木聚糖酶检测底物筛选及玉米芯低聚木糖的制备
作者：刘代云 李萌 田盼
来源：《福建农业科技》2021年第07期
摘 要：為解决木聚糖酶国标底物短缺，并筛选可用于酶法制备低聚木糖的木聚糖酶，首先以甘蔗渣和榉木来源的木聚糖替代GB/T 23874-2009《饲料添加剂木聚糖酶活力的测定》中规定的底物检测木聚糖酶活力，同时采用木霉和毕赤酵母木聚糖酶酶解不同木聚糖底物和玉米芯木聚糖，应用高效液相色谱法对酶解产物组分进行分析，并初步优化玉米芯木聚糖酶解条件。结果表明：与GB/T 23874-2009《饲料添加剂木聚糖酶活力的测定》中规定的底物检测结果相比，榉木木聚糖（Megazyme）检测结果与原底物检测结果基本一
致;而甘蔗渣木聚糖检测结果偏高，但稳定性良好，两者均可替代原底物进行木聚糖酶活力检测。通过高效液相色谱法分析，毕赤酵母木聚糖酶水解不同底物的木二糖含量较木霉木聚糖酶的高，而木糖含量则相反。利用3种木聚糖酶同步水解玉米芯粉木聚糖，毕赤酵母木聚糖酶水解玉米芯粉木聚糖的产物中90%以上为低聚木糖，其中木二糖占72.54%，木三糖占17.86%，接近木聚糖酶Shearzyme 500L水解产物中的低聚木糖比例，高于木霉木聚糖酶，具有制备低聚木糖的应用潜力。毕赤酵母木聚糖酶水解玉米芯木聚糖的最优条件为反应温度60℃、pH 4.8，玉米芯底物浓度0.10 g·mL-1，酶解时间24 h，在此最优条件下玉米芯木聚糖的水解率可达76%。
关键词：木聚糖底物;木聚糖酶;低聚木糖;水解率
中图分类号：TQ 925 文献标志码：A 文章编号：0253-2301（2021）07-0074-08
DOI： 10.ki.fjnykj.2021.07.014
Screening of the Substrates for Xylana Detection and thePreparation of Corncob Xylooligosaccharide
LIU Daiyun， LI Meng， TIAN Pan
（Yichang Dongyangguang Biochemical Pharmaceutical Co.， Ltd.， Yichang， Hubei 443000， China）
Abstract： In order to solve the shortage of xylana substrate and screen the xylana which could be ud for the enzymatic preparation of xylooligosaccharides， firstly， the xylana activity was detected by replacing the substrate specified in GB/T 23874-2009 ″Determination of xylana activity in feed additives″ with xylana derived from bagas and beech. At the same time， The xylana of trichoderma and pichia pastoris was ud to hydrolyze different xylan substrates and corncob xylan by enzyme. The components of the enzymatic hydrolysates were analyzed by HPLC， and the enzymatic hydrolysis conditions of corncob xylan were preliminarily optimized. The results showed that compared with the detection results of the substrate specified in GB/T 23874-2009 ″Determination of xylana activity in feed additives″， the detection results of beech xylan （Megazyme） were basically consistent with that of the original s
ubstrate. The results of bagas xylan were higher， but the stability was good. Both of them could replace the original substrate for the detection of xylana activity. By the analysis of HPLC， compared with trichoderma xylana， the content of xylobio was higher in the hydrolysis of different substrates by pichia pastoris xylana， while the content of xylo was opposite. Three kinds of xylana were ud to hydrolyze corncob xylan simultaneously. More than 90% of the products of corncob xylan hydrolyzed by pichia pastoris xylana were xylooligosaccharide， 72.54% and 17.86% of which was respectively xylobio and xylotrio. being clo to the proportion of xylooligosaccharide in the products hydrolyzed by Shearzyme 500L xylana and higher than that by trichoderma xylana， indicating that it had the application potential in the preparation of xylooligosaccharide. The optimal conditions for the hydrolysis of corncob xylan by pichia pastoris xylana were as follows： reaction temperature of 60℃， pH of 4.8， the concentration of corncob substrate of 0.10 g·mL-1 and hydrolysis time of 24 h. Under the conditions， the hydrolysis rate of corncob xylan could reach 76%.
Key words： Xylan substrate; Xylana; Xylooligosaccharide; Hydrolysis rate
木聚糖酶是一类可以将木聚糖降解成低聚木糖和木糖的水解酶，主要包含内切β1，4木聚糖酶、外切β1，4木聚糖酶和
β木糖苷酶，其中内切木聚糖酶切β1，4糖苷键生成低聚木糖，β木糖苷酶水解低聚木糖释放木糖[1]。木聚糖酶除用于饲料[2-3]、烘焙[4]、造纸[5]等行业外，其中内切木聚糖酶还可用于低聚木糖的制备[6-9]。木聚糖酶来源广，仅饲料添加剂品种目录（2013）中木聚糖酶允许使用产生菌种就有7个，而工业高密度发酵产生菌以长柄木霉[10-11]和毕赤酵母[1]为主，但两种菌种发酵酶活差异较大，组分也存在差异。酶活力可以表征地反应木聚糖酶的水解能力，现通用木聚糖酶活力检测方法为GB/T 23874-2009 《饲料添加剂木聚糖酶活力的测定》[12]，底物为Sigma公司生产的X0627燕麦木聚糖，目前已停止生产。有研究发现以燕麦为底物检测结果要比桦木的高16%以上[13-14]。表明底物对酶活力检测结果有很大影响。由于各厂家使用底物不同，导致检测结果存在较大的差异。这对木聚糖酶的开发、应用及推广造成不利影响。
低聚木糖（XOS）亦称木寡糖，是由2个9个木糖单元，以β1，4糖苷键连接而成的以木二糖和木三糖为主要成分的混合物。XOS2-4指木二糖、木三糖、木四糖之和[15]，木二
糖是低聚木糖最主要的活性成分。現低聚木糖主要用酶法水解木聚糖生产，在工业化生产时所用酶制剂的内切木聚糖酶活力应尽可能高，而β木糖苷酶活力尽可能低，才能保证酶解产物中有较高的低聚木糖。现有研究大多集中在实验室规模，发酵酶活低[15]，已报告的商业化产品诺维信Shearzyme 500L可用于水解甘蔗渣制备低聚木糖[6，16]，但价格偏贵，实际应用生产成本较高。本研究利用不同来源的木聚糖底物检测木聚糖酶活性，以筛选国标底物的替代物。同时利用毕赤酵母和木霉木聚糖酶水解纯度较高的木聚糖底物和玉米芯木聚糖，分析比较2种木聚糖酶的酶解产物组分差异，以筛选适合用于制备低聚木糖的木聚糖酶并优化玉米芯木聚糖酶解条件，旨在为生产上提供参考依据。
