Gly Thr Lys Tyr Ser
435 440 445
Phe Leu Ser Lys Gln Leu Ser Gly Leu Pro Val Leu Gly Thr Phe His
450 455 460
Ser Asn Asp Ile Val Phe Gln Asp Tyr Leu Leu Gly Ser Gly Ser Leu
465 470 475 480
Ile Tyr Asn Asn Ala Phe Ile Ala Phe Ala Thr Asp Leu Asp Pro Asn
485 490 495
Thr Ala Gly Leu Leu Val Lys Trp Pro Glu Tyr Thr Ser Ser Ser Gln
500 505 510
Ser Gly Asn Asn Leu Met Met Ile Asn Ala Leu Gly Leu Tyr Thr Gly
515 520 525
Lys Asp Asn Phe Arg Thr Ala Gly Tyr Asp Ala Leu Phe Ser Asn Pro
530 535 540
Pro Ser Phe Phe Val
545
<210> 11
<211> 549
<212> PRT
<213> 人工序列
<220>
<223> 来自柱状假丝酵母（Candida cylindracea）的包含信号肽的脂肪酶LIP1的L428N突变体
<400> 11
Met Glu Leu Ala Leu Ala Leu Ser Leu Ile Ala Ser Val Ala Ala Ala
1 5 10 15
Pro Thr Ala Thr Leu Ala Asn Gly Asp Thr Ile Thr Gly Leu Asn Ala
20 25 30
Ile Ile Asn Glu Ala Phe Leu Gly Ile Pro Phe Ala Glu Pro Pro Val
35 40 45
Gly Asn Leu Arg Phe Lys Asp Pro Val Pro Tyr Ser Gly Ser Leu Asp
50 55 60
Gly Gln Lys Phe Thr Ser Tyr Gly Pro Ser Cys Met Gln Gln Asn Pro
65 70 75 80
Glu Gly Thr Tyr Glu Glu Asn Leu Pro Lys Ala Ala Leu Asp Leu Val
85 90 95
Met Gln Ser Lys Val Phe Glu Ala Val Ser Pro Ser Ser Glu Asp Cys
100 105 110
Leu Thr Ile Asn Val Val Arg Pro Pro Gly Thr Lys Ala Gly Ala Asn
115 120 125
Leu Pro Val Met Leu Trp Ile Phe Gly Gly Gly Phe Glu Val Gly Gly
130 135 140
Thr Ser Thr Phe Pro Pro Ala Gln Met Ile Thr Lys Ser Ile Ala Met
145 150 155 160
Gly Lys Pro Ile Ile His Val Ser Val Asn Tyr Arg Val Ser Ser Trp
165 170 175
Gly Phe Leu Ala Gly Asp Glu Ile Lys Ala Glu Gly Ser Ala Asn Ala
180 185 190
Gly Leu Lys Asp Gln Arg Leu Gly Met Gln Trp Val Ala Asp Asn Ile
195 200 205
Ala Ala Phe Gly Gly Asp Pro Thr Lys Val Thr Ile Phe Gly Glu Ser
210 215 220
Ala Gly Ser Met Ser Val Met Cys His Ile Leu Trp Asn Asp Gly Asp
225 230 235 240
Asn Thr Tyr Lys Gly Lys Pro Leu Phe Arg Ala Gly Ile Met Gln Ser
245 250 255
Gly Ala Met Val Pro Ser Asp Ala Val Asp Gly Ile Tyr Gly Asn Glu
260 265 270
Ile Phe Asp Leu Leu Ala Ser Asn Ala Gly Cys Gly Ser Ala Ser Asp
275 280 285
Lys Leu Ala Cys Leu Arg Gly Val Ser Ser Asp Thr Leu Glu Asp Ala
290 295 300
Thr Asn Asn Thr Pro Gly Phe Leu Ala Tyr Ser Ser Leu Arg Leu Ser
305 310 315 320
Tyr Leu Pro Arg Pro Asp Gly Val Asn Ile Thr Asp Asp Met Tyr Ala
325 330 335
Leu Val Arg Glu Gly Lys Tyr Ala Asn Ile Pro Val Ile Ile Gly Asp
340 345 350
Gln Asn Asp Glu Gly Thr Phe Phe Gly Thr Ser Ser Leu Asn Val Thr
355 360 365
Thr Asp Ala Gln Ala Arg Glu Tyr Phe Lys Gln Ser Phe Val His Ala
370 375 380
Ser Asp Ala Glu Ile Asp Thr Leu Met Thr Ala Tyr Pro Gly Asp Ile
385 390 395 400
Thr Gln Gly Ser Pro Phe Asp Thr Gly Ile Leu Asn Ala Leu Thr Pro
405 410 415
Gln Phe Lys Arg Ile Ser Ala Val Leu Gly Asp Asn Gly Phe Thr Leu
420 425 430
Ala Arg Arg Tyr Phe Leu Asn His Tyr Thr Gly Gly Thr Lys Tyr Ser
435 440 445
Phe Leu Ser Lys Gln Leu Ser Gly Leu Pro Val Leu Gly Thr Phe His
450 455 460
Ser Asn Asp Ile Val Phe Gln Asp Tyr Leu Leu Gly Ser Gly Ser Leu
465 470 475 480
Ile Tyr Asn Asn Ala Phe Ile Ala Phe Ala Thr Asp Leu Asp Pro Asn
485 490 495
Thr Ala Gly Leu Leu Val Lys Trp Pro Glu Tyr Thr Ser Ser Ser Gln
500 505 510
Ser Gly Asn Asn Leu Met Met Ile Asn Ala Leu Gly Leu Tyr Thr Gly
515 520 525
Lys Asp Asn Phe Arg Thr Ala Gly Tyr Asp Ala Leu Phe Ser Asn Pro
530 535 540
Pro Ser Phe Phe Val
545
<210> 12
<211> 549
<212> PRT
<213> 人工序列
<220>
<223> 来自柱状假丝酵母（Candida cylindracea）的包含信号肽的脂肪酶LIP1的G429F突变体
<400> 12
Met Glu Leu Ala Leu Ala Leu Ser Leu Ile Ala Ser Val Ala Ala Ala
1 5 10 15
Pro Thr Ala Thr Leu Ala Asn Gly Asp Thr Ile Thr Gly Leu Asn Ala
20 25 30
Ile Ile Asn Glu Ala Phe Leu Gly Ile Pro Phe Ala Glu Pro Pro Val
35 40 45
Gly Asn Leu Arg Phe Lys Asp Pro Val Pro Tyr Ser Gly Ser Leu Asp
50 55 60
Gly Gln Lys Phe Thr Ser Tyr Gly Pro Ser Cys Met Gln Gln Asn Pro
65 70 75 80
Glu Gly Thr Tyr Glu Glu Asn Leu Pro Lys Ala Ala Leu Asp Leu Val
85 90 95
Met Gln Ser Lys Val Phe Glu Ala Val Ser Pro Ser Ser Glu Asp Cys
100 105 110
Leu Thr Ile Asn Val Val Arg Pro Pro Gly Thr Lys Ala Gly Ala Asn
115 120 125
Leu Pro Val Met Leu Trp Ile Phe Gly Gly Gly Phe Glu Val Gly Gly
130 135 140
Thr Ser Thr Phe Pro Pro Ala Gln Met Ile Thr Lys Ser Ile Ala Met
145 150 155 160
Gly Lys Pro Ile Ile His Val Ser Val Asn Tyr Arg Val Ser Ser Trp
165 170 175
Gly Phe Leu Ala Gly Asp Glu Ile Lys Ala Glu Gly Ser Ala Asn Ala
180 185 190
Gly Leu Lys Asp Gln Arg Leu Gly Met Gln Trp Val Ala Asp Asn Ile
195 200 205
Ala Ala Phe Gly Gly Asp Pro Thr Lys Val Thr Ile Phe Gly Glu Ser
210 215 220
Ala Gly Ser Met Ser Val Met Cys His Ile Leu Trp Asn Asp Gly Asp
225 230 235 240
Asn Thr Tyr Lys Gly Lys Pro Leu Phe Arg Ala Gly Ile Met Gln Ser
245 250 255
Gly Ala Met Val Pro Ser Asp Ala Val Asp Gly Ile Tyr Gly Asn Glu
260 265 270
Ile Phe Asp Leu Leu Ala Ser Asn Ala Gly Cys Gly Ser Ala Ser Asp
275 280 285
Lys Leu Ala Cys Leu Arg Gly Val Ser Ser Asp Thr Leu Glu Asp Ala
290 295 300
Thr Asn Asn Thr Pro Gly Phe Leu Ala Tyr Ser Ser Leu Arg Leu Ser
305 310 315 320
Tyr Leu Pro Arg Pro Asp Gly Val Asn Ile Thr Asp Asp Met Tyr Ala
325 330 335
Leu Val Arg Glu Gly Lys Tyr Ala Asn Ile Pro Val Ile Ile Gly Asp
340 345 350
Gln Asn Asp Glu Gly Thr Phe Phe Gly Thr Ser Ser Leu Asn Val Thr
355 360 365
Thr Asp Ala Gln Ala Arg Glu Tyr Phe Lys Gln Ser Phe Val His Ala
370 375 380
Ser Asp Ala Glu Ile Asp Thr Leu Met Thr Ala Tyr Pro Gly Asp Ile
385 390 395 400
Thr Gln Gly Ser Pro Phe Asp Thr Gly Ile Leu Asn Ala Leu Thr Pro
405 410 415
Gln Phe Lys Arg Ile Ser Ala Val Leu Gly Asp Leu Phe Phe Thr Leu
420 425 430
Ala Arg Arg Tyr Phe Leu Asn His Tyr Thr Gly Gly Thr Lys Tyr Ser
435 440 445
Phe Leu Ser Lys Gln Leu Ser Gly Leu Pro Val Leu Gly Thr Phe His
450 455 460
Ser Asn Asp Ile Val Phe Gln Asp Tyr Leu Leu Gly Ser Gly Ser Leu
465 470 475 480
Ile Tyr Asn Asn Ala Phe Ile Ala Phe Ala Thr Asp Leu Asp Pro Asn
485 490 495
Thr Ala Gly Leu Leu Val Lys Trp Pro Glu Tyr Thr Ser Ser Ser Gln
500 505 510
Ser Gly Asn Asn Leu Met Met Ile Asn Ala Leu Gly Leu Tyr Thr Gly
515 520 525
Lys Asp Asn Phe Arg Thr Ala Gly Tyr Asp Ala Leu Phe Ser Asn Pro
530 535 540
Pro Ser Phe Phe Val
545
<210> 13
<211> 549
<212> PRT
<213> 人工序列
<220>
<223> 来自柱状假丝酵母（Candida cylindracea）的包含信号肽的脂肪酶LIP1的G429M突变体
<400> 13
Met Glu Leu Ala Leu Ala Leu Ser Leu Ile Ala Ser Val Ala Ala Ala
1 5 10 15
Pro Thr Ala Thr Leu Ala Asn Gly Asp Thr Ile Thr Gly Leu Asn Ala
20 25 30
Ile Ile Asn Glu Ala Phe Leu Gly Ile Pro Phe Ala Glu Pro Pro Val
35 40 45
Gly Asn Leu Arg Phe Lys Asp Pro Val Pro Tyr Ser Gly Ser Leu Asp
50 55 60
Gly Gln Lys Phe Thr Ser Tyr Gly Pro Ser Cys Met Gln Gln Asn Pro
65 70 75 80
Glu Gly Thr Tyr Glu Glu Asn Leu Pro Lys Ala Ala Leu Asp Leu Val
85 90 95
Met Gln Ser Lys Val Phe Glu Ala Val Ser Pro Ser Ser Glu Asp Cys
100 105 110
Leu Thr Ile Asn Val Val Arg Pro Pro Gly Thr Lys Ala Gly Ala Asn
115 120 125
Leu Pro Val Met Leu Trp Ile Phe Gly Gly Gly Phe Glu Val Gly Gly
130 135 140
Thr Ser Thr Phe Pro Pro Ala Gln Met Ile Thr Lys Ser Ile Ala Met
145 150 155 160
Gly Lys Pro Ile Ile His Val Ser Val Asn Tyr Arg Val Ser Ser Trp
165 170 175
Gly Phe Leu Ala Gly Asp Glu Ile Lys Ala Glu Gly Ser Ala Asn Ala
180 185 190
Gly Leu Lys Asp Gln Arg Leu Gly Met Gln Trp Val Ala Asp Asn Ile
195 200 205
Ala Ala Phe Gly Gly Asp Pro Thr Lys Val Thr Ile Phe Gly Glu Ser
210 215 220
Ala Gly Ser Met Ser Val Met Cys His Ile Leu Trp Asn Asp Gly Asp
225 230 235 240
Asn Thr Tyr Lys Gly Lys Pro Leu Phe Arg Ala Gly Ile Met Gln Ser
245 250 255
Gly Ala Met Val Pro Ser Asp Ala Val Asp Gly Ile Tyr Gly Asn Glu
260 265 270
Ile Phe Asp Leu Leu Ala Ser Asn Ala Gly Cys Gly Ser Ala Ser Asp
275 280 285
Lys Leu Ala Cys Leu Arg Gly Val Ser Ser Asp Thr Leu Glu Asp Ala
290 295 300
Thr Asn Asn Thr Pro Gly Phe Leu Ala Tyr Ser Ser Leu Arg Leu Ser
305 310 315 320
Tyr Leu Pro Arg Pro Asp Gly Val Asn Ile Thr Asp Asp Met Tyr Ala
325 330 335
Leu Val Arg Glu Gly Lys Tyr Ala Asn Ile Pro Val Ile Ile Gly Asp
340 345 350
Gln Asn Asp Glu Gly Thr Phe Phe Gly Thr Ser Ser Leu Asn Val Thr
355 360 365
Thr Asp Ala Gln Ala Arg Glu Tyr Phe Lys Gln Ser Phe Val His Ala
370 375 380
Ser Asp Ala Glu Ile Asp Thr Leu Met Thr Ala Tyr Pro Gly Asp Ile
385 390 395 400
Thr Gln Gly Ser Pro Phe Asp Thr Gly Ile Leu Asn Ala Leu Thr Pro
405 410 415
Gln Phe Lys Arg Ile Ser Ala Val Leu Gly Asp Leu Met Phe Thr Leu
420 425 430
Ala Arg Arg Tyr Phe Leu Asn His Tyr Thr Gly Gly Thr Lys Tyr Ser
435 440 445
Phe Leu Ser Lys Gln Leu Ser Gly Leu Pro Val Leu Gly Thr Phe His
450 455 460
Ser Asn Asp Ile Val Phe Gln Asp Tyr Leu Leu Gly Ser Gly Ser Leu
465 470 475 480
Ile Tyr Asn Asn Ala Phe Ile Ala Phe Ala Thr Asp Leu Asp Pro Asn
485 490 495
Thr Ala Gly Leu Leu Val Lys Trp Pro Glu Tyr Thr Ser Ser Ser Gln
500 505 510
Ser Gly Asn Asn Leu Met Met Ile Asn Ala Leu Gly Leu Tyr Thr Gly
515 520 525
Lys Asp Asn Phe Arg Thr Ala Gly Tyr Asp Ala Leu Phe Ser Asn Pro
530 535 540
Pro Ser Phe Phe Val
545
<210> 14
<211> 549
<212> PRT
<213> 人工序列
<220>
<223> 来自柱状假丝酵母（Candida cylindracea）的包含信号肽的脂肪酶LIP1的G429I突变体
<400> 14
Met Glu Leu Ala Leu Ala Leu Ser Leu Ile Ala Ser Val Ala Ala Ala
1 5 10 15
Pro Thr Ala Thr Leu Ala Asn Gly Asp Thr Ile Thr Gly Leu Asn Ala
20 25 30
Ile Ile Asn Glu Ala Phe Leu Gly Ile Pro Phe Ala Glu Pro Pro Val
35 40 45
Gly Asn Leu Arg Phe Lys Asp Pro Val Pro Tyr Ser Gly Ser Leu Asp
50 55 60
Gly Gln Lys Phe Thr Ser Tyr Gly Pro Ser Cys Met Gln Gln Asn Pro
65 70 75 80
Glu Gly Thr Tyr Glu Glu Asn Leu Pro Lys Ala Ala Leu Asp Leu Val
85 90 95
Met Gln Ser Lys Val Phe Glu Ala Val Ser Pro Ser Ser Glu Asp Cys
100 105 110
Leu Thr Ile Asn Val Val Arg Pro Pro Gly Thr Lys Ala Gly Ala Asn
115 120 125
Leu Pro Val Met Leu Trp Ile Phe Gly Gly Gly Phe Glu Val Gly Gly
130 135 140
Thr Ser Thr Phe Pro Pro Ala Gln Met Ile Thr Lys Ser Ile Ala Met
145 150 155 160
Gly Lys Pro Ile Ile His Val Ser Val Asn Tyr Arg Val Ser Ser Trp
165 170 175
Gly Phe Leu Ala Gly Asp Glu Ile Lys Ala Glu Gly Ser Ala Asn Ala
180 185 190
Gly Leu Lys Asp Gln Arg Leu Gly Met Gln Trp Val Ala Asp Asn Ile
195 200 205
Ala Ala Phe Gly Gly Asp Pro Thr Lys Val Thr Ile Phe Gly Glu Ser
210 215 220
Ala Gly Ser Met Ser Val Met Cys His Ile Leu Trp Asn Asp Gly Asp
225 230 235 240
Asn Thr Tyr Lys Gly Lys Pro Leu Phe Arg Ala Gly Ile Met Gln Ser
245 250 255
Gly Ala Met Val Pro Ser Asp Ala Val Asp Gly Ile Tyr Gly Asn Glu
260 265 270
Ile Phe Asp Leu Leu Ala Ser Asn Ala Gly Cys Gly Ser Ala Ser Asp
275 280 285
Lys Leu Ala Cys Leu Arg Gly Val Ser Ser Asp Thr Leu Glu Asp Ala
290 295 300
Thr Asn Asn Thr Pro Gly Phe Leu Ala Tyr Ser Ser Leu Arg Leu Ser
305 310 315 320
Tyr Leu Pro Arg Pro Asp Gly Val Asn Ile Thr Asp Asp Met Tyr Ala
325 330 335
Leu Val Arg Glu Gly Lys Tyr Ala Asn Ile Pro Val Ile Ile Gly Asp
340 345 350
Gln Asn Asp Glu Gly Thr Phe Phe Gly Thr Ser Ser Leu Asn Val Thr
355 360 365
Thr Asp Ala Gln Ala Arg Glu Tyr Phe Lys Gln Ser Phe Val His Ala
370 375 380
Ser Asp Ala Glu Ile Asp Thr Leu Met Thr Ala Tyr Pro Gly Asp Ile
385 390 395 400
Thr Gln Gly Ser Pro Phe Asp Thr Gly Ile Leu Asn Ala Leu Thr Pro
405 410 415
Gln Phe Lys Arg Ile Ser Ala Val Leu Gly Asp Leu Ile Phe Thr Leu
420 425 430
Ala Arg Arg Tyr Phe Leu Asn His Tyr Thr Gly Gly Thr Lys Tyr Ser
435 440 445
Phe Leu Ser Lys Gln Leu Ser Gly Leu Pro Val Leu Gly Thr Phe His
450 455 460
Ser Asn Asp Ile Val Phe Gln Asp Tyr Leu Leu Gly Ser Gly Ser Leu
465 470 475 480
Ile Tyr Asn Asn Ala Phe Ile Ala Phe Ala Thr Asp Leu Asp Pro Asn
485 490 495
Thr Ala Gly Leu Leu Val Lys Trp Pro Glu Tyr Thr Ser Ser Ser Gln
500 505 510
Ser Gly Asn Asn Leu Met Met Ile Asn Ala Leu Gly Leu Tyr Thr Gly
515 520 525
Lys Asp Asn Phe Arg Thr Ala Gly Tyr Asp Ala Leu Phe Ser Asn Pro
530 535 540
Pro Ser Phe Phe Val
545
<210> 15
<211> 549
<212> PRT
<213> 人工序列
<220>
<223> 来自柱状假丝酵母（Candida cylindracea）的包含信号肽的脂肪酶LIP1的V549F突变体
<400> 15
Met Glu Leu Ala Leu Ala Leu Ser Leu Ile Ala Ser Val Ala Ala Ala
1 5 10 15
Pro Thr Ala Thr Leu Ala Asn Gly Asp Thr Ile Thr Gly Leu Asn Ala
20 25 30
Ile Ile Asn Glu Ala Phe Leu Gly Ile Pro Phe Ala Glu Pro Pro Val
35 40 45
Gly Asn Leu Arg Phe Lys Asp Pro Val Pro Tyr Ser Gly Ser Leu Asp
50 55 60
Gly Gln Lys Phe Thr Ser Tyr Gly Pro Ser Cys Met Gln Gln Asn Pro
65 70 75 80
Glu Gly Thr Tyr Glu Glu Asn Leu Pro Lys Ala Ala Leu Asp Leu Val
85 90 95
Met Gln Ser Lys Val Phe Glu Ala Val Ser Pro Ser Ser Glu Asp Cys
100 105 110
Leu Thr Ile Asn Val Val Arg Pro Pro Gly Thr Lys Ala Gly Ala Asn
115 120 125
Leu Pro Val Met Leu Trp Ile Phe Gly Gly Gly Phe Glu Val Gly Gly
130 135 140
Thr Ser Thr Phe Pro Pro Ala Gln Met Ile Thr Lys Ser Ile Ala Met
145 150 155 160
Gly Lys Pro Ile Ile His Val Ser Val Asn Tyr Arg Val Ser Ser Trp
165 170 175
Gly Phe Leu Ala Gly Asp Glu Ile Lys Ala Glu Gly Ser Ala Asn Ala
180 185 190
Gly Leu Lys Asp Gln Arg Leu Gly Met Gln Trp Val Ala Asp Asn Ile
195 200 205
Ala Ala Phe Gly Gly Asp Pro Thr Lys Val Thr Ile Phe Gly Glu Ser
210 215 220
Ala Gly Ser Met Ser Val Met Cys His Ile Leu Trp Asn Asp Gly Asp
225 230 235 240
Asn Thr Tyr Lys Gly Lys Pro Leu Phe Arg Ala Gly Ile Met Gln Ser
245 250 255
Gly Ala Met Val Pro Ser Asp Ala Val Asp Gly Ile Tyr Gly Asn Glu
260 265 270
Ile Phe Asp Leu Leu Ala Ser Asn Ala Gly Cys Gly Ser Ala Ser Asp
275 280 285
Lys Leu Ala Cys Leu Arg Gly Val Ser Ser Asp Thr Leu Glu Asp Ala
290 295 300
Thr Asn Asn Thr Pro Gly Phe Leu Ala Tyr Ser Ser Leu Arg Leu Ser
305 310 315 320
Tyr Leu Pro Arg Pro Asp Gly Val Asn Ile Thr Asp Asp Met Tyr Ala
325 330 335
Leu Val Arg Glu Gly Lys Tyr Ala Asn Ile Pro Val Ile Ile Gly Asp
340 345 350
Gln Asn Asp Glu Gly Thr Phe Phe Gly Thr Ser Ser Leu Asn Val Thr
355 360 365
Thr Asp Ala Gln Ala Arg Glu Tyr Phe Lys Gln Ser Phe Val His Ala
370 375 380
Ser Asp Ala Glu Ile Asp Thr Leu Met Thr Ala Tyr Pro Gly Asp Ile
385 390 395 400
Thr Gln Gly Ser Pro Phe Asp Thr Gly Ile Leu Asn Ala Leu Thr Pro
405 410 415
Gln Phe Lys Arg Ile Ser Ala Val Leu Gly Asp Leu Gly Phe Thr Leu
420 425 430
Ala Arg Arg Tyr Phe Leu Asn His Tyr Thr Gly Gly Thr Lys Tyr Ser
435 440 445
Phe Leu Ser Lys Gln Leu Ser Gly Leu Pro Val Leu Gly Thr Phe His
450 455 460
Ser Asn Asp Ile Val Phe Gln Asp Tyr Leu Leu Gly Ser Gly Ser Leu
465 470 475 480
Ile Tyr Asn Asn Ala Phe Ile Ala Phe Ala Thr Asp Leu Asp Pro Asn
485 490 495
Thr Ala Gly Leu Leu Val Lys Trp Pro Glu Tyr Thr Ser Ser Ser Gln
500 505 510
Ser Gly Asn Asn Leu Met Met Ile Asn Ala Leu Gly Leu Tyr Thr Gly
515 520 525
Lys Asp Asn Phe Arg Thr Ala Gly Tyr Asp Ala Leu Phe Ser Asn Pro
530 535 540
Pro Ser Phe Phe Phe
545
Claims (11)
1.一种天然干酪的制造方法，其特征在于，包括使来自微生物的脂肪酶和来自丝状菌的蛋白酶作用于乳汁的工序。
2.根据权利要求1所述的制造方法，其中，所述来自微生物的脂肪酶包含下述(1)～(3)所示的至少任一种多肽：
(1)多肽，其包含序列号1～15中任一者所示的氨基酸序列、或从序列号1～15所示的氨基酸序列中分别去除第1～15位的氨基酸序列而成的氨基酸序列；
(2)多肽，其是在序列号1～15中任一者所示的氨基酸序列、或从序列号1～15所示的氨基酸序列中分别去除第1～15位的氨基酸序列而成的氨基酸序列中，1个或多个氨基酸残基被替换、添加、插入或缺失而成的，且具有短链脂肪酸选择性；以及
(3)多肽，其相对于序列号1～15中任一者所示的氨基酸序列、或从序列号1～15所示的氨基酸序列中分别去除第1～15位的氨基酸序列而成的氨基酸序列的序列一致性为70％以上，且具有短链脂肪酸选择性。
3.根据权利要求1或2所述的制造方法，其中，所述来自微生物的脂肪酶的使用量相对于所述乳汁中的1g乳脂肪为1LU以上。
4.根据权利要求1～3中任一项所述的制造方法，其中，所述来自丝状菌的蛋白酶的使用量相对于所述来自微生物的脂肪酶1LU为0.02U以上。
5.根据权利要求1～4中任一项所述的制造方法，其中，所述来自丝状菌的蛋白酶的使用量相对于所述乳汁中的1g乳蛋白为0.3U以上。
6.根据权利要求5所述的制造方法，其中，所述来自丝状菌的蛋白酶的使用量相对于所述乳汁中的1g乳蛋白为20U以下。
7.根据权利要求1～6中任一项所述的制造方法，其中，所述来自丝状菌的蛋白酶为来自曲霉属的蛋白酶。
8.一种天然干酪，其特征在于，包含来自微生物的脂肪酶和来自丝状菌的蛋白酶。
9.一种天然干酪制造中的短链脂肪酸游离促进剂，其特征在于，包含来自丝状菌的蛋白酶，
所述天然干酪制造中使用来自微生物的脂肪酶。
10.一种干酪加工品的制造方法，其特征在于，包括通过权利要求1～7中任一项所述的制造方法制造天然干酪的工序；以及
加工所述天然干酪的工序。
11.一种干酪加工品，其特征在于，通过权利要求10所述的制造方法制造。
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| JP2021016920 | 2021-02-04 | ||
| JP2021-016920 | 2021-02-04 | ||
| PCT/JP2022/004515 WO2022168954A1 (ja) | 2021-02-04 | 2022-02-04 | ナチュラルチーズの製造方法 |
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| US (1) | US20240114915A1 (zh) |
| EP (1) | EP4289277A1 (zh) |
| JP (1) | JPWO2022168954A1 (zh) |
| CN (1) | CN116828989A (zh) |
| AU (1) | AU2022216111A1 (zh) |
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| DE3444282A1 (de) | 1984-12-05 | 1986-06-05 | Hoechst Ag, 6230 Frankfurt | Kaesearoma |
| DK46793D0 (da) * | 1993-04-26 | 1993-04-26 | Novo Nordisk As | Enzym |
| US6375947B1 (en) | 1998-11-05 | 2002-04-23 | International Flavors & Fragrances Inc. | Recombinant kid pregastric esterase and methods for its production and use |
| US6406724B1 (en) * | 2000-09-12 | 2002-06-18 | Kraft Foods Holdings, Inc. | Natural biogenerated cheese flavoring system |
| CN1913783A (zh) * | 2004-01-30 | 2007-02-14 | 帝斯曼知识产权资产管理有限公司 | 用于奶酪熟化的羧肽酶 |
| ES2429492T3 (es) | 2008-02-29 | 2013-11-15 | Dsm Ip Assets B.V. | Lipasas con alta especificidad hacia ácidos grasos de cadena corta y usos de las mismas |
| JP6787668B2 (ja) | 2013-12-10 | 2020-11-18 | 天野エンザイム株式会社 | 改変型リパーゼ及びその用途 |
| WO2017211930A1 (en) | 2016-06-10 | 2017-12-14 | Dsm Ip Assets B.V. | Mutant lipase and use thereof |
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| AU2022216111A1 (en) | 2023-08-31 |
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