CN102108347B - 一种提高真菌产纤维素酶酶活的方法 - Google Patents

一种提高真菌产纤维素酶酶活的方法 Download PDF

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CN102108347B
CN102108347B CN2009102430751A CN200910243075A CN102108347B CN 102108347 B CN102108347 B CN 102108347B CN 2009102430751 A CN2009102430751 A CN 2009102430751A CN 200910243075 A CN200910243075 A CN 200910243075A CN 102108347 B CN102108347 B CN 102108347B
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cellulase
enzyme activity
farnesol
fungal
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CN102108347A (zh
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庄国强
马安周
吕迪
郭红光
张洪勋
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Research Center for Eco Environmental Sciences of CAS
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Abstract

本发明涉及提高纤维素酶酶活的新技术方法。本发明通过在真菌纤维素酶发酵生产过程中添加法尼醇的手段提高纤维素酶的酶活。通过添加法尼醇可以使瑞氏木霉产纤维素酶的滤纸酶活提高约30％，使斜卧青霉产纤维素酶的滤纸酶活提高了约46％。本发明涉及的方法简单易行，提高纤维素酶酶活的效果明显，并且该发明适用范围广，可用于多种真菌发酵生产纤维素酶工业。

Description

一种提高真菌产纤维素酶酶活的方法
技术领域：
本发明涉及生物技术领域，具体涉及在真菌纤维素酶发酵生产的过程中通过添加法尼醇来提高纤维素酶的酶活。
背景技术：
纤维素酶是一种重要的资源，广泛地应用在食品、酿造、饲料、纺织等工业技术领域，并且在纤维素生物质资源化的工业技术中作用日益凸显。当前石化能源的日益匮乏和环境污染的背景下，利用可再生纤维素生物质获得纤维基产品和能源显然具有很大的潜力和广阔的市场前景。纤维素生物质资源化过程中关键的一步是纤维素的生物转化过程(纤维素的酶解)，因而提高纤维素酶的酶活是非常重要的。真菌产纤维素酶的分泌量大，纤维素酶酶系完整，是纤维素酶制剂的主要工业生产菌种。目前主要局限于利用传统的方法来提高纤维素酶的酶活，包括通过物理或者化学手段诱变菌种，优化发酵培养基，以及利用分子生物学的手段改造工程菌种等，这些手段在提高纤维素酶酶活方面取得了一定的成果。但是有必要开发新的技术以提高纤维素酶的活力。群体密度感应(quorum sensing)是微生物化学交流的一种方式，当细胞密度达到一定的阈值时会激发和调控相关基因的表达，比如外泌的纤维素酶等。法尼醇是真核生物化学交流的重要群体感应信号分子，但是还未见有利用法尼醇来提高纤维素酶酶活的相关报道。
发明内容：
本发明旨在通过真菌纤维素酶发酵生产的过程中添加法尼醇来提高纤维素酶的酶活，在提高纤维素酶活力方面提供新的技术。
本发明所述的提高真菌产纤维素酶酶活的方法如下：
(1)菌种
产纤维素酶真菌，包括瑞氏木霉、斜卧青霉、拟康木霉、黑曲霉等。
(2)发酵培养基
使用纤维素物质作为碳源，包括微晶纤维素0.4～0.7％；麸皮2～4％；稻草秸秆、玉米秸秆、小麦秸秆等纤维素性生物质2～4％。补加一定量的无机盐溶液成分，其配方为：磷酸二氢钾2.0～4.0g/l，硫酸铵1.0～2.0g/l，MgSO4·7H2O 0.4～0.5g/l，CaCl2·2H2O 10～15mg/l等，pH 5.5。
(3)发酵过程
摇瓶发酵：接种量8～11％，28℃～30℃振荡培养，摇床转速为160～180rpm。
发酵罐：接种量8～11％，发酵温度28℃～30℃，溶氧量20％，发酵前期转速为200～250rpm，发酵后期转速为150～170rpm。
发酵前期加入法尼醇，法尼醇的加入量是0.1～1mM。
本发明依据群体密度感应的机理，在纤维素酶生产发酵的过程中添加法尼醇，调控真菌纤维素酶的分泌和提高纤维素酶的酶活力。实际操作简便可行，效果明显，为工业生产中提高纤维素酶的酶活力提供一种方法。
具体实施方式：
以下将将结合具体实施例对本发明作进一步的说明，目的在于帮助读者更好的理解本发明的精神实质，但不作为对本发明实施范围的限定。
实施例1：
选用斜卧青霉为生产菌种，使用的发酵培养基其配方如下：微晶纤维素0.6％；麸皮3％；玉米秸秆3％。无机盐溶液成分：磷酸二氢钾3.0g/l，硫酸铵2.0g/l，MgSO4·7H2O 0.5g/l，CaCl2·2H2O 15mg/l等，pH 5.5。
取3环平板上活化好的菌种，接种在液体培养基中，30℃振荡培养3天，摇床转速为180rpm，作为种子。将上述种子接种发酵培养基，接种量10％，30℃振荡培养3天，摇床转速为180rpm。发酵培养第3天时加入法尼醇，培养基中法尼醇的终浓度为1mM。不同的发酵时间点取样，测定纤维素酶酶活。滤纸酶活的定义：在50℃，相应pH条件下，1min水解滤纸纤维素底物，产生出相当于1μmol葡萄糖的还原糖量，为一个酶活力单位，以IU/mL(IU/g)表示。滤纸酶活测定时选用的滤纸为whatman公司的No1滤纸，还原糖的检测使用DNS法。以第8天的发酵样品为例，添加法尼醇的培养基中斜卧青霉产纤维素酶的滤纸酶活是4.29IU/mL，其活力较一般情况下斜卧青霉产纤维素酶的滤纸酶活提高了约46％。
实施例2：
选用瑞氏木霉为生产菌种，使用的发酵培养基其配方如下：微晶纤维素0.5％；麸皮3.5％；稻草秸秆粉3％。无机盐溶液成分：磷酸二氢钾3.0g/l，硫酸铵1.0g/l，MgSO4·7H2O 0.5g/l，CaCl2·2H2O 10mg/l等，pH5.5。
取3环平板上活化好的菌种，接种在液体培养基中，30℃振荡培养4天，摇床转速为180rpm，作为种子。将上述种子接种发酵培养基，接种量10％，30℃振荡培养4天，摇床转速为180rpm。发酵培养第4天时加入法尼醇，培养基中法尼醇的终浓度为0.5mM。不同的发酵时间点取样，测定纤维素酶酶活。以第5天的发酵样品为例，添加法尼醇的培养基中瑞氏木霉产纤维素酶的滤纸酶活是3.56IU/mL，其活力较一般情况下瑞氏木霉产纤维素酶的滤纸酶活提高了约30％。

Claims (1)

1.一种提高真菌产纤维素酶酶活的方法，其特征在于，在真菌纤维素酶发酵生产过程中通过添加法尼醇的手段提高纤维素酶的酶活，具体如下：
(1)使用的产纤维素酶真菌菌种是斜卧青霉或瑞氏木霉；
(2)在真菌纤维素酶发酵生产过程中添加法尼醇，具体是发酵的前期添加法尼醇，发酵培养基中法尼醇的浓度是0.1～1mM。
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CN103045484B (zh) * 2011-10-11 2014-12-24 济南圣泉集团股份有限公司 一种产纤维素酶的青霉菌株及其酶解纤维素的应用

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