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    Cellulase variants
    4.
    发明申请
    Cellulase variants 审中-公开
    纤维素酶变体

    公开(公告)号：US20050009166A1

    公开(公告)日：2005-01-13

    申请号：US10919195

    申请日：2004-08-16

    申请人： Kim Andersen ,  Martin Schulein ,  Hanne Dela ,  Lars Christiansen ,  Bo Damgaard ,  Claus Von der Osten

    发明人： Kim Andersen ,  Martin Schulein ,  Hanne Dela ,  Lars Christiansen ,  Bo Damgaard ,  Claus Von der Osten

    IPC分类号： C12N15/09 ,  C11D3/386 ,  C12N1/15 ,  C12N1/21 ,  C12N9/42 ,  C12R1/645 ,  C12S11/00 ,  C12S99/00 ,  D06L4/40 ,  D06M16/00 ,  D21B1/02 ,  D21C5/02 ,  D21H21/24 ,  C07H21/04 ,  C12N15/74

    CPC分类号： C11D3/38645 ,  C12N9/2437 ,  C12Y302/01004

    摘要： The present invention relates to a method for improving the properties of a cellulolytic enzyme by amino acid substitution, deletion or insertion, the method comprising the steps of: a. constructing a multiple alignment of at least two amino acid sequences known to have three-dimensional structures similar to endoglucanase V (EGV) from Humicola insolens known from Protein Data Bank entry 4ENG; b. constructing a homology-built three-dimensional structure of the cellulolytic enzyme based on the structure of the EGV; c. identifying amino acid residue positions present in a distance from the substrate binding cleft of not more than 5 Å; d. identifying surface-exposed amino acid residues of the enzyme; e. identifying all charged or potentially charged amino acid residue positions of the enzyme; f. choosing one or more positions wherein the amino acid residue is to be substituted, deleted or where an insertion is to be provided; and g. carrying out the substitution, deletion or insertion by using conventional protein engineering techniques. Also described are cellulase variants obtained by this method.

    摘要翻译： 本发明涉及通过氨基酸取代，缺失或插入改进纤维素分解酶的性质的方法，该方法包括以下步骤：a。 构建已知具有类似于蛋白质数据库条目4ENG中已知的Humicola insolens的内切葡聚糖酶V（EGV）的三维结构的至少两个氨基酸序列的多重比对; b。 基于EGV的结构构建纤维素分解酶的同源构建的三维结构; C。 鉴定存在于与所述基质结合裂缝一定距离的氨基酸残基位置不大于5埃; d。 鉴定酶的表面暴露的氨基酸残基; e。 鉴定酶的所有带电荷或潜在带电的氨基酸残基位置; F。 选择其中氨基酸残基被取代，缺失或提供插入的一个或多个位置; 和g。 通过使用常规蛋白质工程技术进行取代，缺失或插入。 还描述了通过该方法获得的纤维素酶变体。

    Extracellular expression of cellulose binding domains (CBD) using Bacillus
    5.
    发明授权
    Extracellular expression of cellulose binding domains (CBD) using Bacillus 失效
    使用芽孢杆菌的纤维素结合结构域（CBD）的细胞外表达

    公开(公告)号：US6060274A

    公开(公告)日：2000-05-09

    申请号：US959212

    申请日：1997-10-28

    申请人： Mads Eskelund Bjornvad ,  Martin Schulein ,  Per Lina Jorgensen

    发明人： Mads Eskelund Bjornvad ,  Martin Schulein ,  Per Lina Jorgensen

    IPC分类号： C12N15/09 ,  C07K14/195 ,  C07K14/21 ,  C07K14/33 ,  C12N1/21 ,  C12P21/02 ,  C12Q1/68 ,  C12R1/07 ,  C12P21/06 ,  C12N1/20 ,  C12N15/00

    CPC分类号： C07K14/195 ,  C07K14/21 ,  C07K14/33

    摘要： The present invention relates to Bacillus hosts transformed with a vector comprising a DNA sequence encoding for a cellulose binding domain (CBD) and capable of expressing said sequence, the expressed polypeptide protein consisting essentially of one or more non-catalytic domains; the cellulose binding domain having a molecular weight in the range of from 4 kD to 35 kD and being obtainable from a microorganism or from a plant, preferably from a bacterium or a fungus; the Bacillus host e.g. being one of the species Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilos, and Bacillus amyloliquefaciens; and a Bacillus expression vector carrying an inserted DNA sequence encoding for a cellulose binding domain; and a method for producing a cellulose binding domain polypeptide in a Bacillus host cell.

    摘要翻译： 本发明涉及用包含编码纤维素结合结构域（CBD）并能够表达所述序列的DNA序列的载体转化的芽孢杆菌宿主，所述表达的多肽蛋白质基本上由一个或多个非催化结构域组成; 所述纤维素结合结构域具有4kD至35kD的分子量，可从微生物或植物，优选来自细菌或真菌得到; 芽孢杆菌宿主例如 是枯草芽孢杆菌，地衣芽孢杆菌，巨大芽孢杆菌，嗜热脂肪芽孢杆菌和解淀粉芽孢杆菌之一; 和携带编码纤维素结合结构域的插入DNA序列的芽孢杆菌表达载体; 以及在芽孢杆菌宿主细胞中生产纤维素结合结构域多肽的方法。

    ENDO-BETA-1,4-GLUCANASE FROM BACILLUS
    7.
    发明申请
    ENDO-BETA-1,4-GLUCANASE FROM BACILLUS 有权
    来自BACILLUS的内切-14-GLUCANASE

    公开(公告)号：US20050112749A1

    公开(公告)日：2005-05-26

    申请号：US10479446

    申请日：2002-06-06

    申请人： Helle Outtrup ,  Martin Schulein ,  Mads Eskelund ,  Keith Gibson

    发明人： Helle Outtrup ,  Martin Schulein ,  Mads Eskelund ,  Keith Gibson

    IPC分类号： C11D3/386 ,  C12N9/24 ,  C07H21/04 ,  C12N15/74 ,  C12P19/04

    CPC分类号： C11D3/386

    摘要： An enzyme exhibiting endo-beta-1,4-glucanase activity (EC 3.2.1.4), which is selected from one of a) a polypeptide encoded by the DNA sequence of positions 1 to 2322 of SEQ ID NO:1; b) a polypeptide produced by culturing a cell comprising the sequence of SEQ ID NO:1 under conditions wherein the DNA sequence is expressed; c) an endo-beta-1,4-glucanase enzyme having a sequence of at least 97% identity to the amino acid sequence of position 1 to position 773 of SEQ ID NO:2; and fragments thereof exhibiting endo-beta-1,4-glucanase activity, and d) a polypeptide having endo-beta-1,4-glucanase activity that is encoded by a polynucleo-tide that hybridizes with the nucleotide sequence shown in positions 1-2322 of SEQ ID NO:1, is useful for detergent and textile applications.

    摘要翻译： 显示内β-1,4-葡聚糖酶活性的酶（EC 3.2.1.4），其选自a）由SEQ ID NO：1的1至2322位的DNA序列编码的多肽; b）通过在表达DNA序列的条件下培养包含SEQ ID NO：1的序列的细胞产生的多肽; c）具有与SEQ ID NO：2的位置1至位置773的氨基酸序列具有至少97％同一性的序列的内切-β-1,4-葡聚糖酶; 和表现出内切-β-1,4-葡聚糖酶活性的片段，以及d）具有内切-β-1,4-葡聚糖酶活性的多肽，其与通过多核苷酸编码的多核苷酸杂交，所述多核苷酸与位置1- SEQ ID NO：1的2322可用于洗涤剂和纺织品应用。

    Cell-wall degrading enzyme variants
    9.
    发明授权
    Cell-wall degrading enzyme variants 有权
    细胞壁降解酶变体

    公开(公告)号：US06607902B2

    公开(公告)日：2003-08-19

    申请号：US09910505

    申请日：2001-07-19

    申请人： Sanne O Schrøder Glad ,  Carsten Andersen ,  Martin Schulein ,  Torben Peter Frandsen

    发明人： Sanne O Schrøder Glad ,  Carsten Andersen ,  Martin Schulein ,  Torben Peter Frandsen

    IPC分类号： C12N988

    CPC分类号： C11D3/38636 ,  C12N9/88

    摘要： A variant of a cell-wall degrading enzyme having a beta-helix structure, which variant holds at least one substituent in a position determined by identifying all residues potentially belonging to a stack; characterising the stack as interior or exterior; characterising the stack as polar, hydrophobic or aromatic/heteroaromatic based on the dominating characteristics of the parent or wild-type enzyme stack residues and/or its orientation relative to the beta-helix (interior or exterior); optimizing all stack positions of a stack either to hydrophobic aliphatic amino acids, hydrophobic aromatic or polar amino acids by allowing mutations within one or all positions to amino acids belonging to one of these groups; measuring thermostability of the variants by DSC or an application-related assay such as a Pad-Steam application test; and selecting the stabilized variants. Variant of a wild-type parent pectate lyase (EC 4.2.2.2) having the conserved amino acid residues D111, D141 or E141, D145, K165, R194 and R199 when aligned with the pectate lyase comprising the amino acid sequence of SEQ ID NO: 2 are preferred.

    摘要翻译： 具有β-螺旋结构的细胞壁降解酶的变体，该变体保持在通过鉴定潜在属于堆叠的所有残基而确定的位置中的至少一个取代基; 将堆栈表征为内部或外部; 基于母体或野生型酶堆残基的主导特征和/或其相对于β-螺旋（内部或外部）的取向，将所述叠层表征为极性，疏水性或芳族/杂芳族化合物; 通过在属于这些基团之一的氨基酸的一个或所有位置内允许突变将堆叠的所有堆叠位置优化为疏水性脂族氨基酸，疏水性芳族或极性氨基酸; 通过DSC测量变体的热稳定性或应用相关测定，例如Pad-Steam应用测试; 并选择稳定的变体。 当与包含SEQ ID NO：1的氨基酸序列的果胶酸裂解酶比对时，具有保守氨基酸残基D111，D141或E141，D145，K165，R194和R199的野生型亲本果胶酸裂合酶（EC 4.2.2.2） 2是优选的。