CN106754828A - 一种用于桉木制备低聚木糖的复合酶 - Google Patents
一种用于桉木制备低聚木糖的复合酶 Download PDFInfo
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Abstract
本发明提供一种用于桉木制备低聚木糖的复合酶，包括木聚糖酶和纤维素酶；上述的复合酶还包含有甘露聚糖酶。本发明的复合酶可广泛应用于低聚木糖的生产，能有效提高桉木半纤维素转化低聚木糖的效率，而且复合酶中的纤维素酶和甘露聚糖酶能明显促进木聚糖酶对半纤维素的降解作用，三种酶协同作用，能使木二糖、木三糖和木四糖的总转化率提高12.5％，并且木四糖的转化率也提高了50％，取得了意料不到的技术效果。
Description
技术领域
本发明涉及酶制剂应用技术领域，具体涉及一种用于桉木制备低聚木糖的复合酶及其应用。
背景技术
低聚木糖亦称木寡糖，由2-7个D-木糖以β-1,4-木糖苷键结合而成，以木二糖和木三糖为主，是功能性低聚木糖家族中的一个重要成员，能够促进肠道内双歧杆菌的繁殖，有益于肠胃健康，还有润肠通便，提高免疫力、抗肿瘤，降低血压、血糖、血清胆固醇，防龋齿、清口臭，促进钙的吸收等生物学作用。它的甜度比蔗糖和葡糖糖均低，与麦芽糖差不多，约为蔗糖的40％。低聚木糖对pH值及热的稳定性较好，即使是在酸性条件(pH2.5-7)加热也基本不分解，适合用于酸奶、乳酸菌饮料和碳酸饮料等酸性饮料中。
低聚木糖是由木聚糖经酶解产生的低聚合度寡糖，在商业上主要是以含有木质纤维素如竹笋、树木、农业废弃物(桔杆、棉子、玉米芯、蔗渣等)为原料，用食品级的木聚糖酶水解后,分离精制而得。因此，木寡糖的生产原料来源十分广泛。
随着酶化学的进步和发展，人们发现了许多来源于微生物的水解酶和转移酶等，进一步深入研究，使得各种低聚糖可被廉价地大量生产。特别是随着高活性木聚糖酶的开发，使得在地利用资源如木材、玉米穗轴、棉籽壳、甘蔗渣及稻草等植物体中大量含有的半纤维素-木聚糖可被用来生产具有良好物性及功能性的低聚木糖。
目前制备低聚木糖的方法主要有化学法、发酵法和酶法。化学法原理是通过蒸汽或水中的水合氢离子催化降解木聚糖，具体过程为先将原料经溶剂处理得到木聚糖液，再经蒸汽、水或酸处理得到低聚木糖溶液，此法已应用于实际生产中，存在较严重的废液污染环境，大量碱腐蚀设备，副反应较多，产品安全性不高的问题。发酵法是用酶或微生物直接处理或发酵天然纤维原料生产低聚木糖，存在反应困难、酶回收困难、提取率低的问题。而酶法综合了上述两种方法，扬长避短，具有反应专一性强，易于控制其水解速度及程度，且所得产物分离及精制较为容易等优点，因此逐渐成为生产高品质低聚木糖的主要方法。
酶法制备主要是利用木聚糖酶水解半纤维素原料得到以木二糖、木三糖为主要成分的低聚木糖混合物，但目前这种方法普遍存在低聚木糖收率低的问题。因此，如何提高低聚木糖的收率是目前本领域急需解决的问题。
发明内容
本发明为解决现有技术问题，提供了一种用于桉木制备低聚木糖的复合酶，能有效提高桉木半纤维素转化低聚木糖的效率，提高木二糖、木三糖和木四糖的产量，应用前景广阔。
本发明一方面提供了一种用于桉木制备低聚木糖的复合酶，包括木聚糖酶和纤维素酶。
上述的复合酶还包含有甘露聚糖酶。
上述的复合酶，其各组分及其含量分别为：木聚糖酶4400～22000IU/g，纤维素酶600～1200IU/g，甘露聚糖酶0～600IU/g。
进一步优选地，所述用于桉木制备低聚木糖的复合酶，其各组分及其含量分别为：木聚糖酶22000IU/g，纤维素酶1200IU/g，甘露聚糖酶600IU/g。
本发明还提供了一种利用桉木生产低聚木糖的方法，按如下步骤得到：
1)将桉木首先经过亚硫酸钠高温高压处理，得到纤维素和半纤维素固体；然后将纤维素和半纤维素固体通过高浓度的碱溶液浸提出半纤维素溶液；半纤维素溶液通过陶瓷膜过滤脱碱，得到半纤维素糖液；
2)测定半纤维素糖液中总木聚糖的浓度，调节半纤维素糖液的pH至4.8～5.5备用；
3)按0.5～1g/g木聚糖的比例向半纤维素糖液中加入上述复合酶，50～60℃反应4～8h，10000rpm离心5min，取上清液，即得低聚木糖溶液。
本发明的复合酶可广泛应用于低聚木糖的生产，能有效提高桉木半纤维素转化低聚木糖的效率，而且复合酶中的纤维素酶和甘露聚糖酶能明显促进木聚糖酶对半纤维素的降解作用，三种酶协同作用，能使木二糖、木三糖和木四糖的总转化率提高12.5％，并且木四糖的转化率也提高了50％，取得了意料不到的技术效果。
具体实施方式
实施例1桉木半纤维素糖液的制备
将桉木首先经过亚硫酸钠高温高压处理，脱除部分的木质素，得到纤维素和半纤维素固体；纤维素和半纤维素固体通过高浓度的碱溶液浸提出半纤维素溶液；半纤维素溶液通过陶瓷膜过滤脱碱，得到半纤维素糖液。
将上述半纤维素糖液和12％的稀硫酸按1:1体积比混匀，然后100℃高温处理90min，用碳酸钙调节pH至中性，用离子液相色谱测定半纤维素糖液中木聚糖的总含量，为50克/升；调节半纤维素糖液的pH至4.8-5.5备用。
实施例2木聚糖酶对低聚木糖转化率的影响
将实施例1制得的半纤维素糖液分为5个处理组，每个处理组500mL，分别按0IU/g木聚糖，4400IU/g木聚糖，11000IU/g木聚糖，22000IU/g木聚糖，44000IU/g木聚糖的比例添加木聚糖酶；50℃反应4h，10000rpm离心5min，分别取上清液；用离子液相色谱分别测定其中木二糖、木三糖和木四糖的含量，计算木二糖、木三糖和木四糖的总转化率，分别为0.46％，20％，30％，40％，40％。
上述结果表明，木聚糖酶能有效促进桉木半纤维素转化为低聚木糖，尤其是木二糖、木三糖和木四糖，且随着木聚糖酶添加量的增加，木二糖、木三糖和木四糖这三种低聚木糖的总转化率得到显著提高，当达到22000IU/g木聚糖的添加量时，木二糖、木三糖和木四糖的总转化率达到最高40％，以后再增加木聚糖酶的添加量，上述三种低聚木糖的总转化率不再提高。
实施例3木聚糖酶和纤维素酶对低聚木糖转化率的影响
将实施例1制得的半纤维素糖液分为3个处理组，每个处理组500mL；首先按22000IU/g木聚糖的添加量向每个处理组中加入木聚糖酶；然后再按0IU/g木聚糖，600IU/g木聚糖，1200IU/g木聚糖的添加量向3个处理组中分别加入纤维素酶；同时设置2个对照组，在不添加木聚糖酶的情况下，仅添加纤维素酶，添加量分别为600IU/g木聚糖，1200IU/g木聚糖。
50℃反应4h，10000rpm离心2min，分别取上清液；用离子液相色谱分别测定其中木二糖、木三糖和木四糖的含量，计算木二糖、木三糖和木四糖的总转化率，具体数据见表1。
转化率＝(木二糖+木三糖+木四糖)/总木聚糖含量×100％；
表1木聚糖酶和/或纤维素酶对低聚木糖转化率的影响
从表1的结果可以看出，单独添加纤维素酶的对照组1和2中木二糖、木三糖和木四糖的总转化率为0，说明纤维素酶单独作用对于半纤维素转化低聚木糖没有任何效果；但在木聚糖酶的基础上再添加纤维素酶，却能显著提高上述三种低聚木糖的转化率，与单独添加木聚糖酶的处理组1相比，处理组2和3中木二糖、木三糖和木四糖的总转化率分别提高了7.5％和12.5％，从而说明纤维素酶能与木聚糖酶产生协同促进作用，有效提高半纤维素转化低聚木糖的效率，取得了意料不到的技术效果。
实施例4木聚糖酶、纤维素酶和甘露聚糖酶对低聚木糖转化率的影响
将实施例1制得的半纤维素糖液分为2个处理组，每个处理组500mL；首先按22000IU/g木聚糖的添加量向每个处理组中加入木聚糖酶，按1200IU/g木聚糖的添加量向每个处理组中加入纤维素酶；然后再按0IU/g木聚糖，600IU/g木聚糖的添加量向2个处理组中分别加入甘露聚糖酶；同时设置3个对照组，分别单独添加相同添加量的木聚糖酶，纤维素酶和甘露聚糖酶。
50℃反应4h，10000rpm离心2min，分别取上清液；用离子液相色谱分别测定其中木二糖、木三糖和木四糖的含量，分别计算木四糖的转化率，以及木二糖、木三糖和木四糖的总转化率。具体结果见表2。
表2木聚糖酶、纤维素酶和/或甘露聚糖酶对低聚木糖转化率的影响
从表2的结果可以看出，单独添加纤维素酶和甘露聚糖酶的对照组2和3中，木二糖、木三糖和木四糖的转化率均为0，说明纤维素酶和甘露聚糖酶本身对半纤维素降解没有任何作用。但与单独添加木聚糖酶的对照组1相比，增加了纤维素酶的处理组1中木二糖、木三糖和木四糖的总转化率提高了12.5％，木四糖的转化率提高了25％；与处理组1相比，增加了甘露聚糖酶的处理组2中木二糖、木三糖和木四糖的总转化率提高了11.1％，木四糖的转化率提高了50％。上述结果表明，纤维素酶和/或甘露聚糖酶能明显促进木聚糖酶对半纤维素的降解作用，三种酶协同作用，能有效提高木二糖、木三糖和木四糖的总转化率，并且木四糖的转化率也得到大幅提高，取得了意料不到的技术效果。
上述的结果表明，木聚糖酶、纤维素酶和甘露聚糖酶协同作用能明显促进桉木生产低聚木糖，尤其是木二糖、木三糖和木四糖。
实施例5桉木制备低聚木糖的专用复合酶及其应用
一种用于桉木制备低聚木糖的复合酶，其组分及含量分别为木聚糖酶22000IU/g、纤维素酶1200IU/g和甘露聚糖酶600IU/g。
1)将桉木首先经过亚硫酸钠高温高压处理，得到纤维素和半纤维素固体；然后将纤维素和半纤维素固体通过高浓度的碱溶液浸提出半纤维素溶液；半纤维素溶液通过陶瓷膜过滤脱碱，得到半纤维素糖液；
2)测定半纤维素糖液中总木聚糖的浓度为50克/升；调节半纤维素糖液的pH至4.8～5.5备用；
3)按0.5g/g木聚糖的比例向半纤维素糖液中加入上述复合酶，50℃反应4h，10000rpm离心5min，取上清液，即得低聚木糖溶液。用离子液相色谱分别测定其中木二糖、木三糖和木四糖的含量，计算木二糖、木三糖和木四糖的总转化率以及木四糖的转化率。
结果显示，利用本发明所述专用复合酶制备得到的桉木低聚木糖溶液中，木二糖、木三糖和木四糖的总转化率达到38％，其中木四糖的转化率达到12.2％。
Claims (6)
1.一种复合酶，其特征在于，所述的复合酶包含有木聚糖酶和纤维素酶。
2.如权利要求1所述的复合酶，其特征在于，所述的复合酶还包含有甘露聚糖酶。
3.如权利要求2所述的复合酶，其特征在于，所述的复合酶中各组分及其含量分别为：木聚糖酶4400～22000IU/g，纤维素酶600～1200IU/g，甘露聚糖酶0～600IU/g。
4.如权利要求2所述的复合酶，其特征在于，所述的复合酶中各组分及其含量分别为木聚糖酶22000IU/g，纤维素酶1200IU/g，甘露聚糖酶600IU/g。
5.权利要求1-4任一项所述的复合酶在桉木生产低聚木糖中的应用。
6.一种利用桉木生产低聚木糖的方法，其特征在于，所述的方法包括如下步骤：
1)将桉木首先经过亚硫酸钠高温高压处理，得到纤维素和半纤维素固体；然后将纤维素和半纤维素固体通过高浓度的碱溶液浸提出半纤维素溶液；半纤维素溶液通过陶瓷膜过滤脱碱，得到半纤维素糖液；
2)测定半纤维素糖液中总木糖的浓度，调节半纤维素糖液的pH至4.8～5.5备用；
3)按0.5～1g/g木糖的比例向半纤维素糖液中加入权利要求1-4任一项所述的复合酶，50～60℃反应4～8h，10000rpm离心2min，取上清液，即得低聚木糖溶液。
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