CN1563369A - 黄孢原毛平革菌木质素降解酶非浸没式液体发酵方法 - Google Patents
黄孢原毛平革菌木质素降解酶非浸没式液体发酵方法 Download PDFInfo
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- 241000222393 Phanerochaete chrysosporium Species 0.000 title claims description 14
- 230000001925 catabolic effect Effects 0.000 title 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 8
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 4
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- OEGPRYNGFWGMMV-UHFFFAOYSA-N (3,4-dimethoxyphenyl)methanol Chemical compound COC1=CC=C(CO)C=C1OC OEGPRYNGFWGMMV-UHFFFAOYSA-N 0.000 claims description 4
- 239000001257 hydrogen Substances 0.000 claims description 2
- 229910052739 hydrogen Inorganic materials 0.000 claims description 2
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 7
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- 244000061456 Solanum tuberosum Species 0.000 description 2
- ZMFVLYPTTFPBNG-UHFFFAOYSA-N azane;2,3-dihydroxybutanedioic acid Chemical compound [NH4+].OC(=O)C(O)C(O)C([O-])=O ZMFVLYPTTFPBNG-UHFFFAOYSA-N 0.000 description 2
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Abstract
本发明是一种黄孢原毛平革菌木质素降解酶非浸没式液体发酵方法,属于应用微生物领域。本方法是将黄孢原毛平革菌液体种子接入含载体的液体培养基中,在35~39℃下,在空气环境中振荡培养。所说的固定化培养载体是惰性载体、非惰性载体中的任一种,其形状为0.5~2.5cm的块状体。载体量的选择是使载体的堆积高度高于液体培养基的液面,载体处于非浸没状态,载体堆积高度为液面高度1~5倍。所说的发酵培养基采用碳限制(碳氮比≤8∶1)液体培养基、氮限制(碳氮比≥30∶1)液体培养基中的任一种,酸碱度(pH)控制范围为4.0~6.0,培养基中可含有0~2.5mM藜芦醇。本发明方法发酵条件简单,实施容易,木质素降解酶产量高,降低了发酵成本,提高了发酵的可行性。
Description
技术领域
本发明涉及黄孢原毛平革菌木质素降解酶发酵方法,属于应用微生物领域。
背景技术
黄孢原毛平革菌能够产生并向细胞外分泌木质素降解酶,降解木质素和许多持久性有机污染物,在环境污染治理中具有重要的应用价值。黄孢原毛平革菌木质素降解酶发酵主要分为液体发酵和固态发酵两种形式,与固态发酵相比,液体发酵具有传质效果好、发酵条件容易控制、发酵产物容易回收以及适于放大等优点,但发酵过程中氧的供应会受到较大影响,酶的产率会受到限制。黄孢原毛平革菌木质素降解酶的表达一般在较高的氧分压条件下比较活跃,其木质素降解酶液体发酵一般在纯氧或富氧条件下进行,在空气条件下的研究极少。富氧条件虽有利于黄孢原毛平革菌木质素降解酶的合成,但同时可能引起蛋白酶活力增大,导致木质素降解酶快速分解而不稳定。并且,富氧条件的实施会增大发酵成本,降低发酵的可行性,不利于木质素降解酶大规模生产的实现。目前,尚没有一种成熟的能够在空气环境条件下有效进行的黄孢原毛平革菌木质素降解酶液体发酵形式。
发明内容
本发明的目的是提供一种在空气条件下黄孢原毛平革菌木质素降解酶高水平发酵的方法。
本发明是将黄孢原毛平革菌液体种子接入含载体的液体培养基中,在35~39℃下,在空气环境中振荡培养。
所说的固定化培养载体是惰性载体、非惰性载体中的任一种,其形状为0.5~2.5cm的块状体。载体量的选择是使载体的堆积高度高于液体培养基的液面,载体处于非浸没状态,载体堆积高度为液面高度1~5倍。
所说的发酵培养基采用碳限制(碳氮比≤8∶1)液体培养基、氮限制(碳氮比≥30∶1)液体培养基中的任一种,酸碱度(pH)控制范围为4.0~6.0,培养基中可含有0~2.5mM藜芦醇。
采用本发明方法进行木质素降解酶发酵,不需对通常采用的培养基作改进和优化,不需改变温度和搅拌条件,不需在培养开始或培养过程中通入纯氧或含高浓度氧气的空气,培养系统中较大比重的液体培养基和菌丝在空气中适当的暴露,使发酵过程中菌丝可以得到比较充分的液体营养和氧的供应,发酵条件容易控制,在空气条件下可以发酵获得较高的木质素降解酶活力,并且,产生的木质素降解酶容易收获。本发明方法发酵条件简单,实施容易,木质素降解酶产量高,降低了发酵成本,提高了发酵的可行性。
具体实施方式
实施例1
配制碳限制(碳氮比3.8∶1)培养基,培养基含有葡萄糖5g/L,酒石酸氨4.1g/L,磷酸二氢钾2.0g/L,硫酸镁0.5g/L,氯化钙0.1g/L,维生素B1 1mg/L,以及适量的微量元素。采用0.5cm聚氨酯泡沫块作为固定化培养载体,将载体煮洗、烘干后放入250ml锥形瓶(含100ml培养基)中,载体量为3.0g/250ml锥形瓶,载体堆积高度约为液面高度5倍,将培养基和载体一起在113℃下灭菌30min。将生长在30℃土豆葡萄糖琼脂平板(200g/L土豆汁,20g/L葡萄糖和20g/L琼脂)上的黄孢原毛平革菌BKM-F-1767孢子无菌接入培养基中,接种量为1×105孢子/ml。将锥形瓶置于旋转式摇床上,在转速160rpm和温度37℃条件下在空气环境中进行发酵。培养4d,获得木质素过氧化物酶酶活87.5U/L。
实施例2
配制氮限制(碳氮比152.7∶1)培养基,培养基含有葡萄糖10g/L,酒石酸氨0.2g/L,磷酸二氢钾2.0g/L,硫酸镁0.5g/L,氯化钙0.1g/L,维生素B1 1mg/L,以及适量的微量元素。采用0.5cm聚氨酯泡沫块作为固定化培养载体,将载体煮洗、烘干后放入250ml锥形瓶(含100ml培养基)中,载体量为2.2g/250ml锥形瓶,载体堆积高度约为液面高度3倍,将培养基和载体一起在113℃下灭菌30min。将生长在30℃土豆葡萄糖琼脂平板(200g/L土豆汁,20g/L葡萄糖和20g/L琼脂)上的黄孢原毛平革菌BKM-F-1767孢子无菌接入培养基中,接种量为1×105孢子/ml。将锥形瓶置于旋转式摇床上,在转速160rpm和温度37℃条件下在空气环境中进行发酵。培养4d,获得锰过氧化物酶酶活338.7U/L。
Claims (1)
1.一种黄孢原毛平革菌木质素降解酶非浸没式液体发酵方法,其特征是将黄孢原毛平革菌液体种子接入含载体的液体培养基中,在35~39℃下,在空气环境中振荡培养,所说的固定化培养载体是惰性载体、非惰性载体中的任一种,其形状为0.5~2.5cm的块状体。载体量的选择是使载体的堆积高度高于液体培养基的液面,载体处于非浸没状态,载体堆积高度为液面高度1~5倍,所说的发酵培养基采用碳限制(碳氮比≤8∶1)液体培养基、氮限制(碳氮比≥30∶1)液体培养基中的任一种,酸碱度(pH)控制范围为4.0~6.0,培养基中可含有0~2.5mM藜芦醇。
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101665446B (zh) * | 2009-09-25 | 2012-06-27 | 四川汇科生物技术有限公司 | 辣椒素和辣椒红色素的提取方法 |
US8691194B2 (en) | 2002-12-12 | 2014-04-08 | R.B.T (Rakuto Bio Technologies) Ltd. | Methods of producing lignin peroxidase and its use in skin and hair lightening |
CN105524891A (zh) * | 2016-02-02 | 2016-04-27 | 清华大学 | 一种黄孢原毛平革菌木质素降解酶活力衰减后提高酶活的方法 |
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2004
- 2004-04-02 CN CN 200410003496 patent/CN1563369A/zh active Pending
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8691194B2 (en) | 2002-12-12 | 2014-04-08 | R.B.T (Rakuto Bio Technologies) Ltd. | Methods of producing lignin peroxidase and its use in skin and hair lightening |
US9693946B2 (en) | 2002-12-12 | 2017-07-04 | R.B.T. (Rakuto Bio Technologies) Ltd. | Methods of producing lignin peroxidase and its use in skin and hair lightening |
CN101665446B (zh) * | 2009-09-25 | 2012-06-27 | 四川汇科生物技术有限公司 | 辣椒素和辣椒红色素的提取方法 |
CN105524891A (zh) * | 2016-02-02 | 2016-04-27 | 清华大学 | 一种黄孢原毛平革菌木质素降解酶活力衰减后提高酶活的方法 |
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