CN113215209A - 一种促进木质纤维素酶水解的方法 - Google Patents
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Abstract
本发明公开了一种聚苯乙烯磺酸盐促进木质纤维素酶水解的方法,属于木质纤维素酶解技术领域。该方法为木质纤维素原料经过预处理后进行固液分离,得到的滤渣洗涤至中性后,在加入纤维素酶的同时加入0.05~0.4g/g底物的聚苯乙烯磺酸盐进行酶水解。结果表明,添加少量的聚苯乙烯磺酸盐就能有效提高木质纤维素酶法糖化的产率。其优点是用量少,效率高。而且聚苯乙烯磺酸盐的合成非常方便,而且结构易于控制,这为低成本和高效增益木质纤维素酶水解提供了一种极具希望的新型助剂选择。
Description
技术领域
本发明涉及一种高分子助剂提高木质纤维素原料酶水解效率的方法,属于木质纤维原料生物质转化技术领域。
背景技术
随着全球不断变暖和石化能源的日益枯竭,生物燃料由于其环境友好型和可再生性,近年来一直受广大科研工作者的关注。木质纤维基生物燃料可循环再生,逐步替代石化燃料,可以为我国实现2030年碳达峰和2060年碳中和目标贡献行业力量。木质纤维原料生物质转化的控制步骤之一是木质纤维素的酶水解转化。目前,科研工作者对于纤维素酶水解木质纤维糖化效率的因素和机理越来越深入。很长一段时间,人们认为,木质素作为壁垒阻碍了纤维素酶与纤维素的接触而不利于纤维素酶水解;并且由于木质素碳水化合物复合键的存在,对木质纤维素原料进行预处理不能完全去除木质素。残留的底物木质素主要通过下面三种方式影响纤维素的酶水解效率:一是底物木质素附着在纤维素的表面,降低了纤维素酶的可及性;二是底物木质素与纤维素酶会发生无效吸附,降低了纤维素酶的利用率,这一点得到了广泛地认可;三是在水解液中溶解的木质素或者类木质素结构,尤其是木质素衍生的酚类物质会降低纤维素酶的水解效率。为了提高木质纤维素酶解糖化效率,科研人员主要的研究有两个方面:一方面,加大预处理过程中木质素的脱除,这样不仅增加了预处理成本,而且预处理强度的增加也加大了纤维素、半纤维素的无效降解衍生;另一方面,降低无效吸附,研究人员发现在酶水解体系中添加表面活性剂(如PEG、Tween等)、非催化蛋白(牛血清白蛋白、花生蛋白等),这些表面活性添加剂条件固定,无法通过调节分子量和结构等与生产相适应。近年来,很多研究也发现,可溶性木质素(木质素磺酸盐LS、小分子碱木质素)以及木质素基高分子(木质素接枝PEG等)等均可以提高酶水解效率,这个方面的发现不能用传统木质素壁垒阻碍理论解释木质素对纤维素酶水解的影响。但这些酶解促进剂都存在用量高的缺点,而且对木质素衍生物类酶解促进剂来说,受限于其天然结构,其结构、电荷等等的调控程序复杂、成本较高。
本发明提供一种合成的线性高分子-聚苯乙烯磺酸盐来提高木质纤维素酶水解效率的方法,其优点就是用量少、效率高、结构调控方便。
发明内容
本发明的目的在于提供一种高效率促进木质纤维素酶水解糖化的方法,本发明在木质纤维素原料经过预处理的基础上,在酶水解体系中添加少量的聚苯乙烯磺酸盐提高木质纤维素酶水解糖化的效率。
本发明的技术方法如下:
本发明涉及一种促进木质纤维素酶水解的方法,其特征在于:
步骤(1):酶解促进剂为聚苯乙烯磺酸盐;
步骤(2):木质纤维素原料为绿液预处理的杨木、绿液预处理的麦草、亚硫酸钠-稀硫酸预处理的马尾松,对木质纤维素原料预处理结束后清洗至中性,接着进行盘磨分散;
步骤(3):将步骤(2)的木质纤维素原料加入到醋酸-醋酸钠缓冲液(0.05M,pH4.8)中,得到2%(w/v)的浆料;
步骤(4):然后加入0.05~0.4g/g-底物的聚苯乙烯磺酸盐,最后加入10FPU/g-葡聚糖的纤维素酶进行酶解,其酶解效率比未添加的显著增加,在0.1g/g-底物的添加量的情况下对绿液预处理的杨木的酶解得率能从对照样的39.8%提升到60.3%,比添加量高一倍的木质素磺酸盐的提升性能还高4.6%。
附图说明
图1:绿液预处理杨木在10FPU/g葡聚糖的酶添加量的条件下添加不同商品聚苯乙烯磺酸盐经过72小时酶水解后的葡萄糖转化率。其最佳添加量为0.10g/g底物。
图2:绿液预处理杨木添加聚苯乙烯磺酸盐(PSS 0.05代表PSS的添加量为0.05g/g底物,PSS 0.1代表PSS的添加量为0.1g/g底物)和木质素磺酸盐(LS 0.2代表木质素磺酸盐的添加量为0.2g/g底物)的动态酶水解过程。对比各条曲线可知,PSS和LS相对于不添加任何酶解促进剂的来说,均有较大的促进作用,而相比较PSS和LS而言,PSS在早期的促进作用不如LS明显,其优势是显著增强了酶的活性,可以让酶保持更长的作用时间。
具体实施方式
称取相当于绝干2g的木质纤维素原料(绿液预处理的杨木、绿液预处理的麦草和亚硫酸钠-稀硫酸预处理的马尾松,分别代表阔叶材、禾草类和针叶材),放入100毫升的醋酸-醋酸钠缓冲液(0.05M,pH=4.8)中。然后加入0.05~0.4g/g-底物的(商品或者自制的)聚苯乙烯磺酸盐,最后加入10FPU/g-葡聚糖的纤维素酶(本研究采用的纤维素酶为诺维信公司的CTec2)进行酶解。酶水解时间为72小时,期间和结束后都测定了葡萄糖的转化率,进行比较主要采用72h的最后测定的葡萄糖转化率。
测定葡萄糖浓度的高效液相色谱(HPLC)条件如下:ACQUITY Arc高效液相色谱仪,分析柱为BIO-RAD Aminex HPX-87H Column(300×7.8mm),保护柱为CationH RefillCartridges(30×4.6mm)。流动相:0.05mol/L硫酸溶液,柱温:55℃;流速:0.6mL/min。
商品PSS的72小时葡萄糖转化率效果见图1,其动态酶解效率见图2。
实施例1
称取相当于绝干2g的绿液预处理的杨木,放入100毫升的醋酸-醋酸钠缓冲液(0.05M,pH=4.8)中。然后加入0.05g/g-底物的商品聚苯乙烯磺酸盐(分子量7万),最后加入10FPU/g-葡聚糖的纤维素酶进行酶解。酶水解时间为72小时,测定葡萄糖转化率为54.8%。
实施例2
称取相当于绝干2g的绿液预处理的杨木,放入100毫升的醋酸-醋酸钠缓冲液(0.05M,pH=4.8)中。然后加入0.1g/g-底物的商品聚苯乙烯磺酸盐(分子量7万),最后加入10FPU/g-葡聚糖的纤维素酶进行酶解。酶水解时间为72小时,测定葡萄糖转化率为60.3%。
实施例3
称取相当于绝干2g的绿液预处理的杨木,放入100毫升的醋酸-醋酸钠缓冲液(0.05M,pH=4.8)中。然后加入0.2g/g-底物的商品聚苯乙烯磺酸盐(分子量7万),最后加入10FPU/g-葡聚糖的纤维素酶进行酶解。酶水解时间为72小时,测定葡萄糖转化率为55.7%。
实施例4
称取相当于绝干2g的绿液预处理的杨木,放入100毫升的醋酸-醋酸钠缓冲液(0.05M,pH=4.8)中。然后加入0.4g/g-底物的商品聚苯乙烯磺酸盐(分子量7万),最后加入10FPU/g-葡聚糖的纤维素酶进行酶解。酶水解时间为72小时,测定葡萄糖转化率为43.2%。
实施例5
称取相当于绝干2g的绿液预处理的麦草,放入100毫升的醋酸-醋酸钠缓冲液(0.05M,pH=4.8)中。然后加入0.1g/g-底物的自制的聚苯乙烯磺酸盐(分子量21万),最后加入10FPU/g-葡聚糖的纤维素酶进行酶解。酶水解时间为72小时,测定葡萄糖转化率为67.5%。
实施例6
称取相当于绝干2g的亚硫酸钠-稀硫酸预处理的马尾松原料,放入100毫升的醋酸-醋酸钠缓冲液(0.05M,pH=4.8)中。然后加入0.12g/g-底物的商品聚苯乙烯磺酸盐(分子量7万),最后加入10FPU/g-葡聚糖的纤维素酶进行酶解。酶水解时间为72小时,测定葡萄糖转化率为52.3%。
实施例7
对照组:分别称取相当于绝干2g的绿液预处理的杨木、绿液预处理的麦草和亚硫酸钠-稀硫酸预处理的马尾松,分别放入100毫升的醋酸-醋酸钠缓冲液(0.05M,pH=4.8)中,只加入10FPU/g-葡聚糖的纤维素酶进行酶解,而不加入任何酶解促进剂,酶水解时间为72小时,测定葡萄糖转化率分别为39.8%,42.3和32.9%。
实施例8
对照组:分别称取相当于绝干2g的绿液预处理的杨木原料,分别放入100毫升的醋酸-醋酸钠缓冲液(0.05M,pH=4.8)中,然后加入0.2g/g-底物的木质素磺酸盐,最后加入10FPU/g-葡聚糖的纤维素酶进行酶解,酶水解时间为72小时,测定葡萄糖转化率分别为55.7%。
另外申明,具体实施方式中仅是本发明的较佳实施例而已,并非对本发明作任何形式上的限制,虽然本发明已以较佳实施例揭露如上,然而并非用以限定本发明,任何熟悉本专业的技术人员,在不脱离本发明技术方案的范围内,当可利用上述揭示的技术内容做出些许更动或修饰为等同变化的等效实施例,但凡是未脱离本发明技术方案内容,依据本发明的技术实质对以上的实施例所作的任何简单修改、等同变化与修饰,均仍属于本发明技术方案的范围内。
Claims (1)
1.本发明涉及一种促进木质纤维素酶水解的方法,其特征在于:
步骤(1):酶解促进剂为聚苯乙烯磺酸盐;
步骤(2):木质纤维素原料为绿液预处理的杨木、绿液预处理的麦草、亚硫酸钠-稀硫酸预处理的马尾松,对木质纤维素原料预处理结束后清洗至中性,接着进行盘磨分散;
步骤(3):将步骤(2)的木质纤维素原料加入到醋酸-醋酸钠缓冲液(0.05M,pH 4.8)中,得到2%(w/v)的浆料;
步骤(4):然后加入0.05~0.4g/g-底物的聚苯乙烯磺酸盐,最后加入10FPU/g-葡聚糖的纤维素酶进行酶解,其酶解效率比未添加的显著增加,在0.1g/g-底物的添加量的情况下对绿液预处理的杨木的酶解得率能从对照样的39.8%提升到60.3%,比添加量高一倍的木质素磺酸盐的提升性能还高4.6%。
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