CN113215135A - 一种内切葡聚糖酶在卷烟原料改良中的应用 - Google Patents
一种内切葡聚糖酶在卷烟原料改良中的应用 Download PDFInfo
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Abstract
本发明提供一种内切葡聚糖酶在卷烟原料改良中的应用,具体地,本发明提供一种烟草源枯草芽孢杆菌(Bacillus subtilis)的内切葡聚糖酶Cel‑FX1,并利用基因工程技术使得所述内切葡聚糖酶在大肠杆菌中获得高效表达。相较于天然表达的多肽以及商品化纤维素酶,经本发明的多肽或者含有所述多肽的全培养液配制品或细胞培养组合物处理的烟草原料中纤维素含量显著降低,而还原糖含量明显提高,常规化学成分(例如,烟碱、总氮、淀粉和K)含量同样有下降趋势,并且感官质量也有明显改善。
Description
技术领域
本发明属于烟草加工领域,具体涉及一种内切葡聚糖酶在卷烟原料改良中的应用。
背景技术
纤维素是烟叶的主要成份,可增加烟叶持火力同时会带来枯焦气、木质气等杂气,对吸食口感及烟草本香的透发有显著的负面作用。此外,燃烧过程热解产生的稠环芳烃(PAH)有致癌作用,降低烟叶中的纤维素含量既可以有效提升吸食品质,又可以降低烟气中的有害成份。
现有技术主要集中于利用商品化酶制剂处理烟叶原料,通过纤维素酶或者复合酶制剂处理卷烟原料进而改善原料品质,如CN202010982036.X,CN102631021.A;或者利用产纤维素酶的菌株或其发酵液处理卷烟原料,如CN201710343989.X,CN201611261221.X;而目前商品化酶制剂由于保质及剂型等诸多因素,酶制剂中含有大量辅料,处理效果的稳定性差;使用菌株直接处理原料,过程可控性难把握。
发明内容
本发明提供一种烟草源枯草芽孢杆菌(Bacillus subtilis)的内切葡聚糖酶Cel-FX1,并利用基因工程技术使得所述内切葡聚糖酶在大肠杆菌中获得高效表达。相较于天然表达的多肽以及商品化纤维素酶,经本发明的多肽或者含有所述多肽的全培养液配制品或细胞培养组合物处理的烟草原料中纤维素含量显著降低,而还原糖含量明显提高,常规化学成分(例如,烟碱、总氮、淀粉和K)含量同样有下降趋势,并且感官质量也有明显改善。
因此,在一个方面,本发明提供一种从枯草芽孢杆菌(Bacillus subtilis)中分离的多肽在烟草原料加工或者降低烟草原料中纤维素含量中的用途,其中所述多肽具有纤维素分解活性和/或内切葡聚糖酶活性。
在另一个方面,本发明提供一种多肽在烟草原料加工或者降低烟草原料中纤维素含量中的用途,其中所述多肽具有纤维素分解活性和/或内切葡聚糖酶活性,该多肽具有选自以下的氨基酸序列:
(1)如SEQ ID NO:1所示的氨基酸序列;
(2)与SEQ ID NO:1具有至少80%同一性,优选至少85%同一性、至少90%同一性、至少95%同一性、或至少99%同一性的氨基酸序列;
(3)与SEQ ID NO:1相异在于一个或多个氨基酸残基的置换、缺失或添加的氨基酸序列;
(4)包含如SEQ ID NO:2所示的氨基酸序列,和/或SEQ ID NO:3所示的氨基酸序列;
(5)包含与SEQ ID NO:2具有至少80%同一性,优选至少85%同一性、至少90%同一性、至少95%同一性、或至少99%同一性的氨基酸序列,并且所述氨基酸序列为催化纤维素分解的催化结构域;和/或与SEQ ID NO:3具有至少80%同一性,优选至少85%同一性、至少90%同一性、至少95%同一性、或至少99%同一性的氨基酸序列,并且所述氨基酸序列为结合纤维素底物的结合结构域;和,
(6)包含与SEQ ID NO:2相异在于一个或多个氨基酸残基的置换、缺失或添加的氨基酸序列,和/或与SEQ ID NO:3相异在于一个或多个氨基酸残基的置换、缺失或添加的氨基酸序列。
在另一个方面,本发明提供一种全培养液配制品或细胞培养组合物在烟草原料加工或者降低烟草原料中纤维素含量中的用途,其中所述的全培养液配制品或细胞培养组合物通过以下方法制备得到:
利用宿主细胞诱导表达前文所述的多肽,收集所述多肽,任选地,对收集得到的多肽进行纯化,得到所述全培养液配制品或细胞培养组合物。
在一些实施方案中,所述全培养液配制品或细胞培养组合物酶活力为100-1000U/mL,例如,100-500U/mL,100-400U/mL,100-300U/mL,或100-200U/mL。
在一些实施方案中,所述全培养液配制品或细胞培养组合物中含有0.1-2.0mg/mL所述多肽,例如,含有0.1-1.5mg/mL,0.1-1.0mg/mL,0.1-0.8mg/mL,0.1-0.6mg/mL,0.1-0.4mg/mL,0.2-0.3mg/mL所述多肽。
在一些实施方案中,所述宿主细胞为大肠杆菌,例如大肠杆菌BL21。
在另一个方面,本发明提供一种处理烟草原料或者降低烟草原料中纤维素含量的方法,其包括用前文所述的多肽或全培养液配制品或细胞培养组合物处理所述烟草原料的步骤。
在一些实施方案中,所述方法的特征在于下述的一项或多项:
(1)以50-500U/Kg的量添加所述多肽或全培养液配制品或细胞培养组合物;
(2)用所述多肽、全培养液配制品或细胞培养组合物在25-60℃(例如35-60℃)处理所述烟草原料1-10小时(例如2-5小时);
(3)处理结束后,还包括灭酶的步骤,例如将所述烟草原料置于70-80℃10min-1h(例如,20-30min);
(4)所述烟草原料为片烟、梗丝、薄片或叶组烟丝。
在本文中,所述烟草原料为片烟、梗丝、薄片或叶组烟丝。
术语定义
在本发明中,除非另有说明,否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且,本文中所用的分子遗传学、核酸化学、细胞培养、生物化学、细胞生物学等操作步骤均为相应领域内广泛使用的常规步骤。同时,为了更好地理解本发明,下面提供相关术语的定义和解释。
术语“内切葡聚糖酶”是指内切-1,4-(1,3;1,4)-β-D-葡萄糖4-葡聚糖水解酶(E.C.3.2.1.4),它催化纤维素、纤维素衍生物(例如羧甲基纤维素和羟乙基纤维素)、地衣多糖中的1,4-β-D-糖苷键,混合的β-1,3葡聚糖(例如谷物β-D-葡聚糖或木聚糖)中的β-1,4键,以及含有纤维素其他植物材料的内切水解。可以通过测量底物粘度的降低或通过还原糖测定所确定的还原性末端的增加来确定内切葡聚糖酶活性(Zhang etal.Biotechnology Advances,2006,24:452-481)。可以根据Ghose,Pure andAppl.Chem.1987,59:257-268使用羧甲基纤维素作为底物测定内切葡聚糖酶活性。
典型地,内切葡聚糖酶具有至少两个功能结构域:碳水化合物结合模块(CBM)和催化模块。其中CBM例如被描述于Boraston et al.Biochem.J.2004,382:769-781,Tomme P,Warren RA,Miller RC et al(1995)Cellulose binding domains:classification andproperties.In:Saddler JN,Penner MH(eds)Enzymatic degradation of insolublecarbohydrates.American Chemical Society,Washington,DC,pp 42–161.据信CBM结合至底物可增加酶地催化活性部分地功效。在本文中,纤维素结合结构域是指特异性结合至纤维素底物的CBM亚组。
术语“催化结构域”是指一种酶中包含该酶催化机器的区域。
术语“表达”包括涉及多肽生产的任何步骤,包括但不限于转录、转录后修饰、翻译、翻译后修饰以及分泌。
术语“载体”是指线性或环状DNA分子,该分子包括编码多肽的多核苷酸并且该多核苷酸可操作地与提供用于其表达的控制序列相连接。此处控制序列是指对于表达编码本文所述多肽必须的核酸序列,其相对于编码多肽的多核苷酸是天然的或外源的,或相对于彼此是天然的或外源的,包括但不限于前导子、聚腺苷酸化序列、前肽序列、启动子、信号肽序列、以及转录终止子。
术语“宿主细胞”是指易于用包括本发明的多核苷酸的核酸构建体或表达载体转化、转染、转导等的任何细胞类型,其涵盖由于复制期间发生的突变而与亲本细胞不同的亲本细胞的任何后代。
发明的有益效果
本发明提供了一种烟草源枯草芽孢杆菌(Bacillus subtilis)的内切葡聚糖酶Cel-FX1,并利用基因工程技术使得所述内切葡聚糖酶在大肠杆菌中获得高效表达。相较于天然表达的多肽以及商品化纤维素酶,经本发明的多肽或者含有所述多肽的全培养液配制品或细胞培养组合物处理的烟草原料中纤维素含量显著降低,而还原糖含量明显提高,常规化学成分(例如,烟碱、总氮、淀粉和K)含量同样有下降趋势,并且感官质量也有明显改善。
附图说明
图1:重组表达载体pET-28a/cel-FX1示意图。
图2:SDS-PAGE电泳图,泳道1为Marker(Thermofisher 26616;10-180kDa),泳道2为阴性对照菌(E.coli/pET28a)发酵上清,泳道3为重组菌BL21-celFX1发酵上清(粗酶液CP),泳道4为纯化后本发明所述Cel-FX1内切葡聚糖酶。
图3:Cel-FX1内切葡聚糖酶酶活随温度变化曲线。
图4:Cel-FX1内切葡聚糖酶酶活随pH变化曲线。
具体实施方式
下面通过具体实施例,对本发明的技术方案作进一步的具体说明。应当理解,本发明的实施并不局限于下面的实施例,对本发明所做的任何形式上的变通和/或改变都将落入本发明保护范围。
除非特别指明,本发明中所使用的分子生物学实验方法基本上参照J.Sambrook等人,分子克隆:实验室手册,第2版,冷泉港实验室出版社,1989,以及F.M.Ausubel等人,精编分子生物学实验指南,第3版,John Wiley&Sons,Inc.,1995中所述的方法进行。
在本发明中,实施例中使用的全部材料、试剂、质粒等,如无特殊说明,均可从商业途径得到。其中所有引物均由生工生物工程(上海)股份有限公司合成。下述实施例中的方法,如无特别说明,均为本领域的常规方法。
实施例1:内切葡聚糖酶基因的克隆
材料:细菌基因组DNA提取试剂盒购自上海赛百盛基因技术有限公司;DH5α感受态细胞和BL21(DE3)感受态细胞、硫酸卡那霉素购自北京全式金生物技术有限公司;Isopropyl-β-D-thiogalactopyranoside(IPTG)购自生工生物工程(上海)股份有限公司;DNA聚合酶、各种限制性内切酶DNA连接酶、DNA Marker及BSA Standard Solution购自Takara公司(中国);pET28a质粒购自Novagen公司(Cat.69864-3);质粒提取试剂盒和胶回收试剂盒购自OMEGA公司。
具体操作过程如下:
在NCBI中查找已经公布的枯草芽孢杆菌内切葡聚糖酶的基因信息,利用软件SnapGene2.0.1设计引物;分别添加BamH I和Xho I酶切位点,扩增目的基因片段,具体引物序列如下:
P1:
5'CGCGGATCCATGATGCGAAGGAGGAAAAGATCAGATATGAAAC 3'P2:
5'CCGCTCGAGTTACTAATTTGGTTCTGTTCCCCAAATCAGTTTTCC3'
其中,划线部分为酶切位点。
采用细菌基因组DNA提取试剂盒提取枯草芽孢杆菌FX-1基因组DNA,具体步骤参照所述试剂盒的说明书。
PCR扩增内切葡聚糖酶编码基因cel-FX1;以从醇化烟叶表面分离到枯草芽抱杆菌FX-1全基因组DNA为模板,PCR扩增内切葡聚糖酶基因。PCR反应体系如表1。将上述体系吹打混匀后,12000r/min瞬时离心,然后进行PCR扩增。PCR反应参数:98℃预变性10s,98℃变性10s,64℃退火15s,72℃延伸10s,32个循环后,72℃延伸1min,4℃保存。在1%琼脂糖凝胶上,取5μL LPCR产物80V电压下电泳。以Marker 5000为对照,观察扩增的目的片段大小。
表1 PCR反应体系
cel-FX1基因的纯化采用EZNA Gel Extraction kit-spin protocol胶回收试剂盒,具体步骤参照所述试剂盒的说明书。
pET-28a/cel-FX1表达载体的构建,用限制性内切酶BamH I和Xho I分别双切质粒cel-FX1内切葡聚糖酶基因与表达载体pET28a(+),经琼脂糖凝胶分别回收目的片段与载体大片段,将各反应体系如表2所示,加入0.2mL的EP管中吹打混匀后,12000r/min瞬时离心,16℃过夜连接。
表2连接反应体系
重组质粒pET-28a/cel-FX1在大肠杆菌中的表达,热激转化区20μL连接产物与大肠杆菌感受态BL21(DE3)小心混匀,冰浴30min;42℃水浴热激45s;立即取出冰浴2min;加入900μL无菌LB液体培养基,37℃、180rpm恒温培养2h;吸取转化后的菌液,涂布于含卡那霉素的LB筛选培养基中,37℃恒温培养12-16h,挑取菌斑进行PCR鉴定并送样进行测序鉴定,确认为阳性的克隆子划线至含卡那霉素的LB固体培养基中培养。
实施例2:重组内切葡聚糖酶的诱导表达、纯化及酶学特性分析
(1)重组内切葡聚糖酶的诱导表达
a.挑取阳性克隆单菌落接种至5mL含卡那霉素(50μg/mL)的LB液体培养基中,37℃、180rpm培养12-16h;
b.将5mL菌液转接至含卡那霉素100mL LB液体培养基中,37℃、180rpm继续培养至OD600=0.6-0.7(约2h左右);
c.加入40μL IPTG(终浓度0.4mmol/L),25℃,180rpm低温诱导培养20h;
d.将诱导表达的菌液转移至50mL离心管中,12000rpm,4℃离心15min,弃掉上清,加入30mL PBS缓冲液(pH7.4)充分重悬菌体;
e.放入至盛有冰块的烧杯中,超声波进行破碎(超声15min,工作时间5s,间歇时间5s,6号变幅杆,破碎功率500w);
f.破碎菌液12000rpm,4℃离心10min,所得上清为粗蛋白液(CP),立即使用或4℃保存。
(2)重组内切葡聚糖酶的纯化
a.取出Ni柱,将保存液乙醇流出,再用5倍柱体积PBS缓冲液冲洗Ni柱,以备使用;
b.使his-tag蛋白与Ni柱充分吸附,取上步所获得的粗蛋白液转移至Ni柱中,4℃旋转混匀1-2h;
c.装柱上样,旋转混匀后的层析柱固定于支架上(垂直),打开塞子,让柱内液体流出,每个样品各手机1mL于Ep管中,以备后续使用;
d.用Binding Buffer进行洗涤,约6-7次后,各收集1mL流出液于Ep管中;
e.取出Bradord Dye Reagant试剂,加入200μL试剂于96空板中,加20μL上述收集液,移液枪吹打混匀,检测有无蛋白(若变蓝,则继续重复上述步骤,直至不变蓝即可);
f.加Binding Buffer洗脱至滤液无蛋白析出后,再加入Elution Buffer(按1倍填料体积1次的速度)并收集流出液于新的Ep管中,4℃保存备用;
g.脱盐纯化,检测上述步骤所收集的样液,100μL Bradord Dye Reagant加4μL样品进行反应,若变蓝,则证明含有蛋白样品;
h.将检测存在蛋白样品的样液分别加入到超滤管中,混匀,5000rpm,4℃离30min左右,至内管中样品体积小于1.5mL刻度线,弃外管滤液;
i.往内管中加入PBS缓冲液(pH7.4)2mL左右,用移液枪吹打混匀,将含有蛋白的样品转移至新的Ep管中,4℃保存备用;
j.纯化蛋白浓度检测,采用Bradford法,具体步骤参照BSA Standard Solution试剂使用说明书进行操作;
k.将纯化产物进行SDS-PAGE分析。
Cel-FX1内切葡聚糖酶蛋白浓度检测结果显示,蛋白表达量为0.23mg/mL,发酵上清酶活为187U/mL,SDS-PAGE结果如图2所示:重组酶cel-FX1的蛋白大小量为56kDa左右,氨基酸序列为SEQ ID No:1。
(3)重组内切葡聚糖酶的酶学特性分析
内切葡聚糖酶活力单位定义:每分钟催化产生1μmol还原需的酶量为1个活力单位(U)。
计算公式:酶活力(U)=N1·N2·G·10-3·1.0·106/(180·10·0.2)(μmol/min·mL)
N1:粗酶液稀释倍数;N2:比色时反应液的稀释倍数;10-3:毫克与克的转换;1.0:绘制标准曲线时葡萄糖溶液的体积(mL);106:由mol转化为μmol的系数;180:葡萄糖的分子量;10:酶水解反应时间(min);0.2:测定酶活力时所用粗酶液的体积(mL)。
最适酶活温度的测定
酶活的测定采用DNS法,将0.2mL粗酶液与0.8mL 1%的CMC-Na溶液(pH5.0)以1:4的体积比混合,分别于不同温度(30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃、70℃、75℃)条件下水浴10min,取出冷却至室温,加入1.5mL DNS试剂,迅速混匀后沸水浴反应10min,冷却后,加水定容到10mL,并于一定条件下测定纤维素酶活性,另取稀释酶液0.2mL和0.1mol/L pH 5.0缓冲溶液0.8mL作为空白,不加CMC溶液,同样依上述步骤,用空白调零点,得出酶促反应最适温度。结果如图3所示,重组酶Cel-FX1的最适温度为60℃。
最适酶活pH的测定
将0.2mL粗酶液与0.8mL 1%的不同pH的CMC-Na溶液(pH3.0-8.0)反应测定酶活,得出酶促反应的最适pH。结果如图4所示,重组酶Cel-FX1的最适pH为5.0。
实施例3:内切葡聚糖酶Cel-FX1在卷烟原料改良中的应用
分别取野生型FX-1发酵粗酶液(野生型菌株FX-1发酵24h菌液离心后的上清液),食品级纤维素酶(宁夏和氏璧生物技术有限公司),重组Cel-FX1,重组Cel-ms22(红树林源,海洋三所创新中心实验室提供)使用超纯水配制成100U/mL母液;各组分别称取100g片烟(三明尤溪B3F-2019),超纯水配制各实验组工作液至每组喷施体积为2mL,喷施酶量为按100U/Kg卷烟原料,用小型喷雾器均匀喷施于烟叶表面。将喷施酶液后的烟叶用自封袋包装,置于35℃恒温箱中,处理3h。处理结束后放入75℃烘箱中保持20min进行灭酶。设置喷施等量超纯水按上诉相同操作作为空白对照组。
灭酶后取野生型FX-1发酵粗酶液、食品级纤维素酶(宁夏和氏璧生物技术有限公司)、重组Cel-FX1(烟草源)处理后的10g烟叶浸泡在水中,测定水溶液中还原糖含量、纤维素含量及烟叶化学成份。另外,灭酶后取野生型FX-1发酵粗酶液、食品级纤维素酶(宁夏和氏璧生物技术有限公司)、重组Cel-FX1(烟草源)、重组Cel-ms22(红树林源)处理后的样品于恒温恒湿箱中平衡24h后进行感官评吸(参照YC/T 138-1998烟草及烟草制品感官评价方法)。
表3不同类型酶液处理对烟叶中纤维素及还原糖含量的影响
如表3所示:使用不同酶液处理烟叶均可明显降低样品中的纤维素含量,提高还原糖含量。其中纤维素降低幅度最大的为重组Cel-FX1,达20.95%,野生型酶液次之,商品酶制剂降幅最低仅为18.53%,还原糖增加幅度则为商品酶制剂最高,其主要由于商品酶制剂中含有大量的还原糖类辅料导致。
表4常规化学成份测定结果
如表4所示:经酶制剂处理后,烟碱、总氮、淡粉及K含量均呈现不同层度的下降趋势。
其中,各成分测定方法参照:
YC/T 159-2002烟草及烟草制品水溶性糖的测定连续流动法;
NY/T 3494-2019农业生物质原料纤维素、半纤维素、木质素测定;
GB/T 23355-2009卷烟总粒相物中烟碱的测定气相色谱法;
YC/T 161-2002烟草及烟草制品总氮的测定连续流动法;
YC/T 283-2009烟草及烟草制品淀粉的测定酶水解-离子色谱法;
YC/T 217-2007烟草及烟草制品钾的测定连续流动法。
表5感官评吸结果
如表5所示:经酶制剂处理后的样品,在质感、清雅度及杂气等方面均优于水对照,其中使用烟草源重组Cel-FX1处理后样品感官评析显著优于红树林源重组Cel-ms22,其清甜蜜甜香风格明显,烟香纯正且清雅飘逸,甜感突出,丰富性、透发性、细腻度、杂气、刺激,劲头及余味均明显优于另外4组实验组的处理效果。
SEQ ID NO:1
508aa
MMRRRKRSDMKRSISIFITCLLITLLTMGGMLASPASAAGTKTPVAKNGQLSIKGTQLVNRDGKAVQLK GISSHGLQWYGEYVNKDSLKWLRDDWGITVFRAAMYTADGGYIDNPSVKNKVKEAVEAAKELGIYVIIDWHILNDGN PNQNKEKAKEFFKEMSSLYGNTPNVIYEIANEPNGDVNWKRDIKPYAEEVISVIRKNDPDNIIIVGTGTWSQDVNDA ADDQLKDANVMYALHFYAGTHGQFLRDKANYALSKGAPIFVTEWGTSDASGNGGVFLDQSREWLKYLDSKTISWVNWNLSDKQESSSALKPGASKTGGWRLSDLSASGTFVRENILGTKDSTKDIPETPAKDKPTQENGISVQYRAGDGSMNSN QIRPQLQIKNNGNTTVDLKDVTARYWYNAKNKGQNVDCDYAQLGCGNVTYKFVTLHKPKQGADTYLELGFKNGTLAPGASTGNIQLRLHNDDWSNYAQSGDYSFFKSNTFKTTKKITLYDQGKLIWGTEPN
SEQ ID No.:2
催化结构域
VNRDGKAVQLKGISSHGLQWYGEYVNKDSLKWLRDDWGITVFRAAMYTADGGYIDNPSVKNKVKEAVEA
AKELGIYVIIDWHILNDGNPNQNKEKAKEFFKEMSSLYGNTPNVIYEIANEPNGDVNWKRDIKPYAEEVISVIRKND
PDNIIIVGTGTWSQDVNDAADDQLKDANVMYALHFYAGTHGQFLRDKANYALSKGAPIFVTEWGTSDASGNGGVFLD
QSREWLKYLDSKTISWV
SEQ ID No.:3
结合结构域
VQYRAGDGSMNSNQIRPQLQIKNNGNTTVDLKDVTARYWYNAKNKGQNVDCDYAQLGCGNVTYKFVTLH
KPKQGADTYLELG
SEQ ID NO:4
1527bp
ATGCGAAGGAGGAAAAGATCAGATATGAAACGGTCAATCTCTATTTTTATTACGTGTTTATTGATTACGTTATTGACAATGGGCGGCATGCTGGCTTCGCCGGCATCAGCAGCAGGGACAAAAACGCCAGTAGCCAAGAATGGCCAGCTTAGCATAAAAGGTACACAGCTCGTTAACCGAGACGGTAAAGCGGTACAGCTGAAGGGGATCAGTTCACACGGATTGCAATGGTATGGAGAATATGTCAATAAAGACAGCTTAAAATGGCTGAGGGACGATTGGGGTATCACCGTTTTCCGTGCAGCGATGTATACGGCAGATGGCGGTTATATTGACAACCCGTCCGTGAAAAATAAAGTGAAAGAAGCGGTTGAAGCGGCAAAAGAGCTTGGGATATATGTCATCATTGACTGGCATATCTTAAATGACGGTAATCCAAACCAAAATAAAGAGAAGGCAAAAGAATTCTTCAAGGAAATGTCAAGCCTTTACGGAAACACGCCAAACGTCATTTATGAAATTGCAAACGAACCAAACGGTGATGTGAACTGGAAGCGTGATATTAAACCGTATGCGGAAGAAGTGATTTCCGTTATCCGCAAAAATGATCCAGACAACATCATCATTGTCGGAACCGGTACATGGAGCCAGGATGTGAATGATGCTGCCGATGACCAGCTAAAAGATGCAAACGTTATGTACGCACTTCATTTTTATGCCGGCACACACGGCCAATTTTTACGGGATAAAGCAAACTATGCACTCAGCAAAGGAGCACCTATTTTTGTGACAGAGTGGGGAACAAGCGACGCGTCTGGCAATGGCGGTGTATTCCTTGATCAATCGAGGGAATGGCTGAAATATCTCGACAGCAAGACCATCAGCTGGGTGAACTGGAATCTTTCTGATAAGCAGGAATCATCCTCAGCTTTAAAGCCGGGGGCATCTAAAACAGGCGGCTGGCGGTTGTCAGATTTATCTGCTTCAGGAACATTCGTTAGAGAAAACATTCTCGGCACCAAAGATTCGACGAAGGACATTCCTGAAACGCCAGCAAAAGATAAACCCACACAGGAAAACGGTATTTCTGTACAATACAGAGCAGGGGATGGGAGTATGAACAGCAACCAAATCCGTCCGCAGCTTCAAATAAAAAATAACGGCAATACCACGGTTGATTTAAAAGATGTCACTGCCCGTTACTGGTATAACGCGAAAAACAAAGGCCAAAACGTTGACTGTGACTACGCGCAGCTTGGATGCGGCAATGTGACATACAAGTTTGTGACGTTGCATAAACCAAAGCAAGGTGCAGATACCTATCTGGAACTTGGATTTAAAAACGGAACGCTGGCACCGGGAGCAAGCACAGGGAATATTCAGCTTCGTCTTCACAATGATGACTGGAGCAATTATGCACAAAGCGGCGATTATTCCTTTTTCAAATCAAATACGTTTAAAACAACGAAAAAAATCACATTATATGATCAAGGAAAACTGATTTGGGGAACAGAACCAAATTAGTAA
尽管本发明的具体实施方式已经得到详细的描述,本领域技术人员将会理解,根据已经公开的所有教导,可以对那些细节进行各种修改和替换,这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
SEQUENCE LISTING
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Asp Asp Gln Leu Lys Asp Ala Asn Val Met Tyr Ala Leu His Phe Tyr
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Tyr Ala Leu His Phe Tyr Ala Gly Thr His Gly Gln Phe Leu Arg Asp
180 185 190
Lys Ala Asn Tyr Ala Leu Ser Lys Gly Ala Pro Ile Phe Val Thr Glu
195 200 205
Trp Gly Thr Ser Asp Ala Ser Gly Asn Gly Gly Val Phe Leu Asp Gln
210 215 220
Ser Arg Glu Trp Leu Lys Tyr Leu Asp Ser Lys Thr Ile Ser Trp Val
225 230 235 240
<210> 3
<211> 82
<212> PRT
<213> artificial
<220>
<223> 结合结构域
<400> 3
Val Gln Tyr Arg Ala Gly Asp Gly Ser Met Asn Ser Asn Gln Ile Arg
1 5 10 15
Pro Gln Leu Gln Ile Lys Asn Asn Gly Asn Thr Thr Val Asp Leu Lys
20 25 30
Asp Val Thr Ala Arg Tyr Trp Tyr Asn Ala Lys Asn Lys Gly Gln Asn
35 40 45
Val Asp Cys Asp Tyr Ala Gln Leu Gly Cys Gly Asn Val Thr Tyr Lys
50 55 60
Phe Val Thr Leu His Lys Pro Lys Gln Gly Ala Asp Thr Tyr Leu Glu
65 70 75 80
Leu Gly
<210> 4
<211> 1527
<212> DNA
<213> artificial
<220>
<223> Cel-FX1核酸序列
<400> 4
atgcgaagga ggaaaagatc agatatgaaa cggtcaatct ctatttttat tacgtgttta 60
ttgattacgt tattgacaat gggcggcatg ctggcttcgc cggcatcagc agcagggaca 120
aaaacgccag tagccaagaa tggccagctt agcataaaag gtacacagct cgttaaccga 180
gacggtaaag cggtacagct gaaggggatc agttcacacg gattgcaatg gtatggagaa 240
tatgtcaata aagacagctt aaaatggctg agggacgatt ggggtatcac cgttttccgt 300
gcagcgatgt atacggcaga tggcggttat attgacaacc cgtccgtgaa aaataaagtg 360
aaagaagcgg ttgaagcggc aaaagagctt gggatatatg tcatcattga ctggcatatc 420
ttaaatgacg gtaatccaaa ccaaaataaa gagaaggcaa aagaattctt caaggaaatg 480
tcaagccttt acggaaacac gccaaacgtc atttatgaaa ttgcaaacga accaaacggt 540
gatgtgaact ggaagcgtga tattaaaccg tatgcggaag aagtgatttc cgttatccgc 600
aaaaatgatc cagacaacat catcattgtc ggaaccggta catggagcca ggatgtgaat 660
gatgctgccg atgaccagct aaaagatgca aacgttatgt acgcacttca tttttatgcc 720
ggcacacacg gccaattttt acgggataaa gcaaactatg cactcagcaa aggagcacct 780
atttttgtga cagagtgggg aacaagcgac gcgtctggca atggcggtgt attccttgat 840
caatcgaggg aatggctgaa atatctcgac agcaagacca tcagctgggt gaactggaat 900
ctttctgata agcaggaatc atcctcagct ttaaagccgg gggcatctaa aacaggcggc 960
tggcggttgt cagatttatc tgcttcagga acattcgtta gagaaaacat tctcggcacc 1020
aaagattcga cgaaggacat tcctgaaacg ccagcaaaag ataaacccac acaggaaaac 1080
ggtatttctg tacaatacag agcaggggat gggagtatga acagcaacca aatccgtccg 1140
cagcttcaaa taaaaaataa cggcaatacc acggttgatt taaaagatgt cactgcccgt 1200
tactggtata acgcgaaaaa caaaggccaa aacgttgact gtgactacgc gcagcttgga 1260
tgcggcaatg tgacatacaa gtttgtgacg ttgcataaac caaagcaagg tgcagatacc 1320
tatctggaac ttggatttaa aaacggaacg ctggcaccgg gagcaagcac agggaatatt 1380
cagcttcgtc ttcacaatga tgactggagc aattatgcac aaagcggcga ttattccttt 1440
ttcaaatcaa atacgtttaa aacaacgaaa aaaatcacat tatatgatca aggaaaactg 1500
atttggggaa cagaaccaaa ttagtaa 1527
Claims (6)
1.一种从枯草芽孢杆菌(Bacillus subtilis)中分离的多肽在烟草原料加工或者降低烟草原料中纤维素含量中的用途,其中所述多肽具有纤维素分解活性和/或内切葡聚糖酶活性。
2.一种多肽在烟草原料加工或者降低烟草原料中纤维素含量中的用途,其中所述多肽具有纤维素分解活性和/或内切葡聚糖酶活性,该多肽具有选自以下的氨基酸序列:
(1)如SEQ ID NO:1所示的氨基酸序列;
(2)与SEQ ID NO:1具有至少80%同一性,优选至少85%同一性、至少90%同一性、至少95%同一性、或至少99%同一性的氨基酸序列;
(3)与SEQ ID NO:1相异在于一个或多个氨基酸残基的置换、缺失或添加的氨基酸序列;
(4)包含如SEQ ID NO:2所示的氨基酸序列,和/或SEQ ID NO:3所示的氨基酸序列;
(5)包含与SEQ ID NO:2具有至少80%同一性,优选至少85%同一性、至少90%同一性、至少95%同一性、或至少99%同一性的氨基酸序列,并且所述氨基酸序列为催化纤维素分解的催化结构域;和/或与SEQ ID NO:3具有至少80%同一性,优选至少85%同一性、至少90%同一性、至少95%同一性、或至少99%同一性的氨基酸序列,并且所述氨基酸序列为结合纤维素底物的结合结构域;和,
(6)包含与SEQ ID NO:2相异在于一个或多个氨基酸残基的置换、缺失或添加的氨基酸序列,和/或与SEQ ID NO:3相异在于一个或多个氨基酸残基的置换、缺失或添加的氨基酸序列。
3.一种全培养液配制品或细胞培养组合物在烟草原料加工或者降低烟草原料中纤维素含量中的用途,其中所述的全培养液配制品或细胞培养组合物通过以下方法制备得到:
利用宿主细胞诱导表达权利要求1或2中所述的多肽,收集所述多肽,任选地,对收集得到的多肽进行纯化,得到所述全培养液配制品或细胞培养组合物;
优选地,所述全培养液配制品或细胞培养组合物酶活力为100-1000U/mL,例如,100-500U/mL,100-400U/mL,100-300U/mL,或100-200U/mL;
优选地,所述宿主细胞为大肠杆菌,例如大肠杆菌BL21。
4.权利要求1-3任一项所述的用途,其中所述烟草原料为片烟、梗丝、薄片或叶组烟丝。
5.一种处理烟草原料或者降低烟草原料中纤维素含量的方法,其包括用权利要求1或2中所述的多肽或权利要求3中所述的全培养液配制品或细胞培养组合物处理所述烟草原料的步骤。
6.权利要求5所述的方法,其特征在于下述的一项或多项:
(1)以50-500U/Kg的量添加所述多肽或全培养液配制品或细胞培养组合物;
(2)用所述多肽、全培养液配制品或细胞培养组合物在25-60℃(例如35-60℃)处理所述烟草原料1-10小时(例如2-5小时);
(3)处理结束后,还包括灭酶的步骤,例如将所述烟草原料置于70-80℃10min-1h(例如,20-30min);
(4)所述烟草原料为片烟、梗丝、薄片或叶组烟丝。
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