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1.发明授权Modification of xylanase to improve thermophilicity, alkalophilicity and thermostability 失效改良木聚糖酶以改善嗜热性，嗜碱性和热稳定性
公开(公告)号：US5759840A
公开(公告)日：1998-06-02
申请号：US709912
申请日：1996-09-09
申请人： Wing L. Sung , Makoto Yaguchi , Kazuhiko Ishikawa
发明人： Wing L. Sung , Makoto Yaguchi , Kazuhiko Ishikawa
CPC分类号： C12Y302/01032 , C12N9/248 , C12Y302/01008 , D21C5/005 , C07K2319/00
摘要： Producing a xylanase enzyme of superior performance in the bleaching of pulp. More specifically, a modified xylanase of Family 11 that shows improved thermophilicity, alkalophilicity, and thermostability as compared to the natural xylanase. The modified xylanases contain any of three types of modifications: (1) changing amino acids 10, 27, and 29 of Trichoderma reesei xylanase II or the corresponding amino acids of another Family 11 xylanase, where these amino acids are changed to histidine, methionine, and leucine, respectively; (2) substitution of amino acids in the N-terminal region with amino acids from another xylanase enzyme. In a preferred embodiment, substitution of the natural Bacillus circulans or Trichoderma reesei xylanase with a short sequence of amino acids from Thermomonospora fusca xylanase yielded chimeric xylanases with higher thermophilicity and alkalophilicity; (3) an extension upstream of the N-terminus of up to 10 amino acids. In a preferred embodiment, extension of the N-terminus of the xylanase with the tripeptide glycine-arginine-arginine improved its performance.
摘要翻译： 在木浆漂白中生产出优异的木聚糖酶。 更具体地，与天然木聚糖酶相比，家族11的改性木聚糖酶显示出改善的嗜热性，嗜碱性和热稳定性。 修饰的木聚糖酶包含三种类型的修饰中的任何一种：（1）改变里氏木霉木聚糖酶II的氨基酸10,27和29或另一种家族11木聚糖酶的相应氨基酸，其中这些氨基酸被改变为组氨酸，甲硫氨酸， 和亮氨酸; （2）用来自另一种木聚糖酶的氨基酸取代N-末端区域中的氨基酸。 在一个优选的实施方案中，用热单孢菌木聚糖酶短链氨基酸取代天然环状芽孢杆菌或里氏木霉木聚糖酶得到具有较高嗜热性和嗜碱性的嵌合木聚糖酶; （3）N-末端上游至多10个氨基酸的延伸。 在优选的实施方案中，木聚糖酶的N-末端与三肽甘氨酸 - 精氨酸 - 精氨酸的延伸改善了其性能。
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2.发明授权
公开(公告)号：US5523211A
公开(公告)日：1996-06-04
申请号：US277076
申请日：1994-07-19
IPC分类号： A01N63/02 , C07K14/325 , G01N30/02 , G01N30/34 , G01N33/569 , C12Q1/37 , C12N1/20 , C12Q1/00 , C12Q1/24
CPC分类号： G01N33/56911 , A01N63/02 , C07K14/325 , G01N30/02 , G01N30/34
摘要： A process for identifying proteinaceous protoxins expressed by Bacillus thuringiensis genes is disclosed. According to the process, daughter toxins are first generated by subjecting a protoxin-containing material, such as parasporal crystals of Bacillus thuringiensis, to limited proteolysis with a proteolytic enzyme in an aqueous suspension having a pH above 9.5. The daughter toxins are then separated by high performance anion-exchange liquid chromatography at a constant pH in excess of 10 in an increasing gradient of a salt, preferably sodium chloride. The gradient conditions, which are specific for the column used, are achieved by employing a series of buffers having increasing concentration of the salt and introduced at a predetermined time and rate. The procedure provides a chromatogram showing clearly identifiable peaks of toxins and permits therefore the qualitative and quantitative characterization of the original mixture and isolation of the individual toxins. By this it provides a way of screening and testing new Bacillus thuringiensis isolates, both single--and multigene, and monitoring the level of expression of known genes from a known strain. The digestion and isolation conditions permit the production of the toxins in a biologically fully active state.
摘要翻译： 公开了一种鉴定苏云金芽孢杆菌基因表达的蛋白质原毒素的方法。 根据该方法，首先通过使含有原毒素的材料（例如苏云金芽孢杆菌的寄生虫晶体）在pH高于9.5的水性悬浮液中用蛋白水解酶限制蛋白水解产生子代毒素。 然后通过高效阴离子交换液相色谱法，在不断增加的盐，优选氯化钠梯度下，以恒定的pH超过10分离子体毒素。 对所使用的柱特异的梯度条件通过使用一系列具有增加的盐浓度并以预定时间和速率引入的缓冲液来实现。 该程序提供了一个色谱图，显示了清晰可辨的毒素峰值，因此允许原始混合物的定性和定量表征以及各种毒素的分离。 因此，它提供了一种筛选和测试新型苏云金芽孢杆菌分离株，单基因和多基因的方法，并监测已知菌株已知基因的表达水平。 消化和分离条件允许在生物学上完全活跃的状态下生产毒素。
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3.发明授权Modification of xylanase to improve thermophilicity, alkophilicity and thermostability 失效修饰木聚糖酶以改善嗜热性，亲油性和热稳定性
公开(公告)号：US5866408A
公开(公告)日：1999-02-02
申请号：US47370
申请日：1998-03-25
申请人： Wing L. Sung , Makoto Yaguchi , Kazuhiko Ishikawa
发明人： Wing L. Sung , Makoto Yaguchi , Kazuhiko Ishikawa
IPC分类号： C12N15/09 , C12N9/24 , C12N15/00 , C12N15/56 , C12R1/09 , C12R1/885 , D21C5/00 , D21C9/10 , D21C3/00
CPC分类号： C12Y302/01032 , C12N9/248 , C12Y302/01008 , D21C5/005 , C07K2319/00
摘要： Producing a xylanase enzyme of superior performance in the bleaching of pulp. More specifically, a modified xylanase of Family 11 that shows improved thermophilicity, alkalophilicity, and thermostability as compared to the natural xylanase. The modified xylanases contain any of three types of modifications: (1) changing amino acids 10, 27, and 29 of Trichoderma reesei xylanase II or the corresponding amino acids of another Family 11 xylanase, where these amino acids are changed to histidine, methionine, and leucine, respectively; (2) substitution of amino acids in the N-terminal region with amino acids from another xylanase enzyme. In a preferred embodiment, substitution of the natural Bacillus circulans or Trichoderma reesei xylanase with a short sequence of amino acids from Thermomonospora fusca xylanase yielded chimeric xylanases with higher thermophilicity and alkalophilicity; (3) an extension upstream of the N-terminus of up to 10 amino acids. In a preferred embodiment, extension of the N-terminus of the xylanase with the tripeptide glycine-arginine-arginine improved its performance.
摘要翻译： 在木浆漂白中生产出优异的木聚糖酶。 更具体地，与天然木聚糖酶相比，家族11的改性木聚糖酶显示出改善的嗜热性，嗜碱性和热稳定性。 修饰的木聚糖酶包含三种类型的修饰中的任一种：（1）改变里氏木霉木聚糖酶II的氨基酸10,27和29或另一种家族11木聚糖酶的相应氨基酸，其中这些氨基酸被改变为组氨酸，甲硫氨酸， 和亮氨酸; （2）用来自另一种木聚糖酶的氨基酸取代N-末端区域中的氨基酸。 在一个优选的实施方案中，用热单孢菌木聚糖酶短链氨基酸取代天然环状芽孢杆菌或里氏木霉木聚糖酶得到具有较高嗜热性和嗜碱性的嵌合木聚糖酶; （3）N-末端上游至多10个氨基酸的延伸。 在优选的实施方案中，木聚糖酶的N-末端与三肽甘氨酸 - 精氨酸 - 精氨酸的延伸改善了其性能。
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公开(公告)号：US5405769A
公开(公告)日：1995-04-11
申请号：US44621
申请日：1993-04-08
CPC分类号： C12Y302/01032 , C12N9/248 , C12Y302/01008 , D21C5/005
摘要： The thermostability of the 20,396 dalton Bacillus circulans xylanase was increased by site-directed mutagenesis. The thermostability was conferred by the presence of non-native disulfide bridges, and selected N-terminal mutations. The introduction of these non-native disulfide bridges was accomplished by the examination of the three-dimensional structure of the enzyme, and choosing sites where a favorable geometry for a bridge existed. The N-terminal mutations were constructed on the basis of primary sequence comparison with other family G xylanases. The mutant proteins were examined for their ability to retain enzymatic activity after heating as an indication of increased thermostability. These thermotolerant variants are useful as an alternative to chemical bleaching of Kraft pulp in a pre-bleaching step (bio-bleaching). The pre-bleaching involves temperatures higher than that normally used for these enzymes and accordingly these thermotolerant variants can be advantageously used at this step. Thermotolerant xylanases are also of use in the food processing industry.
摘要翻译： 通过定点诱变增加了20396道尔顿环状芽孢杆菌木聚糖酶的热稳定性。 耐热性由非天然二硫键的存在和选择的N端突变赋予。 这些非天然二硫键的引入是通过检查酶的三维结构，并选择存在桥的有利几何形状的位点来实现的。 基于与其他家族G木聚糖酶的初级序列比较构建N末端突变。 检查突变体蛋白质在加热后保留酶活性的能力，作为增加热稳定性的指标。 这些耐热变体在预漂白步骤（生物漂白）中可用作牛皮纸浆的化学漂白的替代物。 预漂白涉及比通常用于这些酶的温度更高的温度，因此这些耐热变体可有利地用于该步骤。 耐热木聚糖酶也可用于食品加工业。
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5.发明授权Isolation, quantitation and purification of insecticidal proteins from Bacillus thuringiensis 失效苏云金芽孢杆菌的杀虫蛋白的分离，定量和纯化
公开(公告)号：US5356788A
公开(公告)日：1994-10-18
申请号：US102491
申请日：1993-08-05
IPC分类号： A01N63/02 , C07K1/22 , C07K14/325 , C12Q1/04 , C12Q1/37 , C12Q1/68 , G01N30/00 , G01N30/02 , G01N30/34 , G01N33/569 , C12N1/20 , G01N33/567
CPC分类号： C07K14/325 , A01N63/02 , C12Q1/04 , G01N2333/325 , G01N30/02 , G01N30/34 , Y10S435/803
摘要： A process for identifying proteinaceous protoxins expressed by Bacillus thuringiensis genes is disclosed. According to the process, daughter toxins are first generated by subjecting a protoxin-containing material, such as parasporal crystals of Bacillus thuringiensis, to limited proteolysis with a proteolytic enzyme in an aqueous suspension having a pH above 9.5. The daughter toxins are then separated by high performance anion-exchange liquid chromatography at a constant pH in excess of 10 in an increasing gradient of a salt, preferably sodium chloride. The gradient conditions, which are specific for the column used, are achieved by employing a series of buffers having increasing concentration of the salt and introduced at a predetermined time and rate. The procedure provides a chromatogram showing clearly identifiable peaks of toxins and permits therefore the qualitative and quantitative characterization of the original mixture and isolation of the individual toxins. By this it provides means of screening and testing new Bacillus thuringiensis isolates, both single - and multigene, and monitoring the level of expression of known genes from a known strain. The digestion and isolation conditions permit the production of the toxins in a biologically fully active state.
摘要翻译： 公开了一种鉴定苏云金芽孢杆菌基因表达的蛋白质原毒素的方法。 根据该方法，首先通过使含有原毒素的材料（例如苏云金芽孢杆菌的寄生虫晶体）在pH高于9.5的水性悬浮液中用蛋白水解酶限制蛋白水解产生子代毒素。 然后通过高效阴离子交换液相色谱法，在不断增加的盐，优选氯化钠梯度下，以恒定的pH超过10分离子体毒素。 对所使用的柱特异的梯度条件通过使用一系列具有增加的盐浓度并以预定时间和速率引入的缓冲液来实现。 该程序提供了一个色谱图，显示了清晰可辨的毒素峰值，因此允许原始混合物的定性和定量表征以及各种毒素的分离。 因此，它提供了筛选和测试新型苏云金芽孢杆菌分离株单 - 和多基因的方法，并监测已知菌株已知基因的表达水平。 消化和分离条件允许在生物学上完全活跃的状态下生产毒素。
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