CN103501637A - 从植物汁制备异麦芽酮糖的方法 - Google Patents
从植物汁制备异麦芽酮糖的方法 Download PDFInfo
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Abstract
本发明提供了一种使含有蔗糖的植物汁异构化以产生异麦芽酮糖的方法。
Description
技术领域
本发明涉及使含有蔗糖的植物汁异构化生成异麦芽酮糖的方法。
背景技术
借助于异麦芽酮糖合酶(蔗糖葡萄糖基变位酶(Succharose Glucosylmutasen),EC 5.4.99.11)将水溶液中的蔗糖酶促异构化生成异麦芽酮糖是一种已知的方法。
例如,DE1049800描述了一种借助于细菌菌株红色精蛋白杆菌(Protaminobacter rubrum)由含有蔗糖的植物汁发酵制备异麦芽酮糖的方法。可替换地,酶促转化也可以用死生物体进行。在反应结束时,通过离心逐批分离出细胞群,并进一步后处理产物。该方法参见文献H. Schiweck, alimenta 19, 5-16, 1980。
EP0001099描述了一种用尤其是浓汁或稀汁作为底物连续发酵细菌菌株红色精蛋白杆菌(CBS574.77)和普城沙雷菌(Serratia plymuthica)(ATCC 15928)同时将蔗糖异构化为异麦芽酮糖的方法,其中所述底物溶液本身是细胞生长的营养培养基。在反应以后,借助于离心从产物溶液分离出细胞。在与底物培养基不同的培养基中与实际异构化分离培养生物催化上有效的生物体已经被明确证实是不利的。
DE3241788描述了一种用细菌菌株的游离细胞由蔗糖水溶液制备1-O-α-D-吡喃型葡萄糖苷-D-果糖(1-O-α-D-Glucopyranosido-D-Fructose)的方法,所述细菌菌株选自红色精蛋白杆菌(CBD574.77)、普城沙雷菌(ATCC 15928)、粘质沙雷氏菌(Serratia marescens)(NCIB 8285)、肠膜明串珠菌(Leuconostoc mesenteroides)(NRRL-B-512 F)和大黄欧文氏菌(Erwinia rhapontici)(NCPPB 1578)。在所述的方法中,可以使用浓汁,并且所述浓汁同样充当细胞的生长过程的碳源。在固定化的细胞的情况中,则使用纯的蔗糖溶液。
在前述2篇说明书中公开的发酵制备异麦芽酮糖的情况中,存在在无菌条件下使用经济上和能源上有利的浓汁的可能性。但是,这里必须指出的缺点是,部分蔗糖被用于形成细胞群,并且需要昂贵的无菌硬件以及复杂的细胞混悬液与产物流的分离。
EP0983374描述了一种用固定化的细菌菌株同时制备异麦芽酮糖和甜菜碱的方法,所述细菌菌株选自红色精蛋白杆菌(CBD574.77)、普城沙雷菌(ATCC 15928)和大黄欧文氏菌(NCPPB 1578)。该制备方法始于糖蜜,即在制糖制工艺结束时生成的组合物。在该方法中,不能使用浓汁作为底物,并且需要通过例如结晶明确地贫化蔗糖。
WO97/44478描述了一种用活的、固定化的细菌菌株制备异麦芽酮糖的方法,所述细菌菌株选自红色精蛋白杆菌(CBD574.77)、普城沙雷菌(ATCC 15928)和大黄欧文氏菌(ATCC 29284)。该制备方法始于糖蜜或纯蔗糖溶液。
EP0028900描述了一种用细菌菌株大黄欧文氏菌(NCPPB 1578)的固定化细胞制备异麦芽酮糖的方法。该生产方法始于纯的蔗糖溶液与最多15% v/v糖蜜的掺混物。
DE2217628、EP49472、EP3038219和EP91063描述了用固定化的细菌细胞将纯蔗糖酶促转化为异麦芽酮糖的方法。为此,EP0625578使用选自红色精蛋白杆菌(CBS 574.77)、普城沙雷菌(ATCC 15928)、粘质沙雷菌(Serratia marcescens)(NCIB 8285)、肠膜明串珠菌(NRRL-B 512 F(ATCC 1083 a))和大黄欧文氏菌(NCPPB 1578)的细菌菌株。EP0392556和EP1257638描述了选自土生结克雷伯菌(Klebsiella terrigena)JCM 1687、克雷伯菌属(Klebsiella sp. )No. 88(FERM BP-2838)和新加坡克雷伯菌(Klebsiella singaporensis)LX3和LX21的细菌菌株的用途。
使用固定化的细胞的异构化方法描述于DE3133123和EP0915986中,并在固定化方法的范围中用作藻酸钙酶催化剂或离子交换剂。
所有已知的用固定化的细胞将蔗糖异构化为异麦芽酮糖的方法的一个共同点是,仅使用制糖工艺的最终产物,即纯的蔗糖或糖蜜。
所以对与这些过程关联的结晶的过程控制没有高的要求,因为异构化混合物是不复杂的且具有非常确定的组成,从其可以容易地分离出异麦芽酮糖。迄今已知的结晶方法不适合用于由更严重地污染的溶液以令人满意的品质分离出异麦芽酮糖。
因此,本发明的目的是,提供能够由容易获得的蔗糖来源制备纯化的异麦芽酮糖的方法。
发明内容
令人惊奇地,现在已经发现,在使用固定化的酶复合物的条件下使用含有蔗糖的植物汁来制备异麦芽酮糖,能够实现该目的。
因此,本发明提供了制备异麦芽酮糖的方法,所述方法包括下述方法步骤:
A) 使酶复合物、优选固定化的酶复合物与含有蔗糖的溶液接触,所述酶复合物能够催化蔗糖生成异麦芽酮糖的转化;
B) 将至少部分蔗糖异构化为异麦芽酮糖;
C) 分离出所述酶复合物,得到进料溶液,
D) 通过蒸发部分除去所述进料溶液的水,得到异麦芽酮糖-晶体的混悬液,
E) 将所述混悬液分离成含有异麦芽酮糖的固体和母液,和任选地
F) 洗涤所述含有异麦芽酮糖的固体,
其特征在于,作为含有蔗糖的溶液使用选自浓汁或稀汁的至少一种,优选浓汁。
在这方面,本发明有利地提供了含有蔗糖的植物汁作为异构化的底物的用途。
令人惊讶地,尽管事实上所使用的含有蔗糖的溶液是未纯化的并因而是非常复杂的底物混合物,但是通过本发明却实现了与用高纯度蔗糖相同的异构化中的收率和甚至更高的选择性。
本发明的一个优点是,通过增加固定化的细胞的停留时间以及使用粗制的植物汁作为底物,实现了显著更高的资源效率,因为节省了得到高纯度蔗糖的纯化步骤。
本发明的另一个优点是,通过直接使用浓汁,能够节省能量和生产装置(Betriebsmittel)。
本发明的另一个优点是,通过使用浓汁,不需要用于稳定酶活性的任何种类的缓冲溶液或另外的添加剂,而是进行所使用的酶的天然稳定化。
本发明的另一个优点是,与使用纯的蔗糖水溶液相反,使用含有蔗糖的植物汁会防止床的渗漏。
本发明的另一个优点是,通过在异构化过程中使用浓汁,与使用经纯化的蔗糖溶液相比,可以降低生龋的单糖类果糖和葡萄糖的含量。
术语“异麦芽酮糖”应当理解为是指6-O-α-D-吡喃型葡萄糖苷-D-果糖。
术语“酶复合物”应当理解为是指,具有至少一种活性酶的组合物或混合物组合物,其也可以是复合物性质的,例如,活细胞。酶复合物的另外的例子是融合蛋白,其中至少一种活性酶与至少一种另外的多肽连接,但是纯化的酶本身也可以是本发明意义上的酶复合物。
在本发明的上下文中,术语“固定化的酶复合物”应当理解为是指这样的酶复合物:其结合在基质上,或者被基质包封,使得所述酶复合物在水溶液中其自由扩散或其自由运动受到限制,例如减慢。
在本发明的上下文中,术语“稀汁”应当理解为是指,在由糖用甜菜或甘蔗制糖的过程中,在汁纯化以后存在的产物。基于整个汁计,稀汁通常包含10-20重量%的蔗糖。
在本发明的上下文中,术语“浓汁”应当理解为是指经浓缩的“稀汁”,基于整个汁计,其包含超过20重量%的蔗糖。
除非另外说明,所有给出的百分比(%) 是质量百分比。
在根据本发明的方法中,作为含有蔗糖的溶液有利地使用浓汁;如果需要的话,可以用水将其稀释至最多1:5的体积比,基于浓汁:水,从而得到更好的可控的异构化。
具体地,用水稀释浓汁如此来选择,使得在方法步骤A)开始时,基于整个汁计,在浓汁中存在20-80重量%、尤其是30-50重量%的蔗糖浓度。
具体地,可有利地使用糖用甜菜的稀汁或浓汁,其中糖用甜菜的浓汁是特别优选的。
含于酶复合物中的酶优选地是酶类别EC 5.4.99.11中的至少一种蔗糖葡萄糖基变位酶。在根据本发明的方法中,特别优选使用这样的蔗糖葡萄糖基变位酶,其得自:红色精蛋白杆菌、尤其是菌株红色精蛋白杆菌CBS 574.77和具有Seq ID Nr. 5和6的那些;红色精朊杆菌(Protaminobacter ruber)Z12;普城沙雷菌、尤其是菌株普城沙雷菌ATCC 15928;气味沙雷氏菌(Serratia odorifera)、尤其是菌株气味沙雷氏菌4Rx13(Seq ID Nr. 7);粘质沙雷菌、尤其是菌株粘质沙雷菌NCIB 8285;肠膜明串珠菌、尤其是菌株肠膜明串珠菌ATCC 1083 a;大黄欧文氏菌、尤其是菌株大黄欧文氏菌ATCC29283(Seq ID Nr. 1)、NCPPB 1578、DSM 4484(Seq ID Nr. 8)、NX-5(Seq ID Nr. 9)和WAC2928(Seq ID Nr. 10);欧文氏菌属(Erwinia sp.)、尤其是菌株欧文氏菌属D12;放射形土壤杆菌(Agrobacterium radiobacter)、尤其是菌株放射形土壤杆菌MX-232;土生结克雷伯菌、尤其是菌株土生结克雷伯菌JCM 1687;克雷伯菌属、尤其是菌株克雷伯菌属FERM BP-2838、LX3(Seq ID Nr. 11)和NK33-98-8(Seq ID Nr. 12);肺炎克雷伯菌(Klebsiella pneumoniae)、尤其是菌株肺炎克雷伯菌342(Seq ID Nr. 4);新加坡克雷伯菌、尤其是菌株新加坡克雷伯菌LX21;嗜中酸假单胞菌(Pseudomonas mesoacidophila)、尤其是菌株嗜中酸假单胞菌MX-45(Seq ID Nr. 2);分散泛菌(Pantoea dispersa)、尤其是菌株分散泛菌UQ68J(Seq ID Nr. 3);植物生克雷伯菌(Klebsiella planticola)、尤其是菌株植物生克雷伯菌CCRC 19112、MX10和UQ14S(Seq ID Nr. 13);肠杆菌属(Enterobactersp.)、尤其是菌株肠杆菌属FMB-1(Seq ID Nr. 16)、SZ62和Ejp617(Seq ID Nr. 14);维涅兰德固氮菌(Azotobacter vinelandii)DJ(Seq ID Nr. 15),其中红色精蛋白杆菌CBS 574.77和红色精朊杆菌Z12是特别优选的。
所述酶可以以经纯化形式作为多肽来使用。为了易于纯化,这些可以作为融合蛋白存在,其中,例如,使能够易于纯化的标签与所述酶融合,所述标签例如His-标签、Strep-标签、GST-标签或MBP-标签。
在根据本发明的方法中,优选的是,所述酶复合物是整个细胞,所述细胞优选地选自:红色精蛋白杆菌、尤其是菌株红色精蛋白杆菌CBS 574.77;红色精朊杆菌Z12;普城沙雷菌、尤其是菌株普城沙雷菌ATCC 15928;气味沙雷氏菌、尤其是菌株气味沙雷氏菌4Rx13;粘质沙雷菌、尤其是菌株粘质沙雷菌NCIB 8285;肠膜明串珠菌、尤其是菌株肠膜明串珠菌ATCC 1083 a;大黄欧文氏菌、尤其是菌株大黄欧文氏菌ATCC29283、NCPPB 1578、DSM 4484、NX-5和WAC2928;欧文氏菌属、尤其是菌株欧文氏菌属D12;放射形土壤杆菌、尤其是菌株放射形土壤杆菌MX-232;土生结克雷伯菌、尤其是菌株土生结克雷伯菌JCM 1687;克雷伯菌属、尤其是菌株克雷伯菌属FERM BP-2838、LX3和NK33-98-8;肺炎克雷伯菌、尤其是菌株肺炎克雷伯菌342;新加坡克雷伯菌、尤其是菌株新加坡克雷伯菌LX21;嗜中酸假单胞菌、尤其是菌株嗜中酸假单胞菌MX-45;分散泛菌、尤其是菌株分散泛菌UQ68J;植物生克雷伯菌、尤其是菌株植物生克雷伯菌CCRC 19112、MX10和UQ14S;肠杆菌属、尤其是菌株肠杆菌属FMB-1、SZ62和Ejp617;维涅兰德固氮菌DJ,其中红色精蛋白杆菌CBS 574.77和红色精朊杆菌Z12是特别优选的。
酶复合物的固定化例如可以以CLEAs (insoluble crosslinked enzyme aggregates (不可溶交联酶聚集体))的形式进行(Cao, L. 等人, 2000, Cross-linked enzyme aggregates: a simple and effective method for the immobilization of penicillin acylase, Org. Lett., 2: 1361-1264),或者在天然或合成起源的固体载体材料上进行。天然材料例如是多糖诸如藻酸盐、琼脂糖(Agarose)、琼脂糖凝胶(Sepharose)、纤维素及其衍生物(例如DEAE-或CM-纤维素)。也可以使用改性的琼脂糖凝胶,例如被环氧基-活化的、溴氰根-活化的、NHS-活化的、硫醇-活化的琼脂糖凝胶。这些琼脂糖例如是在公司GE Healthcare、BioRad、Sigma和Pierce商购可得的。
作为合成有机聚合物可以使用聚苯乙烯-衍生物、聚丙烯酸酯、尤其是环氧化物活化的丙烯酸树脂珠(Eupergit)、聚甲基丙烯酸酯、聚丙烯酰胺、乙烯基-和烯丙基聚合物、聚酯或聚酰胺。
作为无机载体可以是基于硅氧化物或铝氧化物或其混合物的材料。
酶复合物的固定化还可以通过包封在聚合的多孔凝胶中进行,例如RSi(OCH3)3或者RSi(OCH3)3与Si(OCH3)4 的混合物的疏水性溶胶-凝胶材料(Reetz, M.T.; Zonta, A.; Simpelkamp, J.; Rufinska, A.; Tesche, B. J. Sol-Gel Sci. Technol. 1996, 7, 第35-43页)或多孔的聚合硅胶-凝胶(Elgren, T.M.; Zadvorny, O.A.; Brecht, E.; Douglas, T.; Zorin, N.A.; Maroney, M. J. & Peters, J. W.)。
此外,除了固定化以外,在酶膜反应器中的酶的多次使用是可能的。
在根据本发明的方法中使用的酶复合物,尤其是细胞,优选地被固定在多糖诸如藻酸盐、果胶、角叉菜胶、壳聚糖或聚乙烯醇例如Lentikats或其混合物中,尤其是藻酸盐中;在这点上,参见例如Shimizu, H., 等人(1997) Screening of novel microbial enzymes for the production of biologically and chemically useful compounds,见:Advances in biochemical engineering biotechnology, 第58卷: New Enzymes for Organic Synthesis (Scheper, T., 编) 第45-88页, Springer, New York。
一种对于在根据本发明的方法中使用的酶复合物的任意形式而言特别优选的固定化是,在EP2011865中描述的方法,其中给固定在惰性载体上的酶复合物提供通过氢化硅烷化得到的有机硅涂层。
根据本发明优选的是,在方法步骤B)中,具有4-9.5的pH-值。该pH-值有利地借助于酸、尤其是无机酸来调节,所述酸优选选自硫酸、盐酸或乙酸。
此外,根据本发明优选的是,在方法步骤B)中,具有20-40℃、优选25-35℃的温度,该温度在含有蔗糖的溶液中测得。
在方法步骤C)中,将酶复合物与异麦芽酮糖分离;这例如通过过滤、沉降或离心来进行。因为酶复合物的被固定物特性,该分离相对于未固定的酶变得容易。
由于工艺经济的原因,根据本发明,如果以固体床的形式使用经固定化的酶复合物,是优选的,含有蔗糖的溶液从所述固体床中流过(H. Schiweck, Zuckerind. (1990), 115 (7), 555-565)。相应的以固体床的方法描述于A. Liese, 等人. Processes 在A. Liese, K. Seelbach, C. Wandrey (编), Industrial Biotransformations第2版(2006), Wiley-VCH, Weinheim中。在这样的方法中,方法步骤A)至C) 连续进行,并且从而彼此逐渐变成直至同时进行。
一种根据本发明优选的方法的特征在于,在方法步骤D)中,加入异麦芽酮糖-晶种,尤其是以0.01-10重量% 的量,且特别优选0.1-1重量%,基于在理论上计算出的晶体量计。
已经证实,如果在进料溶液中的音速在1800 m/s至2000 m/s、尤其是1850 m/s至1950 m/s、尤其是1870 m/s至1920 m/s的范围的时间点加入异麦芽酮糖-晶种,对于根据本发明的方法而言是有利的。所述音速使用用于测定音速的商业上惯用的探针进行测量。音速探针的典型生产商是公司Sensotech (Magdeburg, 德国)或者Anton Paar (Graz, 奥地利)。
一种根据本发明优选的方法的特征在于,方法步骤D)在50℃至70℃、优选55℃至65℃、特别优选58℃至62℃的温度范围和在70毫巴至200毫巴、优选100毫巴至180、特别优选110毫巴至140毫巴的压力范围进行。具体地,在这方面优选的是,在根据本发明的方法中,在58℃至62℃的温度范围和在110毫巴至140毫巴的压力范围进行方法步骤D)。
通过改变蒸发度,可以实现不同的固体含量和/或收率;这可以例如通过在方法步骤D)中的停留时间来控制。如果在方法步骤D)中除去如此多的水,使得基于所得到的混悬液计,所述混悬液包含20重量%至70重量%、优选30重量%至65重量%、特别优选40重量%至60重量%的作为固体的异麦芽酮糖,则是有利并因此是优选的。
在方法步骤E)中,可以使用本领域技术人员已知的用于将固体与液体分离的所有方法,例如离心、沉降、滗析、在重力场中分离和过滤。一种根据本发明优选的方法的特征在于,在方法步骤E) 中的分离通过离心来进行。
根据本发明优选的是,在方法步骤F)中的洗涤用水进行,尤其是用具有20℃至90℃、优选30℃至80℃、特别优选50℃至70℃的温度的水。
为了提高异麦芽酮糖的收率和避免产物损失,根据本发明优选的是,将在方法步骤F)中用于洗涤使用过的液体,尤其是用于洗涤使用过的水,供入至方法步骤C)的进料溶液中。
因此,一种根据本发明优选的方法的特征在于,包含异麦芽酮糖和水的方法步骤C)的进料溶液包含在方法步骤F)中用于洗涤使用过的液体;所述进料溶液特别优选包含在方法步骤F)中用于洗涤使用过的液体和含有蔗糖的植物汁的直接异构化产物。
为了实现相同的技术效果,优选的是,根据本发明的方法的特征在于,它额外包括下述方法步骤:
G) 冷却得自方法步骤E) 的母液,得到异麦芽酮糖-晶体的第二混悬液,
H) 将所述混悬液分离成第二含有异麦芽酮糖的固体和第二母液,和任选地
I) 将所述第二含有异麦芽酮糖的固体溶解在方法步骤C)的进料溶液中。
为了能够尽可能能量有利地进行该优选方法,优选的是,除方法步骤D)而外,方法步骤F)也在50℃至70℃、优选55℃至65℃、特别优选58℃至62℃的温度范围和在70毫巴至200毫巴、优选100毫巴至180毫巴、特别优选110毫巴至140毫巴的压力范围进行。具体地,在这方面,优选的是,除方法步骤D)而外,方法步骤F)也在58℃至62℃的温度范围和在110毫巴至140毫巴的压力范围进行。
一种根据本发明的优选方法具体地特征在于,在方法步骤G)中,将得自方法步骤E) 的母液冷却至10℃至30℃、优选至15℃至25℃、特别优选至18℃至22℃,其中压力优选地相应于0.9-1.1巴的环境空气压力。
在根据本发明的优选方法的方法步骤H)中,可以使用与在根据本发明的方法的方法步骤E)中相同的方法用于将固体与液体分离,以得到第二含有异麦芽酮糖的固体和第二母液;方法步骤H) 优选地在10℃至30℃、优选地在15℃至25℃、特别优选地在18℃至22℃进行。
可以将所述第二含有异麦芽酮糖的固体进一步加工成终产物,或者如建议的,在方法步骤I) 中将其加入到方法步骤C)的进料溶液中,其中优选的是,所述第二含有异麦芽酮糖的固体在加入方法步骤C)的进料溶液中以后,以溶解形式存在于该溶液中。
根据本发明优选的是,进行在根据本发明的方法中的方法步骤I)。因此,一种根据本发明优选的方法的特征在于,所述方法步骤C)的进料溶液包含异麦芽酮糖和水;特别优选地,所述进料溶液包含溶解的得自方法步骤H)的第二含有异麦芽酮糖的固体和在方法步骤F)中用于洗涤使用过的液体。
下面列出的实施例示例性描述了本发明,而非意在将其范围由整个说明书和权利要求书给出的本发明限制在实施例中所提及的实施方案中。
下述附图是实施例的组成部分:
图1:结晶的示例性工业过程控制。
实施例:
实施例1:浓汁的异构化
用1-5 ml无菌营养培养基将细胞从菌株红色精蛋白杆菌(CBS574.77)的传代培养物上洗下,所述营养培养基由50 g/kg的蔗糖、15 g/kg的玉米泡胀水、7 g/kg的硫酸铵、0.5 g/kg的酵母浸出物、1 g/kg的硫酸二氢钾、0.41 g/kg的氯化镁七水合物、0.004 g/kg的氯化锰四水合物、0.047 g/kg的柠檬酸铁一水合物和926 g/kg的水组成,需要时将其调节至pH 7.2。该混悬液充当预培养物的接种物,在1 l摇瓶中所述预培养物包含200 ml上述营养物溶液。
在30℃培养20小时以后,以使初始光密度(OD600)为1的方式,用所述预培养物给2- L发酵罐接种,所述发酵罐装有1升无菌生产培养基,所述生产培养基由50 g/kg的蔗糖、15 g/kg的玉米泡胀水、3 g/kg的硫酸铵、4 g/kg的磷酸氢铵、0.5 g/kg的酵母浸出物、1 g/kg的硫酸二氢钾、0.41 g/kg的氯化镁七水合物、0.004 g/kg的氯化锰四水合物、0.047 g/kg的柠檬酸铁一水合物和926 g/kg的水组成,其被调节至pH 7.2。
在30℃、pH 7 (经调节)和30%的pO2 (级联搅拌器,Airflow)下进行发酵。在发酵过程中,补料蔗糖。15-20 h以后,发酵过程结束,可以收获生物质用于固定化。
为此,将得到的混悬液与水和4%浓度的藻酸盐溶液以体积比1:1进行混合。然后将该混悬液通过滴入到2%浓度的CaCl2-溶液中来固定化。用聚乙烯亚胺和戊二醛后固化所得到的珠子。得到的生物催化剂可以在4-10℃保存数周。
将得到的经固定化的细胞填充到可加热的柱反应器中,并将温度调节至20-35℃,并使得自糖用甜菜的具有41重量%蔗糖含量的经稀释的浓汁连续通流穿过。
异构化以后的组成可以参见表1。
果糖 | [重量%] | 1.4% |
葡萄糖 | [重量%] | 0.5% |
蔗糖 | [重量%] | 1.7% |
异麦芽酮糖 | [重量%] | 32.5% |
海藻糖 | [重量%] | 3.8% |
异松三糖 | [重量%] | 1.1% |
水 | [重量%] | 59.0% |
表1. 异构化的浓汁的组成。
实施例2:异构化的浓汁的结晶
阶段1
得自实施例1的异构化的浓汁可用作起始原料。
借助于3 L双层搅拌容器,进行得自异构化的浓汁的异麦芽酮糖的结晶。
在20-25℃的温度下,将2999 g进料溶液引入3 L-结晶器中,并加热至60℃。在60℃的温度下,小心地降低压力,直到观察到沸腾过程。通过升高恒温器温度和轻微调节真空,补偿由于蒸发冷却造成的温度下降。沸腾温度应当保持恒定在60℃。一旦在恒定压力下温度增加,则马上调节真空,使得温度保持在60℃。借助于安装的真空接受管和馏出液接收器,连续确定在此期间的馏出物率(Destillatrate)。在41.5%的馏出物率或1900 m/s的音速时,中断真空,并经由安装的漏斗用10 g具有粒度< 20μm的晶种接种该浓缩物。将容器内容物在搅拌下在60℃保持约30分钟。然后小心地继续进行溶剂的蒸发和实际的蒸发结晶,直到达到48%的蒸发率或2130 m/s的音速。在此时,中断130毫巴的最终真空,并停止蒸发。将混悬液在结晶器中在搅拌下保持另外30分钟直至固-液-分离。
从结晶器中取出1553.5 g混悬液。提供Cepa的过滤篮式离心机用于固-液-分离和晶体洗涤,其具有200 mm的篮直径。在500 rpm的转数下,将1514.6 g混悬液引入过滤篮式离心机中。在排空和填充离心机之间,可计算出38.9 g或2.5% 的质量差。将离心机的转数增加至4000 rpm用于实际的固-液-分离。通过固-液-分离,得到632.6 g母液。理论计算的在滤布上的固体质量是882 g。在预先给定的10%的洗涤水的量的情况下,由此计算出88.2 g理论洗涤水的量。然后在500 rpm的转数下,在离心机上用90 g超纯水洗涤固体。将离心机调节至4000 rpm的转数用于随后的干燥离心。能够除去225.3 g的负载的洗涤水的量。从滤布能够得到574 g经洗涤的固体。所述固体具有3%的残留水分。因此,通过固-液-分离产生153.7 g的质量差或10.1%的相对质量损失。
分离出的固体具有下述组成,其中所给出的水是异麦芽酮糖的结晶水:
表2. 阶段1的经洗涤的固体的组成。
阶段1的分离出的母液具有下述组成:
表3. 阶段1的母液的组成。
基于所得到的质量可以计算基于异麦芽酮糖计的未校正的收率:
可以用实验操作的误差来校正收率:
k1=固-液-分离装料的校正=1.025
k2=固-液-分离期间的校正= 1.101
阶段2
为了提高收率,然后在另一个阶段处理母液。这通过从60℃至约20℃的所谓的蒸发结晶进行。在此在实验中,将包含母液的水溶液返回到结晶器中,并在60℃进行恒温处理,所述母液具有857.9 g的质量。借助于线性冷却斜坡,在这里将温度以0.2 K/min-逐步缓慢地冷却。在57℃的温度下,加入1.8 g具有粒度< 20μm的晶种。该晶种量相对于预期的晶体量的大约0.5-1% 。所述加入可以通过例如晶种在异丙醇中的混悬液来进行。在达到最终温度以后,将混悬液在搅拌下在20℃还恒温处理约30分钟。从结晶器中可取出575.8 g混悬液,并在500 rpm的转数下放入离心机中。这相当于相对于结晶开始时56.8 g的质量差或8.9%的相对误差。将转数增加至4000 rpm用于离心机中的固-液-分离。阶段2没有晶体洗涤,因为将再溶解和再结晶在阶段1的较晚过程中得到的固体。固-液-分离以后,得到334.8 g母液和180.8 g固体。所述固体具有6%的残留水分。在固-液-分离阶段,测得47.9 g的质量差。这相当于8.3%的误差。对于阶段2,也可基于在阶段1中使用的异麦芽酮糖来计算收率:
对于阶段2,同样可以计算校正收率。出来阶段1的校正因子而外,还必须考虑阶段2的额外的校正因子:
k3=装料/排空校正= 1.09
k2=固-液-分离期间的校正= 1.083
。
因此,对于整个过程计算出70.3%的未经校正的总收率,或者用质量平衡误差来校正的82.3%的总收率。
在下面列出了在阶段2中分离出的固体的组成,其中所给出的水是异麦芽酮糖的结晶水。
表4. 得自阶段2的固体的组成。
基于干物质计,阶段2的分离出的母液具有下述组成:
表5. 阶段2的母液的组成。
实施例3:结晶的工业过程控制的实施例
在图1中显示了根据本发明优选的方法的一种可能的工业过程控制:
在过程开始时,使用进料溶液(1) ,以再溶解得自结晶阶段S2 (H)/固-液-分离(I)的未洗过的固体(14)和得自晶体洗涤(E/F)的洗涤水(10)。然后在蒸发结晶S1 (D)中使用混合物(3)。在这里,通过在60℃和130-150毫巴的压力下蒸发相应的水份额(5),逾越异麦芽酮糖的溶解度,由此异麦芽酮糖结晶析出,且可以作为混悬液(4) 从蒸发结晶(D)传送至固-液-分离/晶体洗涤(E/F)。在固-液-分离/晶体洗涤(E/F)中,使异麦芽酮糖晶体与母液(11)分离。用超纯水(7)洗涤带有母液的固体。将洗涤水(10)逆向试验进程再次供至进料流,用于后处理。将经洗涤的晶体(6)运输至产品成型(J)。这里的产品成型可以作为干燥或作为用超纯水(9)溶解处来进行。为了提高收率,将母液(11) 供至冷却结晶处(G)。在这里,将60℃的溶液冷却至20℃的温度。由此逾越异麦芽酮糖的溶解度限值,因此异麦芽酮糖结晶析出,并形成混悬液(12)。使混悬液(12)从冷却结晶处(G) 供至固-液-分离(H)。在这里,使产生的晶体(14)与母液(13)分离。不洗涤晶体。母液(13) 离开该过程。将固体(14) 再次溶解在异麦芽酮糖进料流(1)中用于重结晶。
在过程流程图中,未列出用于结晶阶段可能的分批操作的缓冲液容器等。
1 | 进料溶液 | 8 | 产品 |
2 | 进料溶液+固体S 2 | 9 | 超纯水(根据需要) |
3 | 进料溶液+固体S2 + 洗涤水S1 | 10 | 洗涤水 |
4 | 混悬液S1 | 11 | 母液S1 |
5 | 馏出物S1 | 12 | 混悬液S2 |
6 | 固体S1 | 13 | 母液S2 |
7 | 超纯水 | 14 | 固体S2 |
表6. 物流表。
序列表
<110> Evonik Industries
<120> 从植物汁制备异麦芽酮糖的方法
<130> 201100345
<160> 16
<170> PatentIn 3.5版
<210> 1
<211> 600
<212> PRT
<213> 大黄欧文氏菌
<400> 1
Met Ser Ser Gln Gly Leu Lys Thr Ala Val Ala Ile Phe Leu Ala Thr
1 5 10 15
Thr Phe Ser Ala Thr Ser Tyr Gln Ala Cys Ser Ala Gly Pro Asp Thr
20 25 30
Ala Pro Ser Leu Thr Val Gln Gln Ser Asn Ala Leu Pro Thr Trp Trp
35 40 45
Lys Gln Ala Val Phe Tyr Gln Val Tyr Pro Arg Ser Phe Lys Asp Thr
50 55 60
Asn Gly Asp Gly Ile Gly Asp Leu Asn Gly Ile Ile Glu Asn Leu Asp
65 70 75 80
Tyr Leu Lys Lys Leu Gly Ile Asp Ala Ile Trp Ile Asn Pro His Tyr
85 90 95
Asp Ser Pro Asn Thr Asp Asn Gly Tyr Asp Ile Arg Asp Tyr Arg Lys
100 105 110
Ile Met Lys Glu Tyr Gly Thr Met Glu Asp Phe Asp Arg Leu Ile Ser
115 120 125
Glu Met Lys Lys Arg Asn Met Arg Leu Met Ile Asp Ile Val Ile Asn
130 135 140
His Thr Ser Asp Gln His Ala Trp Phe Val Gln Ser Lys Ser Gly Lys
145 150 155 160
Asn Asn Pro Tyr Arg Asp Tyr Tyr Phe Trp Arg Asp Gly Lys Asp Gly
165 170 175
His Ala Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Glu
180 185 190
Lys Asp Asp Lys Ser Gly Gln Tyr Tyr Leu His Tyr Phe Ala Lys Gln
195 200 205
Gln Pro Asp Leu Asn Trp Asp Asn Pro Lys Val Arg Gln Asp Leu Tyr
210 215 220
Asp Met Leu Arg Phe Trp Leu Asp Lys Gly Val Ser Gly Leu Arg Phe
225 230 235 240
Asp Thr Val Ala Thr Tyr Ser Lys Ile Pro Asn Phe Pro Asp Leu Ser
245 250 255
Gln Gln Gln Leu Lys Asn Phe Ala Glu Glu Tyr Thr Lys Gly Pro Lys
260 265 270
Ile His Asp Tyr Val Asn Glu Met Asn Arg Glu Val Leu Ser His Tyr
275 280 285
Asp Ile Ala Thr Ala Gly Glu Ile Phe Gly Val Pro Leu Asp Lys Ser
290 295 300
Ile Lys Phe Phe Asp Arg Arg Arg Asn Glu Leu Asn Ile Ala Phe Thr
305 310 315 320
Phe Asp Leu Ile Arg Leu Asp Arg Asp Ala Asp Glu Arg Trp Arg Arg
325 330 335
Lys Asp Trp Thr Leu Ser Gln Phe Arg Lys Ile Val Asp Lys Val Asp
340 345 350
Gln Thr Ala Gly Glu Tyr Gly Trp Asn Ala Phe Phe Leu Asp Asn His
355 360 365
Asp Asn Pro Arg Ala Val Ser His Phe Gly Asp Asp Arg Pro Gln Trp
370 375 380
Arg Glu His Ala Ala Lys Ala Leu Ala Thr Leu Thr Leu Thr Gln Arg
385 390 395 400
Ala Thr Pro Phe Ile Tyr Gln Gly Ser Glu Leu Gly Met Thr Asn Tyr
405 410 415
Pro Phe Lys Lys Ile Asp Asp Phe Asp Asp Val Glu Val Lys Gly Phe
420 425 430
Trp Gln Asp Tyr Val Glu Thr Gly Lys Val Lys Ala Glu Glu Phe Leu
435 440 445
Gln Asn Val Arg Gln Thr Ser Arg Asp Asn Ser Arg Thr Pro Phe Gln
450 455 460
Trp Asp Ala Ser Lys Asn Ala Gly Phe Thr Ser Gly Thr Pro Trp Leu
465 470 475 480
Lys Ile Asn Pro Asn Tyr Lys Glu Ile Asn Ser Ala Asp Gln Ile Asn
485 490 495
Asn Pro Asn Ser Val Phe Asn Tyr Tyr Arg Lys Leu Ile Asn Ile Arg
500 505 510
His Asp Ile Pro Ala Leu Thr Tyr Gly Ser Tyr Ile Asp Leu Asp Pro
515 520 525
Asp Asn Asn Ser Val Tyr Ala Tyr Thr Arg Thr Leu Gly Ala Glu Lys
530 535 540
Tyr Leu Val Val Ile Asn Phe Lys Glu Glu Val Met His Tyr Thr Leu
545 550 555 560
Pro Gly Asp Leu Ser Ile Asn Lys Val Ile Thr Glu Asn Asn Ser His
565 570 575
Thr Ile Val Asn Lys Asn Asp Arg Gln Leu Arg Leu Glu Pro Trp Gln
580 585 590
Ser Gly Ile Tyr Lys Leu Asn Pro
595 600
<210> 2
<211> 584
<212> PRT
<213> 嗜中酸假单胞菌
<400> 2
Met Leu Met Lys Arg Leu Phe Ala Ala Ser Leu Met Leu Ala Phe Ser
1 5 10 15
Ser Val Ser Ser Val Arg Ala Glu Glu Ala Val Lys Pro Gly Ala Pro
20 25 30
Trp Trp Lys Ser Ala Val Phe Tyr Gln Val Tyr Pro Arg Ser Phe Lys
35 40 45
Asp Thr Asn Gly Asp Gly Ile Gly Asp Phe Lys Gly Leu Thr Glu Lys
50 55 60
Leu Asp Tyr Leu Lys Gly Leu Gly Ile Asp Ala Ile Trp Ile Asn Pro
65 70 75 80
His Tyr Ala Ser Pro Asn Thr Asp Asn Gly Tyr Asp Ile Ser Asp Tyr
85 90 95
Arg Glu Val Met Lys Glu Tyr Gly Thr Met Glu Asp Phe Asp Arg Leu
100 105 110
Met Ala Glu Leu Lys Lys Arg Gly Met Arg Leu Met Val Asp Val Val
115 120 125
Ile Asn His Ser Ser Asp Gln His Glu Trp Phe Lys Ser Ser Arg Ala
130 135 140
Ser Lys Asp Asn Pro Tyr Arg Asp Tyr Tyr Phe Trp Arg Asp Gly Lys
145 150 155 160
Asp Gly His Glu Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala
165 170 175
Trp Glu Lys Asp Pro Val Thr Gly Gln Tyr Tyr Leu His Tyr Phe Gly
180 185 190
Arg Gln Gln Pro Asp Leu Asn Trp Asp Thr Pro Lys Leu Arg Glu Glu
195 200 205
Leu Tyr Ala Met Leu Arg Phe Trp Leu Asp Lys Gly Val Ser Gly Met
210 215 220
Arg Phe Asp Thr Val Ala Thr Tyr Ser Lys Thr Pro Gly Phe Pro Asp
225 230 235 240
Leu Thr Pro Glu Gln Met Lys Asn Phe Ala Glu Ala Tyr Thr Gln Gly
245 250 255
Pro Asn Leu His Arg Tyr Leu Gln Glu Met His Glu Lys Val Phe Asp
260 265 270
His Tyr Asp Ala Val Thr Ala Gly Glu Ile Phe Gly Ala Pro Leu Asn
275 280 285
Gln Val Pro Leu Phe Ile Asp Ser Arg Arg Lys Glu Leu Asp Met Ala
290 295 300
Phe Thr Phe Asp Leu Ile Arg Tyr Asp Arg Ala Leu Asp Arg Trp His
305 310 315 320
Thr Ile Pro Arg Thr Leu Ala Asp Phe Arg Gln Thr Ile Asp Lys Val
325 330 335
Asp Ala Ile Ala Gly Glu Tyr Gly Trp Asn Thr Phe Phe Leu Gly Asn
340 345 350
His Asp Asn Pro Arg Ala Val Ser His Phe Gly Asp Asp Arg Pro Gln
355 360 365
Trp Arg Glu Ala Ser Ala Lys Ala Leu Ala Thr Val Thr Leu Thr Gln
370 375 380
Arg Gly Thr Pro Phe Ile Phe Gln Gly Asp Glu Leu Gly Met Thr Asn
385 390 395 400
Tyr Pro Phe Lys Thr Leu Gln Asp Phe Asp Asp Ile Glu Val Lys Gly
405 410 415
Phe Phe Gln Asp Tyr Val Glu Thr Gly Lys Ala Thr Ala Glu Glu Leu
420 425 430
Leu Thr Asn Val Ala Leu Thr Ser Arg Asp Asn Ala Arg Thr Pro Phe
435 440 445
Gln Trp Asp Asp Ser Ala Asn Ala Gly Phe Thr Thr Gly Lys Pro Trp
450 455 460
Leu Lys Val Asn Pro Asn Tyr Thr Glu Ile Asn Ala Ala Arg Glu Ile
465 470 475 480
Gly Asp Pro Lys Ser Val Tyr Ser Phe Tyr Arg Asn Leu Ile Ser Ile
485 490 495
Arg His Glu Thr Pro Ala Leu Ser Thr Gly Ser Tyr Arg Asp Ile Asp
500 505 510
Pro Ser Asn Ala Asp Val Tyr Ala Tyr Thr Arg Ser Gln Asp Gly Glu
515 520 525
Thr Tyr Leu Val Val Val Asn Phe Lys Ala Glu Pro Arg Ser Phe Thr
530 535 540
Leu Pro Asp Gly Met His Ile Ala Glu Thr Leu Ile Glu Ser Ser Ser
545 550 555 560
Pro Ala Ala Pro Ala Ala Gly Ala Ala Ser Leu Glu Leu Gln Pro Trp
565 570 575
Gln Ser Gly Ile Tyr Lys Val Lys
580
<210> 3
<211> 598
<212> PRT
<213> 分散泛菌
<400> 3
Met Phe Leu Asn Gly Phe Lys Thr Val Ile Ala Leu Thr Met Ala Ser
1 5 10 15
Ser Phe Tyr Leu Ala Ala Ser Pro Leu Thr Lys Pro Ser Thr Pro Ile
20 25 30
Ala Ala Thr Asn Ile Gln Lys Ser Ala Asp Phe Pro Ile Trp Trp Lys
35 40 45
Gln Ala Val Phe Tyr Gln Ile Tyr Pro Arg Ser Phe Lys Asp Ser Asn
50 55 60
Gly Asp Gly Ile Gly Asp Ile Pro Gly Ile Ile Glu Lys Leu Asp Tyr
65 70 75 80
Leu Lys Met Leu Gly Val Asp Ala Ile Trp Ile Asn Pro His Tyr Glu
85 90 95
Ser Pro Asn Thr Asp Asn Gly Tyr Asp Ile Ser Asp Tyr Arg Lys Ile
100 105 110
Met Lys Glu Tyr Gly Ser Met Ala Asp Phe Asp Arg Leu Val Ala Glu
115 120 125
Met Asn Lys Arg Gly Met Arg Leu Met Ile Asp Ile Val Ile Asn His
130 135 140
Thr Ser Asp Arg His Arg Trp Phe Val Gln Ser Arg Ser Gly Lys Asp
145 150 155 160
Asn Pro Tyr Arg Asp Tyr Tyr Phe Trp Arg Asp Gly Lys Gln Gly Gln
165 170 175
Ala Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Gln Leu
180 185 190
Asp Lys Gln Thr Asp Gln Tyr Tyr Leu His Tyr Phe Ala Pro Gln Gln
195 200 205
Pro Asp Leu Asn Trp Asp Asn Pro Lys Val Arg Ala Glu Leu Tyr Asp
210 215 220
Ile Leu Arg Phe Trp Leu Asp Lys Gly Val Ser Gly Leu Arg Phe Asp
225 230 235 240
Thr Val Ala Thr Phe Ser Lys Ile Pro Gly Phe Pro Asp Leu Ser Lys
245 250 255
Ala Gln Leu Lys Asn Phe Ala Glu Ala Tyr Thr Glu Gly Pro Asn Ile
260 265 270
His Lys Tyr Ile His Glu Met Asn Arg Gln Val Leu Ser Lys Tyr Asn
275 280 285
Val Ala Thr Ala Gly Glu Ile Phe Gly Val Pro Val Ser Ala Met Pro
290 295 300
Asp Tyr Phe Asp Arg Arg Arg Glu Glu Leu Asn Ile Ala Phe Thr Phe
305 310 315 320
Asp Leu Ile Arg Leu Asp Arg Tyr Pro Asp Gln Arg Trp Arg Arg Lys
325 330 335
Pro Trp Thr Leu Ser Gln Phe Arg Gln Val Ile Ser Gln Thr Asp Arg
340 345 350
Ala Ala Gly Glu Phe Gly Trp Asn Ala Phe Phe Leu Asp Asn His Asp
355 360 365
Asn Pro Arg Gln Val Ser His Phe Gly Asp Asp Ser Pro Gln Trp Arg
370 375 380
Glu Arg Ser Ala Lys Ala Leu Ala Thr Leu Leu Leu Thr Gln Arg Ala
385 390 395 400
Thr Pro Phe Ile Phe Gln Gly Ala Glu Leu Gly Met Thr Asn Tyr Pro
405 410 415
Phe Lys Asn Ile Glu Glu Phe Asp Asp Ile Glu Val Lys Gly Phe Trp
420 425 430
Asn Asp Tyr Val Ala Ser Gly Lys Val Asn Ala Ala Glu Phe Leu Gln
435 440 445
Glu Val Arg Met Thr Ser Arg Asp Asn Ser Arg Thr Pro Met Gln Trp
450 455 460
Asn Asp Ser Val Asn Ala Gly Phe Thr Gln Gly Lys Pro Trp Phe His
465 470 475 480
Leu Asn Pro Asn Tyr Lys Gln Ile Asn Ala Ala Arg Glu Val Asn Lys
485 490 495
Pro Asp Ser Val Phe Ser Tyr Tyr Arg Gln Leu Ile Asn Leu Arg His
500 505 510
Gln Ile Pro Ala Leu Thr Ser Gly Glu Tyr Arg Asp Leu Asp Pro Gln
515 520 525
Asn Asn Gln Val Tyr Ala Tyr Thr Arg Ile Leu Asp Asn Glu Lys Tyr
530 535 540
Leu Val Val Val Asn Phe Lys Pro Glu Gln Leu His Tyr Ala Leu Pro
545 550 555 560
Asp Asn Leu Thr Ile Ala Ser Ser Leu Leu Glu Asn Val His Gln Pro
565 570 575
Ser Leu Gln Glu Asn Ala Ser Thr Leu Thr Leu Ala Pro Trp Gln Ala
580 585 590
Gly Ile Tyr Lys Leu Asn
595
<210> 4
<211> 598
<212> PRT
<213> 肺炎克雷伯菌
<400> 4
Met Ser Phe Ile Thr Leu Arg Thr Gly Val Ala Val Ala Leu Ser Ser
1 5 10 15
Leu Met Ile Ser Leu Ala Ser Pro Ala Val Ser Ala Ala Pro Ser Val
20 25 30
Asn Gln Asp Ile Leu Val Gln Lys Glu Ser Glu Tyr Pro Ala Trp Trp
35 40 45
Lys Glu Ala Val Phe Tyr Gln Ile Tyr Pro Arg Ser Phe Lys Asp Thr
50 55 60
Asn Asp Asp Gly Ile Gly Asp Ile Arg Gly Ile Ile Glu Lys Leu Asp
65 70 75 80
Tyr Leu Lys Ser Leu Gly Ile Asp Ala Ile Trp Ile Asn Pro His Tyr
85 90 95
Asp Ser Pro Asn Thr Asp Asn Gly Tyr Asp Ile Ser Asn Tyr Arg Gln
100 105 110
Ile Met Lys Glu Tyr Gly Thr Met Glu Asp Phe Asp Asn Leu Val Ala
115 120 125
Glu Met Lys Lys Arg Asn Met Arg Leu Met Ile Asp Val Val Ile Asn
130 135 140
His Thr Ser Asp Gln His Pro Trp Phe Ile Gln Ser Lys Ser Asp Lys
145 150 155 160
Asn Asn Pro Tyr Arg Asp Tyr Tyr Phe Trp Arg Asp Gly Lys Asp Asn
165 170 175
Gln Pro Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Gln
180 185 190
Lys Asp Ala Lys Ser Gly Gln Tyr Tyr Leu His Tyr Phe Ala Arg Gln
195 200 205
Gln Pro Asp Leu Asn Trp Asp Asn Pro Lys Val Arg Glu Asp Leu Tyr
210 215 220
Ala Met Leu Arg Phe Trp Leu Asp Lys Gly Val Ser Gly Met Arg Phe
225 230 235 240
Asp Thr Val Ala Thr Tyr Ser Lys Ile Pro Gly Phe Pro Asn Leu Thr
245 250 255
Pro Glu Gln Gln Lys Asn Phe Ala Glu Gln Tyr Thr Met Gly Pro Asn
260 265 270
Ile His Arg Tyr Ile Gln Glu Met Asn Arg Glu Val Leu Ser Arg Tyr
275 280 285
Asp Val Ala Thr Ala Gly Glu Ile Phe Gly Val Pro Leu Asp Arg Ser
290 295 300
Ser Gln Phe Phe Asp Arg Arg Arg His Glu Leu Asn Met Ala Phe Met
305 310 315 320
Phe Asp Leu Ile Arg Leu Asp Arg Asp Ser Asn Glu Arg Trp Arg His
325 330 335
Lys Ser Trp Ser Leu Ser Gln Phe Arg Gln Ile Ile Ser Lys Met Asp
340 345 350
Val Thr Val Gly Lys Tyr Gly Trp Asn Thr Phe Phe Leu Asp Asn His
355 360 365
Asp Asn Pro Arg Ala Val Ser His Phe Gly Asp Asp Arg Pro Gln Trp
370 375 380
Arg Glu Ala Ser Ala Lys Ala Leu Ala Thr Ile Thr Leu Thr Gln Arg
385 390 395 400
Ala Thr Pro Phe Ile Tyr Gln Gly Ser Glu Leu Gly Met Ile Asn Tyr
405 410 415
Pro Phe Arg Gln Leu Asn Glu Phe Asp Asp Ile Glu Val Lys Gly Phe
420 425 430
Trp Gln Asp Tyr Val Gln Ser Gly Lys Val Thr Ala Thr Glu Phe Leu
435 440 445
Asp Asn Val Arg Leu Thr Ser Arg Asp Asn Ser Arg Thr Pro Phe Gln
450 455 460
Trp Asn Asp Thr Leu Asn Ala Gly Phe Thr Arg Gly Lys Pro Trp Phe
465 470 475 480
His Ile Asn Pro Asn Tyr Val Glu Ile Asn Ala Glu Arg Glu Glu Thr
485 490 495
Arg Glu Asp Ser Val Leu Asn Tyr Tyr Lys Lys Met Ile Gln Leu Arg
500 505 510
His His Ile Pro Ala Leu Val Tyr Gly Ala Tyr Gln Asp Leu Asn Pro
515 520 525
Gln Asp Asn Thr Val Tyr Ala Tyr Thr Arg Thr Leu Gly Asn Glu Arg
530 535 540
Tyr Leu Val Val Val Asn Phe Lys Glu Tyr Pro Val Arg Tyr Thr Leu
545 550 555 560
Pro Ala Asn Asp Ala Ile Glu Glu Val Val Ile Asp Thr Gln Gln Gln
565 570 575
Ala Thr Ala Pro His Ser Thr Ser Leu Ser Leu Ser Pro Trp Gln Ala
580 585 590
Gly Val Tyr Lys Leu Arg
595
<210> 5
<211> 558
<212> PRT
<213> 红色精蛋白杆菌
<400> 5
Thr Ile Pro Lys Trp Trp Lys Glu Ala Val Phe Tyr Gln Val Tyr Pro
1 5 10 15
Arg Ser Phe Lys Asp Thr Asn Gly Asp Gly Ile Gly Asp Ile Asn Gly
20 25 30
Ile Ile Glu Lys Leu Asp Tyr Leu Lys Ala Leu Gly Ile Asp Ala Ile
35 40 45
Trp Ile Asn Pro His Tyr Asp Ser Pro Asn Thr Asp Asn Gly Tyr Asp
50 55 60
Ile Arg Asp Tyr Arg Lys Ile Met Lys Glu Tyr Gly Thr Met Glu Asp
65 70 75 80
Phe Asp Arg Leu Ile Ser Glu Met Lys Lys Arg Asn Met Arg Leu Met
85 90 95
Ile Asp Val Val Ile Asn His Thr Ser Asp Gln Asn Glu Trp Phe Val
100 105 110
Lys Ser Lys Ser Ser Lys Asp Asn Pro Tyr Arg Gly Tyr Tyr Phe Trp
115 120 125
Lys Asp Ala Lys Glu Gly Gln Ala Pro Asn Asn Tyr Pro Ser Phe Phe
130 135 140
Gly Gly Ser Ala Trp Gln Lys Asp Glu Lys Thr Asn Gln Tyr Tyr Leu
145 150 155 160
His Tyr Phe Ala Lys Gln Gln Pro Asp Leu Asn Trp Asp Asn Pro Lys
165 170 175
Val Arg Gln Asp Leu Tyr Ala Met Leu Arg Phe Trp Leu Asp Lys Gly
180 185 190
Val Ser Gly Leu Arg Phe Asp Thr Val Ala Thr Tyr Ser Lys Ile Pro
195 200 205
Asp Phe Pro Asn Leu Thr Gln Gln Gln Leu Lys Asn Phe Ala Ala Glu
210 215 220
Tyr Thr Lys Gly Pro Asn Ile His Arg Tyr Val Asn Glu Met Asn Lys
225 230 235 240
Glu Val Leu Ser His Tyr Asp Ile Ala Thr Ala Gly Glu Ile Phe Gly
245 250 255
Val Pro Leu Asp Gln Ser Ile Lys Phe Phe Asp Arg Arg Arg Asp Glu
260 265 270
Leu Asn Ile Ala Phe Thr Phe Asp Leu Ile Arg Leu Asp Arg Asp Ser
275 280 285
Asp Gln Arg Trp Arg Arg Lys Asp Trp Lys Leu Ser Gln Phe Arg Gln
290 295 300
Ile Ile Asp Asn Val Asp Arg Thr Ala Gly Glu Tyr Gly Trp Asn Ala
305 310 315 320
Phe Phe Leu Asp Asn His Asp Asn Pro Arg Ala Val Ser His Phe Gly
325 330 335
Asp Asp Arg Pro Gln Trp Arg Glu Pro Ser Ala Lys Ala Leu Ala Thr
340 345 350
Leu Thr Leu Thr Gln Arg Ala Thr Pro Phe Ile Tyr Gln Gly Ser Glu
355 360 365
Leu Gly Met Thr Asn Tyr Pro Phe Lys Ala Ile Asp Glu Phe Asp Asp
370 375 380
Ile Glu Val Lys Gly Phe Trp His Asp Tyr Val Glu Thr Gly Lys Val
385 390 395 400
Lys Ala Asp Glu Phe Leu Gln Asn Val Arg Leu Thr Ser Arg Asp Asn
405 410 415
Ser Arg Thr Pro Phe Gln Trp Asp Gly Ser Lys Asn Ala Gly Phe Thr
420 425 430
Ser Gly Lys Pro Trp Phe Lys Val Asn Pro Asn Tyr Gln Glu Ile Asn
435 440 445
Ala Val Ser Gln Val Thr Gln Pro Asp Ser Val Phe Asn Tyr Tyr Arg
450 455 460
Gln Leu Ile Lys Ile Arg His Asp Ile Pro Ala Leu Thr Tyr Gly Thr
465 470 475 480
Tyr Thr Asp Leu Asp Pro Ala Asn Asp Ser Val Tyr Ala Tyr Thr Arg
485 490 495
Ser Leu Gly Ala Glu Lys Tyr Leu Val Val Val Asn Phe Lys Glu Gln
500 505 510
Met Met Arg Tyr Lys Leu Pro Asp Asn Leu Ser Ile Glu Lys Val Ile
515 520 525
Ile Asp Ser Asn Ser Lys Asn Val Val Lys Lys Asn Asp Ser Leu Leu
530 535 540
Glu Leu Lys Pro Trp Gln Ser Gly Val Tyr Lys Leu Asn Gln
545 550 555
<210> 6
<211> 629
<212> PRT
<213> 红色精蛋白杆菌
<400> 6
Met Pro Arg Gln Gly Leu Lys Thr Ala Leu Ala Ile Phe Leu Thr Thr
1 5 10 15
Ser Leu Cys Ile Ser Cys Gln Gln Ala Phe Gly Thr Gln Gln Pro Leu
20 25 30
Leu Asn Glu Lys Ser Ile Glu Gln Ser Lys Thr Ile Pro Lys Trp Trp
35 40 45
Lys Glu Ala Val Phe Tyr Gln Val Tyr Pro Arg Ser Phe Lys Asp Thr
50 55 60
Asn Gly Asp Gly Ile Gly Asp Ile Asn Gly Ile Ile Glu Lys Leu Asp
65 70 75 80
Tyr Leu Lys Ala Leu Gly Ile Asp Ala Ile Trp Ile Asn Pro His Tyr
85 90 95
Asp Ser Pro Asn Thr Asp Asn Gly Tyr Asp Ile Arg Asp Tyr Arg Lys
100 105 110
Ile Met Lys Glu Tyr Gly Thr Met Glu Asp Phe Asp Arg Leu Ile Ser
115 120 125
Glu Met Lys Lys Arg Asn Met Arg Leu Met Ile Asp Val Val Ile Asn
130 135 140
His Thr Ser Asp Gln Asn Glu Trp Phe Val Lys Ser Lys Ser Ser Lys
145 150 155 160
Asp Asn Pro Tyr Arg Gly Tyr Tyr Phe Trp Lys Asp Ala Lys Glu Gly
165 170 175
Gln Ala Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Gln
180 185 190
Lys Asp Glu Lys Thr Asn Gln Tyr Tyr Leu His Tyr Phe Ala Lys Gln
195 200 205
Gln Pro Asp Leu Asn Trp Asp Asn Pro Lys Val Arg Gln Asp Leu Tyr
210 215 220
Ala Met Leu Arg Phe Trp Leu Asp Lys Gly Val Ser Gly Leu Arg Phe
225 230 235 240
Asp Thr Val Ala Thr Tyr Ser Lys Ile Pro Asp Phe Pro Asn Leu Thr
245 250 255
Gln Gln Gln Leu Lys Asn Phe Ala Ala Glu Tyr Thr Lys Gly Pro Asn
260 265 270
Ile His Arg Tyr Val Asn Glu Met Asn Lys Glu Val Leu Ser His Tyr
275 280 285
Asp Ile Ala Thr Ala Gly Glu Ile Phe Gly Val Pro Leu Asp Gln Ser
290 295 300
Ile Lys Phe Phe Asp Arg Arg Arg Asp Glu Leu Asn Ile Ala Phe Thr
305 310 315 320
Phe Asp Leu Ile Arg Leu Asp Arg Asp Ser Asp Gln Arg Trp Arg Arg
325 330 335
Lys Asp Trp Lys Leu Ser Gln Phe Arg Gln Ile Ile Asp Asn Val Asp
340 345 350
Arg Thr Ala Gly Glu Tyr Gly Trp Asn Ala Phe Phe Leu Asp Asn His
355 360 365
Asp Asn Pro Arg Ala Val Ser His Phe Gly Asp Asp Asp Arg Pro Gln
370 375 380
Trp Arg Glu Pro Ser Ala Lys Ala Leu Ala Thr Leu Thr Leu Thr Gln
385 390 395 400
Arg Ala Thr Pro Phe Ile Tyr Gln Gly Ser Glu Leu Gly Met Thr Asn
405 410 415
Tyr Pro Phe Lys Ala Ile Asp Glu Phe Asp Asp Ile Glu Val Lys Gly
420 425 430
Phe Trp His Asp Tyr Val Glu Thr Gly Lys Val Lys Ala Asp Glu Phe
435 440 445
Leu Gln Asn Val Arg Leu Thr Ser Arg Asp Asn Ser Arg Thr Pro Phe
450 455 460
Gln Trp Asp Gly Ser Lys Asn Ala Gly Phe Thr Ser Gly Lys Pro Trp
465 470 475 480
Phe Lys Val Asn Pro Asn Tyr Gln Glu Ile Asn Ala Val Ser Gln Val
485 490 495
Thr Gln Pro Asp Ser Val Phe Asn Tyr Tyr Arg Gln Leu Ile Lys Ile
500 505 510
Arg His Asp Ile Pro Ala Leu Thr Tyr Gly Thr Tyr Thr Asp Leu Asp
515 520 525
Pro Ala Asn Asp Ser Val Tyr Ala Tyr Thr Arg Ser Leu Gly Ala Glu
530 535 540
Lys Tyr Leu Val Val Val Asn Phe Lys Glu Gln Met Met Arg Tyr Lys
545 550 555 560
Leu Pro Asp Asn Leu Ser Ile Glu Lys Val Ile Ile Asp Ser Asn Ser
565 570 575
Lys Asn Val Val Lys Lys Asn Asp Ser Leu Leu Glu Leu Lys Pro Trp
580 585 590
Gln Ser Gly Val Tyr Lys Thr Lys Ser Ile Asn Leu Ile Val Thr Pro
595 600 605
Asn Asn Val Asn Ile Leu Lys Leu Leu Lys Pro Ala Phe Tyr Ala Gly
610 615 620
Phe Phe Ser Ala Lys
625
<210> 7
<211> 600
<212> PRT
<213> 气味沙雷氏菌
<400> 7
Met Pro Arg Gln Gly Leu Lys Thr Ala Leu Ala Ile Phe Leu Thr Thr
1 5 10 15
Ser Leu Ser Val Ser Cys Gln Gln Ala Leu Gly Thr Gln Gln Pro Leu
20 25 30
Leu Asn Glu Lys Ser Ile Glu Gln Ser Lys Thr Ile Pro Lys Trp Trp
35 40 45
Lys Glu Ala Val Phe Tyr Gln Val Tyr Pro Arg Ser Phe Lys Asp Thr
50 55 60
Asn Gly Asp Gly Ile Gly Asp Ile Lys Gly Ile Ile Glu Lys Leu Asp
65 70 75 80
Tyr Leu Lys Ala Leu Gly Ile Asp Ala Ile Trp Ile Asn Pro His Tyr
85 90 95
Asp Ser Pro Asn Thr Asp Asn Gly Tyr Asp Ile Arg Asp Tyr Arg Lys
100 105 110
Ile Met Lys Glu Tyr Gly Thr Met Glu Asp Phe Asp Arg Leu Ile Ser
115 120 125
Glu Met Lys Lys Arg Asn Met Arg Leu Met Ile Asp Val Val Ile Asn
130 135 140
His Thr Ser Asp Gln Asn Glu Trp Phe Val Lys Ser Lys Ser Ser Lys
145 150 155 160
Asp Asn Pro Tyr Arg Gly Tyr Tyr Phe Trp Lys Asp Ala Lys Glu Gly
165 170 175
Gln Ala Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Gln
180 185 190
Lys Asp Glu Lys Thr Asn Gln Tyr Tyr Leu His Tyr Phe Ala Lys Gln
195 200 205
Gln Pro Asp Leu Asn Trp Asp Asn Pro Lys Val Arg Gln Asp Leu Tyr
210 215 220
Ala Met Leu Arg Phe Trp Leu Asp Lys Gly Val Ser Gly Leu Arg Phe
225 230 235 240
Asp Thr Val Ala Thr Tyr Ser Lys Ile Pro Asp Phe Pro Asn Leu Thr
245 250 255
Gln Gln Gln Leu Lys Asn Phe Ala Ala Glu Tyr Thr Lys Gly Pro Asn
260 265 270
Ile His Arg Tyr Val Asn Glu Met Asn Arg Glu Val Leu Ser His Tyr
275 280 285
Asp Ile Ala Thr Ala Gly Glu Ile Phe Gly Val Pro Leu Asp Gln Ser
290 295 300
Ile Lys Phe Phe Asp Arg Arg Arg Asp Glu Leu Asn Ile Ala Phe Thr
305 310 315 320
Phe Asp Leu Ile Arg Leu Asp Arg Asp Ser Asp Gln Arg Trp Arg Arg
325 330 335
Lys Glu Trp Lys Leu Ser Gln Phe Arg Gln Val Ile Asp Asn Val Asp
340 345 350
Arg Thr Ala Gly Glu Tyr Gly Trp Asn Ala Phe Phe Leu Asp Asn His
355 360 365
Asp Asn Pro Arg Ala Val Ser His Phe Gly Asp Asp Arg Pro Gln Trp
370 375 380
Arg Glu Pro Ser Ala Lys Ala Leu Ala Thr Leu Thr Leu Thr Gln Arg
385 390 395 400
Ala Thr Pro Phe Ile Tyr Gln Gly Ser Glu Leu Gly Met Thr Asn Tyr
405 410 415
Pro Phe Lys Ala Ile Asp Glu Phe Asp Asp Ile Glu Val Lys Gly Phe
420 425 430
Trp His Asp Tyr Val Glu Thr Gly Lys Val Lys Ala Asp Glu Phe Leu
435 440 445
Gln Asn Val Arg Leu Thr Ser Arg Asp Asn Ser Arg Thr Pro Phe Gln
450 455 460
Trp Asp Thr Ser Lys Asn Ala Gly Phe Thr Ser Gly Lys Pro Trp Phe
465 470 475 480
Lys Val Asn Pro Asn Tyr Gln Glu Ile Asn Ala Val Ser Gln Val Ala
485 490 495
Gln Pro Asp Ser Val Phe Asn Tyr Tyr Arg Gln Leu Ile Lys Ile Arg
500 505 510
His Asn Ile Pro Ala Leu Thr Tyr Gly Thr Tyr Thr Asp Leu Asp Pro
515 520 525
Ala Asn Asp Ser Val Tyr Ala Tyr Thr Arg Ser Leu Gly Ala Glu Lys
530 535 540
Tyr Leu Val Val Val Asn Phe Gln Glu Gln Val Met Arg Tyr Lys Leu
545 550 555 560
Pro Asp Asn Leu Ser Ile Glu Lys Val Ile Ile Glu Ser Asn Ser Lys
565 570 575
Asn Val Val Lys Lys Asn Asp Ala Leu Leu Glu Leu Lys Pro Trp Gln
580 585 590
Ser Gly Val Tyr Lys Leu Asn Gln
595 600
<210> 8
<211> 600
<212> PRT
<213> 大黄欧文氏菌
<400> 8
Met Ser Ser Gln Gly Leu Lys Thr Ala Val Ala Ile Phe Leu Ala Thr
1 5 10 15
Thr Phe Ser Ala Thr Ser Tyr Gln Ala Cys Ser Ala Gly Pro Asp Thr
20 25 30
Ala Pro Ser Leu Thr Val Gln Gln Ser Asn Ala Leu Pro Thr Trp Trp
35 40 45
Lys Gln Ala Val Phe Tyr Gln Val Tyr Pro Arg Ser Phe Lys Asp Thr
50 55 60
Asn Gly Asp Gly Ile Gly Asp Leu Asn Gly Ile Ile Glu Asn Leu Asp
65 70 75 80
Tyr Leu Lys Lys Leu Gly Ile Asp Ala Ile Trp Ile Asn Pro His Tyr
85 90 95
Asp Ser Pro Asn Thr Asp Asn Gly Tyr Asp Ile Arg Asp Tyr Arg Lys
100 105 110
Ile Met Lys Glu Tyr Gly Thr Met Glu Asp Phe Asp Arg Leu Ile Ser
115 120 125
Glu Met Lys Lys Arg Asn Met Arg Leu Met Ile Asp Ile Val Ile Asn
130 135 140
His Thr Ser Asp Gln His Ala Trp Phe Val Gln Ser Lys Ser Gly Lys
145 150 155 160
Asn Asn Pro Tyr Arg Asp Tyr Tyr Phe Trp Arg Asp Gly Lys Asp Gly
165 170 175
His Ala Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Glu
180 185 190
Lys Asp Asp Lys Ser Gly Gln Tyr Tyr Leu His Tyr Phe Ala Lys Gln
195 200 205
Gln Pro Asp Leu Asn Trp Asp Asn Pro Lys Val Arg Gln Asp Leu Tyr
210 215 220
Asp Met Leu Arg Phe Trp Leu Asp Lys Gly Val Ser Gly Leu Arg Phe
225 230 235 240
Asp Thr Val Ala Thr Tyr Ser Lys Ile Pro Asn Phe Pro Asp Leu Ser
245 250 255
Gln Gln Gln Leu Lys Asn Phe Ala Glu Glu Tyr Thr Lys Gly Pro Lys
260 265 270
Ile His Asp Tyr Val Asn Glu Met Asn Arg Glu Val Leu Ser His Tyr
275 280 285
Asp Ile Ala Thr Ala Gly Glu Ile Phe Gly Val Pro Leu Asp Lys Ser
290 295 300
Ile Lys Phe Phe Asp Arg Arg Arg Asn Glu Leu Asn Ile Ala Phe Thr
305 310 315 320
Phe Asp Leu Ile Arg Leu Asp Arg Asp Ala Asp Glu Arg Trp Arg Arg
325 330 335
Lys Asp Trp Thr Leu Ser Gln Phe Arg Lys Ile Val Asp Lys Val Asp
340 345 350
Gln Thr Ala Gly Glu Tyr Gly Trp Asn Ala Phe Phe Leu Asp Asn His
355 360 365
Asp Asn Pro Arg Ala Val Ser His Phe Gly Asp Asp Arg Pro Gln Trp
370 375 380
Arg Glu His Ala Ala Lys Ala Leu Ala Thr Leu Thr Leu Thr Gln Arg
385 390 395 400
Ala Thr Pro Phe Ile Tyr Gln Gly Ser Glu Leu Gly Met Thr Asn Tyr
405 410 415
Pro Phe Lys Lys Ile Asp Asp Phe Asp Asp Val Glu Val Lys Gly Phe
420 425 430
Trp Gln Asp Tyr Val Glu Thr Gly Lys Val Lys Ala Glu Glu Phe Leu
435 440 445
Gln Asn Val Arg Gln Thr Ser Arg Asp Asn Ser Arg Thr Pro Phe Gln
450 455 460
Trp Asp Ala Ser Lys Asn Ala Gly Phe Thr Ser Gly Thr Pro Trp Leu
465 470 475 480
Lys Ile Asn Pro Asn Tyr Lys Glu Ile Asn Ser Ala Asp Gln Ile Asn
485 490 495
Asn Pro Asn Ser Val Phe Asn Tyr Tyr Arg Lys Leu Ile Asn Ile Arg
500 505 510
His Asp Ile Pro Ala Leu Thr Tyr Gly Ser Tyr Ile Asp Leu Asp Pro
515 520 525
Asp Asn Asn Ser Val Tyr Ala Tyr Thr Arg Thr Leu Gly Ala Glu Lys
530 535 540
Tyr Leu Val Val Ile Asn Phe Lys Glu Glu Val Met His Tyr Thr Leu
545 550 555 560
Pro Gly Asp Leu Ser Ile Asn Lys Val Ile Thr Glu Asn Asn Ser His
565 570 575
Thr Ile Val Asn Lys Asn Asp Arg Gln Leu Arg Leu Glu Pro Trp Gln
580 585 590
Ser Gly Ile Tyr Lys Leu Asn Pro
595 600
<210> 9
<211> 550
<212> PRT
<213> 大黄欧文氏菌
<400> 9
Met Arg Ser Thr Pro His Trp Lys Glu Ala Val Val Tyr Gln Val Tyr
1 5 10 15
Pro Arg Ser Phe Met Asp Ser Asn Gly Asp Gly Thr Gly Asp Leu Asn
20 25 30
Gly Ile Ile Ser Lys Leu Asp Tyr Leu Gln Gln Leu Gly Ile Thr Leu
35 40 45
Leu Trp Leu Ser Pro Val Tyr Arg Ser Pro Met Asp Asp Asn Gly Tyr
50 55 60
Asp Ile Ser Asp Tyr Glu Glu Ile Ala Asp Ile Phe Gly Ser Met Ser
65 70 75 80
Asp Met Glu Arg Leu Ile Ala Glu Ala Lys Ala Arg Asp Ile Gly Ile
85 90 95
Leu Met Asp Leu Val Val Asn His Thr Ser Asp Glu His Pro Trp Phe
100 105 110
Ile Asp Ala Leu Ser Ser Lys Asn Ser Ala Tyr Arg Asp Phe Tyr Ile
115 120 125
Trp Arg Ala Pro Ala Ala Asp Gly Gly Pro Pro Asp Asp Ser Arg Ser
130 135 140
Asn Phe Gly Gly Ser Ala Trp Thr Leu Asp Glu Ala Ser Gly Glu Tyr
145 150 155 160
Tyr Leu His Gln Phe Ser Thr Arg Gln Pro Asp Leu Asn Trp Glu Asn
165 170 175
Pro Arg Val Arg Glu Ala Ile His Ala Met Met Asn Arg Trp Leu Asp
180 185 190
Lys Gly Ile Gly Gly Phe Arg Met Asp Val Ile Asp Leu Ile Gly Lys
195 200 205
Glu Val Asp Pro Gln Ile Met Ala Asn Gly Arg His Pro His Leu Tyr
210 215 220
Leu Gln Gln Met Asn Arg Ala Thr Phe Gly Pro Arg Gly Ser Val Thr
225 230 235 240
Val Gly Glu Thr Trp Ser Ala Thr Pro Glu Asp Ala Leu Leu Tyr Ser
245 250 255
Ala Glu Glu Arg Gln Glu Arg Gln Glu Leu Thr Met Val Phe Gln Phe
260 265 270
Glu His Ile Lys Leu Phe Trp Asp Glu Gln Tyr Gly Lys Trp Cys Asn
275 280 285
Gln Pro Phe Asp Leu Leu Arg Phe Lys Ala Val Ile Asp Lys Trp Gln
290 295 300
Thr Ala Leu Ala Asp His Gly Trp Asn Ser Leu Phe Trp Ser Asn His
305 310 315 320
Asp Leu Pro Arg Ala Val Ser Lys Phe Gly Asp Asp Gly Glu Tyr Arg
325 330 335
Val Val Ser Ala Lys Met Leu Ala Thr Ala Leu His Cys Leu Lys Gly
340 345 350
Thr Pro Tyr Ile Tyr Gln Gly Glu Glu Ile Gly Met Thr Asn Val Asn
355 360 365
Phe Ala Asp Ile Asp Asp Tyr Arg Asp Ile Glu Ser Leu Asn Leu Tyr
370 375 380
Gln Glu Arg Ile Ala Glu Gly Met Ser His Glu Ala Met Met Arg Gly
385 390 395 400
Ile His Ala Asn Gly Pro Asp Asn Ala Arg Thr Pro Met Gln Trp Thr
405 410 415
Ala Val His Met Pro Gly Leu Pro Pro Val Ser Pro Gly Leu Arg Leu
420 425 430
Ile Leu Thr Ser Gly Gln Trp Asn Val Ala Ala Ala Leu Asp Asp Pro
435 440 445
Asp Ser Val Phe Tyr His Tyr Gln Lys Leu Val Ala Leu Arg Lys Gln
450 455 460
Leu Pro Leu Leu Val His Gly Asp Phe Arg Gln Ile Val Val Glu His
465 470 475 480
Pro Gln Val Phe Ala Trp Leu Arg Thr Leu Gly Glu Gln Thr Leu Val
485 490 495
Val Ile Asn Asn Phe Thr Arg Asp Ala Val Met Leu Ala Ile Pro Asp
500 505 510
Asn Leu Gln Ser Gln Gln Gly Arg Cys Leu Ile Asn Asn Tyr Ala Pro
515 520 525
Arg Glu Gln Leu Glu Pro Ile Met Glu Leu Gln Pro Tyr Glu Ser Phe
530 535 540
Ala Leu Leu Ile Glu Arg
545 550
<210> 10
<211> 632
<212> PRT
<213> 大黄欧文氏菌
<220>
<221> misc_feature
<222> (236)..(236)
<223> Xaa可以是任意天然存在的氨基酸
<220>
<221> misc_feature
<222> (449)..(449)
<223> Xaa可以是任意天然存在的氨基酸
<400> 10
Met Ser Ser Gln Glu Leu Lys Ala Ala Val Ala Ile Phe Leu Ala Thr
1 5 10 15
Thr Phe Ser Ala Thr Ser Tyr Gln Ala Cys Ser Ala Gly Pro Asp Thr
20 25 30
Ala Pro Ser Leu Thr Val Gln Gln Ser Asn Ala Leu Pro Thr Trp Trp
35 40 45
Lys Gln Ala Val Phe Tyr Gln Val Tyr Pro Arg Ser Phe Lys Asp Thr
50 55 60
Asn Gly Asp Gly Ile Gly Asp Leu Asn Gly Ile Ile Glu Asn Leu Asp
65 70 75 80
Tyr Leu Lys Lys Leu Gly Ile Asp Ala Ile Trp Ile Asn Pro His Tyr
85 90 95
Asp Ser Pro Asn Thr Asp Asn Gly Tyr Asp Ile Arg Asp Tyr Arg Lys
100 105 110
Ile Met Lys Glu Tyr Gly Thr Met Glu Asp Phe Asp Arg Leu Ile Ser
115 120 125
Glu Met Lys Lys Arg Asn Met Arg Leu Met Ile Asp Ile Val Ile Asn
130 135 140
His Thr Ser Asp Gln His Ala Trp Phe Val Gln Ser Lys Ser Gly Lys
145 150 155 160
Asn Asn Pro Tyr Arg Asp Tyr Tyr Phe Trp Arg Asp Gly Lys Asp Gly
165 170 175
His Ala Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Glu
180 185 190
Lys Asp Asp Lys Ser Gly Gln Tyr Tyr Leu His Tyr Phe Ala Lys Gln
195 200 205
Gln Pro Asp Leu Asn Trp Asp Asn Pro Lys Val Arg Gln Asp Leu Tyr
210 215 220
Asp Met Leu Arg Phe Trp Leu Asp Lys Gly Val Xaa Gly Leu Arg Phe
225 230 235 240
Asp Thr Val Ala Thr Tyr Ser Lys Ile Pro Asn Phe Pro Asp Leu Ser
245 250 255
Gln Gln Gln Leu Lys Asn Phe Ala Glu Glu Tyr Thr Lys Gly Pro Lys
260 265 270
Ile His Asp Tyr Val Asn Glu Met Asn Arg Glu Val Leu Ser His Tyr
275 280 285
Asp Ile Ala Thr Ala Gly Glu Ile Phe Gly Val Pro Leu Asp Lys Ser
290 295 300
Ile Lys Phe Phe Asp Arg Arg Arg Asn Glu Leu Asn Ile Ala Phe Thr
305 310 315 320
Phe Asp Leu Ile Arg Leu Asp Arg Asp Ala Asp Glu Arg Trp Arg Arg
325 330 335
Lys Asp Trp Thr Leu Ser Gln Phe Arg Lys Ile Val Asp Lys Val Asp
340 345 350
Gln Thr Ala Gly Glu Tyr Gly Trp Asn Ala Phe Phe Leu Asp Asn His
355 360 365
Asp Asn Pro Arg Ala Val Ser His Phe Gly Asp Asp Arg Pro Gln Trp
370 375 380
Arg Glu His Ala Ala Lys Ala Leu Ala Thr Leu Thr Leu Thr Gln Arg
385 390 395 400
Ala Thr Pro Phe Ile Tyr Gln Gly Ser Glu Leu Gly Met Thr Asn Tyr
405 410 415
Pro Phe Lys Lys Ile Asp Asp Phe Asp Asp Val Glu Val Lys Gly Phe
420 425 430
Trp Gln Asp Tyr Val Glu Thr Gly Lys Val Lys Ala Glu Glu Phe Leu
435 440 445
Xaa Asn Val Arg Gln Thr Ser Arg Asp Asn Ser Arg Thr Pro Phe Gln
450 455 460
Trp Asp Ala Ser Lys Asn Ala Gly Phe Thr Ser Gly Thr Pro Trp Leu
465 470 475 480
Lys Ile Asn Pro Asn Tyr Lys Glu Ile Asn Ser Ala Asp Gln Ile Asn
485 490 495
Asn Pro Asn Ser Val Phe Asn Tyr Tyr Arg Lys Leu Ile Asn Ile Arg
500 505 510
His Asp Ile Pro Ala Leu Thr Tyr Gly Ser Tyr Ile Asp Leu Ala Pro
515 520 525
Asp Asn Asn Ser Val Tyr Ala Tyr Thr Arg Thr Phe Gly Ala Glu Lys
530 535 540
Tyr Leu Val Val Ile Asn Phe Lys Glu Glu Val Met His Tyr Thr Leu
545 550 555 560
Pro Gly Asp Leu Ser Ile Asn Lys Val Ile Thr Glu Asn Asn Ser His
565 570 575
Thr Ile Val Asn Lys Asn Asp Val Glu Asp Pro Arg Gly Ala Thr Ser
580 585 590
Val Cys Ser Pro Phe Gln Ala Gln Lys Arg Pro Gly Asp Pro Gly Tyr
595 600 605
Ser Ala Ala His Ser Ile Arg Phe Leu Pro Arg Phe Phe Ala Ser Tyr
610 615 620
Arg Gly Asp Ile His Ala Phe Lys
625 630
<210> 11
<211> 598
<212> PRT
<213> 克雷伯菌属
<400> 11
Met Ser Phe Val Thr Leu Arg Thr Gly Val Ala Val Ala Leu Ser Ser
1 5 10 15
Leu Ile Ile Ser Leu Ala Cys Pro Ala Val Ser Ala Ala Pro Ser Leu
20 25 30
Asn Gln Asp Ile His Val Gln Lys Glu Ser Glu Tyr Pro Ala Trp Trp
35 40 45
Lys Glu Ala Val Phe Tyr Gln Ile Tyr Pro Arg Ser Phe Lys Asp Thr
50 55 60
Asn Asp Asp Gly Ile Gly Asp Ile Arg Gly Ile Ile Glu Lys Leu Asp
65 70 75 80
Tyr Leu Lys Ser Leu Gly Ile Asp Ala Ile Trp Ile Asn Pro His Tyr
85 90 95
Asp Ser Pro Asn Thr Asp Asn Gly Tyr Asp Ile Ser Asn Tyr Arg Gln
100 105 110
Ile Met Lys Glu Tyr Gly Thr Met Glu Asp Phe Asp Ser Leu Val Ala
115 120 125
Glu Met Lys Lys Arg Asn Met Arg Leu Met Ile Asp Val Val Ile Asn
130 135 140
His Thr Ser Asp Gln His Pro Trp Phe Ile Gln Ser Lys Ser Asp Lys
145 150 155 160
Asn Asn Pro Tyr Arg Asp Tyr Tyr Phe Trp Arg Asp Gly Lys Asp Asn
165 170 175
Gln Pro Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Gln
180 185 190
Lys Asp Ala Lys Ser Gly Gln Tyr Tyr Leu His Tyr Phe Ala Arg Gln
195 200 205
Gln Pro Asp Leu Asn Trp Asp Asn Pro Lys Val Arg Glu Asp Leu Tyr
210 215 220
Ala Met Leu Arg Phe Trp Leu Asp Lys Gly Val Ser Gly Met Arg Phe
225 230 235 240
Asp Thr Val Ala Thr Tyr Ser Lys Ile Pro Gly Phe Pro Asn Leu Thr
245 250 255
Pro Glu Gln Gln Lys Asn Phe Ala Glu Gln Tyr Thr Met Gly Pro Asn
260 265 270
Ile His Arg Tyr Ile Gln Glu Met Asn Arg Lys Val Leu Ser Arg Tyr
275 280 285
Asp Val Ala Thr Ala Gly Glu Ile Phe Gly Val Pro Leu Asp Arg Ser
290 295 300
Ser Gln Phe Phe Asp Arg Arg Arg His Glu Leu Asn Met Ala Phe Met
305 310 315 320
Phe Asp Leu Ile Arg Leu Asp Arg Asp Ser Asn Glu Arg Trp Arg His
325 330 335
Lys Ser Trp Ser Leu Ser Gln Phe Arg Gln Ile Ile Ser Lys Met Asp
340 345 350
Val Thr Val Gly Lys Tyr Gly Trp Asn Thr Phe Phe Leu Asp Asn His
355 360 365
Asp Asn Pro Arg Ala Val Ser His Phe Gly Asp Asp Arg Pro Gln Trp
370 375 380
Arg Glu Ala Ser Ala Lys Ala Leu Ala Thr Ile Thr Leu Thr Gln Arg
385 390 395 400
Ala Thr Pro Phe Ile Tyr Gln Gly Ser Glu Leu Gly Met Thr Asn Tyr
405 410 415
Pro Phe Arg Gln Leu Asn Glu Phe Asp Asp Ile Glu Val Lys Gly Phe
420 425 430
Trp Gln Asp Tyr Val Gln Ser Gly Lys Val Thr Ala Thr Glu Phe Leu
435 440 445
Asp Asn Val Arg Leu Thr Ser Arg Asp Asn Ser Arg Thr Pro Phe Gln
450 455 460
Trp Asn Asp Thr Leu Asn Ala Gly Phe Thr Arg Gly Lys Pro Trp Phe
465 470 475 480
His Ile Asn Pro Asn Tyr Val Glu Ile Asn Ala Glu Arg Glu Glu Thr
485 490 495
Arg Glu Asp Ser Val Leu Asn Tyr Tyr Lys Lys Met Ile Gln Leu Arg
500 505 510
His His Ile Pro Ala Leu Val Tyr Gly Ala Tyr Gln Asp Leu Asn Pro
515 520 525
Gln Asp Asn Thr Val Tyr Ala Tyr Thr Arg Thr Leu Gly Asn Glu Arg
530 535 540
Tyr Leu Val Val Val Asn Phe Lys Glu Tyr Pro Val Arg Tyr Thr Leu
545 550 555 560
Pro Ala Asn Asp Ala Ile Glu Glu Val Val Ile Asp Thr Gln Gln Gln
565 570 575
Ala Ala Ala Pro His Ser Thr Ser Leu Ser Leu Ser Pro Trp Gln Ala
580 585 590
Gly Val Tyr Lys Leu Arg
595
<210> 12
<211> 598
<212> PRT
<213> 克雷伯菌属 NK33-98-8
<400> 12
Met Ser Phe Val Thr Leu Arg Thr Gly Val Ala Val Ala Leu Ser Ser
1 5 10 15
Leu Ile Ile Ser Leu Ala Cys Pro Ala Val Ser Ala Ala Pro Ser Leu
20 25 30
Asn Gln Asp Ile His Val Gln Lys Glu Ser Glu Tyr Pro Ala Trp Trp
35 40 45
Lys Glu Ala Val Phe Tyr Gln Ile Tyr Pro Arg Ser Phe Lys Asp Thr
50 55 60
Asn Asp Asp Gly Ile Gly Asp Ile Arg Gly Ile Ile Glu Lys Leu Asp
65 70 75 80
Tyr Leu Lys Ser Leu Gly Ile Asp Ala Ile Trp Ile Asn Pro His Tyr
85 90 95
Asp Ser Pro Asn Thr Asp Asn Gly Tyr Asp Ile Ser Asn Tyr Arg Gln
100 105 110
Ile Met Lys Glu Tyr Gly Thr Met Glu Asp Phe Asp Asn Leu Val Ala
115 120 125
Glu Met Lys Lys Arg Asn Met Arg Leu Met Ile Asp Val Val Ile Asn
130 135 140
His Thr Ser Asp Gln His Pro Trp Phe Ile Gln Ser Lys Ser Asp Lys
145 150 155 160
Asn Ser Pro Tyr Arg Asn Tyr Tyr Phe Trp Arg Asp Gly Lys Asp Asn
165 170 175
Gln Pro Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Gln
180 185 190
Lys Asp Val Lys Ser Gly Gln Tyr Tyr Leu His Tyr Phe Ala Arg Gln
195 200 205
Gln Pro Asp Leu Asn Trp Asp Asn Pro Lys Val Arg Glu Asp Leu Tyr
210 215 220
Ala Met Leu Arg Phe Trp Leu Asp Lys Gly Val Ser Gly Met Arg Phe
225 230 235 240
Asp Thr Val Ala Thr Tyr Ser Lys Ile Pro Gly Phe Pro Asn Leu Thr
245 250 255
Pro Glu Gln Gln Lys Asn Phe Ala Glu Gln Tyr Thr Met Gly Pro Asn
260 265 270
Ile His Arg Tyr Ile Gln Glu Met Asn Arg Glu Val Leu Ser Arg Tyr
275 280 285
Asp Val Ala Thr Ala Gly Glu Ile Phe Gly Val Pro Leu Asp Arg Ser
290 295 300
Ser Gln Phe Phe Asp Arg Arg Arg His Glu Leu Asn Met Ala Phe Met
305 310 315 320
Phe Asp Leu Ile Arg Leu Asp Arg Asp Ser Asn Glu Arg Trp Arg His
325 330 335
Lys Ser Trp Ser Leu Ser Gln Phe Arg Gln Ile Ile Ser Lys Met Asp
340 345 350
Val Thr Val Gly Lys Tyr Gly Trp Asn Thr Phe Phe Leu Asp Asn His
355 360 365
Asp Asn Pro Arg Ala Val Ser His Phe Gly Asp Asp Arg Pro Gln Trp
370 375 380
Arg Glu Ala Ser Ala Lys Ala Leu Ala Thr Ile Thr Leu Thr Gln Arg
385 390 395 400
Ala Thr Pro Phe Ile Tyr Gln Gly Ser Glu Leu Gly Met Thr Asn Tyr
405 410 415
Pro Phe Arg Gln Leu Asn Glu Phe Asp Asp Ile Glu Val Lys Gly Phe
420 425 430
Trp Gln Asp Tyr Val Gln Ser Gly Lys Val Thr Ala Thr Glu Phe Leu
435 440 445
Asp Asn Val Arg Leu Thr Ser Arg Asp Asn Ser Arg Thr Pro Phe Gln
450 455 460
Trp Asn Asp Thr Leu Asn Ala Gly Phe Thr Arg Gly Lys Pro Trp Phe
465 470 475 480
His Ile Asn Pro Asn Tyr Val Glu Ile Asn Ala Glu Arg Glu Glu Thr
485 490 495
Arg Glu Asp Ser Val Leu Asn Tyr Tyr Lys Lys Met Ile Gln Leu Arg
500 505 510
His His Ile Pro Ala Leu Val Tyr Gly Ala Tyr Gln Asp Leu Asn Pro
515 520 525
Gln Asp Asn Thr Val Tyr Ala Tyr Thr Arg Thr Leu Gly Asn Glu Arg
530 535 540
Tyr Leu Val Val Val Asn Phe Glu Glu Tyr Pro Val Arg Tyr Thr Leu
545 550 555 560
Pro Ala Asn Asp Ala Ile Glu Glu Val Val Ile Asp Thr Gln Gln Gln
565 570 575
Ala Thr Ala Pro His Ser Thr Ser Leu Ser Leu Ser Pro Trp Gln Ala
580 585 590
Gly Val Tyr Lys Leu Arg
595
<210> 13
<211> 598
<212> PRT
<213> 植物生克雷伯菌
<400> 13
Met Ser Phe Ile Thr Leu Arg Thr Gly Val Ala Val Ala Leu Ser Ser
1 5 10 15
Leu Met Ile Ser Gln Ala Cys Pro Ala Val Ser Ala Ala Pro Ser Val
20 25 30
Asn Gln Asn Ile His Val His Lys Glu Ser Glu Tyr Pro Ala Trp Trp
35 40 45
Lys Glu Ala Val Phe Tyr Gln Ile Tyr Pro Arg Ser Phe Lys Asp Thr
50 55 60
Asn Asp Asp Gly Ile Gly Asp Ile Arg Gly Ile Ile Glu Lys Leu Asp
65 70 75 80
Tyr Leu Lys Ser Leu Gly Ile Asp Ala Ile Trp Ile Asn Pro His Tyr
85 90 95
Asp Ser Pro Asn Thr Asp Asn Gly Tyr Asp Ile Ser Asn Tyr Arg Gln
100 105 110
Ile Met Lys Glu Tyr Gly Thr Met Glu Asp Phe Asp Asn Leu Val Ala
115 120 125
Glu Met Lys Lys Arg Asn Met Arg Leu Met Ile Asp Val Val Ile Asn
130 135 140
His Thr Ser Asp Gln His Pro Trp Phe Ile Gln Ser Lys Ser Asp Lys
145 150 155 160
Asn Asn Pro Tyr Arg Asp Tyr Tyr Phe Trp Arg Asp Gly Lys Asp Asn
165 170 175
Gln Pro Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Gln
180 185 190
Lys Asp Ala Lys Ser Gly Gln Tyr Tyr Leu His Tyr Phe Ala Arg Gln
195 200 205
Gln Pro Asp Leu Asn Trp Asp Asn Pro Lys Val Arg Glu Asp Leu Tyr
210 215 220
Ala Met Leu Arg Phe Trp Leu Asp Lys Gly Val Ser Ser Met Arg Phe
225 230 235 240
Asp Thr Val Ala Thr Tyr Ser Lys Ile Pro Gly Phe Pro Asn Leu Thr
245 250 255
Pro Glu Gln Gln Lys Asn Phe Ala Glu Gln Tyr Thr Met Gly Pro Asn
260 265 270
Ile His Arg Tyr Ile Gln Glu Met Asn Arg Lys Val Leu Ser Arg Tyr
275 280 285
Asp Val Ala Thr Ala Gly Glu Ile Phe Gly Val Pro Leu Asp Arg Ser
290 295 300
Ser Gln Phe Phe Asp Pro Arg Arg His Glu Leu Asn Met Ala Phe Met
305 310 315 320
Phe Asp Leu Ile Arg Leu Asp Arg Asp Ser Asn Glu Arg Trp Arg His
325 330 335
Lys Ser Trp Ser Leu Ser Gln Phe Arg Gln Ile Ile Ser Lys Met Asp
340 345 350
Val Thr Val Gly Lys Tyr Gly Trp Asn Thr Phe Phe Leu Asp Asn His
355 360 365
Asp Asn Pro Arg Ala Val Ser His Phe Gly Asp Asp Arg Pro Gln Trp
370 375 380
Arg Glu Ala Ser Ala Lys Ala Leu Ala Thr Ile Thr Leu Thr Gln Arg
385 390 395 400
Ala Thr Pro Phe Ile Tyr Gln Gly Ser Glu Leu Gly Met Thr Asn Tyr
405 410 415
Pro Phe Arg Gln Leu Asn Glu Phe Asp Asp Ile Glu Val Lys Gly Phe
420 425 430
Trp Gln Asp Tyr Val Gln Ser Gly Lys Val Thr Ala Thr Glu Phe Leu
435 440 445
Asp Asn Val Arg Leu Thr Ser Arg Asp Asn Ser Arg Thr Pro Phe Gln
450 455 460
Trp Asn Asp Thr Leu Asn Ala Gly Phe Thr Arg Gly Lys Pro Trp Phe
465 470 475 480
His Ile Asn Pro Asn Tyr Val Glu Ile Asn Ala Glu Arg Glu Glu Thr
485 490 495
Arg Glu Asp Ser Val Leu Asn Tyr Tyr Lys Lys Met Ile Gln Leu Arg
500 505 510
His His Ile Pro Ala Leu Val Tyr Gly Ala Tyr Gln Asp Leu Asn Pro
515 520 525
Gln Asp Asn Thr Val Tyr Ala Tyr Thr Arg Thr Leu Gly Asn Glu Arg
530 535 540
Tyr Leu Val Val Val Asn Phe Lys Glu Tyr Pro Val Arg Tyr Thr Leu
545 550 555 560
Pro Ala Asn Asp Ala Ile Glu Glu Val Val Ile Asp Thr Gln Gln Gln
565 570 575
Ala Thr Ala Pro His Ser Thr Ser Leu Ser Leu Ser Pro Trp Gln Ala
580 585 590
Gly Val Tyr Lys Leu Arg
595
<210> 14
<211> 599
<212> PRT
<213> 欧文氏菌属 Ejp617
<400> 14
Met Ser Arg Phe Thr Leu Ser Thr Val Met Ala Leu Leu Val Ala Pro
1 5 10 15
Val Leu Ser Val Leu Pro Gly Gln Val Met Ala Gly Lys Met Asp Met
20 25 30
Ala Thr Thr Gln Leu Ala Gln Lys Ser Asp Asp Phe Pro Ala Trp Trp
35 40 45
Lys Gln Ala Val Phe Tyr Gln Val Tyr Pro Arg Ser Phe Lys Asp Thr
50 55 60
Asn Gly Asp Gly Ile Gly Asp Leu Lys Gly Ile Ile Glu Lys Leu Asp
65 70 75 80
Tyr Leu Lys Arg Leu Gly Val Asp Ala Ile Trp Ile Asn Pro His Tyr
85 90 95
Asp Ser Pro Asn Thr Asp Asn Gly Tyr Asp Ile Arg Asp Tyr Arg Lys
100 105 110
Ile Met Lys Glu Tyr Gly Thr Met Glu Asp Phe Asp Arg Leu Ile Ala
115 120 125
Glu Met Asn Lys Arg Asn Met Arg Leu Met Ile Asp Ile Val Ile Asn
130 135 140
His Thr Ser Asp Gln His Ser Trp Phe Val Gln Ser Lys Gly Ser Lys
145 150 155 160
Asp Asn Pro Tyr Arg Asp Tyr Tyr Phe Trp Arg Asp Gly Lys Asn Gly
165 170 175
Gln Pro Pro Asn Asn Tyr Pro Ser Phe Phe Gly Gly Ser Ala Trp Lys
180 185 190
Lys Glu Asp Asn Ser Gly Gln Tyr Tyr Leu His Tyr Phe Ala Thr Gln
195 200 205
Gln Pro Asp Leu Asn Trp Asp Asn Pro Lys Val Arg Glu Asp Leu Tyr
210 215 220
Ala Met Leu Arg Phe Trp Leu Asp Lys Gly Val Ala Gly Leu Arg Phe
225 230 235 240
Asp Thr Val Ala Thr Tyr Ala Lys Ile Pro Gly Phe Pro Asp Leu Thr
245 250 255
Pro Gln Gln Arg Lys Asn Phe Ala Arg Thr Tyr Thr Glu Gly Pro Ser
260 265 270
Ile His Arg Tyr Ile Lys Glu Met His Gln Gln Val Phe Ser His Tyr
275 280 285
Asn Ile Ala Thr Ala Gly Glu Ile Phe Gly Val Pro Leu Glu Lys Ser
290 295 300
Ile Asp Tyr Phe Asp Arg Arg Arg Gly Glu Leu Asn Ile Ala Phe Thr
305 310 315 320
Phe Asp Leu Ile Arg Leu Asp Arg Gly Val Glu Glu Arg Trp Arg Gln
325 330 335
Lys Ala Trp Ser Leu Thr Asp Phe Arg Gln Thr Ile Asp Lys Val Asp
340 345 350
Arg Val Ala Gly Lys Tyr Gly Trp Asn Ala Phe Phe Leu Asp Asn His
355 360 365
Asp Asn Pro Arg Ala Val Ser His Phe Gly Asp Asp Arg Pro Gln Trp
370 375 380
Arg Gln Ala Ser Ala Lys Ala Leu Ala Thr Leu Met Ile Thr Gln Arg
385 390 395 400
Ala Thr Pro Phe Ile Tyr Gln Gly Ser Glu Leu Gly Met Thr Asn Tyr
405 410 415
Pro Phe Lys Ser Ile Ala Asp Phe Asp Asp Ile Glu Val Lys Gly Phe
420 425 430
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Claims (15)
1.制备异麦芽酮糖的方法,其包括下述方法步骤:
A) 使酶复合物与含有蔗糖的溶液接触,所述酶复合物能够催化蔗糖转化成异麦芽酮糖;
B) 将至少部分蔗糖异构化为异麦芽酮糖;
C) 分离出所述酶复合物,得到进料溶液,
D) 通过蒸发,部分除去所述进料溶液的水,得到异麦芽酮糖-晶体的混悬液,
E) 将所述混悬液分离成含有异麦芽酮糖的固体和母液,和任选地
F) 洗涤所述含有异麦芽酮糖的固体,
其特征在于,
作为含有蔗糖的溶液使用选自浓汁或稀汁的至少一种,优选浓汁。
2.根据权利要求1所述的方法,其特征在于,
作为含有蔗糖的溶液使用用水稀释过的浓汁,基于整个汁计,其具有20-80重量%、尤其是30-50重量%的蔗糖浓度。
3.根据权利要求1或2所述的方法,其特征在于,
含于酶复合物中的酶是酶类别EC 5.4.99.11中的至少一种蔗糖葡萄糖基变位酶,特别得自下述菌的一种:红色精蛋白杆菌、尤其是菌株红色精蛋白杆菌CBS 574.77和具有Seq ID Nr. 5和6的那些;红色精朊杆菌Z12;普城沙雷菌、尤其是菌株普城沙雷菌ATCC 15928;气味沙雷氏菌、尤其是菌株气味沙雷氏菌4Rx13(Seq ID Nr. 7);粘质沙雷菌、尤其是菌株粘质沙雷菌NCIB 8285;肠膜明串珠菌、尤其是菌株肠膜明串珠菌ATCC 1083 a;大黄欧文氏菌、尤其是菌株大黄欧文氏菌ATCC29283(Seq ID Nr. 1)、NCPPB 1578、DSM 4484(Seq ID Nr. 8)、NX-5(Seq ID Nr. 9)和WAC2928(Seq ID Nr. 10);欧文氏菌属、尤其是菌株欧文氏菌属D12;放射形土壤杆菌、尤其是菌株放射形土壤杆菌MX-232;土生结克雷伯菌、尤其是菌株土生结克雷伯菌JCM 1687;克雷伯菌属、尤其是菌株克雷伯菌属FERM BP-2838、LX3(Seq ID Nr. 11)和NK33-98-8(Seq ID Nr. 12);肺炎克雷伯菌、尤其是菌株肺炎克雷伯菌342(Seq ID Nr. 4);新加坡克雷伯菌、尤其是菌株新加坡克雷伯菌LX21;嗜中酸假单胞菌、尤其是菌株嗜中酸假单胞菌MX-45(Seq ID Nr. 2);分散泛菌、尤其是菌株分散泛菌UQ68J(Seq ID Nr. 3);植物生克雷伯菌、尤其是菌株植物生克雷伯菌CCRC 19112、MX10和UQ14S(Seq ID Nr. 13);肠杆菌属、尤其是菌株肠杆菌属FMB-1(Seq ID Nr. 16)、SZ62和Ejp617(Seq ID Nr. 14);维涅兰德固氮菌DJ(Seq ID Nr. 15)。
4.根据前述权利要求中的至少一项所述的方法,其特征在于,
所述酶复合物是细胞,尤其是选自得自下述菌的细胞:红色精蛋白杆菌、尤其是菌株红色精蛋白杆菌CBS 574.77;红色精朊杆菌Z12;普城沙雷菌、尤其是菌株普城沙雷菌ATCC 15928;气味沙雷氏菌、尤其是菌株气味沙雷氏菌4Rx13;粘质沙雷菌、尤其是菌株粘质沙雷菌NCIB 8285;肠膜明串珠菌、尤其是菌株肠膜明串珠菌ATCC 1083 a;大黄欧文氏菌、尤其是菌株大黄欧文氏菌ATCC29283、NCPPB 1578、DSM 4484、NX-5和WAC2928;欧文氏菌属、尤其是菌株欧文氏菌属D12;放射形土壤杆菌、尤其是菌株放射形土壤杆菌MX-232;土生结克雷伯菌、尤其是菌株土生结克雷伯菌JCM 1687;克雷伯菌属、尤其是菌株克雷伯菌属FERM BP-2838、LX3和NK33-98-8;肺炎克雷伯菌、尤其是菌株肺炎克雷伯菌342;新加坡克雷伯菌、尤其是菌株新加坡克雷伯菌LX21;嗜中酸假单胞菌、尤其是菌株嗜中酸假单胞菌MX-45;分散泛菌、尤其是菌株分散泛菌UQ68J;植物生克雷伯菌、尤其是菌株植物生克雷伯菌CCRC 19112、MX10和UQ14S;肠杆菌属FMB-1、SZ62和Ejp617;维涅兰德固氮菌DJ。
5.根据权利要求4所述的方法,其特征在于,
所述细胞被固定在多糖诸如藻酸盐、果胶、角叉菜胶、壳聚糖或聚乙烯醇,例如Lentikats,或它们的混合物中,尤其是藻酸盐中。
6.根据前述权利要求中的至少一项所述的方法,其特征在于,
在方法步骤B)中,存在4-9.5的pH-值。
7.根据前述权利要求中的至少一项所述的方法,其特征在于,
在方法步骤B)中,存在20-40℃的温度。
8.根据前述权利要求中的至少一项所述的方法,其特征在于,
以固体床的形式使用经固定化的酶复合物,所述含有蔗糖的溶液通过所述固体床流过。
9.根据前述权利要求中的至少一项所述的方法,其特征在于,
方法步骤D) 在50℃至70℃、优选55℃至65℃、特别优选58℃至62℃的温度范围和在70毫巴至200毫巴、优选100毫巴至180、特别优选110毫巴至140毫巴的压力范围进行。
10.根据前述权利要求中的至少一项所述的方法,其特征在于,
在方法步骤D)中,加入异麦芽酮糖-晶种,尤其是在进料溶液中的音速在1800 m/s至2000 m/s、尤其是1850 m/s至1950 m/s、尤其是1870 m/s至1920 m/s的范围的时间点。
11.根据前述权利要求中的至少一项所述的方法,其特征在于,
用水进行在方法步骤F)中的洗涤。
12.根据前述权利要求中的至少一项所述的方法,其特征在于,
在方法步骤F)中用于洗涤使用过的液体被供至方法步骤D)的进料溶液。
13.根据前述权利要求中的至少一项所述的方法,其特征在于,
所述方法额外包括下述方法步骤:
G) 冷却得自方法步骤E) 的母液,得到异麦芽酮糖-晶体的第二混悬液,
H) 将所述混悬液分离成第二含有异麦芽酮糖的固体和第二母液,和任选地
I) 将所述第二含有异麦芽酮糖的固体加入到方法步骤C)的进料溶液中。
14.根据权利要求13所述的方法,其特征在于,
在方法步骤G)中,将得自方法步骤E) 的母液冷却至10℃至30℃,优选地冷却至15℃至25℃,特别优选地冷却至18℃至22℃。
15.根据权利要求13或14所述的方法,其特征在于,
方法步骤C)的进料溶液包含溶解的得自方法步骤H)的第二含有异麦芽酮糖的固体,和在方法步骤F)中用于洗涤使用过的液体。
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DE3213107A1 (de) | 1982-04-07 | 1983-10-13 | Süddeutsche Zucker AG, 6800 Mannheim | Verfahren zur herstellung von isomaltulose (6-o-(alpha)-d-glucopyranosido-d-fructose) mit hilfe von immobilisierten bakterienzellen |
DE3241788A1 (de) | 1982-11-11 | 1984-05-17 | Süddeutsche Zucker AG, 6800 Mannheim | Verfahren zur herstellung von 1-0-(alpha)-d-glucopyranosido-d-fructose und verwendung als suessungsmittel |
DE3725974A1 (de) | 1987-08-05 | 1989-02-16 | Messerschmitt Boelkow Blohm | Einsatz fuer ein zumindest teilweise aus kunststoff bestehendes bauteil |
CN1030530C (zh) * | 1989-02-03 | 1995-12-20 | 广东省微生物研究所 | 固定化α-葡糖基转移酶制备帕拉金糖 |
JPH02273192A (ja) | 1989-04-13 | 1990-11-07 | Meito Sangyo Kk | イソマルチユロースの製造方法 |
DE59303142D1 (de) | 1993-05-06 | 1996-08-08 | Suedzucker Ag | Süssungsmittel, Verfahren zur Herstellung desselben sowie dessen Verwendung |
DE9321600U1 (de) * | 1993-05-06 | 2000-04-06 | Südzucker Aktiengesellschaft Mannheim/Ochsenfurt, 68165 Mannheim | Süssungsmittel |
FI104563B (fi) | 1996-05-17 | 2000-02-29 | Xyrofin Oy | Menetelmä ja kantaja isomaltuloosin tuottamiseksi immobilisoitujen mikro-organismien avulla |
FI105048B (fi) | 1997-05-22 | 2000-05-31 | Xyrofin Oy | Menetelmä isomaltuloosin ja muiden tuotteiden valmistamiseksi |
CN1434861A (zh) | 2000-02-15 | 2003-08-06 | 分子农业生物学院 | 克雷伯氏菌属的细菌分离物以及从其中分离的异麦芽酮糖合酶基因 |
DE102007031689A1 (de) | 2007-07-06 | 2009-01-08 | Evonik Goldschmidt Gmbh | Enzympräparate |
-
2011
- 2011-05-05 DE DE102011100772A patent/DE102011100772A1/de not_active Ceased
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2012
- 2012-02-08 BR BR112013028369A patent/BR112013028369A2/pt not_active IP Right Cessation
- 2012-02-08 AU AU2012251903A patent/AU2012251903A1/en not_active Abandoned
- 2012-02-08 CN CN201280021859.XA patent/CN103501636A/zh active Pending
- 2012-02-08 EP EP12702557.5A patent/EP2704594B1/de not_active Not-in-force
- 2012-02-08 WO PCT/EP2012/052096 patent/WO2012150051A1/de active Application Filing
- 2012-05-04 EP EP12717789.7A patent/EP2704595A1/de not_active Withdrawn
- 2012-05-04 WO PCT/EP2012/058228 patent/WO2012150332A1/de active Application Filing
- 2012-05-04 CN CN201280021863.6A patent/CN103501637A/zh active Pending
- 2012-05-04 BR BR112013028410A patent/BR112013028410A2/pt not_active IP Right Cessation
- 2012-05-04 AU AU2012251596A patent/AU2012251596A1/en not_active Abandoned
- 2012-05-04 TW TW101116148A patent/TW201307565A/zh unknown
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
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CN112566512A (zh) * | 2018-08-22 | 2021-03-26 | 帝斯曼知识产权资产管理有限公司 | 作为食物和营养补充剂的蔗糖异构酶 |
CN113621601A (zh) * | 2021-06-24 | 2021-11-09 | 浙江工业大学 | 蔗糖异构酶突变体、编码基因及其应用 |
CN113621601B (zh) * | 2021-06-24 | 2023-06-30 | 浙江工业大学 | 蔗糖异构酶突变体、编码基因及其应用 |
CN114703211A (zh) * | 2022-02-16 | 2022-07-05 | 李宪臻 | 一种融合卷曲螺旋结构域的蔗糖异构酶活性包涵体 |
CN116179631A (zh) * | 2023-04-27 | 2023-05-30 | 山东百龙创园生物科技股份有限公司 | 一种异麦芽酮糖晶体及其制备方法 |
CN116179631B (zh) * | 2023-04-27 | 2023-11-28 | 山东百龙创园生物科技股份有限公司 | 一种异麦芽酮糖晶体及其制备方法 |
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WO2012150332A1 (de) | 2012-11-08 |
DE102011100772A1 (de) | 2012-11-08 |
BR112013028369A2 (pt) | 2017-03-01 |
EP2704595A1 (de) | 2014-03-12 |
BR112013028410A2 (pt) | 2019-09-24 |
TW201307565A (zh) | 2013-02-16 |
AU2012251903A1 (en) | 2013-10-24 |
AU2012251596A1 (en) | 2013-10-24 |
EP2704594B1 (de) | 2016-04-27 |
CN103501636A (zh) | 2014-01-08 |
WO2012150051A1 (de) | 2012-11-08 |
EP2704594A1 (de) | 2014-03-12 |
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