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1. 发明专利

JP2011217746A Surfactant-free polymerase 审中-公开
标题翻译：无表面活性剂聚合物
公开(公告)号：JP2011217746A
公开(公告)日：2011-11-04
申请号：JP2011085864
申请日：2011-04-08
申请人： F Hoffmann La Roche Ag , エフ.ホフマン−ラロシュアーゲーF. Hoffmann−La Roche Aktiengesellschaft
发明人： FISCHER ULRIKE , GREIF MICHAEL , SOBEK HARALD , THALHOFER JOHANN-PETER
IPC分类号： C12N15/09 , C12N9/12 , C12Q1/68
CPC分类号： C12Q1/686 , C12N9/1252 , C12Q2527/137 , C12Q2521/101
摘要： PROBLEM TO BE SOLVED: To provide an improved polymerase composition with optimized performance in a polymerase chain reaction (PCR).SOLUTION: The thermostable DNA polymerase composition completely free from surfactant contains 10-50 mM Tris/HCl, 0.05-0.2 mM EDTA, 0.5-2 mM DTT, 50-200 mM potassium chloride, and 20-80% glycerol.
摘要翻译：待解决的问题：提供在聚合酶链反应（PCR）中具有优化性能的改进的聚合酶组合物。解决方案：完全不含表面活性剂的热稳定DNA聚合酶组合物含有10-50mM Tris / HCl，0.05-0.2mM EDTA，0.5-2mM DTT，50-200mM氯化钾和20-80％甘油。版权所有（C）2012，JPO＆INPIT

2. 发明专利

JP2010051314A Stabilization of thermolysin in aqueous solution 有权
标题翻译：水溶液在水溶液中的稳定性
公开(公告)号：JP2010051314A
公开(公告)日：2010-03-11
申请号：JP2009193788
申请日：2009-08-25
申请人： F Hoffmann La Roche Ag , エフ.ホフマン−ラロシュアーゲーF. Hoffmann−La Roche Aktiengesellschaft
发明人： HOELKE WERNER , LIEHRE ANTJE , THALHOFER JOHANN-PETER , WEBER MARKUS
IPC分类号： C12N9/54
CPC分类号： C12N9/54 , C12N9/96 , C12Y304/24027
摘要： PROBLEM TO BE SOLVED: To provide a method for bringing thermolysin to a stabilized form in its aqueous solution, and to provide a composition therefor. SOLUTION: The method includes a first step (P) of mixing an aqueous solvent and a thermolysin-containing solid preparation together to make a first solution; wherein the first solution contains (i) buffering salt(s) enabling the solution to be maintained at pH 4.5-9, (ii) one or more salts, and (iii) thermolysin, wherein the aggregate concentration of one or more salts including the buffering salt(s) in the first solution is about 0.1-500 mM. The method includes, in addition, a second step (Q) of adding further salt(s) selected from the group consisting of measured amounts of NaCl, NaBr, NaNO 3 , NaI, KCl, LiCl, MgCl 2 , CaCl 2 and mixtures thereof to the first solution to dissolve the further salt(s) to effect preparing a second solution in which the thermolysin thus dissolved exists in a stabilized form. COPYRIGHT: (C)2010,JPO&INPIT
摘要翻译：待解决的问题：提供一种使溶血素在其水溶液中稳定化形式的方法，并提供其组合物。解决方案：该方法包括将含水溶剂和含嗜热菌素的固体制剂混合在一起以制备第一溶液的第一步骤（P）; 其中所述第一溶液含有（i）缓冲盐使所述溶液保持在pH 4.5-9，（ii）一种或多种盐，和（iii）嗜热菌蛋白，其中一种或多种盐的总浓度包括第一溶液中的缓冲盐为约0.1-500mM。该方法还包括另外添加另外选自NaCl，NaBr，NaNO 3 SB，NaI，KCl，LiCl，MgCl的量的另外的盐的步骤（Q）将其与其第一溶液混合以溶解另外的盐，从而制备第二溶液，其中所述溶解酶由此溶解，以稳定的形式存在。版权所有（C）2010，JPO＆INPIT

3. 发明专利

JP2004000170A Preparation of weakly active or inactive alkaline phosphatase mutant, and its expression in yeast 有权
公开(公告)号：JP2004000170A
公开(公告)日：2004-01-08
申请号：JP2003080470
申请日：2003-03-24
申请人： F Hoffmann La Roche Ag , エフ.ホフマン−ラロシュアーゲーF. Hoffmann−La Roche Aktiengesellschaft
发明人： MUELLER RAINER , THALHOFER JOHANN-PETER , GEIPEL FRANK , HOELKE WERNER , KIRSCHBAUM THOMAS
IPC分类号： C12N15/09 , C12N1/19 , C12N9/16 , C12N15/55 , C12R1/84
CPC分类号： C12N9/16
摘要： PROBLEM TO BE SOLVED: To provide a method for preparing a weakly active or inactive alkaline phosphatase mutant and a method for expressing the mutant in an yeast. SOLUTION: The invention relates to a method for the recombinant production or expression of eukaryotic alkaline phosphatase (AP) mutants in yeast cells wherein the specifically introduced mutations result in a reduction in specific activity of AP by at least a factor 1:100. The invention also relates to a method for inserting the corresponding nucleic acid sequences into a vector for expression in methylotrophic yeast strains and it relates to the corresponding vectors and host strains. COPYRIGHT: (C)2004,JPO

4. 发明专利

JP2010252793A Stabilized aqueous alpha-galactosidase composition and related method to the same 有权
标题翻译：稳定的水性α-乳糖苷酶组合物及其相关方法
公开(公告)号：JP2010252793A
公开(公告)日：2010-11-11
申请号：JP2010099666
申请日：2010-04-23
申请人： F Hoffmann La Roche Ag , エフ.ホフマン−ラロシュアーゲーF. Hoffmann−La Roche Aktiengesellschaft
发明人： HOELKE WERNER , MUELLER RAINER , SCHMIDT MANFRED , SOBEK HARALD , THALHOFER JOHANN-PETER
IPC分类号： C12N9/96 , C12N9/24 , C12N15/09
CPC分类号： C07K14/435
摘要： PROBLEM TO BE SOLVED: To provide an aqueous composition containing a protein having enzymatic activity of an alpha-galactosidase.
SOLUTION: This method for stabilizing the aqueous composition containing the protein having the enzymatic activity of the alpha-galactosidase is provided. The method for preparing the purified aqueous composition containing the protein having the enzymatic activity of the alpha-galactosidase is also provided.
COPYRIGHT: (C)2011,JPO&INPIT
摘要翻译：待解决的问题：提供含有具有α-半乳糖苷酶的酶活性的蛋白质的含水组合物。解决方案：提供了用于稳定含有具有α-半乳糖苷酶的酶活性的蛋白质的含水组合物的方法。还提供了制备含有具有α-半乳糖苷酶的酶活性的蛋白质的纯化含水组合物的方法。版权所有（C）2011，JPO＆INPIT

5. 发明专利

JP2010046074A Carboxypeptidase b expressed by recombination, and purification of the same 有权
标题翻译：通过重新表达的羧氧化酶B及其相似物的纯化
公开(公告)号：JP2010046074A
公开(公告)日：2010-03-04
申请号：JP2009238318
申请日：2009-10-15
申请人： F Hoffmann La Roche Ag , エフ.ホフマン−ラロシュアーゲーF. Hoffmann−La Roche Aktiengesellschaft
发明人： GLASER STEPHAN , GEIPEL FRANK , KIRSCHBAUM THOMAS , REXER BERNHARD , THALHOFER JOHANN-PETER , MUELLER RAINER , GIESSEL CLAUDIA , ECKSTEIN HELLMUT , WOLF ELVIRA
IPC分类号： C12N9/48 , C12N15/09 , C12N9/98
CPC分类号： C12N9/48 , C07K2319/02 , C07K2319/21
摘要： PROBLEM TO BE SOLVED: To provide a method for producing a protein exhibiting carboxypeptidase B activity from a zymogen.
SOLUTION: A vector comprises a nucleotide sequence encoding a pre-protein consisting of a rat pro-carboxypeptidase B fused with a histidine tag and signal peptide at its N-terminal. The protein without substantially containing the carboxypeptidase B pro-peptide, having the carboxypeptidase B activity and lacking C-terminal tyrosine is obtained by a method for transforming a microorganic host organism by the above vector, culturing it, secreting a histidine-tagged pro-carboxypeptidase B in culturing medium, immobilizing the same on a particle metal chelate-compatible matrix bindable with the histidine tag, releasing the protein having the carboxypeptidase B activity into a liquid phase by a trypsin treatment, and purifying the protein having the carboxypeptidase B activity from the liquid phase.
COPYRIGHT: (C)2010,JPO&INPIT
摘要翻译：待解决的问题：提供从酶原产生显示羧肽酶B活性的蛋白质的方法。解决方案：载体包含编码由组氨酸标签融合的大鼠前羧肽酶B和其N-末端的信号肽组成的前蛋白质的核苷酸序列。通过用上述载体转化微生物宿主生物体的方法，培养它们，分泌组氨酸标记的前羧肽酶，获得具有羧肽酶B活性且缺乏C-末端酪氨酸的基本上不含羧肽酶B前肽的蛋白质 B在培养基中，将其固定在可与组氨酸标签结合的颗粒金属螯合物相容性基质上，通过胰蛋白酶处理将具有羧肽酶B活性的蛋白质释放到液相中，并从其中纯化具有羧肽酶B活性的蛋白质液相。版权所有（C）2010，JPO＆INPIT

6. 发明专利

JP2005168501A Recombinantly expressed carboxypeptidase b and purification thereof 有权
标题翻译：重组表达的羧化酶B及其纯化
公开(公告)号：JP2005168501A
公开(公告)日：2005-06-30
申请号：JP2004350397
申请日：2004-12-02
申请人： F Hoffmann La Roche Ag , エフ.ホフマン−ラロシュアーゲーF. Hoffmann−La Roche Aktiengesellschaft
发明人： GLASER STEPHAN , GEIPEL FRANK , KIRSCHBAUM THOMAS , REXER BERNHARD , THALHOFER JOHANN-PETER , MUELLER RAINER , GIESSEL CLAUDIA , ECKSTEIN HELLMUT , WOLF ELVIRA
IPC分类号： C12N15/09 , C12N9/48
CPC分类号： C12N9/48 , C07K2319/02 , C07K2319/21
摘要： PROBLEM TO BE SOLVED: To provide a method for producing a protein with carboxypeptidase B activity from a zymogen, a method for separating the protein from the zymogen; and to provide a method for separating the protein from the zymogen under a nondenaturing condition. SOLUTION: A method to produce the protein comprising the steps of (a) providing a vector comprising a rat pro-carboxypeptidase B that is fused to a Histidine-tag and a signal peptide; (b) transforming a microbial host organism with the vector; (c) the host organism expresses the pre-protein and secretes the Histidine-tagged pro-carboxypeptidase B; (d) immobilizing the secreted peptidase B on a particulate metal chelate affinity matrix; (e) incubating the matrix in a buffer containing trypsin, releasing the protein with carboxypeptidase B activity into the liquid phase; (f) separating the liquid phase containing the protein from the matrix; and (g) purification of the protein obtained from the liquid phase. COPYRIGHT: (C)2005,JPO&NCIPI
摘要翻译：待解决的问题：提供从酶原产生具有羧肽酶B活性的蛋白质的方法，从酶原分离蛋白质的方法; 并提供在非变性条件下从酶原分离蛋白质的方法。解决方案：一种产生蛋白质的方法，包括以下步骤：（a）提供包含与组氨酸标签和信号肽融合的大鼠前羧肽酶B的载体; （b）用载体转化微生物宿主生物; （c）宿主生物表达前蛋白并分泌组氨酸标记的前羧肽酶B; （d）将分泌的肽酶B固定在金属螯合亲和基质颗粒上; （e）在含有胰蛋白酶的缓冲液中培养基质，将羧肽酶B活性释放到液相中; （f）将含有蛋白质的液相与基质分离; 和（g）从液相中获得的蛋白质的纯化。版权所有（C）2005，JPO＆NCIPI

7. 发明专利

JP2011224002A New t7 rna polymerase variant with enhanced thermostability 有权
标题翻译：新型T7 RNA聚合酶具有增强的热稳定性
公开(公告)号：JP2011224002A
公开(公告)日：2011-11-10
申请号：JP2011090825
申请日：2011-04-15
申请人： F Hoffmann La Roche Ag , エフ.ホフマン−ラロシュアーゲーF. Hoffmann−La Roche Aktiengesellschaft
发明人： GREIF MICHAEL , MUELLER RAINER , RUDOLPH CHRISTIAN , RUF ARMIN , SCHMIDT MANFRED , SOBEK HARALD , THALHOFER JOHANN-PETER
IPC分类号： C12N15/09 , C12N1/15 , C12N1/19 , C12N1/21 , C12N5/10 , C12N9/12 , C12P19/34
CPC分类号： C12N9/1247 , C12P19/34 , C12Y207/07006
摘要： PROBLEM TO BE SOLVED: To provide an improved variant of T7 RNA polymerase by introducing new mutation leading to improved thermostability of the enzyme.SOLUTION: There is provided a variant of T7 RNA polymerase leading to improved thermostability by introducing a new mutation at positions Val426, Ser633, Val650, Thr654, Ala702, Val795 and combinations thereof.
摘要翻译：要解决的问题：通过引入导致酶的热稳定性的新突变来提供T7RNA聚合酶的改进变体。解决方案：提供T7RNA聚合酶的变体，通过在Val426，Ser633，Val650，Thr654，Ala702，Val795及其组合的位置引入新的突变来提高热稳定性。版权所有（C）2012，JPO＆INPIT

8. 发明专利

JP2004201688A Heat-labile desoxyribonculease i variants 有权
公开(公告)号：JP2004201688A
公开(公告)日：2004-07-22
申请号：JP2003422697
申请日：2003-12-19
申请人： F Hoffmann La Roche Ag , エフ.ホフマン−ラロシュアーゲーF. Hoffmann−La Roche Aktiengesellschaft
发明人： MUELLER RAINER , KIRSCHBAUM THOMAS , SUPPMANN BERNHARD , SCHOEN HELMUT , ENGH RICHARD , HOFFMANN ARTUR , THALHOFER JOHANN-PETER , SIEDEL JOACHIM , ENGEL WOLF-DIETER
IPC分类号： C12N15/09 , C12N1/16 , C12N1/19 , C12N9/16 , C12N9/22 , C12N15/55 , C12N15/63 , C12Q1/34 , C12R1/84
CPC分类号： C12N9/22
摘要： PROBLEM TO BE SOLVED: To provide desoxyribonuclease with increased thermolability compared to bovine pancreatic desoxyribonuclease I, and to provide a method with a high cost-efficiency, for producing the desoxyribonuclease by non-animal host organisms. SOLUTION: The bovine pancreatic desoxyribonuclease I variant has a specific amino acid residue of the bovine pancreatic desoxyribonuclease I substituted with a different amino acid, and has activity of the desoxyribonuclease I. The method for producing the same uses a base sequence encoding the variant. A Pichia pastoris strain encodes a preprotein comprising the variant and a signal peptide, and contains a Pichia pastoris AOX1 promoter or a specific base sequence connected to the promoter element, wherein the base sequence encoding the preprotein contains a chromosome obtained by carrying out fusion of the base sequence of the bovine pancreatic desoxyribonuclease I. COPYRIGHT: (C)2004,JPO&NCIPI

9. 发明专利

JP2004194657A Recombinant bovine pancreatic deoxyriboribonuclease i having high specific activity 有权
公开(公告)号：JP2004194657A
公开(公告)日：2004-07-15
申请号：JP2003419939
申请日：2003-12-17
申请人： F Hoffmann La Roche Ag , エフ.ホフマン−ラロシュアーゲーF. Hoffmann−La Roche Aktiengesellschaft
发明人： MUELLER RAINER , SUPPMANN BERNHARD , THALHOFER JOHANN-PETER , RONNING STEPHANIE , SCHOEN HELMUT
IPC分类号： C12N15/09 , C12N9/22 , C12N9/88 , C12N15/55 , C12N15/81 , C12P19/34 , C12R1/72
CPC分类号： Y02P20/52
摘要： PROBLEM TO BE SOLVED: To provide a substitutional supply source for bovine pancreatic deoxyribonulcease I, having a high cost effect. SOLUTION: A method for producing a bovine pancreatic protein includes (a) a process for providing a vector containing a nucleotide sequence which encodes a preprotein comprising the bovine pancreatic protein and a signal peptide, (b) another process for transforming a methylotrophic yeast strain by the vector, (c) a third process for culturing the transformed methylotrophic yeast strain in a growth medium containing nutrients and methanol, wherein the methylotrophic yeast strain expresses the bovine pancreatic protein and secretes the protein in the growth medium, and (d) the other process for purifying the bovine pancreatic protein from the growth medium, so that the bovine pancreatic protein purified in the process (d) has a deoxyribonulcease specific activity of at least 6,000 units per mg of the purified protein. COPYRIGHT: (C)2004,JPO&NCIPI

10. 发明专利

JP2009291195A Improved purification of collagenase from clostridium histolyticum liquid culture 有权
标题翻译：从胶原蛋白液体培养物中提取胶原蛋白的改进
公开(公告)号：JP2009291195A
公开(公告)日：2009-12-17
申请号：JP2009133447
申请日：2009-06-02
申请人： F Hoffmann-La Roche Ag , エフホフマン−ラロッシュアクチェンゲゼルシャフト
发明人： ECKSTEIN HELLMUT , FISCHER MICHAELA , HOELKE WERNER , LIEHRE ANTJE , SUPPMANN BERNHARD , THALHOFER JOHANN-PETER
IPC分类号： C12N9/52 , C07K1/18 , C07K1/20
CPC分类号： C12N9/52 , C07K14/33
摘要： PROBLEM TO BE SOLVED: To provide a method for purifying Clostridium histolyticum collagenase type I and type II proteins from a complex mixture. SOLUTION: The method for purifying Clostridium histolyticum collagenase includes carrying out the purification by performing precipitation with ammonium sulfate, hydrophobic interaction chromatography, cation exchange chromatography, and anion chromatography. Conditions are provided which lead to a stabilized partially purified preparation and the precipitation step. The preparations provide exceptionally pure and intact collagenase type I and type II proteins which are enzymatically active. The blends of the two isolated proteins are provided. The use of the purified collagenase proteins or blends thereof for treating a tissue sample in vitro are also provided. COPYRIGHT: (C)2010,JPO&INPIT
摘要翻译：待解决的问题：提供从复合混合物中纯化溶组织梭状芽胞杆菌胶原酶I型和II型蛋白的方法。解决方案：用于纯化溶组织梭菌胶原酶的方法包括通过用硫酸铵进行沉淀，疏水相互作用层析，阳离子交换层析和阴离子色谱法进行纯化。提供了导致稳定的部分纯化制剂和沉淀步骤的条件。该制剂提供非常纯的和完整的胶原酶I型和II型蛋白质，其具有酶促活性。提供了两种分离的蛋白质的共混物。还提供了纯化的胶原酶蛋白或其混合物用于在体外处理组织样品的用途。版权所有（C）2010，JPO＆INPIT

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