CN107460180B - 一种米曲霉和厚垣镰孢霉混合发酵生产纤维素酶的方法 - Google Patents
一种米曲霉和厚垣镰孢霉混合发酵生产纤维素酶的方法 Download PDFInfo
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Abstract
本发明公开了一种米曲霉和厚垣镰孢霉混合发酵生产纤维素酶的方法,厚垣镰孢霉与米曲霉接种于蔗渣固体培养基上,混合培养发酵,培养温度为20~50℃,pH 4.0~10.0,纤维素酶产量达到最高时结束发酵培养。本发明将米曲霉和厚垣镰孢霉混合培养,米曲霉产高转苷活性β‑葡萄糖苷酶可以合成龙胆二糖,龙胆二糖可以刺激厚垣镰孢霉合成纤维素酶,本发明通过米曲霉和厚垣镰孢霉的混合培养,优化了纤维素酶系组成,可显著提高纤维素酶系的整体酶活力。
Description
技术领域
本发明涉及纤维素酶生产技术领域,尤其是一种米曲霉和厚垣镰孢霉混合发酵生产纤维素酶的方法。
背景技术
纤维素酶(β-1,4-葡聚糖-4-葡聚糖水解酶)是降解纤维素生成葡萄糖的一组酶的总称,它不是单体酶,而是起协同作用的多组分酶系,是一种复合酶,主要由外切β-葡聚糖酶、内切β-葡聚糖酶和β-葡萄糖苷酶等组成,还有很高活力的木聚糖酶。β-葡萄糖苷酶可以水解纤维二糖解除这种反馈抑制作用,提高纤维素酶的降解效率,在纤维素的降解中起着非常关键的作用。
木霉属是主要的纤维素酶工业生产菌株, 可以产高活力的葡聚糖内切酶和葡聚糖外切酶,但存在着β-葡萄糖苷酶分泌量不足及酶活偏低的缺陷,导致水解过程中纤维素寡糖积累,并强烈反馈抑制葡聚糖内切酶和葡聚糖外切酶的活性。筛选产高β-葡萄糖苷酶酶活的菌株,对纤维素资源的综合利用及其在其他领域的应用十分重要。
β-葡萄糖苷酶在多种生物转化中发挥重要作用,可以糖化农业纤维素材料发酵生产燃料乙醇;提高啤酒和白酒的出酒率,改善啤酒的口味;也是水解水果和发酵产品前体物质的糖苷类香气成分的关键酶,增加酒类和果汁产品中的风味物质含量,提高食品芳香性。
米曲霉属半知菌亚门,丝孢纲,丝孢目,从梗孢科,曲霉属真菌中的一个常见种。米曲霉是一类产复合酶的菌株,除产蛋白酶外,还可产淀粉酶、糖化酶、纤维素酶、植酸酶等。已有报道表明米曲霉具有葡聚糖外切酶和葡聚糖内切酶活性,酶活性高,耐高温,可以显著提高纤维素酶系的降解能力,改善纤维素酶系的协同作用。
但目前尚未见镰孢霉属和米曲霉协同发酵生产纤维素酶的相关报道。
发明内容
本发明提供了一种米曲霉和厚垣镰孢霉混合发酵生产纤维素酶的方法,可有效提高纤维素酶的酶活。
为实现上述目的,本发明的技术方案为:
一种米曲霉和厚垣镰孢霉混合发酵生产纤维素酶的方法,其特征在于:厚垣镰孢霉与米曲霉接种于蔗渣固体培养基上,混合培养发酵,培养温度为20~50℃,pH 4.0~10.0,纤维素酶产量达到最高时结束发酵培养。
进一步的,所述厚垣镰孢霉为厚垣镰孢霉HML278,所述米曲霉为米曲霉HML366。
优选的,厚垣镰孢霉HML278先接种在蔗渣固体培养基上,36~48h后再接种米曲霉HML366。更优选的,厚垣镰孢霉HML278先接种在蔗渣固体培养基上,48h后再接种米曲霉HML366。
优选的,所述厚垣镰孢霉HML278的孢子数量大于等于米曲霉HML366的孢子数量。
本发明提供了以上所述的米曲霉和厚垣镰孢霉混合发酵生产纤维素酶的方法在产纤维素酶方面的应用。
进一步的,本发明还提供了以上所述的米曲霉和厚垣镰孢霉混合发酵生产纤维素酶的方法在分解蔗渣纤维素方面的应用。
更进一步的应用为:选用微波法、灭菌锅气爆法、1% w/w磷酸浸泡法中一种或两种以上方法预处理甘蔗渣,加入麸皮和营养盐液,先接种厚垣镰孢霉HML278孢子,20~50℃培养,在48h后再接种米曲霉HML366孢子,48h后再补充氮源,分解甘蔗渣中的纤维素获得葡萄糖。
厚垣镰孢霉HML278产外切葡聚糖酶和内切葡聚糖酶的能力较强,但是产β-葡萄糖苷酶的能力较弱导致纤维二糖和纤维寡糖的积累,致使纤维素酶的总体酶活不高,而米曲霉HML366延迟接种,高浓度纤维二糖和纤维寡糖可以诱导米曲霉HML366产β-葡萄糖苷酶,米曲霉HML366产生的β-葡萄糖苷酶具有较高转苷活性,可以合成龙胆二糖,龙胆二糖是强烈的产纤维素酶诱导物,龙胆二糖可以诱导厚垣镰孢霉HML278产纤维素酶,高活性β-葡萄糖苷酶也可以分解纤维二糖和纤维寡糖,消除高浓度纤维二糖和纤维寡糖对厚垣镰孢霉HML278的葡聚糖外切酶和葡聚糖内切酶产生反馈抑制,进而诱导厚垣镰孢霉HML278进一步产生纤维素酶;综上可见,米曲霉HML366和厚垣镰孢霉HML278混合培养,以厚垣镰孢霉HML278产纤维素酶为主, 米曲霉HML366产β-葡萄糖苷酶为辅,可显著提高纤维素酶系的整体酶活力。
具体实施方式
以下将结合具体实施例对本发明作进一步说明,但本发明的保护范围不限于以下实施例。
(1)菌种保存:
米曲霉HML366和厚垣镰孢霉HML278菌种PDA斜面4℃保存;
(2)米曲霉HML366纯化β-葡萄糖苷酶BG HG2的方法
包括以下步骤:S1.将筛选得到的米曲霉HML366加入10 mL生理盐水制成孢子液,取107个孢子转入蔗渣固体培养基中进行固态发酵,蔗渣固体培养基:6 g蔗渣,4 g麸皮,30mL Mandels 营养液,30℃培养 4 d;培养物加入200 mL无菌ddH2O,40℃恒温水浴1~3h浸提,四层纱布过滤,6000 r/min离心10min得到粗酶液,收集上清酶液4℃保存备用;S2.在4℃条件下,用上清酶液进行非变性电泳活性胶回收:8wt%分离胶, 4wt%浓缩胶,恒定电压为50V;S3.电泳结束后剪一小条电泳胶用β-葡萄糖苷酶特异性底物活性染色,显色后用蒸馏水冲洗停止反应,以这条有黑色沉淀的活性蛋白条带为指示胶,把其余胶带上对应的有β-葡萄糖苷酶的胶带剪下,放预冷研钵中磨碎后用柠檬酸-柠檬酸盐缓冲液(20 mM、pH 4.8)4℃浸提 12h,5000 Da超滤管4000 r/min离心20 min以浓缩脱盐,取上清液;S4.上清液用阴离子交换柱Mono Q 10/100 GL洗脱分离, 0.12 M NaCl 缓冲液浓度时洗出的第1蛋白峰包含β-葡萄糖苷酶 BG HG1,在0.34 M NaCl缓冲液浓度时洗出的第2蛋白峰包含β-葡萄糖苷酶 BG HG2。
(3)新纤维素酶协同作用分解蔗渣纤维素实验
以Whatman 1号滤纸作为底物,测定能反映多组分纤维素酶的协同作用的滤纸酶活力(Filter paper assay,FPase)。
分A、B、C、D四类管,各加入1×6cm的whatman1号滤纸放入试管中,再加入2 mL0.02 M、pH4.8的HAC-NaAC缓冲液。
A管:加入0.5 mL厚垣镰孢霉HML278纤维素酶液;
B管:加入0.5 mL米曲霉HML366纤维素酶液;
C管:加入0.5 mL厚垣镰孢霉HML278纤维素酶液和1mg(26.8 U)米曲霉HML366纯化β-葡萄糖苷酶BG HG2的混合液;
D管:加入0.5 mL厚垣镰孢霉HML278纤维素酶液和1mg(476.9 U)米曲霉HML366纯化木聚糖酶XynH1。
A、B、C、D类管分别做三个平行实验,在50℃水浴中保温60 min后取出,沸水浴5min后冷水浴灭酶活,用 DNS法测定还原糖含量,计算滤纸酶活(FPase)。
(4)新纤维素酶协同作用分解蔗渣纤维素实验结果见表1:
表 1 新纤维素酶协同作用降解蔗渣纤维素
实验结果显示,厚垣镰孢霉HML278纤维素酶液原始滤纸酶活(FPase)达到3.2 U /mL,米曲霉HML366原始FPase酶活 1.2 U /mL,厚垣镰孢霉HML278纤维素酶液原始FPase酶活较高。
厚垣廉孢霉存在β-葡萄糖苷酶活力较低的不足,导致纤维二糖和纤维寡糖积累,降低酶解效率。增加米曲霉HML366纯化β-葡萄糖苷酶BG HG2后,优化纤维素酶系各组分的相对含量,充分发挥纤维素酶系之间的协调作用,FPase酶活提高56.2℅,达到5.0 U /mL。
(5)蔗渣的预处理
称取10g洗净风干的蔗渣,加入盛有90 mLl%(w/w)H3PO4的500mL的烧杯中,放入微波炉中高火处理20min,滤干后用自来水清洗数次直至pH值呈中性,70℃干燥至恒重,将蔗渣加入盛有90 mL ddH2O的500mL的烧杯中,在高压蒸汽灭菌锅中121℃、压力0.145Mpa处理0.5 h,70℃干燥至恒重,得处理后的蔗渣。
(6)蔗渣固体培养基的制作:
6 g处理后的蔗渣,4 g麸皮,30 mL Mandels 营养液,置于500 mL 锥形烧瓶混匀,每天翻动两次,30℃培养 4 d。
(7)按(6)配方制作蔗渣固体培养基,先接种107个厚垣镰孢霉HML278孢子,30℃培养,不同培养瓶分别在24h、36h、48h后接种107个米曲霉HML366孢子,48h后每培养瓶补充0.14 g (NH4)2SO4,另外再分别单独接种厚垣镰孢霉HML278 107个孢子和单独接种米曲霉HML366 107个孢子在一样实验条件进行对照培养。分别做三个平行实验。
从第4天开始每天取样, 4℃条件下4000 r/min离心10min,取上清粗酶液测定滤纸酶活(FPase)和β-葡萄糖苷酶活力(β-glucosidase),测定3个平行样品取平均值。
米曲霉HML366生长速度较快,厚垣镰孢霉HML278生长较慢。实验表明,米曲霉HML366延迟48h后接种酶活较高,厚垣镰孢霉HML278先接种生长48h后已经能在蔗渣固体培养基上正常生长,再接种生活力更强的米曲霉HML366,不相互抑制,这两个菌种兼容性好。24h和36h接种米曲霉HML366的培养瓶,推测是厚垣镰孢霉HML278尚未在全部蔗渣固体培养基恢复生长,接种生活力更强的米曲霉HML366可能影响厚垣镰孢霉HML278生长。取厚垣镰孢霉HML278生长48h后接种米曲霉HML366的滤纸酶活(FPase)和β-葡萄糖苷酶活力(β-glucosidase)测定结果作为混合菌种发酵的测定结果,实验结果见表2:
表 2 厚垣镰孢霉HML278和米曲霉HML366混合发酵产酶时间表(U /mL)
实验结果表2表明,混合发酵酶液的滤纸酶活(FPase)和β-葡萄糖苷酶活力都强于单独发酵培养的菌种所产的酶液。
厚垣镰孢霉HML278所产的葡聚糖外切酶和葡聚糖内切酶酶活较高,β-葡萄糖苷酶活力低,先于米曲霉HML366培养48h,导致纤维二糖和纤维寡糖的积累。米曲霉HML366延迟48h接种,高浓度纤维二糖和纤维寡糖诱导米曲霉HML366产β-葡萄糖苷酶,β-葡萄糖苷酶活力在第5天开始明显增加,第6天达到最大值,为22.64 U/mL。β-葡萄糖苷酶活也高于厚垣镰孢霉HML278单一培养(6.87 U/mL)和米曲霉HML366单一培养酶活(12.86 U/mL)。
米曲霉HML366在该培养条件下,可以产高转苷活性的β-葡萄糖苷酶BG HG2,可以合成龙胆二糖,龙胆二糖是强烈的产纤维素酶诱导物,龙胆二糖可以诱导厚垣镰孢霉HML278产纤维素酶,高活性β-葡萄糖苷酶也可以分解纤维二糖和纤维寡糖,消除高浓度纤维二糖和纤维寡糖对葡聚糖外切酶和葡聚糖内切酶的反馈抑制。混合发酵酶液滤纸酶活(FPase)在4d后开始迅速增加,在第6天达到最大值,为3.82 U/mL,高于厚垣镰孢霉HML278单一培养(2.56 U/mL),第7天起,活力开始减退,到第八天酶活减退更快,导致纤维素酶失活的原因还可能因为培养时间过长,培养基营养成分不足,蛋白质水解酶水解先期培养的厚垣镰孢霉HML278细胞,导致细胞破碎自溶。
本实验充分证明米曲霉HML366和厚垣镰孢霉HML278混合培养产酶是优于单一菌种培养产酶的。厚垣镰孢霉HML278产外切葡聚糖酶和内切葡聚糖酶的能力较强,但是产β-葡萄糖苷酶的能力较弱,致使纤维素酶的总体酶活不高。由于厚垣镰孢霉HML278所产的活力低导致纤维二糖和纤维寡糖的积累,米曲霉HML366延迟48h后接种,高浓度纤维二糖和纤维寡糖可以诱导米曲霉HML366产β-葡萄糖苷酶。米曲霉HML366产β-葡萄糖苷酶具有较高转苷活性,可以合成龙胆二糖,可诱导厚垣镰孢霉HML278产生纤维素酶。可见以厚垣镰孢霉HML278产纤维素酶为主, 米曲霉HML366产β-葡萄糖苷酶为辅,米曲霉HML366和厚垣镰孢霉HML278混合培养可显著提高纤维素酶系的整体酶活力。
Claims (5)
1. 一种米曲霉和厚垣镰孢霉混合发酵生产纤维素酶的方法,其特征在于:厚垣镰孢霉与米曲霉接种于蔗渣固体培养基上,混合培养发酵,培养温度为30℃,蔗渣固体培养基中蔗渣pH 呈中性,纤维素酶产量达到最高时结束发酵培养;
所述厚垣镰孢霉为厚垣镰孢霉HML278,所述米曲霉为米曲霉HML366;
厚垣镰孢霉HML278先接种在蔗渣固体培养基上,36~48h后再接种米曲霉HML366;
接种米曲霉HML366时,所述厚垣镰孢霉HML278的孢子数量大于等于米曲霉HML366的孢子数量。
2.根据权利要求1所述的米曲霉和厚垣镰孢霉混合发酵生产纤维素酶的方法,其特征在于:厚垣镰孢霉HML278先接种在蔗渣固体培养基上,48h后再接种米曲霉HML366。
3.如权利要求1或2所述的米曲霉和厚垣镰孢霉混合发酵生产纤维素酶的方法在产纤维素酶方面的应用。
4.如权利要求1或2所述的米曲霉和厚垣镰孢霉混合发酵生产纤维素酶的方法在分解蔗渣纤维素方面的应用。
5.如权利要求4所述的应用,其特征在于:选用微波法、灭菌锅气爆法、1% w/w磷酸浸泡法中一种或两种以上方法预处理甘蔗渣。
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