CN1914315A - 具有葡聚糖、淀粉、齿斑葡聚糖、菊糖和呋喃果聚糖水解活性的蛋白,编码该蛋白的基因,表达该蛋白的细胞及其生产方法 - Google Patents
具有葡聚糖、淀粉、齿斑葡聚糖、菊糖和呋喃果聚糖水解活性的蛋白,编码该蛋白的基因,表达该蛋白的细胞及其生产方法 Download PDFInfo
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Abstract
本发明公开了具有葡聚糖、淀粉、齿斑葡聚糖、菊糖和呋喃果聚糖水解活性,氨基酸序列为SEQ.ID.No.1的酶,以及编码该酶的基因和表达该酶的转化细胞。此外,本发明还公开了生产能够降解葡聚糖、淀粉、齿斑葡聚糖、菊糖和呋喃果聚糖的酶的方法,该方法包括培养细胞、在细胞中表达该酶以及酶的纯化。含有该酶的组合物提供用于除去糖生产过程中的葡聚糖或多糖污染物。由于具有这种降解活性,该酶不仅在牙齿护理工业中具有广泛的应用,包括抗牙菌斑组合物和漱口液,而且也可以用于除去糖生产过程中的葡聚糖或多糖污染物。
Description
发明背景
1、发明领域
本发明涉及了能够水解葡聚糖、淀粉、齿斑葡聚糖、菊糖和呋喃果聚糖的酶,该酶的基因,表达该酶的细胞及其生产方法。更具体来说,本发明涉及了一种酶,它不仅由于其抑制牙菌斑的形成和降解以前形成的牙菌斑的能力而可以用在抗牙菌斑组合物或漱口液中,而且由于其水解葡聚糖的良好能力而可以用于除去糖生产过程中的葡聚糖,此外还涉及了编码该酶的基因,表达该酶的细胞以及生产该酶的方法。
2、相关技术描述
牙菌斑是在牙齿上沉积的生物膜,来自于牙齿表面的微生物定居。牙菌斑的主体由被称为葡聚糖(不溶性葡聚糖)的细菌产生的细胞外多糖构成,它也被称为齿斑葡聚糖,能够增强定居。该多糖总数达到牙菌斑干重的大约20%,是导致龋齿的重要因素。对变体链球菌(Streptococcus mutans)产生的葡聚糖进行的结构研究表明,不溶性葡聚糖中的葡萄糖部分彼此之间主要通过α-1,3-、α-1,4-、和α-1,6-D-糖苷键连接。因此,有效地消除牙菌斑需要齿斑葡聚糖、淀粉和葡聚糖水解活性。
按照常规,防止牙菌斑和龋齿的形成主要依靠抑制口腔中的变体链球菌的生长。由于这一点,具有抗变体链球菌生长活性的化合物例如抗菌剂或氟,被包含在口腔产品例如牙膏或漱口液中。氟是一种流行的抗龋洞化合物,因为它能够抑制变体链球菌的生长,但是它也能够引起牙齿氟中毒(在牙釉质中形成色斑)以及副作用例如强烈的毒性和空气污染。已经进行了另一种预防龋齿的尝试,就是使用酶例如葡聚糖酶;但是,它的效果还没有得到证实。
美国专利No.5,741,773提供了含有具有抗牙菌斑和抗龋齿活性的配糖巨肽的牙粉组合物。这种常规技术目的在于抑制引起龋齿的细菌的生长。但是,还没有建议防止牙菌斑的形成或水解以前形成的牙菌斑。
授权于本发明人的美国专利No.6,485,953(对应于韩国专利No.10-0358376)提出使用能够水解多种结构多糖的DXAMase来抑制牙菌斑的形成并降解以前形成的牙菌斑。除了能够水解多种多糖的酶之外,生产该酶的微生物(斯氏油脂酵母(Lipomyces starkeyi)KFCC-11077)和含有该酶的组合物也被公开了。
然而,对于能够更有效地抑制牙菌斑的形成并水解以前形成的牙菌斑的新酶的需求仍然存在。
在韩国专利申请No.10-2001-48442中,本发明人还提出由韩国专利No.10-0358376的微生物(斯氏油脂酵母(Lipomyces starkeyi)KFCC-11077)产生的酶DXAMase,由于其高度的葡聚糖降解活性,可以用于除去葡聚糖。
因此,在本技术领域内存在着明显的需求,开发具有葡聚糖降解活性的新酶,其活性足够用于在糖生产过程中除去葡聚糖。
发明概述
因此,在本发明的形成过程中一直考虑着现有技术中存在的上述问题,并且本发明的目的是提供新的酶,它具有防止牙菌斑的形成和降解以前形成的牙菌斑的活性以及良好的葡聚糖水解活性,以及提供编码该酶的基因。
本发明的另一个目的是提供带有该基因的菌株。
本发明的另一个目的是提供生产该酶和该基因的方法。
本发明的另一个目的是提供含有该酶的可以在工业中使用的组合物。
本发明的一个方面提供了含有SEQ.ID.No.1的氨基酸序列,具有葡聚糖、淀粉、齿斑葡聚糖、菊糖和呋喃果聚糖水解活性的蛋白,该蛋白的衍生物或片段。
本发明的另一方面提供了具有SEQ.ID.No.2序列,编码该蛋白、该蛋白的衍生物或片段的基因,该基因的衍生物或片段。
本发明的另一方面提供了表达该基因的转化细胞。
本发明的另一方面提供了生产具有葡聚糖、淀粉、齿斑葡聚糖、菊糖和呋喃果聚糖水解活性的酶的方法,包括:培养细胞;在培养的细胞中表达该酶;以及纯化表达的酶。
附图简述
根据下面的详细描述以及附图,将会更清楚地理解本发明的上述和其它的目的、特征和优点,其中:
图1显示了本发明的从斯氏油脂酵母(Lipomyces starkeyi)(LSD1)中获得的糖水解酶的氨基酸序列以及编码该氨基酸序列的2052bp的核苷酸序列,其中下划线的是用于在载体中克隆蛋白的PCR引物;
图2是将本发明的LSD的活性和稳定性对pH值制作的图;
图3是将本发明的LSD的活性和稳定性对温度制作的图;
图4是TLC结果的照片,显示了在进行酶失活之前和之后本发明的LSD的酶活性(分别为1-5道和6-10道),其中分析的样品是淀粉(1道和6道)、葡聚糖(2道和7道)、齿斑葡聚糖(3道和8道)、呋喃果聚糖(4道和9道)和菊糖(5道和10道),以及将酶提取物与样品反应后的一系列麦芽糖糊精(Mn道)和一系列异麦芽糖糊精(IMn道);以及
图5显示了本发明的酶以及青霉菌(penicillium)葡聚糖酶与羟基磷灰石的结合能力。
优选实施方案描述
编码本发明的糖水解酶(即聚糖酶(LSD))的基因的获取是从在含有葡聚糖的培养基中培养斯氏油脂酵母(Lipomyces starkeyi),并从该微生物中分离poly(A)+RNA开始的。接着,从目前已知的葡聚糖酶编码的基因中获得共同的氨基酸序列,在此序列信息的基础上构建了含有预计的保守区的引物,然后用这些引物进行PCR扩增。PCR产物大约1.1kb长,被用于5’RACE和3’RACE以获得完整的葡聚糖酶基因。在通过PCR扩增后,将该基因克隆到酿酒酵母(Saccharomycescerevisiae)载体pYES2,并转化到酿酒酵母中。经过转化的细胞在含有蓝色葡聚糖和半乳糖的培养基中生长。在蓝色背景上周围形成透明晕圈的菌落被选取(酿酒酵母INVSc1),从该酿酒酵母转化子获得了带有所需基因的重组克隆(pYLSD1)。
已知斯氏油脂酵母能够产生降解葡聚糖的内切葡聚糖酶(EC3.2.1.11)和降解淀粉的α-淀粉酶。该微生物已经被应用于食品中,尚未报道能产生抗生素或其它有毒代谢物。
已知大多数由微生物产生的葡聚糖酶,除了几种来自细菌的之外,都是诱导酶。在美国专利No.5,229,277中首先报道的斯氏油脂酵母ATCC7 4054产生葡聚糖酶和淀粉酶,其性质已经被公开。此外也已经报道了该菌从蔗糖和淀粉产生低分子量的葡聚糖。在这些发现的基础上,本发明人在2002年10月11日获得了韩国专利No.10-0358376(对应于日期为2002年11月26日的美国专利No.6,485,953),该专利涉及能够水解葡聚糖和淀粉两者的DXAMase酶,生产该酶的微生物(鉴定为斯氏油脂酵母KFCC-11077),以及含有该酶的组合物。
本发明从基因(lsd1)表达的酶能够水解淀粉和齿斑葡聚糖(不溶性葡聚糖)以及葡聚糖。此外,本发明的聚糖酶被发现主要将葡聚糖降解为葡萄糖、异麦芽糖和异麦芽三糖,同时还产生少量的分枝戊糖和己糖。
作为牙菌斑组成成分的呋喃果聚糖和菊糖类型的果聚糖可以被本发明的聚糖酶降解。
因此,通过本发明的聚糖酶可以完成葡聚糖的有效降解,不论它是可溶的还是不溶性的。因为可以通过抑制细菌的定居和葡聚糖的聚集而防止牙菌斑的形成和除去以前形成的牙菌斑,聚糖酶可以用于预防龋齿。据推断,聚糖酶具有保持在牙齿上的能力,正如在关于该酶是否能够结合与牙釉质成分相似的羟基磷灰石的试验中所证实的那样。
此外,本发明还涉及了带有编码聚糖酶基因的新的微生物。该微生物是一株酿酒酵母,保藏在位于韩国Daejeon市Yusung Gu的韩国典型培养物保藏中心(KCTC),登记号KCTC10574BP,保藏日期是2003年12月24日。
此外,本发明涉及了生产聚糖酶的方法。首先,通过细胞培养扩增pYLSD1克隆。在从培养物中收获后,将细胞用玻璃珠破碎,从其中分离聚糖酶。pYLSD1编码的聚糖酶的性质基本上与斯氏油脂酵母KFCC-11077中的相同。
用作RNA分离和聚糖酶基因筛选的DNA供体的斯氏油脂酵母KFCC-11077,产生的聚糖酶具有葡聚糖酶和淀粉酶活性。
为了提供所需的DNA,将斯氏油脂酵母KFCC-11077在LMD培养基中通气培养,该培养基含有1%(w/w)葡聚糖、1%(v/v)无机盐溶液和0.3%(w/v)酵母提取物。无机盐溶液含有2%(w/v)MgSO4·7H2O、0.1%(w/v)NaCl、0.1%(w/v)FeSO4·7H2O、0.1%(w/v)MnSO4·H2O和0.13%(w/v)CaCl2·2H2O。在本发明中,通用DNA操作和DNA测序是用大肠杆菌DH5α和pGEM-T easy载体(Promega,USA)进行的。
作为pYLSD1的宿主细胞,酿酒酵母INVSc1被培养在YPD培养基中(酵母提取物10g/l,蛋白胨20g/l,葡萄糖20g/l)以便表达聚糖酶。用于酿酒酵母培养的YPG培养基中添加了合成的右旋糖(SD)和合成的补充物。
含有本发明的酶的组合物可以广泛用于各种口腔护理领域,包括抗牙菌斑组合物、漱口液、牙膏等。由于其降解多糖例如葡聚糖和淀粉的能力,本发明的酶也可以有效地用于在糖生产过程中除去葡聚糖。此外,含有本发明的酶的组合物可以应用于食品中,例如口香糖、饮料、牛奶等,本领域的专业技术人员可以容易地确定其成分。
通过下面的实施例可以获得对本发明更好的理解,提出这些实施例是为了说明的目的,而不对本发明构成限制。
实施例1:从斯氏油脂酵母中分离poly A+RNA
将斯氏油脂酵母接种到LMD培养基中。在28℃培养36小时后(达到指数生长期中期),将培养物于6500xg离心,得到细胞沉淀。将该沉淀悬浮在GIT缓冲液(4M异硫柠檬酸胍,25mM柠檬酸钠(pH7.0),0.5%月桂基肌氨酸的0.1%DEPC处理的水,0.1M 2-巯基乙醇)中,并与酸洗过的玻璃珠和等体积的苯酚(pH4.0)混合。将混合物振荡2分钟,然后离心。在上清液中加入异丙醇沉淀总RNA。通过使用oligotex树脂(Oligotex mRNA试剂盒,Quiagen)形成oligotex-mRNA复合物,从总RNA制备物中纯化mRNA。
实施例2:LSD1的RT-PCR扩增
为了合成第一链cDNA,使用0.5g从斯氏油脂酵母分离的总RNA,在存在修饰的寡聚脱氧胸苷引物T18NN(5’-GAGAGAGAGAGAGAGAGAGAACTAGTCTCGAGTTTTTTTTTTTTTTTTTT-3’)的情况下进行逆转录。10μl第一链cDNA用于扩增一部分编码聚糖酶的碱基序列。参考在葡聚糖酶中已知的7个保守区设计了一对简并引物DC-F和DC-R。引物DC-F(5’-ACCTGGCA(T/C)AG(A/G)(A/T/G)(A/C)(C/A)-3’)和DC-R(5’-G(G/C)(C/T)(T/G)CC(G/C)ACCTGCTT(A/G)TA-3’)的设计分别是基于肽序列TWWH(D/N)(N/S/T)(保守区I)和YKQVG(S/A)(保守区V)。使用这些引物组进行PCR,得到了大约1.1kb的推断的聚糖酶基因片段。使用AccPrepTM凝胶提取试剂盒(Bioneer,Korea)从琼脂糖凝胶中纯化PCR产物,然后将纯化的DNA片段与pGEM-T easy载体(Promega,USA)进行连接。DNA测序在韩国基础科学研究所进行。为了获得完整的聚糖酶基因,在1.1kb DNA片段的信息的基础上进行了RACE(cDNA末端的快速扩增)。关于这一点,5’-RACE和3’-RACE取决于5’-full RACE Core Set和3’-full RACE Core Set(都来自日本Takara公司),从而获得完整大小的为聚糖酶编码的cDNA。通过5’-RACE获得了180bp的PCR产物,而通过3’-RACE获得了900bp的PCR产物。因此获得了总长大约2kb的聚糖酶基因(lsd1)。
实施例3:聚糖酶基因的碱基和氨基酸测序
通过碱裂解方法制备了质粒DNA用于碱基测序。使用ABI PRISMCycle测序试剂盒(Perkin Elmer Corp.USA),在GeneAmp 9600热循环仪DNA测序系统(型号373-18,Applied Biosystems,USA)中进行了碱基测序。在图1和SEQ.ID.No.1和2中给出了碱基测序结果。
在含有聚糖酶基因的DNA片段中发现了一个由1824碱基对组成的开放阅读框。该开放阅读框开始于位于获得的碱基序列的第42位核苷酸的起始密码子(ATG),结束于位于第1868位核苷酸的终止密码子(TGA)。对应于结构基因的推断的蛋白由608个氨基酸残基组成,计算分子量为67.6kDa。
实施例4:重组质粒pYLSD1的构建以及用其转化酿酒酵母
将斯氏油脂酵母在YPG中培养并收获,按照Schwartz和Cantor的方法分离基因组DNA。
使用一组合成引物DX-F:5’-GTCCCTTGAGCTCCCAAC-3’(序列表3)和DX-R:5’-TCAACTAGAATTCATGAACTTCC-3’(序列表4),以对应于聚糖酶基因(lsd1)的DNA片段作为模板,在存在Taq DNA聚合酶的情况下进行了PCR扩增,扩增进行了30个循环,每个循环为在94℃变性1分钟、52℃退火1分钟和72℃延伸2分钟。将PCR产物与pGEM-T easy载体连接,用其进行转化。从转化的细胞制备的质粒用EcoRI处理以切下PCR产物,然后将其与pYES2载体(Invitrogen,USA)连接。关于这一点,载体首先用EcoRI消化并用CIAP处理以防止自连。使用电转化方法将获得的重组质粒转染到酿酒酵母中。在SC培养基中生长的转化子的筛选使用了诱导培养基(2%半乳糖,0.3%蓝色葡聚糖,缺乏尿嘧啶)。当接种有转化子的SC平板在30℃培养2到6天后,如果它们含有重组质粒,在菌落周围就会形成由葡聚糖水解而产生的晕圈。在蓝色背景上其周围形成了透明晕圈的菌落被选出,带有所需基因的克隆被命名为pYLSD1。
实施例5:表达聚糖酶基因的细菌的筛选
进行了半乳糖诱导以检查上清液中克隆的活性。将选取的克隆接种到50ml含有2%半乳糖和1%葡萄糖的SC液体培养基中,接种的量达到OD600=1,然后在30℃培养72小时。通过离心(5000rpm,5分钟)收获细胞,悬浮在5ml 20mM柠檬酸/磷酸缓冲液(pH5.5)中,然后在存在0.1g 0.45mm玻璃珠的情况下剧烈振荡3分钟以进行细胞破碎。将细胞裂解液以6000rpm离心2分钟,小心回收上清液。将上清液与聚乙二醇(PEG,分子量150,000-200,000)在4℃反应至浓缩体积,并从其中除去葡萄糖、二糖和寡糖。将PEG浓缩液对20mM柠檬酸/磷酸缓冲液(pH5.5)透析到原始体积。透析溶液作为粗酶液,为测定蛋白活性,将其与等体积1%葡聚糖混合。在反应16小时后测定活性。
实施例6:酶活性分析
通过二辛可宁酸铜方法测定酶的还原值。具体来说,将100μl二辛可宁酸铜加入到100μl酶溶液中,在80℃反应35分钟,然后冷却大约15分钟。在560nm测量吸收值。聚糖酶的葡聚糖酶活性通过测量将粗酶液与2%葡聚糖缓冲液在37℃反应15分钟时产生的异麦芽糖的量来确定。一个单位的葡聚糖酶活性被定义为与葡聚糖在37℃反应1分钟时产生1μmol异麦芽糖所需的酶量。
实施例7:聚糖酶的最适pH、温度和稳定性分析
通过测量酶与葡聚糖在pH4.1-7.7的范围内反应16小时后的葡聚糖酶活性以分析聚糖酶的葡聚糖酶活性的最适pH。酶的pH稳定性是酶在每种缓冲液中于22℃保温3小时后测定的。
酶的最适温度是通过测量已经在不同温度(10-60℃)下保温16小时的酶的反应速度来确定的。为了确定酶的温度稳定性,测量酶在不同温度(10-60℃)下保温3小时后的残余活性。
发现LSD酶在pH5.5时显示了最适葡聚糖酶活性,在pH5.0-5.7时维持了最适活性的80%或以上(图2,表1)。
表1
pH对聚糖酶活性和稳定性的影响
葡聚糖酶活性 | |
最适pH | 5.5 |
稳定的pH范围 | 5.0-5.7 |
在表1中,稳定的pH意味着在该pH范围内酶的残余活性是起始活性的80%或以上。
此外,酶在温度低于37℃时显示了起始活性的80%或以上,最适活性是在37℃时(图3,表2)。
表2
温度对聚糖酶稳定性的影响
葡聚糖酶活性 | |
稳定的温度范围 | ≤37℃ |
在表2中,稳定的温度范围意味着在该温度范围内酶的残余活性是起始活性的80%或以上。
实施例8:葡聚糖酶对不同底物的降解活性
检测了粗酶液对不同底物的降解活性(图4)。为了进行水解活性试验,除了葡聚糖之外,还制备了各种聚合物的1%水溶液,包括葡聚糖、淀粉、呋喃果聚糖(β-2,6-连接的D-果糖聚合物)、菊糖(β-2,1-连接的D-果糖聚合物)和齿斑葡聚糖(α-1,3-连接的D-葡萄糖聚合物)。酶与葡聚糖的反应产生了0.1%葡萄糖、19.3%异麦芽糖、24.2%异麦芽三糖和17.0%异麦芽四糖,同时还产生了分枝寡糖。因此,聚糖酶相信在与葡聚糖反应时充当内切葡聚糖酶。在存在聚糖酶的情况下,发现淀粉几乎完全被降解成葡萄糖。
此外,通过与不同的聚合物进行反应分析了从克隆pYLDS1表达的聚糖酶的水解活性。尽管比较低,检测到的聚糖酶的水解活性不仅能够作用于α-1,3-D-糖苷键连接的聚合物例如齿斑葡聚糖,而且能够作用于β-连接的聚合物例如菊糖。测量到的聚糖酶的水解活性以对葡聚糖为100%计算,对淀粉是54%,对齿斑葡聚糖是8%、对呋喃果聚糖是3%,和对菊糖是7%。
表3
聚糖酶对不同底物的相对活性
底物 | 相对活性(%) | |
母细胞(斯氏油脂酵母)的聚糖酶 | LSD1聚糖酶 | |
葡聚糖 | 100 | 100 |
淀粉 | 92 | 54 |
齿斑葡聚糖 | 16 | 8 |
呋喃果聚糖 | 22 | 3 |
菊糖 | 18 | 7 |
实施例9:葡聚糖酶与羟基磷灰石(HA)的结合
由于其与骨骼直接结合的能力,磷酸钙陶瓷被广泛用于骨骼的替代物。其中,羟基磷灰石(HA)是最适合用于人造骨骼和牙齿研究的,因为它显示出与在牙齿中发现的天然存在的磷灰石相似的结晶学性质。为此,采用了HA作为测试酶与牙齿键合能力的材料。将HA(Bio-Gel HTP,Bio-Rad Laboratories,Richmond CA)悬浮在10mM磷酸缓冲液(pH6.8)中。酶也被独立地悬浮在同样的缓冲液中。将200μlHA悬浮液与同样体积的酶悬浮液混合,然后将混合液放置60分钟以便HA将酶吸附到其上。在将游离的酶洗掉后,用10、50、100、200、300、400和500mM的磷酸缓冲液(pH6.8)进行洗脱,洗脱缓冲液中都含有1mM NaCl。收集后,分析酶洗脱级分的聚糖酶活性。
正如在图5中看到的那样,聚糖酶被300mM羟基磷灰石洗脱。还可以了解到聚糖酶在磷灰石上的残余量高于青霉菌葡聚糖酶的残余量。总之,这些结果表明聚糖酶与羟基磷灰石发生强烈的结合,因此能够留在牙齿上。
正如在本文前面所描述的那样,本发明的斯氏油脂酵母突变株产生的聚糖酶是一种大约70kDa的单一蛋白,当对其PCR产物进行碱基测序分析时发现有一个由1824bp核苷酸组成的开放阅读框。推测的结构基因蛋白由608个氨基酸残基组成,分子量为大约67.6kDa。
聚糖酶与葡聚糖反应获得的最终产物只有内切葡聚糖酶的典型产物。所需的酶降解葡聚糖主要产生葡萄糖、异麦芽糖、异麦芽三糖和异麦芽四糖,同时还产生了分枝戊糖。此外,发现了酶对多种碳水化合物具有降解活性,包括α-1,3-D-糖苷连接的聚合物以及β-连接的果聚糖例如呋喃果聚糖和菊糖。
因此,由于具有上述的降解活性,本发明的酶不仅能够在牙齿护理工业中发现广泛的应用,包括抗牙菌斑组合物和漱口液,而且也可以用于除去糖生产过程中的葡聚糖或多糖污染物。
尽管出于说明的目的公开了本发明的优选实施方案,本技术领域的专业人员将会认识到对本发明进行各种修改、补充和替代,而不背离权利要求中公开的本发明的范围和精神仍是可能的。
序列表
<110>利分扎株式会社(Lifenza Co.,Ltd.)
<120>具有葡聚糖、淀粉、齿斑葡聚糖、菊糖和呋喃果聚糖水解活性的蛋白,编码该蛋白
的基因,表达该蛋白的细胞及其生产方法(Protein with activity of hydrolyzing
dextran,starch,mutan,inulin and levan,gene encoding the same,cell
expressing the same,and production method thereof)
<130>SCT063527-47
<150>KR2004-0006185
<151>2004-01-30
<160>4
<170>KopatentIn 1.71
<210>1
<211>608
<212>PRT
<213>Artificial Sequence
<220>
<223>S.cerevisiae/pYES2-LSD1
<400>1
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Leu Ala Asp Arg Asn Gln Phe Tyr Asp Ser Phe Val Tyr Glu Ser Ile
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Pro Arg Asn Gly Asn Gly Arg Ile Tyr Ser Pro Thr Asp Pro Pro Asn
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Ser Asn Thr Leu Asn Ser Ser Ile Asp Asp Gly Ile Ser Ile Glu Pro
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Lys Asn Ala Leu Val Ile Phe Ala Ser Pro Phe Leu Pro Arg Asp Met
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Ile Asn Asn Gly Asp Trp Gly Ser Lys Pro Ile Leu Tyr Phe Pro Pro
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Gly Val Tyr Trp Met Asn Glu Asp Thr Ser Gly Asn Pro Gly Lys Leu
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Ala Pro Gly Ala Tyr Val Lys Gly Ala Ile Glu Tyr Phe Thr Lys Gln
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530 535 540
Pro Asp Gly Leu Gln Thr Asn Pro Ile Gly Ile Gly Glu Ser Ile Ile
545 550 555 560
Pro Ala Ala Ser Gly Cys Thr Met Asp Leu Glu Ile Thr Asn Trp Thr
565 570 575
Val Lys Gly Gln Lys Val Thr Met Gln Asn Phe Gln Ser Gly Ser Leu
580 585 590
Gly Gln Phe Asp Ile Asp Gly Ser Tyr Trp Gly Gln Trp Ser Ile Asn
595 600 605
<210>2
<211>2052
<212>DNA
<213>Artificial Sequence
<220>
<223>S.cerevisiae/pYLSD1
<400>2
tgggtgtgtc ccttgctctg ccaacgttgt tgattgtttt catgacatta atctacgtgc 60
cttcaatatt tacaatggtc ccctcaatca cacggattgt actggttaac attctgttgg 120
cgacgttggt tttgggagct gcagtccttc cacgagacaa cagaactgtt tgcgggagtc 180
aactctgcac atggtggcac gactccggcg agataaacac cggtactcct gtacaggcag 240
gaaacgttcg acaatcccga aagtactctg tccatgtgag cctggeagac cgtaaccaat 300
tctacgactc tttcgtatat gaatcgatac ctaggaacgg caatggcaga atttattctc 360
ccaccgaccc acctaacagc aatacattga atagtagcat tgacgacggt atatcaatcg 420
aaccatctct cggcatcaac atggcttggt cccagttcga atatagacga gatgtcgaca 480
ttaagattac tacaatcgat ggctcaatat tggatggccc tttggacatt gttattcggc 540
cgacttctgt taagtactca gtcaaaagat gtgtgggtgg tatcattatt agagtccctt 600
atgatcccaa tggtcgaaaa ttctctgttg agttaaagag tgacctttac agttacctct 660
ccgacggttc gcaatatgtg acctctggag ggagcgtggt tggtgtggag ccaaaaaatg 720
ccctggtgat ctttgccagc cctttcttgc cacgggatat ggttcctcat atgacaccac 780
acgacaccca gacaatgaag ccgggcccaa tcaataatgg ggactggggt tcaaagccta 840
tactctactt cccgcctggc gtatactgga tgaacgagga tacctctggt aaccccggga 900
agctcggctc aaatcatatg cggctggatc ccaataccta ctgggtccat ctagccccag 960
gagcctatgt gaaaggagcc attgagtatt tcacgaagca aaatttctat gcaacgggtc 1020
atggcgttct ctcaggtgag aactatgttt atcaggccaa tgcagctgat aactactatg 1080
ccgtcaagag tgatggcaca agcttgagaa tgtggtggca caacaacctt ggaggcggtc 1140
aaacatggtt ttgcatgggg cccaccatta atgcaccgcc gtttaatacg atggacttca 1200
acggaaactc taatatttcc agccggatta gtgactataa gcaggttggc gcttattttt 1260
tccaaacaga cggaccggag atctacgagg acagtgttgt ccatgacgtc ttctggcatg 1320
ttaatgatga tgccatcaag acatattatt ccggagcttc aatttcacga gcaaccatct 1380
ggaagtgtca caatgacccg atcatacaga tgggctggac gtcacgaaat ctcaccggaa 1440
tcagcattga taacctgcac gtcatccaca cgagatattt caaatctgaa acagtggttc 1500
cttcagcaat cattggagcg tctccattct acgcaagtgg aatgactgtt gatcccagcg 1560
agtccatcag catgaccatc tctaacgtgg tgtgtgaggg tctatgcccc tcactgttcc 1620
gtatcactcc gcttcagagc tacaacaacc ttgttgtcaa gaacgtggcc tttcccgatg 1680
gactgcagac aaatccaatc ggaataggag agagcattat accagcagct tccggctgta 1740
caatggactt ggaaatcaca aactggaccg tcaaaggaca aaaagtcacc atgcaaaact 1800
ttcagtccgg gtcaettggc cagttcgata tcgatggttc atactggggt caatggtcca 1860
taaactaaag ctattcccat tcacctgagt attttcgtgg gttcaatgag ttcttgttac 1920
tgatggggcc cttgctagtg gtaaaagtag agggacttgt cctcgccggg cgccaaggaa 1980
gttcatgtct tctagttgaa tagtatttgt ttcttctctc tcgttaaaaa aaaaaaaaaa 2040
aaaaaaaaaa aa 2052
<210>3
<211>18
<212>DNA
<213>Artificial Sequence
<220>
<223>L.starkeyi DX-F primer(sense)
<400>3
gtcccttgag ctcccaac 18
<210>4
<211>23
<212>DNA
<213>Artificial Sequence
<220>
<223>L.starkeyi DX-R primer(antisense)
<400>4
tcaactagaa ttcatgaact tcc 23
Claims (10)
1.含有SEQ.ID.No.1的氨基酸序列,具有水解葡聚糖、淀粉、齿斑葡聚糖、菊糖和呋喃果聚糖活性的蛋白,其衍生物或片段。
2.SEQ.ID.No.2的基因,编码权利要求1的蛋白,其衍生物或片段,以及该基因的衍生物或片段。
3.表达权利要求2的基因,其衍生物或片段的转化细胞。
4.权利要求3的转化细胞,其中的细胞是原核细胞或真核细胞。
5.权利要求3或4的转化细胞,其中的细胞是2003年12月24日保藏的、登记号为KCTC10574BP的大肠杆菌BL21(DE3)pLysS。
6.生产具有葡聚糖、淀粉、齿斑葡聚糖、菊糖和呋喃果聚糖水解活性的酶的方法,包括:
培养权利要求3的细胞;
在培养的细胞中表达酶;以及
纯化被表达的酶。
7.通过权利要求6的方法生产的酶。
8.含有权利要求7的酶的组合物。
9.权利要求8的组合物,其中的组合物用于在糖生产过程中除去葡聚糖。
10.权利要求8的组合物,其中的组合物用于消除牙菌斑或用作漱口液。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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KR1020040006185A KR20050078077A (ko) | 2004-01-30 | 2004-01-30 | 뮤탠, 이눌린 및 레반을 분해하는 단백질, 그 단백질을코딩하는 유전자, 그 발현 세포 및 상기 단백질의 생산 방법 |
KR1020040006185 | 2004-01-30 |
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CN1914315A true CN1914315A (zh) | 2007-02-14 |
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CNA2005800037448A Pending CN1914315A (zh) | 2004-01-30 | 2005-01-27 | 具有葡聚糖、淀粉、齿斑葡聚糖、菊糖和呋喃果聚糖水解活性的蛋白,编码该蛋白的基因,表达该蛋白的细胞及其生产方法 |
Country Status (6)
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US (1) | US20070140989A1 (zh) |
EP (1) | EP1716231A4 (zh) |
JP (1) | JP2007519418A (zh) |
KR (2) | KR20050078077A (zh) |
CN (1) | CN1914315A (zh) |
WO (1) | WO2005073368A1 (zh) |
Families Citing this family (3)
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JP2007295806A (ja) * | 2006-04-27 | 2007-11-15 | Nagase & Co Ltd | 核内受容体リガンドの分析方法、それに用いる培地、形質転換酵母およびキット |
CN105102617A (zh) * | 2013-04-05 | 2015-11-25 | 诺维信公司 | 具有右旋糖酐酶活性的多肽与编码它们的多核苷酸 |
WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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US5643758A (en) * | 1987-03-10 | 1997-07-01 | New England Biolabs, Inc. | Production and purification of a protein fused to a binding protein |
US5229277A (en) * | 1991-03-05 | 1993-07-20 | Louisiana State University Board Of Supervisors | Process for the production of dextran polymers of controlled molecular size and molecular size distributions |
JP2575258B2 (ja) * | 1992-02-19 | 1997-01-22 | 住友ゴム工業株式会社 | 印刷用オフセットブランケット |
AU685181B2 (en) * | 1993-12-14 | 1998-01-15 | Centro De Ingenieria Genetica Y Biotecnologia | Dextranase enzyme, method for its production and DNA encoding the enzyme |
US5741773A (en) * | 1996-04-26 | 1998-04-21 | Colgate Palmolive Company | Storage stable dentifrice composition containing an antibacterial casein glycomacropeptide adjuvant |
WO2001066570A1 (en) * | 2000-03-09 | 2001-09-13 | Doman Kim | Enzyme capable of hydrolyzing plaque, microorganism producing thesame, and a composition comprising the same |
US6485953B1 (en) * | 1999-03-09 | 2002-11-26 | Lifenza Co. Ltd. | Enzyme capable of hydorlyzing plaque, microorganism producing the same, and a composition comprising the same |
AU2001286275A1 (en) * | 2001-08-25 | 2003-03-10 | Lifenza Co., Ltd. | Enzyme with the removal activities of the plaques, dna sequence encoding said enzyme, the expressing host cell and methods for producing and purifying said enzyme |
-
2004
- 2004-01-30 KR KR1020040006185A patent/KR20050078077A/ko not_active Application Discontinuation
-
2005
- 2005-01-27 WO PCT/KR2005/000234 patent/WO2005073368A1/en active Application Filing
- 2005-01-27 US US10/588,140 patent/US20070140989A1/en not_active Abandoned
- 2005-01-27 KR KR1020067017493A patent/KR100809090B1/ko not_active IP Right Cessation
- 2005-01-27 JP JP2006550947A patent/JP2007519418A/ja active Pending
- 2005-01-27 CN CNA2005800037448A patent/CN1914315A/zh active Pending
- 2005-01-27 EP EP05710834A patent/EP1716231A4/en not_active Withdrawn
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KR100809090B1 (ko) | 2008-03-03 |
KR20050078077A (ko) | 2005-08-04 |
KR20060114026A (ko) | 2006-11-03 |
US20070140989A1 (en) | 2007-06-21 |
JP2007519418A (ja) | 2007-07-19 |
EP1716231A4 (en) | 2008-03-26 |
WO2005073368A1 (en) | 2005-08-11 |
EP1716231A1 (en) | 2006-11-02 |
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