CN115011647A - 一种生产纤维素酶的方法及其在蔗渣纤维素中的应用 - Google Patents
一种生产纤维素酶的方法及其在蔗渣纤维素中的应用 Download PDFInfo
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Abstract
本发明公开了一种生产纤维素酶的方法,利用一株保藏编号为CGMCC No.20746的木霉菌株A‑8(Trichoderma sp.A‑8)菌株发酵生产纤维素酶,通过将木霉菌株A‑8菌株接种到发酵培养基中培养,然后离心取上清液来获得纤维素酶。该纤维素酶应用于甘蔗渣的酶促水解时,主要是通过对酸预处理或碱预处理后的甘蔗渣酶促水解来实现,所述菌株产纤维素酶的最pH4,最适温度30-35℃。所述菌株的种子液接种于发酵培养基中,在优化条件下培养5d后,离心分离得到不清液,即为酶液。
Description
技术领域
本发明属于微生物发酵领域,具体是一种生产纤维素酶的方法及其在蔗渣纤维素中的应用。
背景技术
纤维素是植物细胞壁的主要成分。甘蔗渣中纤维素是一种不分枝的线性多糖,以β-D-吡喃型葡萄糖为单体,由葡萄糖亚基通过β-1,4-糖苷键连接在一起构成,纤维素分子中不同的纤维素链之间主要通过氢键和范德华力相互作用使得纤维素呈现规则或不规则排列。纤维素以两种形式存在,包括更易受酶作用的非晶态和结晶形式,即高度有序的结构使其难以降解。
自然界中微生物可以作为纤维素酶的有效生产者,真菌、细菌、放线菌等都可以在某一特定条件下产纤维素酶。微生物降解纤维素分细菌和真菌两种形式,细菌是先将生物质表面进行破坏,然后再由外向内进行生长和产酶,一步步降解纤维素;而真菌则是先从内部开始进行降解,其菌丝能够吸附在纤维素顶端,沿着顶端进入纤维素的内部进行生长并分泌纤维素酶,实现由纤维素内部开始向外部逐渐进行降解。商业纤维素酶大多数来源于真菌菌株分泌的胞外酶,据相关报道曲霉属和木霉属已为全世界纤维素酶市场提供20%的纤维素酶产品。
甘蔗渣是甘蔗制糖工业过程中的主要副产品,是由甘蔗经过机械破碎、多辊压榨以及进行固-液(蔗汁)分离后留下的甘蔗茎固体纤维残渣,是一种可再生的、丰富的资源,适合生产生物燃料和化学品等生物基产品,是传统石油基燃料的理想替代品,也是生物炼制概念的支柱。甘蔗渣纤维素材料既节能又环保,在降解纤维素生物质的多种方法中,生物法较行之有效,纤维素的降解和转化利用的是微生物的次级代谢产物纤维素酶,但是在工业生产上并没有得到广泛的应用,主要原因就是在实际应用方面存在菌株产酶能力弱且质量不稳定,纤维素转化率低、酶成本及生产成本高等问题,因此迫切需要寻找具有高效纤维素酶生产潜力的菌株。
发明内容
本发明所要解决的技术问题是提供一种木霉发酵生产纤维素酶的方法,及其在甘蔗渣酶促水解中的应用。
为实现上述目的,本发明采用了以下的技术方案:一株纤维素酶产生菌木霉菌株A-8,保藏于中国普通微生物菌种保藏管理中心CGMCC No.20746,地址为:北京市朝阳区北辰西路1号院3号,保藏时间为2020年10月26日。
一种木霉发酵生产纤维素酶的方法,将木霉菌株A-8接种到发酵培养基中培养,然后离心取上清液,上清液即为纤维素酶粗酶液。
所述接种的接种量为5-8%。
所述的发酵培养基按以下方法制备:取CMC-Na 10g、(NH4)2SO44g、KH2PO4 2g、MgSO4·7H2O 0.5g,蛋白胨10g,牛肉膏5g,加超纯水至1000mL混合搅拌均匀,pH值自然,然后再121℃湿热灭菌30min。
所述培养的培养时间3-8d,温度20-40℃。
所述离心的条件是4℃下,离心转速为6000r/min,离心时间10min。
所述的纤维素酶在降解甘蔗渣中的应用,包括以下步骤:
(1)甘蔗渣预处理,采用酸液或碱液预处理的方式,对甘蔗渣进行预处理,收集固体残留物;
(2)酶促水解:以甘蔗渣为碳源和底物制得甘蔗渣发酵培养基,以5-8%的孢子接种量,在20-40℃、200r/min条件下分别发酵培养5-8d,通过离心分离水解产物和残余物,离心速度为8000r/min离心时间为10min,收集水解上清液,水解产物主要为葡萄糖、木糖和阿拉伯糖。
所述的酸预处理按以下方法进行:按固液比1:6加入浓度为0.4%体积分数H2SO4溶液,温度控制在150℃,处理时间为15min。
所述的碱预处理按以下方法进行:按固液比1:6加入浓度为2%体积分数 NaOH溶液,温度控制在170℃,处理时间为60min。
所述甘蔗渣是含量为1%纤维素的甘蔗渣。
所述的发酵培养在pH 4.0条件下进行。
本发明获得的有益效果是:
1.提供了一种木霉菌株A-8发酵生产纤维素酶的方法,并且操作简单,获得的纤维素酶活性高。
2.获得的纤维素酶在降解甘蔗渣时降解效率高,成本低。
具体实施方式
下面对本发明进行描述。本实施例使用的甘蔗渣是由洋浦南华糖业集团有限公司在广西上林糖厂提供的,并于40℃烘箱中烘干使其含水量低于10%。使用高速粉碎机将其粉碎后取20目到40目筛分得到的甘蔗渣(甘蔗渣粒径 0.5-1.0mm),于室温下保存在密封袋中备用。
实施例1
本实施例是木霉菌株A-8进行发酵产纤维素酶的一个实施例。
在装液量为50mL的250mL三角瓶中接种8%的孢子液(107个/mL), 30℃、200r/min发酵培养,发酵周期5d,发酵结束后,对发酵液进行离心(4℃,6000r/min,10min)取上清液,即为粗酶液。
实施例2
本实施例是利用木霉菌株A-8应用到甘蔗渣酶解的一个实施例。以甘蔗渣 (1%纤维素底物(w/v))为碳源和底物制得甘蔗渣发酵培养基,以8%的孢子接种量,在30℃、200r/min条件下分别发酵培养5d。最后,通过离心分离水解产物和残余物,离心速度为8000r/min离心时间为10min,收集水解上清液,主要水解产物为葡萄糖、木糖和阿拉伯糖。
Claims (9)
1.一株木霉菌株A-8菌株在降解甘蔗渣中的应用,其特征在于,包括以下步骤:
(1)将木霉菌株A-8菌株接种到发酵培养基中培养,然后离心取上清液,上清液即为纤维素酶粗酶液;
(2)甘蔗渣预处理,采用酸液或碱液预处理的方式,对甘蔗渣进行预处理,收集固体残留物;
(3)酶促水解:以甘蔗渣为碳源和底物制得甘蔗渣发酵培养基,以5-8%的孢子接种量,在20-40℃、200r/min条件下分别发酵培养5-8d,通过离心分离水解产物和残余物,离心速度为8000r/min离心时间为10min,收集水解上清液,水解产物主要为葡萄糖、木糖和阿拉伯糖。
2.根据权利要求1所述的纤维素酶在降解甘蔗渣中的应用,其特征在于,所述的酸预处理按以下方法进行:按固液比1:6加入浓度为0.4%体积分数H2SO4溶液,温度控制在150℃,处理时间为15min。
3.根据权利要求1所述的纤维素酶在降解甘蔗渣中的应用,其特征在于,所述的碱预处理按以下方法进行:按固液比1:6加入浓度为2%体积分数NaOH溶液,温度控制在170℃,处理时间为60min。
4.根据权利要求1所述的纤维素酶在降解甘蔗渣中的应用,其特征在于,所述甘蔗渣含1%纤维素。
5.根据权利要求1所述的纤维素酶在降解甘蔗渣中的应用,其特征在于,所述的发酵培养在pH 4.0条件下进行。
6.根据权利要求1所述的纤维素酶在降解甘蔗渣中的应用,其特征在于,步骤(1)所述接种的接种量为5-8%。
7.根据权利要求1所述的纤维素酶在降解甘蔗渣中的应用,其特征在于,步骤(1)所述的发酵培养基按以下方法制备:取CMC-Na 10g、(NH4)2SO44g、KH2PO42g、MgSO4·7H2O 0.5g,蛋白胨10g,牛肉膏5g,加超纯水至1000mL混合搅拌均匀,pH值自然,然后再121℃湿热灭菌30min。
8.根据权利要求1所述的纤维素酶在降解甘蔗渣中的应用,其特征在于,步骤(1)所述培养的培养时间3-8d,温度20-40℃。
9.根据权利要求1所述的纤维素酶在降解甘蔗渣中的应用,其特征在于,步骤(1)所述离心的条件是4℃下,离心转速为6000r/min,离心时间10min。
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