CN114032189B - 一种降解木质素的融合子菌株r3及其应用 - Google Patents
一种降解木质素的融合子菌株r3及其应用 Download PDFInfo
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Abstract
本发明公开了一种降解木质素的融合子菌株R3及其应用。本发明利用PEG诱导方法对亲本菌株X1、X19进行融合,通过筛选出高效稳定的降解木质素融合子并优化其产酶条件,所得融合子菌株R3保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 2021420,保藏地址为武汉市武昌区珞珈武汉大学保藏中心。该融合子菌株R3可应用在木质素酶的制备以及木质素的降解中。本发明融合子菌株R3与亲本相比,三种木质素降解酶酶活比值更均衡,协同降解效果更优。
Description
技术领域
本发明属于生物技术领域,尤其涉及一种降解木质素的融合子菌株R3及 其应用。
背景技术
木质素是一类常见的难降解物质,主要作为农作物废弃秸秆和造纸工业废 水存在,导致资源的严重浪费和环境污染。与传统的物理、化学方法相比,能 耗低、清洁度高和对环境友好的生物法降解木质素正成为研究热点。由于细菌 具有来源广、生长快、便于工程改造且易于工业化生产等优势。因此,在自然 界中存在木质素的降解菌中,细菌降解研究最具前景。
据报道,目前起降解作用的细菌主要有厌氧梭菌、不动杆菌属、黄杆菌属、 微球菌属等,该类细菌产生一系列的木质素生物降解酶,主要包括漆酶、锰过 氧化物酶和木质素过氧化物酶,通过木质素降解酶的协同作用,木质素高聚物 断裂结构单元间连接键,从而分解为低分子物质。但是,现有降解细菌对木质 素的降解时间较长,效率较低,限制了这类细菌的实际应用。
发明内容
本发明的目的在于提供一种降解木质素的融合子菌株R3及其应用,旨在 解决现有降解细菌的降解率非常低的问题。
本发明是这样实现的,一种枯草芽孢杆菌-巨大芽孢杆菌融合子R3,该融 合子R3(枯草芽孢杆菌Bacillus subtilis和巨大芽孢杆菌Bacillus megaterium de Bary的融合子R3)于2021年4月21号保藏于中国典型培养物保藏中心,保 藏编号为CCTCC NO:M2021420,保藏地址为武汉市武昌区珞珈武汉大学保
本发明进一步公开了上述降解木质素的融合子菌株R3在制备木质素酶中 的应用。
优选地,所述木质素酶包括过氧化物酶、锰过氧化物酶、漆酶。
本发明进一步公开了上述降解木质素的融合子菌株R3在降解木质素中的 应用。
本发明克服现有技术的不足,提供一种降解木质素的融合子菌株R3及其 应用。本发明所用的亲本菌株X1、X19经16S rRNA基因序列测序及序列比对, 分别为枯草芽孢杆菌和巨大芽孢杆菌。两亲本均从盐城大丰麋鹿自然保护区麋 鹿粪便样品中筛得。在此基础上,本发明利用PEG诱导方法对上述亲本菌株 X1、X19进行融合,通过筛选出高效稳定的降解木质素融合子并优化其产酶条 件,其过程包括菌株的分离纯化及初筛、菌株的复筛、PEG法诱导融合、融合 子的筛选、遗传稳定性检测和产酶条件优化,该融合子菌株R3保藏于中国典型培养物保藏中心,保藏编号为CCTCC M 2021420R3,保藏地址为武汉市武昌 区珞珈武汉大学保藏中心,邮编为430072。
相比于现有技术的缺点和不足,本发明具有以下有益效果:
(1)本发明制备且培养条件优化后的融合子与亲本相比,三种木质素降解 关键酶(过氧化物酶(LiP)、锰过氧化物酶(MnP)、漆酶(Lac))的酶活 比值更均衡,协同降解效果更好;
(2)本发明融合子菌株R3具有较强氧耐受性,在木质素降解方面具有很 好的开发利用价值,可为后续的工业化生产提供可靠的菌种资源支撑;
(3)本发明融合子菌株R3对水稻和青菜种子萌发及早期幼苗生长的研究, 表明融合子菌株R3的生物安全性较高。
附图说明
图1是亲本X1的苯胺蓝平板脱色结果;
图2是亲本X19的苯胺蓝平板脱色结果;
图3是亲本菌株(X1、X19)的菌种鉴定结果;
图4是融合子菌株R3的菌落形态;
图5是融合子菌株R3木质素酶活力测定结果;
图6是融合子菌株R3的传代培养;
图7是不同氧环境下融合子菌株R3木质素降解率;
图8是融合子菌株R3的环境耐受性测试;
图9是融合子菌株R3在水稻中的生物安全性检测;
图10是融合子菌株R3的青菜中的生物安全性检测。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及实 施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅 仅用以解释本发明,并不用于限定本发明。
实施例
一、亲本菌株的分离纯化及初筛
1、样品前处理
将在大丰麋鹿自然保护区采集的麋鹿新鲜便样装入无菌玻璃瓶后,放置冰 盒保存,并在2h内带回实验室进行处理。
超净工作台内称取2g粪便样品,置于50mL灭菌小烧杯中,加入20mL 的无菌磷酸盐缓冲液(pH 7.2~7.4)。玻璃棒捣碎粪便样品并充分搅拌,用3 层灭菌纱布对样液进行粗过滤,去除其中大颗粒杂质,将过滤后的样品液取6 mL于离心管中,500rpm/min离心5min,取上层微浑浊液体于试管中,作为 10-1浓度,用无菌水梯度稀释10-2~10-5浓度。
2、菌株初筛
通过LB培养基对菌株进行富集培养,划线纯化直至获得纯菌株。将纯化 后的菌株接种到木质素初筛培养基上,于37℃恒温培养,观察其生长情况。
LB培养基(1L):蛋白胨10g,酵母粉5g,NaCl 10g,固体培养基添加 琼脂20g,pH调至7.0左右。
木质素初筛培养基的配制方法:木质素磺酸钠3g,(NH4)2SO42 g,K2HPO4 1g,MgSO40.2 g,CaCl20.1 g,MnSO40.02 g,KH2PO41 g,FeSO40.05 g,pH 调至7.0左右。
二、菌株的复筛
通过LB培养基对菌株进行富集培养,将浓度梯度的样品液取50μL均匀 涂布于培养平皿上,恒温培养箱中37℃培养2~3d,挑取单一菌落划线于分离 平板上,并通过反复平板划线进行分离,纯化直至获得纯菌株。
将已经分离纯化并保存的菌株分别接种到木质素初筛培养基上,作3个平 行样,于37℃恒温培养,观察其生长情况。
木质素降解菌产生的木质素过氧化物酶(LiP)和锰过氧化物酶(MnP)能够 使苯胺蓝平板脱色,根据是否脱色来判断两种酶的存在,从而筛选出亲本菌株, 透明圈较大的亲本菌株有X1、X19(图1、图2)。
苯胺蓝筛选培养基(1L):酵母粉10g,葡萄糖10g,苯胺蓝0.1g,琼脂 18g,pH7.0左右。
三、PEG法诱导融合
(1)亲本处理:取酶活较高的2株菌(X1为枯草芽孢杆菌、X19为巨大 芽孢杆菌(图3))的菌液5mL,4000r/min条件下离心10min,弃上清,向 离心管中滴加4mL磷酸缓冲液,不断震荡摇匀,使菌株悬浮于该液中。
(2)脱壁:取5mL菌悬液,加入3mL溶菌酶溶液,摇匀后于37℃恒 温水浴锅中水浴保温处理,5000r/min离心5min,弃去上清液,得到原生质体, 用配制好的蔗糖溶液洗涤除酶,将原生质体悬浮于稳渗剂中(0.6mol/L蔗糖溶 液),待用。
(3)融合操作:分别取制备好的两亲本的原生质体溶液2mL,混匀后静 置5~10min,加入1.8mL 40%的PEG 6000和0.2mL 0.1mol/L CaCl2混匀, 置37℃恒温水浴锅中促融30min后2500r/min离心10min,向沉淀中加入2mL 稳渗剂,充分混合摇匀。
四、融合子的筛选
取0.2mL融合液,均匀涂布在固体筛选培养基上,37℃恒温培养2~3d, 检出融合子。观察融合子的外观形状大小、边缘质地,菌落周边及顶部颜色的 变化,菌株生长情况,以及菌落的分布。筛选出融合子菌株R3(图4),通过苯胺蓝固体培养基褪色情况分析木质素降解能力,并进行相关酶活测定(图5)。
五、产酶条件优化
(1)R3的活化与传代培养
将-20℃,40%甘油冻存的细菌R3,于4℃自然解冻,涂布接种到固体LB 培养基中进行活化,在37℃下恒温培养2~3d,观察,重复三次,设置空白对照 组。2~3d后挑取单菌落接种到液体LB培养基中进行传代培养,重复三次,设 置空白对照组,在37℃、200r/min的恒温振荡培养箱中培养3~4d,连续传代, 备用。同时,将部分菌液与50%甘油按体积比1:1转移至冻存管,-20℃保存。经过连续传代培养,融合子菌株R3遗传性状稳定(图6)。
(2)产酶条件优化:影响木质素降解酶活性的因素主要有碳源、氮源、温 度、pH等。本发明采用四因素四水平试验设计(如下表1所示),优化融合子 菌株R3的培养条件,拟提高菌种的木质素酶活力。
表1.正交试验设计表
六、环境耐受性测试
本发明以高效木质素降解细菌融合子菌株R3为材料,采用对照方法,仅 改变氧这一因素,保持正交优化后的碳源、氮源、温度和pH等其他环境因素 不变,测定其在不同氧环境下的生长情况及木质素降解率(图7、图8)。
七、生物安全性测定
(1)预处理:将水稻和青菜种子在75%乙醇中浸泡,连续匀速搅拌1min, 重复两次。用蒸馏水冲洗3次,无菌水冲洗4次,晒干,备用。
(2)浸种:4个稀释梯度的菌液和无菌水中分别浸泡水稻100粒种子、青 菜100粒种子。
(3)培养:在种子分别浸种24h,36h,48h后,在无菌条件下,用镊子夹 取各浓度下的水稻种子和青菜种子各30粒,移至带有湿润滤纸的培养皿中,重 复三次。于室温下,一天的太阳光照为光源培养一定天数。青菜种子每隔24h 观察记录一次并补充水分,水稻种子每隔48h观察记录一次并补充水分,保持整个体系在培养过程中始终质量守恒。
由本发明融合制备出的R3对处理后的水稻种子和青菜种子发芽率没有显 著性影响,但发芽指数却明显提高。同时增强了植物细胞的保水能力,提高了 水稻和青菜的抗逆性,且能检测出POD酶活性(图9、图10),有一定的生物安全性。
效果实施例
本发明利用细胞融合技术从麋鹿肠道中获得了一株木质素降解能力较高的 融合子菌株R3。培养条件优化后的融合子菌株R3测得Lip、Mnp和Lac的酶 活分别为69.00U/L、130U/L、20.00U/L,三种木质素酶活分别提高了1.03、 1.65和1.22倍,均超过了亲本X1、X19。
本发明中融合子菌株R3能将木质素作为唯一碳源进行生长,并且能分泌 Lip、Mnp和Lac等3种重要的木质素降解酶,具备木质素降解能力,且融合 子菌株R3所含的木质素降解酶酶活比值更均衡。
本发明中融合子R3在不同氧环境下均能生长良好,其中,在有氧条件下 生长模型的拟合方程为:y=-0.24767+0.03439x-1.10348x2(R2=0.94852),在厌 氧条件下生长模型的拟合方程为:y=-0.28027+0.04355x-1.63108x2(R2=0.95483)。
本发明中融合子R3在木质素磺酸钠为唯一碳源的液体培养基中,发酵5d 时对木质素的降解率达到28%。与文献报道中从堆肥分离到的木质素降解细菌 MZ-9发酵6d的降解率20.51%,及从牛粪中分离筛选出产芽孢的木质素降解 菌发酵24d的降解率24%相比,融合子R3短时间内降解效果更加明显。
因此,融合子菌株R3是一株高效的木质素降解菌株,其遗传性能稳定, 环境耐受性强,在木质素降解方面具有很好的开发利用价值,可为后续的工业 化生产提供可靠的菌种资源支撑。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发 明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明 的保护范围之内。
Claims (3)
1.一种枯草芽孢杆菌-巨大芽孢杆菌融合子R3,其特征在于,该融合子R3保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO:M 2021420,保藏地址为武汉市武昌区珞珈武汉大学保藏中心,邮编为430072。
2.权利要求1所述的枯草芽孢杆菌-巨大芽孢杆菌融合子R3在制备木质素酶中的应用,所述木质素酶为木质素过氧化物酶、锰过氧化物酶、漆酶。
3.权利要求1所述的枯草芽孢杆菌-巨大芽孢杆菌融合子R3在降解木质素中的应用。
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