RU2008118356A - Применение и получение стабильной при хранении нейтральной металлопротеиназы - Google Patents

Применение и получение стабильной при хранении нейтральной металлопротеиназы Download PDF

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RU2008118356A
RU2008118356A RU2008118356/13A RU2008118356A RU2008118356A RU 2008118356 A RU2008118356 A RU 2008118356A RU 2008118356/13 A RU2008118356/13 A RU 2008118356/13A RU 2008118356 A RU2008118356 A RU 2008118356A RU 2008118356 A RU2008118356 A RU 2008118356A
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metalloproteinase
neutral metalloproteinase
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Эндрю ШО (US)
Эндрю ШО
Луиз УОЛЛЭЙС (US)
Луиз УОЛЛЭЙС
Дэвид А. ЭСТЕЛЛ (US)
Дэвид А. ЭСТЕЛЛ
Рональд ХОММЕС (NL)
Рональд ХОММЕС
Санг-Киу ЛИ (US)
Санг-Киу ЛИ
Юджин Стивен САДЛОВСКИ (US)
Юджин Стивен САДЛОВСКИ
Хироси ОХ (US)
Хироси ОХ
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Джененкор Интернэшнл, Инк. (Us)
Джененкор Интернэшнл, Инк.
Дзе Проктер Энд Гэмбл Компани (US)
Дзе Проктер Энд Гэмбл Компани
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Abstract

1. Выделенная нейтральная металлопротеиназа, обладающая улучшенной стабильностью при хранении, где указанная нейтральная металлопротеиназа представляет собой нейтральную металлопротеиназу Bacillus. ! 2. Нейтральная металлопротеиназа по п.1, где указанный Bacillus представляет собой B. amyloliquefaciens. ! 3. Нейтральная металлопротеиназа по п.1, где указанная нейтральная металлопротеиназа обладает по меньшей мере 45% идентичностью аминокислот с нейтральной металлопротеиназой, содержащей SEQ ID NO:3 или SEQ ID NO:18. ! 4. Нейтральная металлопротеиназа по п.1, где указанная нейтральная металлопротеиназа содержит аминокислотную последовательность, указанную в SEQ ID NO:3 или SEQ ID NO:18. ! 5. Нуклеотидная последовательность, кодирующая по меньшей мере часть нейтральной металлопротеиназы по п.1, где указанная нуклеотидная последовательность выбрана из SEQ ID NO:1, 2, 12 и 13. ! 6. Экспрессирующий вектор, содержащий нуклеотидную последовательность, кодирующую аминокислотную последовательность, указанную в SEQ ID NO:3. ! 7. Клетка-хозяин, содержащая экспрессирующий вектор по п.6. ! 8. Стабильная при хранении нейтральная металлопротеиназа, полученная из указанной клетки-хозяина по п.7, где указанную нейтральную металлопротеиназу кодирует указанный экспрессирующий вектор. ! 9. Выделенная нейтральная металлопротеиназа, обладающая иммунологической перекрестной реактивностью с нейтральной металлопротеиназой по п.1. ! 10. Аминокислотная последовательность, указанная в SEQ ID NO:18. ! 11. Аминокислотная последовательность, указанная в SEQ ID NO:3. ! 12. Аминокислотная последовательность по п.10, где указанная последовательность содержит по меньшей мере одну замену аминокислоты, выполнен�

Claims (49)

1. Выделенная нейтральная металлопротеиназа, обладающая улучшенной стабильностью при хранении, где указанная нейтральная металлопротеиназа представляет собой нейтральную металлопротеиназу Bacillus.
2. Нейтральная металлопротеиназа по п.1, где указанный Bacillus представляет собой B. amyloliquefaciens.
3. Нейтральная металлопротеиназа по п.1, где указанная нейтральная металлопротеиназа обладает по меньшей мере 45% идентичностью аминокислот с нейтральной металлопротеиназой, содержащей SEQ ID NO:3 или SEQ ID NO:18.
4. Нейтральная металлопротеиназа по п.1, где указанная нейтральная металлопротеиназа содержит аминокислотную последовательность, указанную в SEQ ID NO:3 или SEQ ID NO:18.
5. Нуклеотидная последовательность, кодирующая по меньшей мере часть нейтральной металлопротеиназы по п.1, где указанная нуклеотидная последовательность выбрана из SEQ ID NO:1, 2, 12 и 13.
6. Экспрессирующий вектор, содержащий нуклеотидную последовательность, кодирующую аминокислотную последовательность, указанную в SEQ ID NO:3.
7. Клетка-хозяин, содержащая экспрессирующий вектор по п.6.
8. Стабильная при хранении нейтральная металлопротеиназа, полученная из указанной клетки-хозяина по п.7, где указанную нейтральную металлопротеиназу кодирует указанный экспрессирующий вектор.
9. Выделенная нейтральная металлопротеиназа, обладающая иммунологической перекрестной реактивностью с нейтральной металлопротеиназой по п.1.
10. Аминокислотная последовательность, указанная в SEQ ID NO:18.
11. Аминокислотная последовательность, указанная в SEQ ID NO:3.
12. Аминокислотная последовательность по п.10, где указанная последовательность содержит по меньшей мере одну замену аминокислоты, выполненную в положении, эквивалентном положению в нейтральной металлопротеиназе, содержащей аминокислотную последовательность, указанную в SEQ ID NO:18, где указанные положения выбраны из положений 1, 3, 4, 5, 6, 11, 12, 13, 14, 16, 21, 23, 24, 25, 31, 32, 33, 35, 36, 38, 44, 45, 46, 47, 48, 49, 50, 51, 54, 55, 58, 59, 60, 61, 62, 63, 65, 66, 69, 70, 76, 85, 86, 87, 88, 90, 91, 92, 96, 97, 98, 99, 100, 102, 109, 110, 111, 112, 113, 115, 117, 119, 127, 128, 129, 130, 132, 135, 136, 137, 138, 139, 140, 146, 148, 151, 152, 153, 154, 155, 157, 158, 159, 161, 162, 169, 173, 178, 179, 180, 181, 183, 184, 186, 190, 191, 192, 196, 198, 199, 200, 202, 203, 204, 205, 210, 211, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 228, 229, 237, 239, 240, 243, 244, 245, 248, 252, 253, 260, 261, 263, 264, 265, 267, 269, 270, 273, 277, 280, 282, 283, 284, 285, 286, 288, 289, 290, 292, 293, 296, 297 и 299.
13. Выделенный вариант нейтральной металлопротеиназы, обладающий аминокислотной последовательностью, содержащей по меньшей мере одну замену аминокислоты, выполненную в положении, эквивалентном положению в нейтральной металлопротеиназе, содержащей аминокислотную последовательность, указанную в SEQ ID NO:18.
14. Выделенный вариант нейтральной металлопротеиназы по п.13, где указанные замены выполнены в положениях, эквивалентных положениям 1, 3, 4, 5, 6, 11, 12, 13, 14, 16, 21, 23, 24, 25, 31, 32, 33, 35, 36, 38, 44, 45, 46, 47, 48, 49, 50, 51, 54, 55, 58, 59, 60, 61, 62, 63, 65, 66, 69, 70, 76, 85, 86, 87, 88, 90, 91, 92, 96, 97, 98, 99, 100, 102, 109, 110, 111, 112, 113, 115, 117, 119, 127, 128, 129, 130, 132, 135, 136, 137, 138, 139, 140, 146, 148, 151, 152, 153, 154, 155, 157, 158, 159, 161, 162, 169, 173, 178, 179, 180, 181, 183, 184, 186, 190, 191, 192, 196, 198, 199, 200, 202, 203, 204, 205, 210, 211, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 228, 229, 237, 239, 240, 243, 244, 245, 248, 252, 253, 260, 261, 263, 264, 265, 267, 269, 270, 273, 277, 280, 282, 283, 284, 285, 286, 288, 289, 290, 292, 293, 296, 297 и 299 нейтральной металлопротеиназы, содержащей аминокислотную последовательность, указанную в SEQ ID NO:18.
15. Выделенный вариант нейтральной металлопротеиназы по п.14, где указанная протеиназа содержит по меньшей мере одну мутацию, выбранную из
Figure 00000001
Figure 00000002
Figure 00000003
16. Выделенный вариант нейтральной металлопротеиназы по п.13, где указанная протеиназа содержит множественные мутации, выбранные из
Figure 00000004
Figure 00000005
Figure 00000006
17. Выделенный вариант нейтральной металлопротеиназы по п.13, где указанная протеиназа содержит множественные мутации, выбранные из
Figure 00000007
Figure 00000008
Figure 00000009
18. Выделенный вариант нейтральной металлопротеиназы по п.13, где указанная протеиназа содержит множественные мутации, выбранные из
Figure 00000010
Figure 00000011
19. Вариант нейтральной металлопротеиназы, обладающий улучшенной производительностью по сравнению с нейтральной металлопротеиназой B. amyloliquefaciens дикого типа.
20. Вариант нейтральной металлопротеиназы по п.19, где указанная улучшенная производительность включает в себя улучшенную термостабильность по сравнению с нейтральной металлопротеиназой B. amyloliquefaciens дикого типа.
21. Вариант нейтральной металлопротеиназы по п.19, где указанная улучшенная производительность включает в себя улучшенную производительность в условиях более низкого или более высокого pH по сравнению с нейтральной металлопротеиназой B. amyloliquefaciens дикого типа.
22. Вариант нейтральной металлопротеиназы по п.19, где указанная улучшенная производительность включает в себя улучшенную устойчивость к автолизу по сравнению с нейтральной металлопротеиназой B. amyloliquefaciens дикого типа.
23. Способ получения фермента, обладающего активностью нейтральной металлопротеиназы, включающий в себя трансформацию клетки-хозяина экспрессирующим вектором, содержащим полинуклеотид, обладающий по меньшей мере 70% идентичностью последовательности с SEQ ID NO:3, и культивирование указанной трансформированной клетки-хозяина в условиях, подходящих для продуцирования указанной нейтральной металлопротеиназы.
24. Способ по п.23, дополнительно включающий в себя стадию сбора указанной продуцированной нейтральной металлопротеиназы.
25. Способ по п.23, где указанная клетка-хозяин представляет собой разновидность Bacillus.
26. Выделенный зонд из нуклеиновой кислоты, содержащий последовательность от 4 до 150 нуклеотидов, по существу, идентичных соответствующему фрагменту SEQ ID NO:1, 2, 12 и/или 13.
27. Выделенный зонд из нуклеиновой кислоты по п.26, где указанный зонд используют для детекции последовательности нуклеиновой кислоты, кодирующей фермент, обладающий металлопротеиназной активностью.
28. Последовательность нуклеиновой кислоты по п.27, где указанная последовательность нуклеиновой кислоты получена из Bacillus sp.
29. Композиция, содержащая по меньшей мере одну нейтральную металлопротеиназу, полученную из B. amyloliquefaciens, где указанная композиция дополнительно содержит по меньшей мере один ион кальция и/или ион цинка.
30. Композиция, содержащая по меньшей мере одну нейтральную металлопротеиназу, полученную из B. amyloliquefaciens, где указанная композиция дополнительно содержит по меньшей мере один стабилизатор.
31. Композиция по п.30, где указанный стабилизатор выбран из группы, состоящей из боракса, глицерина, ионов цинка, ионов кальция и формата кальция.
32. Композиция по п.30, где указанный стабилизатор представляет собой по меньшей мере один конкурентный ингибитор, стабилизирующий по меньшей мере одну указанную нейтральную металлопротеиназу в присутствии анионного поверхностно-активного вещества.
33. Очищающая композиция, содержащая выделенную нейтральную металлопротеиназу по п.1.
34. Очищающая композиция по п.33, где указанная очищающая композиция представляет собой моющее средство.
35. Очищающая композиция по п.33, дополнительно содержащая по меньшей мере один дополнительный фермент или производное фермента, выбранные из протеаз, амилаз, липаз, маннаназ, пектиназ, кутиназ, оксидоредуктаз, гемицеллюлаз и целлюлаз.
36. Очищающая композиция по п.34, где указанная композиция содержит по меньшей мере приблизительно 0,0001 мас.% указанной нейтральной металлопротеиназы.
37. Очищающая композиция по п.36, где указанная композиция содержит приблизительно от 0,001 до 0,5 мас.% указанной нейтральной металлопротеиназы.
38. Очищающая композиция по п.36, где указанная композиция дополнительно содержит по меньшей мере один вспомогательный ингредиент.
39. Очищающая композиция по п.31, дополнительно содержащая достаточное количество модификатора pH для обеспечения точного pH композиции приблизительно от 3 до 5, где композиция является в основном свободной от материалов, гидролизующихся при pH приблизительно от pH 3 до pH 5.
40. Очищающая композиция по п.39, где указанные материалы, гидролизующиеся при pH приблизительно от pH 3 до pH 5, содержат по меньшей мере одно поверхностно-активное вещество.
41. Очищающая композиция по п.40, где указанное поверхностно-активное вещество представляет собой поверхностно-активное вещество алкилсульфат натрия, содержащее группу этиленоксида.
42. Очищающая композиция по п.33, где указанная композиция представляет собой жидкость.
43. Очищающая композиция по п.33, дополнительно содержащая по меньшей мере один устойчивый к кислоте фермент.
44. Способ очистки, включающий в себя стадию контактирования поверхности и/или изделия, содержащего ткань, с очищающей композицией по п.33.
45. Способ по п.44, дополнительно включающий в себя стадию ополаскивания указанной поверхности и/или материала после контакта указанной поверхности или материала с указанной очищающей композицией.
46. Способ по п.44, где указанная поверхность и/или изделие, содержащее ткань, содержит загрязнение травой, и указанная стадия контактирования включает в себя контактирование указанного загрязнения травой с указанной очищающей композицией.
47. Композиция корма для животных, содержащая выделенную нейтральную металлопротеиназу по п.1.
48. Композиция для обработки ткани, содержащая выделенную нейтральную металлопротеиназу по п.1.
49. Композиция для обработки кожи, содержащая выделенную нейтральную металлопротеиназу по п.1.
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