CN113416663A - 一种协同发酵小麦秸秆制备高品质生物饲料的方法 - Google Patents
一种协同发酵小麦秸秆制备高品质生物饲料的方法 Download PDFInfo
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- CN113416663A CN113416663A CN202110627093.0A CN202110627093A CN113416663A CN 113416663 A CN113416663 A CN 113416663A CN 202110627093 A CN202110627093 A CN 202110627093A CN 113416663 A CN113416663 A CN 113416663A
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Abstract
本发明属于生物饲料领域,具体涉及一种协同降解发酵小麦秸秆转化成高品质生物饲料的方法。首先将来源于白囊耙齿菌的木质素过氧化物酶基因优化后导入缺陷型粟酒裂殖酵母中,得到木质素过氧化物酶‑粟酒裂殖酵母重组工程菌;然后以重组工程菌联合粗糙脉孢菌、发酵乳杆菌、产阮假丝酵母以及产锰过氧化物酶的S.pombe‑pREP‑mnp发酵菌种,并添加葡萄糖、氮源、漆酶和葡萄糖氧化酶以及小分子物质进行菌酶协同混菌发酵秸秆,得到高品质生物饲料。本发明合理利用农业废弃物,促进秸秆中木质纤维素转化,提高秸秆饲料中真蛋白含量、降解其中木质纤维素含量,短时间内显著提升发酵饲料的品质和营养价值,获得优质秸秆发酵饲料。
Description
技术领域
本发明属于生物饲料领域,具体涉及一种协同降解发酵小麦秸秆转化成高品质生物饲料的方法。
背景技术
随着我国粮食产量每年增长,秸秆产量也激增,中国每年产生约9亿吨秸秆。由于缺乏科学、合理的应用技术,这些秸秆多作为农业废弃物被丢弃。随着我国现代化农业进程不断推进,秸秆资源合理、高效利用成为当前研究重点。我国也是畜牧业大国,每年产生大量的优质牧草饲料需求,当前国内大量的优质牧草依旧依靠进口。将秸秆转化为优质生物饲料既能缓解当前秸秆资源浪费现状又能解决当前优质牧草饲料短缺的问题。
由于秸秆中含有大量的木质纤维素,秸秆饲料具有质感硬、口感差、蛋白含量低的缺点。木质纤维素主要由纤维素、半纤维素和木质素构成。当前市场上已有各种纤维素酶、半纤维素酶相关酶制剂,除漆酶和葡萄糖氧化酶外,木质素降解酶商品化制剂尚属空白,尤其降解木质素能力最强的木质素过氧化物酶的商品化进程还需进一步研究。受限于市场上酶制剂现状,当前生物饲料发酵过程中较重视纤维素和半纤维素的降解,以木质素为突破口的发酵工艺报道较少,特别是菌、酶联合小分子物质协同发酵制备满足高蛋白含量、低木质纤维素含量要求的生物饲料的研究更是少见。
发明内容
针对现有技术存在的不足,本发明构建一株重组木质素过氧化物酶酵母工程菌,并提供一种短时间内提升秸秆营养价值、制备优质秸秆生物饲料的安全、高效的发酵方式。
为实现上述发明目的,本发明采取的技术方案如下:
(1)将来源于白囊耙齿菌(Irpex lacteus,I.lacteus)的木质素过氧化物酶(Lignin peroxidase,LiP)编码序列(CDS)按照粟酒裂殖酵母密码子偏爱性进行密码子优化,获得编码木质素过氧化物酶的基因,核苷酸序列如SEQ ID NO.1所示;
(2)以步骤(1)中所述编码木质素过氧化物酶的基因为模板,进行PCR扩增,与穿梭质粒载体pREP线性化片段连接后转化到大肠杆菌受体菌中,得到重组质粒经醋酸锂转化法导入缺陷型粟酒裂殖酵母中,获得重组木质素过氧化物酶酵母工程菌,为 S.pombe-pREP-lip;
(3)协同降解发酵小麦秸秆转化为高品质生物饲料原料:首先以小麦秸秆为原料,添加葡萄糖、氮源、漆酶和葡萄糖氧化酶以及金属离子、酸类小分子物质,然后接种粗糙脉孢菌(N.crassa)、发酵乳杆菌(L.fermentum)进行一期发酵,然后再添加步骤(2)中所述的S.pombe-pREP-lip、胞外分泌锰过氧化物酶的S.pombe-pREP-mnp以及产朊假丝酵母(C.utilis)进行二期发酵,所得产物即为高蛋白含量、低木质纤维素含量的优质生物饲料原料。
优选的,步骤(2)中所述重组穿梭质粒为pREP-lip。
优选的,步骤(2)中所述大肠杆菌是6-磷酸葡萄糖胺合成酶缺陷型大肠杆菌,需要在含有葡萄糖胺的培养基上生长,无抗性基因,具有生物安全性。
优选的,步骤(2)中所述缺陷型粟酒裂殖酵母是6-磷酸果糖氨基转移酶缺陷型粟酒裂殖酵母,需要在含葡萄糖胺的培养基中继续生长,是具有生物安全性的食品级酵母菌。
优选的,步骤(3)中所述胞外分泌锰过氧化物酶的S.pombe-pREP-mnp制备步骤为:
S1、将来源于白囊耙齿菌(Irpex lacteus)的锰过氧化物酶基因序列根据粟酒裂殖酵母密码子偏爱性进行优化,得到编码锰过氧化物酶的基因,核苷酸序列如SEQ ID NO.2所示;
S2、以S1中所述的编码锰过氧化物酶的基因为模板,进行PCR扩增,与穿梭质粒载体pREP连接后转化到大肠杆菌感受态细胞中,抽提获得重组穿梭质粒后,利用电转化法导入到缺陷型粟酒裂殖酵母感受态细胞中,得到重组锰过氧化物酶酵母工程菌,即 S.pombe-pREP-mnp。
优选的,步骤(3)中所述N.crassa、S.pombe-pREP-lip、S.pombe-pREP-mnp、L.fermentum 和C.utilis的总接种量为1-15%,所述N.crassa、S.pombe-pREP-lip、S.pombe-pREP-mnp、 L.fermentum和C.utilis的接种体积比例为2:2:2:1:1;所述菌种的接种使用的均为活化培养后的发酵种子液;所述一期、二期发酵体系的含水量均为40%-70%。
优选的,步骤(3)中所述氮源为尿素和硫酸铵,其中尿素添加量为小麦秸秆质量的0.5%-4%;硫酸铵的添加量为小麦秸秆质量的1%-4%。
优选的,步骤(3)中所述葡萄糖添加量为小麦秸秆质量的0.1%-2%;所述金属离子为MnSO4、MgSO4、K2HPO4和ZnSO4,其中ZnSO4添加量为小麦秸秆质量的0.5%-4%, K2HPO4添加量为小麦秸秆质量的0.1%-1%,MgSO4添加量为小麦秸秆质量的1%-4%, MnSO4添加量为小麦秸秆质量的0.1%-1%;葡萄糖氧化酶(GOD)添加量为小麦秸秆质量的1%-4%,漆酶(Lac)添加量为小麦秸秆质量的1%-4%;所述酸类小分子物质为柠檬酸和丙二酸,其中柠檬酸添加量为小麦秸秆质量的1%-4%,丙二酸添加量为小麦秸秆质量的 0.1%-1%。
优选的,步骤(3)中所述漆酶的酶活力为10 000U/g,葡萄糖氧化酶的酶活力为200U/g。
优选的,步骤(3)中所述一期发酵温度为25-37℃,发酵时间为1-4d,二期发酵温度为25-37℃,发酵时间为1-4d。
其中,漆酶和葡萄糖氧化酶购自夏盛(北京)生物技术开发有限公司,为普通饲料级商业化酶制剂;S.pombe-pREP-mnp为本实验室构建;粗糙脉孢菌(CGMCC3.1613)、产阮假丝酵母(CGMCC2.3047)和发酵乳杆菌(CGMCC1.20290)购自中国普通微生物菌种保藏管理中心。
本发明的优点和技术效果是:
(1)自然界中白囊耙齿菌产酶条件难控制,产酶量低,且生长速度缓慢,直接将其用于发酵会提升发酵过程中污染的风险,同时增加发酵的时间成本和生产成本。本发明提供一种具有生物安全性的食品级重组木质素过氧化物酶重组酵母工程菌,可以快速获得大量木质素过氧化物酶,实现木质素过氧化物酶大规模生产。同时,本发明构建的重组酵母工程菌选用营养缺陷型筛选标记,其本身不含任何抗性基因,保证了发酵饲料菌种的生物安全性。
(2)制备秸秆生物饲料的关键在于如何提升其营养价值,降低木质素含量、提升真蛋白含量是提升营养价值的重要内容。一直以来,制备秸秆饲料研究的焦点多集中于降解其中木质纤维素,提高产物适口性;在降解木质纤维素方面,当前的研究多重视纤维素、半纤维素降解,以木质素降解为突破口的研究报道不多。多因素协同发酵以木质素降解为突破口、同步提升产物真蛋白含量的研究更少。本发明首次协同N.crassa、S.pombe-pREP-lip、 S.pombe-pREP-mnp和L.fermentum、C.utilis等发酵菌株,并与多种酶制剂和小分子物质联合,采用分步发酵的方式协同发酵小麦秸秆,提高发酵产物真蛋白含量、降解其中木质纤维素。发酵产物木质素少,真蛋白多,满足优质生物饲料标准。
(3)本发明提供了一种安全、高效的制备优质秸秆生物饲料的发酵工艺,短时间内提升发酵饲料产品品质,为小麦秸杆发酵转化为高品质生物饲料产业化提供保障。
附图说明
图1是载体和目的基因PCR产物片段电泳图。
图2是S.pombe-pREP-lip胞外分泌蛋白SDS-PAGE电泳图。
图3是S.pombe-pREP-lip胞外分泌蛋白Western Blot图。
图4是S.pombe-pREP-lip胞外分泌蛋白随培养时间变化图。
图5是S.pombe-pREP-lip胞外LiP活性随培养时间变化图。
图6温度对重组LiP酶活力的影响。
图7pH对重组LiP酶活力的影响。
图8重组LiP的温度耐受性。
图9重组LiP的pH耐受性。
具体实施方式
以下实施例中进一步定义本发明,根据以上的描述和这些实施例,本领域技术人员可以确定本发明的基本特征,并且在不偏离本发明精神和范围的情况下,可以对本发明做出各种修改和改变,以使其使用各种用途和条件。下述实施例中所使用的实验原料如无特殊说明,均可通过商业途径得到。除特殊注明外,本发明所采用的均为该领域现有技术。
实施例1:
将JGI数据库(https://jgi.doe.gov)Irpex lacteus CCBAS Fr.238 617/93v1.0中LiP(Protein ID:1589173)的氨基酸序列如SEQ ID NO.3所示。将其编码序列按照粟酒裂殖酵母密码子偏爱性进行密码子优化,密码子优化后的LiP基因的核苷酸序列如SEQ NO.1所示。
设计引物扩增目的基因序列,正向引物序列如SEQ ID NO.4所示,反向引物序列如SEQ ID NO.5所示,PCR扩增目的基因线性化片段(条件为:94℃30s,60℃30s,72℃60 s,30个循环)。以穿梭质粒pREP为模板设计引物,正向引物序列如SEQ ID NO.6所示,反向引物序列如SEQ ID NO.7所示,PCR扩增载体线性化片段(98℃10s,60℃15s, 72℃50s,30个循环)。回收目的片段并测定产物浓度。图1为载体和目的基因的PCR 产物片段图,根据ClonExpress MultiS One step Cloning Kit试剂盒连接上述PCR产物片段。连接产物导入6-磷酸葡萄糖胺合成酶缺陷型大肠杆菌中,在LB固体培养基(不含葡萄糖胺)上筛选阳性转化子,从转化子中提取质粒并测序,将正向***且无基因突变的质粒命名为pREP-lip。pREP-lip经醋酸锂转化法导入6-磷酸果糖氨基转移酶缺陷型粟酒裂殖酵母中,在YES(不含葡萄糖胺)固体培养基筛选阳性转化子,提取阳性转化子基因组DNA,设计特异性引物,正向引物序列如SEQ ID NO.8所示,反向引物序列如SEQ ID NO.9所示,进行基因组PCR(条件为:94℃30s,60℃30s,72℃60s,30个循环),PCR产物经测序验证未发生基因突变的阳性转化子即为重组木质素过氧化物酶酵母工程菌,命名为 S.pombe-pREP-lip。
所述6-磷酸葡萄糖胺合成酶缺陷型大肠杆菌为E.coli strain DH10B利用I-Red介导的重组酶***去除6-磷酸葡萄糖胺合成酶基因(glmS)获得;所述6-磷酸果糖氨基转移酶缺陷型粟酒裂殖酵母为S.pombe YHL6381(h+,his-D1,leu l-32,ura4-D18,ade6-M210)利用kanmx6介导的基因敲除和Cre-loxP标记移除***敲除6-磷酸果糖氨基合成酶基因(gfa) 获得;以上两种缺陷型菌株的构建方法参考Guogan Wu等 (https://doi.org/10.1371/journal.pone.0017082)。
实施例2:
本实施例测定重组LiP的酶学性质。
将实施例1中得到的S.pombe-pREP-lip在YES液体培养基中扩大培养,收集培养液。培养液经超滤管浓缩后,SDS-PAGE和Western Blot验证粟酒裂殖酵母胞外分泌目的蛋白,图2为粟酒裂殖酵母胞外分泌蛋白SDS-PAGE电泳图,图3为粟酒裂殖酵母胞外分泌蛋白Western Blot图;结合图2和图3可以看出,重组LiP在粟酒裂殖酵母中胞外表达,且表观分子量为37kDa左右。胞外分泌蛋白量随培养时间的变化如图4所示,从图中可以看出S.pombe-pREP-lip胞外分泌蛋白量在培养第6d达到峰值,超过6d以后胞外分泌蛋白量呈下降趋势,图5为S.pombe-pREP-lip胞外分泌LiP酶活力随培养时间变化曲线,从图中可知,胞外LiP酶活力与胞外分泌蛋白量变化曲线基本一致,胞外LiP酶活力在培养第6d 达到峰值。
藜芦醇氧化法测定S.pombe-pREP-lip胞外液中重组LiP酶活随时间变化曲线,如图5 所示。同样的方法测定LiP最适温度、最适pH以及不同条件下的耐受性。30℃时,在不同pH条件下测定LiP酶活力,以最高值为100%,其他酶活力与最高值的比值为相对酶活力;当缓冲液pH为3.5时,在不同温度条件下测定LiP活力,以最高值为100%,其他酶活力与最高值的比值为相对酶活力;在37℃条件下,使蛋白液分别在不同pH缓冲液中保持1h,测定酶活后计算相对酶活力;将37℃、pH为3.5时的酶活力记为100%,在pH为 3.5时,使蛋白液在不同温度下保持1h,测定酶活后计算相对酶活力。重组LiP最适温度为37℃,最适pH为3.5,在20-50℃,pH为2.0-4.0范围内有较好的稳定性。
图6为重组LiP的最适pH测定图,从图中可以看出重组LiP最适pH为3.5;图7为重组LiP的最适温度测定图,从图中可以看出重组LiP最适温度为37℃;图8为重组LiP 的pH耐受性图,从图中可以看出重组LiP在pH为2.0-4.0范围相对酶活力能保持在50%以上;图9为重组LiP的温度耐受性图,从图中可以看出重组LiP在温度为20-50℃范围内相对酶活力能保持在50%以上,具有较好的温度耐受性。
一期发酵条件筛选:
实施例3:
本实施例中S.pombe-pREP-lip、S.pombe-pREP-mnp接种量为2%,N.crassa接种量为2%,L.fermentium接种量为1%,C.utilis接种量为1%;所述菌种的接种使用的均为发酵种子液;菌种的发酵种子液制备方法如下:
N.crassa:取冻存的N.crassa,接种于PDA固体培养基斜面上,30℃恒温培养箱静置培养3d至产孢丰富,在超净工作台中,8层纱布过滤,灭菌水反复冲洗PDA斜面的N.crassa成熟孢子,洗脱液即为孢子悬液,使用血球计数板计数,调节至孢子数量为1×109个/mL;接着以1%接种量将N.crassa孢子悬液接到PDA培养液中,120rpm,30℃,恒温摇床培育72h,得到N.crassa发酵种子液。
S.pombe-pREP-lip、S.pombe-pREP-mnp:在超净工作台中取少量菌液划线于YES固体培养基上,30℃恒温培养箱中静置培养3d;挑取单菌落接种于YES液体培养基中,200rpm,30℃,恒温摇床培育72h,得到S.pombe-pREP-lip发酵种子液和S.pombe-pREP-mnp发酵种子液。
L.fermentium:冻存的L.fermentium以1%接种量接种于MRS液体培养基中,37℃、静置培养至OD600为0.8-1.2,得到L.fermentium发酵种子液。
C.utilis:取冻存的C.utilis在斜面培养基上划线,30℃恒温培养箱中培养12h,从斜面培养基表面挑取单个菌落接种于液体麦芽汁培液中,180rpm、30℃恒温摇床培育12h,得到C.utilis发酵种子液。
①:称取小麦秸秆10g,控制氮源为秸秆质量1%的尿素,其中选择碳源为葡萄糖,控制葡萄糖添加量分别为秸秆质量的0.1%、0.5%、1%、2%、4%,固态发酵总含水量为60%,搅拌均匀后于30℃恒温恒湿培养箱中发酵72h。表1是不同浓度葡萄糖添加量时产物中真蛋白含量对比表。
表1不同葡萄糖添加量时发酵产物真蛋白含量
从表中可以看出,当葡萄糖添加量逐渐提升,真蛋白积累量也呈现上升趋势,当葡萄糖添加量超过1%时,真蛋白含量增长速度减慢;综合考虑认为葡萄糖最适添加量为1%。②:称取小麦秸秆10g,控制碳源为秸秆质量的1%葡萄糖,控制尿素添加量分别为秸秆质量的0.1%、0.5%、1%、2%、4%,固态发酵总含水量为60%,搅拌均匀后于30℃恒温恒湿培养箱中发酵72h;发酵结束产物真蛋白含量如表2所示:
表2不同尿素添加量时发酵产物真蛋白含量
当尿素添加量为1%时,真蛋白含量最高。当超过1%时,随着尿素添加量提高,真蛋白积累量开始减少;分析认为,发酵过程中过量的尿素分解产生氨气,进而形成铵态氮,导致pH升高,不利于菌体生长,因此需要控制尿素的添加量。
③:在葡萄糖(秸秆质量的1%)为碳源,尿素(秸秆质量的1%)为氮源的基础上,在添加尿素的基础上添加硫酸铵作为复合氮源,控制硫酸铵添加量分别为秸秆质量的:0.5%、1%、2%、4%、8%,固态发酵总含水量为60%,搅拌均匀后于30℃恒温恒湿培养箱中发酵72h。发酵结束产物真蛋白含量如表3所示:
表3不同硫酸铵添加量时发酵产物真蛋白含量
1%尿素复配2%硫酸铵时,真蛋白积累量最高;且两者复配明显高于仅使用尿素(1%) 作为氮源时发酵积累的真蛋白量。
实施例4:
在葡萄糖(秸秆质量的1%)为碳源,尿素(秸秆质量的1%)、硫酸铵(秸秆质量的2%)为氮源的基础上,测定不同金属离子对发酵小麦秸秆产真蛋白的影响。
发酵过程中分别添加不同量的MgSO4(添加量分别为秸秆质量的0.1%、0.5%、1%、 2%、4%)、ZnSO4(添加量分别为秸秆质量的0.1%、0.5%、1%、2%、4%)、K2HPO4 (添加量分别为秸秆质量的0.1%、0.5%、1%、2%、4%)、MnSO4(添加量分别为秸秆质量的0.1%、0.5%、1%、2%、4%),固态发酵总含水量为60%,搅拌均匀后于30℃恒温恒湿培养箱中发酵72h,发酵结束,产物中真蛋白含量如表4:
表4不同金属离子添加量时发酵产物真蛋白含量
MgSO4添加量为2%时对产真蛋白最有效,ZnSO4添加量为1%时对产真蛋白最有效,K2HPO4添加量为0.5%时对产真蛋白最有效,MnSO4添加量为0.5%时对产真蛋白最有效。
实施例5:
在葡萄糖(秸秆质量的1%)为碳源,尿素(秸秆质量的1%)、硫酸铵(秸秆质量的2%)为氮源的基础上,添加不同添加量的ZnSO4、K2HPO4和MgSO4复配,固态发酵总含水量为60%,搅拌均匀后于30℃恒温恒湿培养箱中发酵72h。发酵结束产物中真蛋白含量如下表5所示:
表5不同发酵条件下产物真蛋白含量
当发酵体系中同时添加以上三种金属离子,且ZnSO4添加量为秸秆质量的1%,K2HPO4添加量为秸秆质量的0.5%,MgSO4添加量为秸秆质量的2%时,发酵产物中真蛋白含量为 11.06%,相较于未发酵原料(真蛋白含量为1.81%),发酵产物真蛋白含量提升了511%。
实施例6:
在前期实施例3-5最优条件的基础上,即在葡萄糖(秸秆质量的1%)为碳源,尿素(秸秆质量的1%)、硫酸铵(秸秆质量的2%)为氮源,MgSO4添加量为秸秆质量的2%,ZnSO4添加量为秸秆质量的1%,K2HPO4添加量为秸秆质量的0.5%的基础上;分别添加GOD、 Lac、柠檬酸、丙二酸;并控制发酵过程中GOD(添加量为秸秆质量的0.1%、0.5%、1%、2%、4%)、Lac(添加量为秸秆质量的0.1%、0.5%、1%、2%、4%)、柠檬酸(添加量为秸秆质量的0.1%、0.5%、1%、2%、4%)、丙二酸(添加量为秸秆质量的0.1%、0.5%、1%、 2%、4%)添加量不同,固态发酵总含水量为60%,搅拌均匀后于30℃恒温恒湿培养箱中发酵72h。
发酵结束后测定发酵产物中木质纤维素降解率,结果如表6所示:
表6不同发酵条件下木质素降解率
随着GOD、Lac添加量逐渐增加,产物中木质素降解率均呈现先增加后下降的趋势,且二者均为添加量为2%时木质素降解率达到最大值;当柠檬酸添加量在0.1%到2%范围内,随着柠檬酸添加量逐渐增大,木质素降解率随之提升,当柠檬酸添加量在2%-4%范围内,随着柠檬酸添加量增加,木质素降解率提升不显著;丙二酸在添加量为0.5%时,木质素降解率最高,随着丙二酸添加量继续增加,木质素降解率开始下降。
实施例7:
在葡萄糖(秸秆质量的1%)为碳源,尿素(秸秆质量的1%)、硫酸铵(秸秆质量的2%)为氮源,MgSO4添加量为秸秆质量的2%,ZnSO4添加量为秸秆质量的1%,K2HPO4添加量为秸秆质量的0.5%的基础上;共同添加GOD、Lac、丙二酸;其中不同添加量的 GOD、Lac、丙二酸复配发酵小麦秸秆,固态发酵总含水量为60%,搅拌均匀后于30℃恒温恒湿培养箱中发酵72h。发酵产物木质素降解率、真蛋白含量如表7所示:
表7不同发酵条件下木质素讲解率
发酵体系中同时添加GOD、Lac、丙二酸,且GOD添加量为秸秆质量的2%,Lac添加量为秸秆质量的2%,丙二酸添加量为秸秆质量的0.5%时,发酵产物中木质素降解率为65.96%,此时真蛋白含量11.13%,比秸秆原始真蛋白增加了514.9%。
说明:以上实施例仅用以说明本发明而非限制本发明所描述的技术方案。因此,尽管本说明书参考上述的各个实施例对本发明进行了详细的说明,但是本领域的普通技术人员应当理解,仍然可以对本发明进行修改或同等替换,而一切不脱离本发明的精神和范围的技术方案及其改进,其均应涵盖在本发明的权利要求范围内。
本发明涉及序列如下:
1.SEQ ID NO.1
ATGCATCATCATCATCATCATCCTGCTCCTCAAGATGCTCAAGTTAATTGTGGTGG TGGTCGTTTTGTTAAAAATGCTGCTTGTTGTGCTTGGTTTCCTGTTTTGGATGATATTC AAGAAAATTTGTTTTCTGGTTCTTTATGTGCTGAAGAAGCTCATGAAGCTCTTCGTTT GACTTTTCATGATGCTATTGCTGCTGAACGTGAGGGTAAATTTGGTGGTGGTGGTGCT GATGGTTCTATTCTTGCTTTTTCTGATATTGAAACTTCTTTTGCTGCTAACTTTGGTTTGGATTTTACTACTGAAGCTTTTATTCCTTTTGCTCTTGCTCATAAAGTTTCTTTTGGAGAT TTTGTTCAATTTGCTGGTGCTGTTGGTGTTTCTAATTGTATTGGTGGTCCTCGTTTACA ATTTCTTGCTGGTCGTTCTAATAATTCTCGTCCTTCTCCTGATAATCTTGTTCCTGAACC TACTGATTCTGCTGAAAAGATTTTTGAACGTTTGCAAGATATTGGTTTTTCTCCTATTG AAGTTGTTCATCTTCTTACTGCTCATACTGTTTCTGCTCAATATGAAGTTGATACTGATG TTGCTGGTTCTCCTTTTGATTCTACTCCTTCTTCTTTTGATAACCAATTTTTCGTTGAAT CTTTGCTTAAGGGTACTGCTTTTACTGGTAATGGTCAAGGTGGTGAAGTTACTTCTCCT ATTCCTGGTGAATTTCGTCTTCAATCTGATTTTGCTATTTCTCGTGATTCTCGTACTGCT TGTGAATGGCAATCTCTTGTTACTAATCATGCTAACATGGTTTCTAAGTTTGAAACTGT TATGGCTAAGCTTGCTACTGTTGGTCAAAACCCTAACAATTTGATTGATTGTTCTGATG TTATTCCTGTTCCTCCTGCTGCTAAAGTTACTACTGGTTCTTTTCCTCCTGGTAAATCTA AAGCTGATGTTCAATCTGCTTGTGCTGCTACTCCTTTTCCTAATCTTGCTACTCAACCT GGTCCTGTTACTTCTGTTCTTCCTGTTACTGCTTAA
2.SEQ ID NO.2
ATGGTGCGTCGTGTTACCTGCCCGGACGGTGTGAACACCGCGACCAACGCGGCG TGCTGCAGCCTGTTTGCGGTTCGTGACGATATCCAGCAAAACCTGTTTGACAACGGC CAGTGCGGCGAGGATGTGCACGAAAGCTTCCGTCTGAGCTTTCACGACGCGATCGGC ATTAGCCCGAAGATTGCGGCGACCGGTCAATTTGGTGGCGGTGGCGCGGATGGCAGC ATCATTCTGTTCGAGGAAATTGAGACCAACTTTCACGCGAACATCGGTGTGGACGAG ATTGTTGATGAACAGAAACCGTTTATCGCGCGTCACAACATTACCCCGGGCGACTTCA TCCAATTTGCGGCGGCGGTGGGCGTTAGCAACTGCCCGGGTGCGCCGCGTCTGGACT TCTTTCTGGGTCGTCCGGCGGCGACCCAGCCGGCGCCGGATAAGACCGTGCCGGAGC CGTTCGACACCGTTGATACCATTCTGGAACGTTTCGCGGATGCGGGTAACTTTACCCC GGCGGAAGTGGTTGCGCTGCTGGTTAGCCACACCATTGCGGCGGCGGACGAAGTTGA TCCGACCATTCCGGGTACCCCGTTTGACAGCACCCCGGAAGTGTTTGATAGCCAGTTC TTTGTTGAAACCCAACTGCGTGGTACCGGTTTTCCGGGTACCGCGGGTAACCAAGGT GAGGTGGAAAGCCCGCTGGCGGGTGAACTGCGTCTGCAAAGCGACAGCGAACTGGCGCGTGATGCGCGTACCGCGTGCGAGTGGCAGAGCTTTGTTGGTAACCAGCAAAAGAT CCAGACCGCGTTCAAGGCGGCGTTTCAAAAAATGGCGGTGCTGGGCGTTGACACCA GCAAAATGGTGGATTGCAGCGAGCTGATCCCGGTTCCGCCGGAACTGAAGATTACCG CGGCGCACTTCCCGGCGGGTAAAACCAACGCGGACGTTGAACAAGCGTGCGCGAGCACCCCGTTTCCGACCCTGAGCACCGATCCGGGTCCGGCGACCAGCGTGGCGCCGGTT CCGCCGAGCTAA
3.SEQ ID NO.3
MAFQSLFTLVALAAAVVAVPAPQDAQVNCGGGRFVKNAACCAWFPVLDDIQENLF SGSLCAEEAHEALRLTFHDAIAAEREGKFGGGGADGSILAFSDIETSFAANFGLDFTTEAF IPFALAHKVSFGDFVQFAGAVGVSNCIGGPRLQFLAGRSNNSRPSPDNLVPEPTDSAEKIF ERLQDIGFSPIEVVHLLTAHTVSAQYEVDTDVAGSPFDSTPSSFDNQFFVESLLKGTAFTG NGQGGEVTSPIPGEFRLQSDFAISRDSRTACEWQSLVTNHANMVSKFETVMAKLATVGQ NPNNLIDCSDVIPVPPAAKVTTGSFPPGKSKADVQSACAATPFPNLATQPGPVTSVLPVTA *
4.SEQ ID NO.4
人工序列:5-TCCTTTTACCCCCCGGATCCTTAATGATGATGATGAT GATG AGCAGTAACAGGAAGAACAGAAGTAACA-3;
5.SEQ ID NO.5
人工序列:5-TCGTATCCGCTCAGTTTATGATGCCTGCTCCTCA
AGATGCTCAA-3;
6.SEQ ID NO.6
人工序列:5-GGATCCGGGGGGTAAAAGGAAT-3;
7.SEQ ID NO.7
人工序列:5-CATAAACTGAGCGGATACGACG-3;
8.SEQ ID NO.8
人工序列:5-GCAGCAGGAGGAACAGGAAT-3;
9.SEQ ID NO.9
人工序列:5-GTGGTGGTGCTGATGGTTCT-3 。
序列表
<110> 江苏大学
<120> 一种协同发酵小麦秸秆制备高品质生物饲料的方法
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1041
<212> DNA
<213> 白囊耙齿菌(Irpex lacteus)
<400> 1
atgcatcatc atcatcatca tcctgctcct caagatgctc aagttaattg tggtggtggt 60
cgttttgtta aaaatgctgc ttgttgtgct tggtttcctg ttttggatga tattcaagaa 120
aatttgtttt ctggttcttt atgtgctgaa gaagctcatg aagctcttcg tttgactttt 180
catgatgcta ttgctgctga acgtgagggt aaatttggtg gtggtggtgc tgatggttct 240
attcttgctt tttctgatat tgaaacttct tttgctgcta actttggttt ggattttact 300
actgaagctt ttattccttt tgctcttgct cataaagttt cttttggaga ttttgttcaa 360
tttgctggtg ctgttggtgt ttctaattgt attggtggtc ctcgtttaca atttcttgct 420
ggtcgttcta ataattctcg tccttctcct gataatcttg ttcctgaacc tactgattct 480
gctgaaaaga tttttgaacg tttgcaagat attggttttt ctcctattga agttgttcat 540
cttcttactg ctcatactgt ttctgctcaa tatgaagttg atactgatgt tgctggttct 600
ccttttgatt ctactccttc ttcttttgat aaccaatttt tcgttgaatc tttgcttaag 660
ggtactgctt ttactggtaa tggtcaaggt ggtgaagtta cttctcctat tcctggtgaa 720
tttcgtcttc aatctgattt tgctatttct cgtgattctc gtactgcttg tgaatggcaa 780
tctcttgtta ctaatcatgc taacatggtt tctaagtttg aaactgttat ggctaagctt 840
gctactgttg gtcaaaaccc taacaatttg attgattgtt ctgatgttat tcctgttcct 900
cctgctgcta aagttactac tggttctttt cctcctggta aatctaaagc tgatgttcaa 960
tctgcttgtg ctgctactcc ttttcctaat cttgctactc aacctggtcc tgttacttct 1020
gttcttcctg ttactgctta a 1041
<210> 2
<211> 1035
<212> DNA
<213> 白囊耙齿菌(Irpex lacteus)
<400> 2
atggtgcgtc gtgttacctg cccggacggt gtgaacaccg cgaccaacgc ggcgtgctgc 60
agcctgtttg cggttcgtga cgatatccag caaaacctgt ttgacaacgg ccagtgcggc 120
gaggatgtgc acgaaagctt ccgtctgagc tttcacgacg cgatcggcat tagcccgaag 180
attgcggcga ccggtcaatt tggtggcggt ggcgcggatg gcagcatcat tctgttcgag 240
gaaattgaga ccaactttca cgcgaacatc ggtgtggacg agattgttga tgaacagaaa 300
ccgtttatcg cgcgtcacaa cattaccccg ggcgacttca tccaatttgc ggcggcggtg 360
ggcgttagca actgcccggg tgcgccgcgt ctggacttct ttctgggtcg tccggcggcg 420
acccagccgg cgccggataa gaccgtgccg gagccgttcg acaccgttga taccattctg 480
gaacgtttcg cggatgcggg taactttacc ccggcggaag tggttgcgct gctggttagc 540
cacaccattg cggcggcgga cgaagttgat ccgaccattc cgggtacccc gtttgacagc 600
accccggaag tgtttgatag ccagttcttt gttgaaaccc aactgcgtgg taccggtttt 660
ccgggtaccg cgggtaacca aggtgaggtg gaaagcccgc tggcgggtga actgcgtctg 720
caaagcgaca gcgaactggc gcgtgatgcg cgtaccgcgt gcgagtggca gagctttgtt 780
ggtaaccagc aaaagatcca gaccgcgttc aaggcggcgt ttcaaaaaat ggcggtgctg 840
ggcgttgaca ccagcaaaat ggtggattgc agcgagctga tcccggttcc gccggaactg 900
aagattaccg cggcgcactt cccggcgggt aaaaccaacg cggacgttga acaagcgtgc 960
gcgagcaccc cgtttccgac cctgagcacc gatccgggtc cggcgaccag cgtggcgccg 1020
gttccgccga gctaa 1035
<210> 3
<211> 358
<212> PRT
<213> 白囊耙齿菌(Irpex lacteus)
<400> 3
Met Ala Phe Gln Ser Leu Phe Thr Leu Val Ala Leu Ala Ala Ala Val
1 5 10 15
Val Ala Val Pro Ala Pro Gln Asp Ala Gln Val Asn Cys Gly Gly Gly
20 25 30
Arg Phe Val Lys Asn Ala Ala Cys Cys Ala Trp Phe Pro Val Leu Asp
35 40 45
Asp Ile Gln Glu Asn Leu Phe Ser Gly Ser Leu Cys Ala Glu Glu Ala
50 55 60
His Glu Ala Leu Arg Leu Thr Phe His Asp Ala Ile Ala Ala Glu Arg
65 70 75 80
Glu Gly Lys Phe Gly Gly Gly Gly Ala Asp Gly Ser Ile Leu Ala Phe
85 90 95
Ser Asp Ile Glu Thr Ser Phe Ala Ala Asn Phe Gly Leu Asp Phe Thr
100 105 110
Thr Glu Ala Phe Ile Pro Phe Ala Leu Ala His Lys Val Ser Phe Gly
115 120 125
Asp Phe Val Gln Phe Ala Gly Ala Val Gly Val Ser Asn Cys Ile Gly
130 135 140
Gly Pro Arg Leu Gln Phe Leu Ala Gly Arg Ser Asn Asn Ser Arg Pro
145 150 155 160
Ser Pro Asp Asn Leu Val Pro Glu Pro Thr Asp Ser Ala Glu Lys Ile
165 170 175
Phe Glu Arg Leu Gln Asp Ile Gly Phe Ser Pro Ile Glu Val Val His
180 185 190
Leu Leu Thr Ala His Thr Val Ser Ala Gln Tyr Glu Val Asp Thr Asp
195 200 205
Val Ala Gly Ser Pro Phe Asp Ser Thr Pro Ser Ser Phe Asp Asn Gln
210 215 220
Phe Phe Val Glu Ser Leu Leu Lys Gly Thr Ala Phe Thr Gly Asn Gly
225 230 235 240
Gln Gly Gly Glu Val Thr Ser Pro Ile Pro Gly Glu Phe Arg Leu Gln
245 250 255
Ser Asp Phe Ala Ile Ser Arg Asp Ser Arg Thr Ala Cys Glu Trp Gln
260 265 270
Ser Leu Val Thr Asn His Ala Asn Met Val Ser Lys Phe Glu Thr Val
275 280 285
Met Ala Lys Leu Ala Thr Val Gly Gln Asn Pro Asn Asn Leu Ile Asp
290 295 300
Cys Ser Asp Val Ile Pro Val Pro Pro Ala Ala Lys Val Thr Thr Gly
305 310 315 320
Ser Phe Pro Pro Gly Lys Ser Lys Ala Asp Val Gln Ser Ala Cys Ala
325 330 335
Ala Thr Pro Phe Pro Asn Leu Ala Thr Gln Pro Gly Pro Val Thr Ser
340 345 350
Val Leu Pro Val Thr Ala
355
<210> 4
<211> 69
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tccttttacc ccccggatcc ttaatgatga tgatgatgat gagcagtaac aggaagaaca 60
gaagtaaca 69
<210> 5
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tcgtatccgc tcagtttatg atgcctgctc ctcaagatgc tcaa 44
<210> 6
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ggatccgggg ggtaaaagga at 22
<210> 7
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cataaactga gcggatacga cg 22
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gcagcaggag gaacaggaat 20
<210> 9
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gtggtggtgc tgatggttct 20
Claims (10)
1.一种协同发酵小麦秸秆制备高品质生物饲料的方法,其特征在于,步骤如下:
(1)将来源于白囊耙齿菌的木质素过氧化物酶编码序列按照粟酒裂殖酵母密码子偏爱性进行密码子优化,获得编码木质素过氧化物酶的基因,核苷酸序列如SEQ ID NO.1所示;
(2)以步骤(1)中所述编码木质素过氧化物酶的基因为模板,进行PCR扩增,与穿梭质粒载体pREP线性化片段连接后转化到大肠杆菌受体菌中,得到重组质粒经醋酸锂转化法导入缺陷型粟酒裂殖酵母中,获得重组木质素过氧化物酶酵母工程菌,为S.pombe-pREP-lip;
(3)协同降解发酵小麦秸秆转化为高品质生物饲料原料:首先以小麦秸秆为原料,添加葡萄糖、氮源、漆酶和葡萄糖氧化酶以及金属离子、酸类小分子物质,然后接种粗糙脉孢菌、发酵乳杆菌进行一期发酵,然后再添加步骤(2)中所述的S.pombe-pREP-lip、胞外分泌锰过氧化物酶的S.pombe-pREP-mnp以及产朊假丝酵母进行二期发酵,发酵后即得生物饲料。
2.根据权利要求1所述的协同发酵小麦秸秆制备高品质生物饲料的方法,其特征在于,步骤(2)中,所述重组穿梭质粒为pREP-lip;所述的大肠杆菌是6-磷酸葡萄糖胺合成酶缺陷型大肠杆菌。
3.根据权利要求1所述的协同发酵小麦秸秆制备高品质生物饲料的方法,其特征在于,步骤(2)中所述的缺陷型粟酒裂殖酵母是6-磷酸果糖氨基转移酶缺陷型粟酒裂殖酵母。
4.根据权利要求1所述的协同发酵小麦秸秆制备高品质生物饲料的方法,其特征在于,步骤(3)中所述胞外分泌锰过氧化物酶的S.pombe-pREP-mnp制备步骤为:
S1、将来源于白囊耙齿菌的锰过氧化物酶基因序列根据粟酒裂殖酵母密码子偏爱性进行优化,得到编码锰过氧化物酶的基因,核苷酸序列如SEQ ID NO.2所示;
S2、以S1中所述的编码锰过氧化物酶的基因为模板,进行PCR扩增,与穿梭质粒载体pREP连接后转化到大肠杆菌感受态细胞中,抽提获得重组穿梭质粒后,利用电转化法导入到缺陷型粟酒裂殖酵母感受态细胞中,得到重组锰过氧化物酶酵母工程菌,即S.pombe-pREP-mnp。
5.根据权利要求1所述的协同发酵小麦秸秆制备高品质生物饲料的方法,其特征在于,步骤(3)中所述N.crassa、S.pombe-pREP-lip、S.pombe-pREP-mnp、L.fermentum和C.utilis的总接种量为1-15%,所述N.crassa、S.pombe-pREP-lip、S.pombe-pREP-mnp、L.fermentum和C.utilis的接种体积比例为2:2:2:1:1;所述菌种的接种使用的均为活化培养后的发酵种子液;所述一期、二期发酵体系的总含水量均为40%-70%。
6.根据权利要求1所述的协同发酵小麦秸秆制备高品质生物饲料的方法,其特征在于,步骤(3)中所述氮源为尿素和硫酸铵,其中尿素添加量为小麦秸秆质量的0.5%-4%;硫酸铵的添加量为小麦秸秆质量的1%-4%。
7.根据权利要求1所述的协同发酵小麦秸秆制备高品质生物饲料的方法,其特征在于,步骤(3)中所述葡萄糖添加量为小麦秸秆质量的0.1%-2%;所述金属离子为MnSO4、MgSO4、K2HPO4和ZnSO4,其中ZnSO4添加量为小麦秸秆质量的0.5%-4%,K2HPO4添加量为小麦秸秆质量的0.1%-1%,MgSO4添加量为小麦秸秆质量的1%-4%,MnSO4添加量为小麦秸秆质量的0.1%-1%;葡萄糖氧化酶添加量为小麦秸秆质量的1%-4%,漆酶添加量为小麦秸秆质量的1%-4%;所述酸类小分子物质为柠檬酸和丙二酸,其中柠檬酸添加量为小麦秸秆质量的1%-4%,丙二酸添加量为小麦秸秆质量的0.1%-1%。
8.根据权利要求1所述的协同发酵小麦秸秆制备高品质生物饲料的方法,其特征在于,步骤(3)中所述漆酶的酶活力为10 000U/g,葡萄糖氧化酶的酶活力为200U/g。
9.根据权利要求8所述的协同发酵小麦秸秆制备高品质生物饲料的方法,其特征在于,步骤(3)中所述一期发酵温度为25-37℃,发酵时间为1-4d,二期发酵温度为25-37℃,发酵时间为1-4d。
10.根据权利要求1-9任一所述方法制备的高品质生物饲料。
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