CN116064487B - 一种固定化里氏木霉高产纤维素酶的方法 - Google Patents
一种固定化里氏木霉高产纤维素酶的方法 Download PDFInfo
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Abstract
本发明公开了一种固定化里氏木霉高产纤维素酶的方法。通过采用一定密度、孔径和外观直径的聚氨酯泡沫作为里氏木霉吸附的固定化载体,利于系统传质作用,可减少工业放大生产过程中的能耗。将利用改性剂聚醚酰亚胺改性后的固定化载体置于生物反应器的固定化载体区域使其自由悬浮更好的吸附里氏木霉,再通过调节发酵培养中的碳氮源和发酵方式,调控菌体的生长,减少菌体量过多对传质带来的影响,同时也促进了酶活的提升,有利于实现长期稳定的固定化连续发酵纤维素酶。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一种固定化里氏木霉高产纤维素酶的方法。
背景技术
纤维素酶是可以降解木质纤维素生成葡萄糖的一组酶的总称,是秸秆类废弃物工业化处理重要用酶,也广泛应用于饲料、食品、医药、环保等领域,潜力很大。目前主要利用里氏木霉通过生物发酵进行规模生产,但存在周期长、酶活低等技术瓶颈。早在20世纪80年代末便兴起了固定化发酵相关研究,固定化技术不仅可以简化生产流程,减少设备投资,缩短产酶周期,降低成本等,且适合于半连续或连续化生产,具有很大的优势。相关研究的固定化载体有棉纤维、秸秆(文献H.Esterbauer,W.Steiner,I.Labudova,A.Hermann,M.Hayn,Production of Trichoderma cellulase in laboratory and pilot scale,BioresourceTechnology,1991,36:51-65)、多孔塑料载体(文献:夏黎明、余世袁、程芝.固定化里氏木霉制备纤维素酶的研究.南京林业大学学报,1994,17:1-5)等。但截止目前,未有相关固定化技术应用于产业化的应用报道。主要原因可能有:1)工业化放大过程中固定化反应器内部结构设计不理想;2)固定化载体带来的传质障碍;3)搅拌环境载体使用寿命短;4)固定化发酵体系菌体生长不稳定等。
发明内容
本发明所要解决的技术问题是针对现有技术的不足,提供了一种固定化里氏木霉高产纤维素酶的方法。
为了解决上述技术问题,本发明公开了一种固定化里氏木霉高产纤维素酶的方法。
将里氏木霉种子液接种于装有固定化载体的生物反应器中连续发酵产纤维素酶;
其中,所述的固定化载体是使用改性剂改性后的聚氨酯泡沫;所述的改性剂为多巴胺、聚乙烯亚胺或聚醚酰亚胺中的任意一种或几种的组合。
其中,所述的里氏木霉为ATCC 26921。
其中,所述的里氏木霉种子液的制备方法为将里氏木霉斜面菌种接种于种子液培养基中摇瓶培养;
具体的,所述的种子液的组成如下:葡萄糖18g/L,乳糖12g/L,酵母膏2g/L,玉米浆10g/L,硫酸铵6g/L,硫酸镁0.5g/L,氯化钙0.5g/L,pH4.2;
具体的,所述的摇瓶培养条件为:28℃、220r/min培养24-28小时。
其中,所述的接种,其接种量为5-20%v/v,优选为12%v/v。
其中,所述的固定化载体,其载体直径为8-24mm,载体孔径为1-4mm,直径与孔径比值为4-24,载体密度为0.72-1.02g/cm3;
优选为,载体直径8-24mm,载体孔径1-4mm,直径与孔径比值4-8,载体密度为0.83-0.91g/cm3。
其中,所述的固定化载体为将聚氨酯泡沫置于30-100℃0.1-1%的改性剂水溶液中浸泡1-6小时后用水冲洗,再60-100℃烘干得到。
具体的,所述的改性剂为聚醚酰亚胺。
其中,所述的固定化载体在连续发酵过程中,其载体量为1-10g/L,优选为4-5g/L。
其中,所述的连续发酵,其初始发酵培养基体积为生物反应器体积的45-65%,当发酵还原糖降低至1-3g/L时流加诱导物,控制残糖水平在2-4g/L,连续发酵5-7天后,进行换液处理,排出发酵醪,补充新鲜的发酵培养基,重复3-6批次;
优选的,其初始发酵培养基体积为生物反应器体积的60%,当发酵还原糖降低至1-3g/L时流加诱导物,控制残糖水平在2-4g/L,连续发酵6天后,进行换液处理,排出发酵醪,补充新鲜的发酵培养基,重复6批次。
具体的,所述的初始发酵培养基的组成如下:乳糖10-20g/L,葡萄糖0-10g/L,玉米浆10-35g/L,硫酸铵2-8g/L,硫酸镁0.1-0.8g/L,硫酸亚铁0.01-0.1g/L,硫酸锰0.001-0.05g/L,氯化钙0.1-1g/L,磷酸二氢钾0.1-1g/L,VB1 00-0.01g/L,pH3.8-5.0;
优选的,初始发酵培养基为:乳糖15g/L,葡萄糖6g/L,玉米浆24g/L,硫酸铵3g/L,硫酸镁0.6g/L,硫酸亚铁0.06g/L,硫酸锰0.01g/L,氯化钙0.2g/L,磷酸二氢钾0.6g/L,VB10.005g/L,pH4.2;
其中,所述的连续发酵,其条件为:通气比0.2-1vvm,转速250-550r/min,压力0.05-0.1Mpa,溶氧量10-35%
优选的,发酵培养条件为:通气量0.2-0.8vvm,搅拌转速300-550r/min,压力0.05-0.08Mpa,溶氧量25%。
其中,所述的诱导物为乳糖和槐糖混合液,其中,溶剂为水,乳糖与槐糖的质量比为1-4:6-9,乳糖和槐糖的总质量与水的质量体积比为10-50g:100mL;所述的诱导物流加速度为50升罐50-180mL/H;
优选的,所述的诱导物为乳糖和槐糖混合液,其中,溶剂为水,乳糖与槐糖的质量比为4:6,乳糖和槐糖的总质量与水的质量体积比为30g:100mL;所述的诱导物流加速度为50升罐50-180mL/H。
其中,所述的新鲜的发酵培养基,其组分如下:乳糖15g/L,葡萄糖6g/L,玉米浆24g/L,硫酸铵3g/L,硫酸镁0.6g/L,硫酸亚铁0.06g/L,硫酸锰0.01g/L,氯化钙0.2g/L,磷酸二氢钾0.6g/L,VB1 0.005g/L,pH4.2或乳糖10g/L,玉米浆10g/L,硫酸铵4g/L,硫酸镁0.6g/L,硫酸亚铁0.06g/L,硫酸锰0.01g/L,氯化钙0.2g/L,磷酸二氢钾0.6g/L,pH3.9;
优选的,所述的新鲜的发酵培养基,其组分为:乳糖10g/L,玉米浆10g/L,硫酸铵4g/L,硫酸镁0.6g/L,硫酸亚铁0.06g/L,硫酸锰0.01g/L,氯化钙0.2g/L,磷酸二氢钾0.6g/L,pH3.9。
其中,所述的生物反应器包括:搅拌桨叶、圆筒状钢丝网、平面钢丝网和爆气盘;
具体的,所述的圆筒状钢丝网将反应器内部分隔为搅拌区域和固定化载体区域;所述的圆筒状钢丝网底端垂直连接平面钢丝网;所述的搅拌桨叶置于搅拌区域内;所述的平面钢丝网的下方设置爆气盘。
具体的,将固定化载体置于固定化载体区域中,圆筒状钢丝网的孔径和平面钢丝网的孔径是固定化载体直径的1/3-2/3,优选为1/2。
有益效果:
(1)本技术采用的改性后的载体吸附效果好;对密度的设计,利于系统传质作用,可减少工业放大生产过程中的能耗。
(2)本技术通过调节碳氮源浓度,调控菌体生长,减少菌体量过多对传质带来的影响,同时也促进了酶活提升,有利于实现长期稳定的固定化连续发酵。
附图说明
下面结合附图和具体实施方式对本发明做更进一步的具体说明,本发明的上述和/或其他方面的优点将会变得更加清楚。
图1为生物反应器结构图。其中1:搅拌桨叶;2:圆筒状钢丝网;3:平面钢丝网;4:载体;5:爆气盘;6:固定化载体区域;7:搅拌区域
具体实施方式
下述实施例中所述实验方法,如无特殊说明,均为常规方法;所述试剂和材料,如无特殊说明,均可从商业途径获得。
实施例1:不同载体球直径与孔径对菌体吸附发酵的影响
(1)菌种:里氏木霉
(2)种子液制备:刮取一环里氏木霉ATCC 26921的斜面菌种至装有100mL种子培养液的500mL摇瓶中,28℃、220r/min培养24-28小时,获得里氏木霉ATCC 26921种子液。
其中,种子培养液配方为葡萄糖18g/L,乳糖12g/L,酵母膏2g/L,玉米浆10g/L,硫酸铵6g/L,硫酸镁0.5g/L,氯化钙0.5g/L,pH4.2。每100mL种子培养液分装至500mL摇瓶中,121℃灭菌20分钟。
(3)发酵:将里氏木霉ATCC 26921种子液按12%v/v的接种量接种至5L三角瓶中,28℃、180r/min发酵培养28小时。
其中,发酵培养基为乳糖15g/L,葡萄糖6g/L,玉米浆24g/L,硫酸铵3g/L,硫酸镁0.6g/L,硫酸亚铁0.06g/L,硫酸锰0.01g/L,氯化钙0.2g/L,磷酸二氢钾0.6g/L,VB10.005g/L,pH4.2。每1L发酵培养基分装至5L三角瓶,每瓶5L三角瓶中装有不同尺寸的固定化载体5g/L(表1)。
(4)检测:称量固定化载体吸附菌体量(固定化载体吸附的菌体烘干后称重,减去初始固定化载体质量,即为吸附菌体量),计算单位固定化载体质量下的吸附菌体量。并测量纤维素酶的滤纸酶活:纤维素酶在一定温度和条件(在50℃,pH4.8,反应30min的条件下,每分钟降解滤纸释放1μmol葡萄糖所需的酶量,定义为一个滤纸纤维素酶,以U表示)下,将滤纸水解,释放出还原糖。在碱性煮沸条件下,3,5-二硝基水杨酸与还原糖发生显色反应,其颜色深浅与还原糖含量成正比。在540nm处测其吸光度,可以得到还原糖的量,计算出纤维素酶的滤纸酶活,以此代表纤维素酶的酶活力,结果如表1所示。
表1不同尺寸的固定化载体的单位吸附菌体量及酶活
固定化载体球直径mm | 8 | 8 | 15 | 15 | 15 | 24 | 24 | 24 | 24 |
固定化载体孔径mm | 1 | 2 | 1 | 2 | 3 | 1 | 2 | 3 | 4 |
比值 | 8 | 4 | 15 | 7.5 | 5 | 24 | 12 | 8 | 6 |
单位吸附菌体量g/g | 1.08 | 0.39 | 1.44 | 1.21 | 0.47 | 2.17 | 1.86 | 1.63 | 1.46 |
酶活U/mL | 18.23 | 19.82 | 16.34 | 20.22 | 21.78 | 9.01 | 12.32 | 16.54 | 22.25 |
通过比较不同尺寸的固定化载体对吸附和发酵的影响,发现孔径越小越有利于菌体吸附,但传质变差,从而导致纤维素酶酶活较低。当增大固定化载体外观直径,提高孔径时,吸附菌体量相对较高,酶活液较大。所以将固定化载体外观直径与孔径比值控制在4-8时,可显著提升菌体发酵水平。
实施例2:不同载体量对菌体吸附发酵的影响
种子液培养和发酵同实施例1,载体量按1-10g/L添加(固定化载体外观直径15mm,孔径2mm),分析不同载体量下吸附菌体量和酶活的差异性,结果如表2所示。
表2不同载体量下吸附菌体量和酶活的差异性结果
载体量g/L | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
单位吸附菌体量g/g | 0.78 | 1.03 | 1.16 | 1.10 | 1.18 | 1.34 | 1.67 | 1.87 | 1.79 | 1.83 |
酶活u/mL | 23.67 | 22.54 | 21.23 | 21.17 | 20.60 | 17.12 | 12.10 | 6.23 | 5.53 | 3.20 |
如表2所示,载体量越大,菌体吸附量也越大,但却不利于酶活提升,尤其在载体量超过5g/L后,酶活下降显著。但载体量越低,对于下批次发酵影响较大,所以载体添加量控制在2-6g/L。
实施例3:固定化载体改性对菌体吸附的影响
(1)固定化载体改性处理:将固定化载体置于60℃0.1-1%的改性剂水溶液中(改性剂为多巴胺、聚乙烯亚胺或聚醚酰亚胺)处理4小时。处理结束后,纯水冲洗,并60-100℃烘干。
(2)种子液培养和发酵:同实施例1,载体量按5g/L添加(固定化载体外观直径15mm,孔径2mm),分析不同改性剂对固定化载体吸附能力的差异性,结果如表3所示。
表3不同改性剂对固定化载体吸附能力的差异性
结果发现,采用氨基类物质进行表面处理,在高温反应下,改性后的固定化载体对吸附菌体量有明显提升。相比较而言,采用聚醚酰亚胺效果较佳,在60-80℃下,相比于30℃低温时,固定化载体对吸附菌体量提升了23-26%,比聚乙烯亚胺的吸附菌体量提升了14%以上,比多巴胺的吸附菌体量提升了8%以上。
实施例4:不同载体密度(改性后)对发酵的影响
(1)构建固定化生物反应器:生物反应器包括搅拌桨叶1、圆筒状钢丝网2、平面钢丝网3和爆气盘5。利用圆筒状钢丝网2将反应器内部分隔为搅拌区域7和固定化载体区域6;同时在圆筒状钢丝网2底端垂直连接平面钢丝网3(钢丝网孔径是固定化载体直径的1/2),在平面钢丝网3的下方设置爆气盘5。将搅拌桨叶1置于搅拌区域7内,将固定化载体4置于固定化载体区域6中,实现固定化载体在底层平面钢丝网3上方和搅拌区域7之外进行自由流动。
(2)种子培养液和发酵培养基同实施例1,将实施例1中的三角瓶更换为50L固定化生物反应器。向固定化载体区域6添加4g/L不同密度的聚醚酰亚胺改性的固定化载体(固定化载体外观直径15mm,孔径2mm),装液量60%v/v,发酵条件为:通气量0.2-0.8vvm,搅拌转速300-550r/min,压力0.05-0.08Mpa,溶氧量25%。用搅拌桨叶1不停地搅拌发酵液,当发酵还原糖至1-3g/L时以50-180mL/H的速度流加诱导物,诱导物为30%的乳糖和槐糖(乳糖和槐糖的质量比为4:6)混合液,残糖水平控制在2-4g/L。发酵6天左右,然后进行换液处理,排出发酵醪,补充新鲜的发酵培养基,重复3批次。分析不同密度的聚醚酰亚胺改性的固定化载体对吸附菌体量和酶活的差异性,结果如表4所示。
表4不同密度的聚醚酰亚胺改性载体对吸附菌体量和酶活的差异性结果
从上表4可以发现,当聚醚酰亚胺改性的固定化载体密度低于0.8g/cm3时,菌体吸附量低,根据发酵过程现象,可以明显观察到载体处于漂浮液面层的状态,传质效果较差。当聚醚酰亚胺改性的固定化载体密度高于0.91g/cm3时,菌体吸附量大,发酵过程悬浮效果较差。在3个发酵批次中,载体密度过低或者过高,酶活下降趋势都很显著。当聚醚酰亚胺改性的固定化载体密度为0.83-0.91g/cm3时,三批次酶活均较高,但是也有下降趋势,主要原因可能是载体的加入影响传质,降低了搅拌对菌丝体剪切作用,有利于菌体生长,导致纤维素酶分泌途径减弱。
实施例5:调节碳氮源浓度,提升纤维素酶酶活提升
通过实施例4发现,如不改变配方,随着发酵批次增多,游离菌体量会越来越高,不利于菌体分泌纤维素酶。所以在进行发酵换液处理时,通过在新的培养基中控制葡萄糖浓度和总氮源量,使得理论转化成菌体浓度控制在18g/L以下。故将新鲜的发酵培养基配方调整为:乳糖10g/L,玉米浆10g/L,硫酸铵4g/L,硫酸镁0.6g/L,硫酸亚铁0.06g/L,硫酸锰0.01g/L,氯化钙0.2g/L,磷酸二氢钾0.6g/L,pH3.9。按实施例4的发酵条件和聚醚酰亚胺改性的固定化载体(载体外观直径15mm,孔径2mm)4g/L的添加量(载体密度0.91g/cm3)发酵6批次,结果如下表5所示。
表5新发酵培养基配方对聚醚酰亚胺改性的固定化载体的吸附菌体量和酶活的影响
通过6批次发酵数据可以发现,减少利于菌体生长的碳氮源,可以有效降低菌体量,同时也改善了传质效果,有利于酶活的提高,发酵6批次稳定性较好,酶活提升显著。
实施例6:与游离发酵对比
游离发酵的种子培养液和发酵条件同实施例4,仅不加载体,发酵6天,结果表6所示。
表6不同发酵模式下酶活及游离菌体量结果
发酵模式 | 酶活u/mL | 游离菌体量g/L |
游离发酵 | 239.14 | 17.72 |
固定化载体发酵 | 287.89 | 15.33 |
相比较于游离发酵,固定化载体发酵,其酶活提升了20%以上,游离菌体量也明显下降,不仅减少了对原料的消耗,也节约了菌种培养的时间,经济效果显著。
本发明提供了一种固定化里氏木霉高产纤维素酶的方法的思路及方法,具体实现该技术方案的方法和途径很多,以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。本实施例中未明确的各组成部分均可用现有技术加以实现。
Claims (10)
1.一种固定化里氏木霉生产纤维素酶的方法,其特征在于,将里氏木霉种子液接种于装有固定化载体的生物反应器中连续发酵产纤维素酶;
其中,所述的固定化载体是使用改性剂改性后的聚氨酯泡沫,具体是将聚氨酯泡沫置于30-100℃ 0.1-1%的改性剂水溶液中浸泡1-6小时后用水冲洗,再60-100℃烘干得到;所述的改性剂为聚醚酰亚胺;
其中,所述的固定化载体,其载体直径为8-24 mm,载体孔径为1-4 mm,直径与孔径比值为4-8,载体密度为0.83-0.91 g/cm3;
其中,所述的固定化载体在连续发酵过程中,其载体量为4-5 g/L;
其中,所述的连续发酵,其初始发酵培养基体积为生物反应器体积的45-65%,当发酵还原糖降低至1-3g/L时流加诱导物,控制残糖水平在2-4g/L,连续发酵5-7天后,进行换液处理,排出发酵醪,补充新鲜的发酵培养基,重复3-6批次;
其中,所述的初始发酵培养基的组成如下:乳糖10-20g/L,葡萄糖0-10g/L,玉米浆10-35g/L,硫酸铵2-8g/L,硫酸镁0.1-0.8g/L,硫酸亚铁0.01-0.1g/L,硫酸锰0.001-0.05g/L,氯化钙0.1-1g/L,磷酸二氢钾0.1-1g/L,VB1 00-0.01g/L,pH3.8-5.0;
其中,所述的连续发酵,其条件为:通气比0.2-1vvm,转速250-550r/min,压力0.05-0.1Mpa,溶氧量10-35%;
其中,所述的诱导物为乳糖和槐糖混合液,其中,溶剂为水,乳糖与槐糖的质量比为1-4: 6-9,乳糖和槐糖的总质量与水的质量体积比为10-50g:100mL;所述的诱导物流加速度为50升罐50-180 mL/H;
其中,所述的新鲜的发酵培养基,其组分如下:乳糖10g/L,玉米浆10g/L,硫酸铵4g/L,硫酸镁0.6g/L,硫酸亚铁0.06g/L,硫酸锰0.01g/L,氯化钙0.2g/L,磷酸二氢钾0.6g/L,pH3.9。
2.根据权利要求1所述的方法,其特征在于,所述的里氏木霉为ATCC 26921。
3.根据权利要求1所述的方法,其特征在于,里氏木霉种子液的制备方法为将里氏木霉斜面菌种接种于种子液培养基中摇瓶培养;
其中,所述的种子液的组成如下:葡萄糖18g/L,乳糖12g/L,酵母膏2g/L,玉米浆10g/L,硫酸铵6g/L,硫酸镁0.5g/L,氯化钙0.5g/L,pH4.2;
其中,所述的摇瓶培养条件为:28℃、220r/min培养24-28小时。
4.根据权利要求1所述的方法,其特征在于,所述的接种,其接种量为5-20% v/v。
5.根据权利要求1所述的方法,其特征在于,所述的连续发酵,其初始发酵培养基体积为生物反应器体积的60%,当发酵还原糖降低至1-3g/L时流加诱导物,控制残糖水平在2-4g/L,连续发酵6天后,进行换液处理,排出发酵醪,补充新鲜的发酵培养基,重复6批次。
6.根据权利要求1所述的方法,其特征在于,所述的初始发酵培养基的组成如下:乳糖15g/L,葡萄糖6g/L,玉米浆24g/L,硫酸铵3g/L,硫酸镁0.6g/L,硫酸亚铁0.06g/L,硫酸锰0.01g/L,氯化钙0.2g/L,磷酸二氢钾0.6g/L,VB1 0.005g/L,pH4.2。
7.根据权利要求1所述的方法,其特征在于,所述的连续发酵,发酵培养条件为:通气量0.2-0.8vvm,搅拌转速300-550r/min,压力0.05-0.08Mpa,溶氧量25%。
8.根据权利要求1所述的方法,其特征在于,所述的诱导物为乳糖和槐糖混合液,其中,溶剂为水,乳糖与槐糖的质量比为4:6,乳糖和槐糖的总质量与水的质量体积比为30g:100mL;所述的诱导物流加速度为50升罐50-180 mL/H。
9.根据权利要求1所述的方法,其特征在于,所述的生物反应器包括:搅拌桨叶(1)、圆筒状钢丝网(2)、平面钢丝网(3)和爆气盘(5);
其中,所述的圆筒状钢丝网(2)将反应器内部分隔为搅拌区域(7)和固定化载体区域(6);所述的圆筒状钢丝网(2)底端垂直连接平面钢丝网(3);所述的搅拌桨叶(1)置于搅拌区域(7)内;所述的平面钢丝网(3)的下方设置爆气盘(5)。
10.根据权利要求9所述的方法,其特征在于,将固定化载体(4)置于固定化载体区域(6)中,圆筒状钢丝网(2)的孔径和平面钢丝网(3)的孔径是固定化载体直径的1/3-2/3。
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