CN105647944A - 一种来源于教酒链霉菌(Streptomyces chartreusis)L1105的木聚糖酶基因及其木聚糖酶 - Google Patents
一种来源于教酒链霉菌(Streptomyces chartreusis)L1105的木聚糖酶基因及其木聚糖酶 Download PDFInfo
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Abstract
本发明公开了一种来源于教酒链霉菌(Streptomyces?chartreusis?L1105)的木聚糖酶Xyn?A的基因,包括其核酸和蛋白质/多肽序列。该基因编码的木聚糖酶全长共有1393个碱基对,G,C含量占68%,编码460个氨基酸,分子量约为48.2kDa,等电点pI为9.04。含有一段信号肽序列。该酶具有F/10糖苷水解酶的典型结构,能翻译一段大小为10kDa的纤维素结合域(CBD)。DNS法测得该酶活为19.7±1.2U/mL。作为一种碱性的木聚糖酶,该酶及其作用产物具备在造纸工业,纺织工业,食品加工,生物能源,饲料工业,化妆品,医药,保健品,酿酒,农作物生产,以及各类生物反应器中的应用价值。
Description
技术领域
本发明涉及一种来源于教酒链霉菌(StreptomyceschartreusisL1105)的木聚糖酶基因及其产物木聚糖酶的应用。
背景技术
木聚糖(xylan)是一种异质多糖(heteropolysaeeharides),是β-D-吡喃型木糖单元(xylopyranoseunits)通过β-1,4-糖苷键连接形成。植物半纤维素中含量最多的就是木聚糖,干重占植物细胞的15~35%。
木聚糖酶是糖苷水解酶的一种,包括β-1,4-内切木聚糖酶(EC3.2.1.8),β-木糖苷酶(EC3.2.1.37),α-L-呋喃型阿拉伯糖苷酶(EC3.2.1.55),α-葡萄糖醛酸酶(Ee3.2.2.1),乙酰木聚糖脂酶(EC3.2.1.6),酚酸脂酶。目前研究最多的木聚糖酶是β-1,4-内切木聚糖酶,其水解大分子的主链,生成木寡糖和木糖,是木聚糖降解酶中的主力。
目前,木聚糖酶最主要的来源是由微生物生产,且同一种微生物可以产生不止一种木聚糖酶,不同的微生物产生的木聚糖酶之间也存在差异。
糖苷水解酶根据催化区的保守氨基酸和疏水区的不同分为58个族,大部分木聚糖酶属于F/10或G/11族,其中F/10族的木聚糖分子量大多大于30kDa,结构多样,除了催化区域一般还含有多种非催化区域如纤维素结合域(CBD)等,能较快的水解木聚糖生成低聚木糖和木糖。G/11族木聚糖酶结构简单,水解木聚糖的速度慢,产物多为木寡糖。目前大部分传统筛选方式得到的木聚糖酶多属于G/11族。而F/10族的木聚糖不但能催化木聚糖水解,还能水解纤维素,具有纤维素酶活,称之为交叉活性。
F/10族的木聚糖为多区域结构除了含有催化区域以外还含有非催化结构域,如纤维素结合域(CBD),木聚糖结合域(Xylan-bingdingDomain,XBD)等,通常,这些非催化结构域并不能催化水解反应,但是却能影响与不溶性底物的反应,能使木聚糖酶附着到纤维素上,主要是对不溶性底物的亲和力产生变化,对酶活和其他酶学性质如底物特异性没有影响。
纤维素结构域普遍存在于纤维素酶,半纤维素酶,木聚糖酶和其他水解酶中,目前经过确定的CBD结构已经超过100种,根据氨基酸相似性,用疏水性聚类方法将CBD结构分为10个家族。研究表明有些酶不只含有一个而是含有两个甚至更多CBD结构,这些结构域共同作用于底物,使酶的亲和性更好。有研究发现,将热纤梭菌(Clostridiumthermocellum)的CBD接到CD的N-端或C-端,对木聚糖的水解能力不变,说明CBD的位置不影响对底物的附着。另外,CBD还与木聚糖酶的热稳定性有关,提高最适反应温度。
木聚糖广泛的存在于自然界中,是植物半纤维素的重要组成部分,作为重要的可再生资源,能否有效的利用木聚糖具有巨大工业价值。木聚糖酶能专一的降解木聚糖,且安全高效,利用木聚糖酶降解木聚糖是目前利用这一资源的有效手段,受到广泛的重视,称为近年科研工作者的研究热点。木聚糖酶在被广泛的应用到食品、饲料、纸浆助漂、医药、纺织、能源等行业中,并发挥重要作用。目前,木聚糖在食品中主要被应用于焙烤工业,酿酒工业,低聚木糖生产等领域。
虽然木聚糖酶的工业中的应用十分广泛,但是木聚糖酶自身存在的一些问题也制约着其更大范围的应用。首先,天然菌株在生产木聚糖酶的过程中,自身往往会分泌除木聚糖酶以外的较多杂蛋白质,这些蛋白质中很少能协同木聚糖酶水解木聚糖酶,如菌株的分泌蛋白酶,对木聚糖酶的产量会有很大的影响。分离纯化发酵液中的木聚糖酶是一个费时费力的工作,且成本很高,如何能用更经济快捷的方法获得单一的木聚糖酶是工业应用研究的重点。构建工程菌株是一种有效的手段。其次,木聚糖酶的生物活性是建立在合适的反应体系中,由于目前大部分的木聚糖酶在比较温和的条件下才能获得最佳的催化结果,而在造纸工业,食品加工,纺织工业,饲料工业,生物能源等工业生产中常常用到高温高压等极限条件,这也制约着木聚糖酶的进一步应用。
发明内容
本发明从来源于教酒链霉菌(StreptomyceschartreusisL1105)的木聚糖酶获得木聚糖酶基因多核苷序列如SEQ-1所示:ATGGCGACCCGCACATCCATCGATCCGCCTCCCGAGCGCCGGCCCCGGCGCTCGCGCGTCCGCACCGCACTGGCCCTCGGCCTGGCCGGCCTGCTCGGCGCGACCGGCGTCGGCGCCCTCACCGGCACCGCGCAGGCCGCGAGCACCCTCGGCGCGGCGGCAGCCGAGAAGGGCCGTTACTTCGGCGCCGCCGTCGCCGCGAACCACCTGGGGGAGAGCGCGTACGTCTCGACGCTCAACACCGAGTTCAACTCGGTGACCCCGGAGAACGAGATGAAGTGGGACGCCACCGAGCGCACCCGCGGCACCTTCACCTTCGGCTCCGCCGACCAGGTCGTGAACCACGCCCAGAGCCAGGGCATGAAGGTCCGCGGCCACACCCTGGTCTGGCACTCGCAGTTACCGAGCTGGGTCGCGGGACTCGGCACCGCCGACCTCAGATCGGCGATGAACAATCACATCACCCAGGTCATGCAGCACTACAAGGGCAAGATCCACTCCTGGGACGTCGTCAACGAGGCATTCCAGGACGGCAGCAGTGGTGCGCGGCGCAGCTCGCCCTTCCAGGACAAGCTCGGCAACGGCTTCATCGAGGAGGCGTTCCGCACCGCCCGGGCCGCCGACCCGGCCGCCAAGCTCTGCTACAACGACTACAACACCGACGGCGTCAACGCGAAGAGCAATGCCGTCTACAACCTGGTCAGGGACTTCAAGTCCCGTGGCGTGCCGATCGACTGCGTGGGCTTCCAGTCCCACTTCAACAGCGCCTCGCCGGTCCCCTCCGACTACCAGGCCAACCTCCAGCGCTTCGCCGACCTCGGCGTGGACGTGCAGATCACCGAGCTGGACATCGAGGGCTCGGGCACCGCACAGGCCAACAGCTACACCAATGTGGTCCGCGCCTGTCTGGCCGTCTCGCGCTGCACCGGCATCACCGTCTGGGGCGTCACCGACAAGTACTCCTGGCGCGCGAGCGGAACGCGCCTGCTGTTCGACGGCAACTACGCCAAGAAGCCGGCGTACACAGCGACGCTGACCGCTCTCGGCGGCTCCTCCTCCGGCGGCGGCACACCCGGCGCCGCCTGCACGGCCACCTACAGCAAGGCCGAGGAGTGGAGTGACCGCTTCAACGGCAAGGTCACCATCACCGCCGGGAGTTCGGCGATCAGCGCCTGGACCGTGAACGTCACGGTGAACTCCCCGCAGAAGGTCTCCACGACCTGGAACGGCACGCCCAGCTGGGACAGCAGCGGCAACGTGATGACGATGAAGTCGAACGGCAACGGCAGCCTGGCCGCCGGGGCTTCCACCAGCTTCGGCTTCACCGTCATGAAGAACGGCAGCAGCGCGACCCCGGCGATCGGTTCCTGCACGGCGTCCTGA
本发明从来源于教酒链霉菌(StreptomyceschartreusisL1105)的木聚糖酶获得木聚糖酶基因多肽序列如SEQ-2所示:
MATRTSIDPPPERRPRRSRVRTALALGLAGLLGATGVGALTGTAQAASTLGAAAAEKGRYFGAAVAANHLGESAYVSTLNTEFNSVTPENEMKWDATERTRGTFTFGSADQVVNHAQSQGMKVRGHTLVWHSQLPSWVAGLGTADLRSAMNNHITQVMQHYKGKIHSWDVVNEAFQDGSSGARRSSPFQDKLGNGFIEEAFRTARAADPAAKLCYNDYNTDGVNAKSNAVYNLVRDFKSRGVPIDCVGFQSHFNSASPVPSDYQANLQRFADLGVDVQITELDIEGSGTAQANSYTNVVRACLAVSRCTGITVWGVTDKYSWRASGTRLLFDGNYAKKPAYTATLTALGGSSSGGGTPGAACTATYSKAEEWSDRFNGKVTITAGSSAISAWTVNVTVNSPQKVSTTWNGTPSWDSSGNVMTMKSNGNGSLAAGASTSFGFTVMKNGSSATPAIGSCTAS*
本发明从来源于教酒链霉菌(StreptomyceschartreusisL1105)的木聚糖酶获得木聚糖酶基因信号多肽序列如SEQ-3所示:
MATRTSIDPPPERRPRRSRVRTALALGLAGLLGATGVGALTGTAQA
本发明从来源于教酒链霉菌(StreptomyceschartreusisL1105)的木聚糖酶获得木聚糖酶基因纤维素结合域(CBD)多肽序列如SEQ-4所示:
SKAEEWSDRFNGKVTITAGSSAISAWTVNVTVNSPQKVSTTWNGTPSWDSSGNVMTMKSNGNGSLAAGASTSFGFTVMKNGSSATPAIGSCTAS*
附图说明
图1是教酒链霉菌基因组总DNA图。
图2是编码木聚糖酶的保守序列图。
图3是HiTail-PCR过程简图。
图4是基因SEQ-1的3′端侧翼序列电泳图。其中M:marker;1:LAD1/SP3-1预扩增,AC1/SP3-2为一轮扩增;2:LAD2/SP3-1预扩增,AC1/SP3-2为一轮扩增;3:LAD3/SP3-1预扩增,AC1/SP3-2为一轮扩增;4:LAD4/SP3-1预扩增,AC1/SP3-2为一轮扩增。
图5是基因SEQ-1的5′端侧翼序列电泳图。其中M,marker;1,LAD1/SP5-2为第一轮扩增,AC1/SP5-3为第二轮扩增;2,LAD3/SP5-2为第一轮扩增,AC1/SP5-3第二轮扩增;3,LAD1/SP5-1为第一轮扩增,AC1/SP5-2为第二轮扩增;3,LAD3/SP5-1为第一轮扩增,AC1/SP5-2为第二轮扩增。
图6是xynA核酸数据库同源性比较图。
图7是XynA酶信号肽预测图。
图8是氨基酸保守功能区图。
图9是教酒链霉菌L1105纯化过程的电泳(a)及酶谱(b)图。其中M,蛋白标样;1,粗酶液的蛋白带;2,50-80%硫酸铵盐析所得的蛋白带;3,纯酶;4,粗酶液酶谱;5,盐析后样品酶谱;6,纯酶酶谱
具体实施方式
本发明以下结合具体实例作进一步说明,但并不是限制本发明。下面结合附图对本发明进一步说明。实施例中未注明具体条件的实验方法,通常可按常规条,如J.萨姆布鲁克(Sambrook)等编写的《分子克隆实验指南第三版》中所述的条件,或按照试剂盒生产商所建议的条件进行。本领域相关的技术人员可以借助实施例更好地理解和掌握本发明。
普通试剂:刚果红;卡那霉素(美国AMRESCO);琼脂糖(西班牙Biowest);氨苄青霉素(美国AMRESCO);桦木木聚糖(美国Sigma);LB固体培养基;木聚糖-刚果红筛选平板;NaCl洗脱液:1M。
生化试剂:细菌基因组提取试剂盒(美国OMEGA);胶回收试剂盒(OMEGA,美国);LaTaqDNA聚合酶(withGCbuffer);限制性内切酶EcoRI、XhoI;1kbDNALadderMarker(日本TAKARA);LaTaqDNA聚合酶(withGCbuffer)(TAKARA,日本);200bpDNAladder(美国Biomega);PCR扩增引物(北京奥科鼎盛合成);200bpDNAladder(美国Biomega)
菌株:教酒链霉菌(Streptomyceschartreusis)L1105;大肠杆菌DH5a和Transtetta(DE3)感受态细胞购自全式金公司
载体:T载体:pMD18-T购于TAKARA公司;pET-28a(+)购自Merk公司
T载体用测序引物:
M13+序列:5’-GTTTTCCCAGTCACGAC-3’
M13-序列:5’-CAGGAAACAGCTATGAC-3’
实施例1:教酒链霉菌(StreptomyceschartreusisL1105)的木聚糖酶XynA的基因克隆
1.1基因组DNA提取
将教酒链霉菌L1105接种于LB液体培养基中,30℃,150rpm培养72h,吸取菌液1.5mL置于2mL离心管中10000×g室温离心5min,得到菌体。提取细菌基因组,得到教酒链霉菌(StreptomyceschartreusisL1105)的木聚糖酶基因组总DNA(图1)。
1.2简并引物设计
在NCBI蛋白数据库(http://www.ncbi.nlm.nih.gov/)中输入木聚糖酶N端15个氨基酸序列ASTLGAAAAEKGRYF(Ala-Ser-Thr-Leu-Gly-Ala-Ala-Ala-Ala-Glu-Lys-Gly-Arg-Tyr-Phe)进行Blastn比对,获得与教酒链霉菌L1105木聚糖酶基因同源性较高的来源于链霉菌属的10条编码木聚糖酶的基因序列,在高同源性区域内设计引物。分析得到该类木聚糖酶的保守序列区(图2)。
1.3PCR扩增核心序列(xynA-Core)
以基因组为模板,通过保守区域设计引物进行PCR(表1)。PCR反应条件:PCR反应条件:94℃,4min(预变性);94℃,30s(变性),62℃,30s(褪火),72℃,60s(延伸),30个循环;72℃延伸10min。
表1简并引物序列
Table1PrimersforxynA-core
注:R=A/G,S=C/G,W=A/T,B=C/G/T,N=A/C/G/T
根据两个引物之间的距离估计PCR产物的大小,挑选大小合适条带进行胶回收。连接到T载体pMD18-T上,将上述转化的后的阳性DH5a克隆接种于1ml的LB培养基,交上海生工生物技术公司进行DNA序列测定,,使用通用测序引物M13+和M13-,测序获得到一个约450bpDNA核心序列片段。然后核酸序列在NCBI上进行比对,结果表明序列与登录号为HE971709.1,AF194024,AB110643.1木聚糖酶基因序列具有很高的同源性,初步确定得到的核心序列能编码木聚糖酶,为木聚糖酶核心序列(xynA-core)。
>xynA-Core
CACCTGGGGGAGAGCGCGTACGTCTCGACGCTCAACACCGAGTTCAACTCGGTGACCCCGGAGAACGAGATGAAGTGGGACGCCACCGAGCGCACCCGCGGCACCTTCACCTTCGGCTCCGCCGACCAGGTCGTGAACCACGCCCAGAGCCAGGGCATGAAGGTCCGCGGCCACACCCTGGTCTGGCACTCGCAGTTACCGAGCTGGGTCGCGGGACTCGGCACCGCCGACCTCAGATCGGCGATGAACAATCACATCACCCAGGTCATGCAGCACTACAAGGGCAAGATCCACTCCTGGGACGTCGTCAACGAGGCATTCCAGGACGGCAGCAGTGGTGCGCGGCGCAGCTCGCCCTTCCAGGACAAGCTCGGCAACGGCTTCATCGAGGAGGCGTTCCGCACCGCCCGGGCCGCCGACCCGGCCGCCAAGCTCTGCTACAACGACTACAACACCGACGGCGTCAACGCGAAGAGCAATGCCGTCTACAACCTGGTCAGGGACTTCAAGTCCCGTGGCGTGCCGATCGACTGCGTGGGCTTCCAGTCCCACTTCAACAGCGCCTCGCCGGTCCCCTCCGACTACCAGGCCAACCTCCAGCGCTTCGCCGACCTCGGCGTGGACGTGCAGATCACCGAGCTGGACATCGAGGGCTCGGGCACCGCACAGGCCAACAGCTACACCAATGTGGTCCGCGCCTGTCTGGCCGTCTCGCGCTGCACCGGCATCACCGTCTGGGGCGTCACCGACAAGTACTCCTGGCGCGCGAGCGGAACGCCCCTGC
1.4巢式-PCR(HiTail-PCR)扩增基因全长
巢式PCR是一种获得已知序列侧翼序列的有效手段,采用改进过的巢式PCR,HiTail-PCR对xynA-core的侧翼序列进行扩增。根据以上获得的约450bpDNA核心序列片段,在核心序列内部设计三个高褪火温度的特异引物,配合通用引物LAD,AC进行PCR,经过三轮反应后得到特异性较强的片段。
巢式PCR扩增5’端侧翼序列的过程如图3所示。在核心序列内部设计3个引物:Sp5-1、Sp5-2、Sp5-3,这三个引物的距离依次接近5’端。首先使用引物Sp5-1与简并引物LAD1进行一次预扩增(Pre-amplification),接下来以预扩增的产物为模板,用引物Sp5-2和AC1进行一次扩增(Primary),最后用Sp5-3和AC1引物,以一次扩增PCR产物为模板进行二次扩增(Secondary)。3’端扩增采用相同的步骤。核心序列内进行Tail-PCR设计扩增木聚糖酶基因上游和下游引物如表2所示。
表2侧翼序列所用到的引物
Table2.PrimersforFlankingsecqence
注:R=A/G,S=C/G,W=A/T,B=C/G/T,N=A/C/G/T
预扩增使用Sp3-1,LAD1,以预扩增反应产物为模板,使用引物Sp3-2和AC1进行一次扩增,电泳检测发现,通过一次扩增已经能获得特异性较高的条带(图4)。图中可以看到泳道2和泳道4分别出现了1000bp和2000bp大小的两条亮带,切胶回收,连T克隆载体测序。得到3’端侧翼序列xynA-3’。
Sp5-1、Sp5-2特异性引物分别于与LAD1、AC1引物配对,Sp3-2、Sp5-3特异性引物分别于与LAD1、AC1引物配对,进行HiTail-PCR,通过两轮反应即获得了特异性较好的条带(图5)。图中泳道1,3分别出现了大小约为1200bp左右的亮带,切胶回收,连T克隆载体测序,得到5’端侧翼序列xynA-5’。
回收电泳中亮度高的条带,连接T载体,通用引物M13测序,分别得到3’端、5’端的侧翼序列,因为特异性引物是在核心序列内部设计得到,这样侧翼序列会和核心序列有一段是重叠的,用DNAMAN软件分析找到这些重叠序列,进而将三段基因拼接到一起,最终得到一段长为1383bp基因全长序列SEQ-1。
实施例2:重组木聚糖酶XynA蛋白分析
2.1pET28a(+)表达载体的构建:
2.1.1PCR扩增基因xynA
根据xynA设计引物,综合考虑表达载体的多克隆位点(MCS)和xynA序列,在xynA两端引入酶切位点EcoRI和XhoI。PCR反应体系参考表2.1。PCR循环条件为:94℃,4min;94℃,30s;68℃,30s;72℃,90s;共30个循环,最后72℃延伸10min。PCR产物切胶回收。引物由北京奥科鼎盛公司合成,引物见表3。
表3克隆xynA的引物
Table3PrimersforxynA
2.1.2重组质粒pET28a-xyn4的构建
将表达载体pET28a和带有酶切位点的xynA用EcoRI和XhoI分别进行双酶切,37℃温浴4h。切胶回收xynA和切开的表达载体。T4DNA连接酶连接xynA和线性pET28a(+),摩尔比3∶1,16℃中连接12h,获得大肠杆菌表达质粒pET28a-xynA。按《分子克隆实验指南第三版》中所述方法热激转化大肠杆菌DH5a菌株″转化得到的阳性克隆用于培养及木聚糖酶表达。
2.2重组XynA酶分析
2.2.1同源性比对
在NCBI上对xynA序列进行BLAST比对,共找到51条与xynA同源性在70%以上的序列(图6),相比较同源性较高的是来自菌株Streptomycesmegasporus(HM003041.1),Streptomyceshalstedii(U41627.1),Streptomyceschattanoogensis(AF121864.2)木聚糖酶的基因,同源性分别达到81%,81%和74%,而且和木聚糖酶的同源性最高,说明该基因也是木聚糖酶,另一方面说明该基因比较新颖,具有较高的研究价值。
2.2.2信号肽预测
将xynA翻译成为氨基酸序列,通过在线信号肽预测网站(http://www.cbs.dtu.dk/services/SignalP)对其进行分析,结果见图7,信号肽序列预测结果表明该酶是一个具有信号肽的分泌蛋白。
2.2.3氨基酸保守功能区分析
蛋白质保守功能区分析采用蛋白质保守序列(CDD)数据库(http://www.ncbi.nlm.nih.Gov/structure/cdd/cdd.shtml.)来预测,结果见图8,表明该酶具有典型的第10族糖苷水解酶保守区域的典型结构。
此外,该基因还编码一段大小为10kDa的纤维素结合域(CBD)。去掉CBD区域,木聚糖酶分子量大小为34kDa。
对木聚糖基因全长xynA进行生物信息学分析,xynA全长共有1393碱基对,G,C含量占68%。DNAMAN软件分析,该基因能编码460个氨基酸如SEQ-2,预测分子量约为48.2kDa,等电点pI为9.04。且含有一段信号肽序列为SEQ-3。去掉该信号肽序列,剩余氨基酸分子量约为43.6kDa,等电点pI=8.19,与天然菌株产的一个约44kDa的木聚糖酶大小相同。分析蛋白质保守序列,结果表明该酶具有F/10糖苷水解酶的典型结构,并能翻译一段大小为10kDa的纤维素结合域,肽序列为SEQ-4。去掉CBD区域,木聚糖酶分子量大小为34kDa。
2.2.4木聚糖酶活性分析
粗粗酶液经硫酸铵分级沉淀、CMSFF柱层析进行纯化,得到了电泳纯分子量为44kDa的木聚糖酶,该过程中蛋白质、总酶活力、比活力、纯化倍数及回收率的变化见表4.3,经过40%~80%的硫酸铵沉淀,木聚糖酶纯化了2.6倍,回收到总酶活的82.9%。经过层析柱CMSFF后纯化了3.79倍,回收到5.3%的酶活。
各步的纯度电泳图及酶谱图见图9。经过两步纯化在分子量为44kDa的地方得到电泳纯的蛋白。酶谱分析显示,此条带具有木聚糖酶活性。
大肠杆菌重组子诱导产酶后经过超声破壁,使得木聚糖酶溶解到上清中采用DNS法测定木聚糖酶活力。具体操作如下:用0.05M,pH值为5.3的柠檬酸缓冲液稀释上清液样品,取100pl样品加入至900p11%的桦木木聚糖底物中,于55℃反应smin,加入1ml的DNS反应终止液,于沸水中显色15min,加入Im140%的酒石酸钾钠溶液,于540nm下测定吸光度,计算得到木聚糖酶酶活。DNS法测的该酶活力为19.7±1.2U/mL。
Claims (12)
1.一种木聚糖酶的核苷酸序列,其序列细节为SEQ-1。
2.这个序列的至少90%的同源。
3.一种木聚糖酶的多肽,其序列细节为SEQ-2。
4.这个序列的至少90%的同源。
5.一种木聚糖酶的信号肽,其序列细节为SEQ-3。
6.这个序列的至少90%的同源。
7.一种木聚糖酶的纤维素结合域(CBD),其序列细节为SEQ-4。
8.这个序列的至少90%的同源。
9.包含权利1,2,3,4,5,6,7,8的重组载体。
10.权利9的大肠杆菌的重组表达系统。
11.类似于权利10在乳酸杆菌,酿酒酵母,芽孢杆菌(如枯草芽孢杆菌),曲霉(如黑曲霉或者米曲霉)中的表达应用。
12.权利1,2,3,4,5,6,7,8,9,10,11在造纸工业,食品加工,纺织工业,饲料工业,生物能源,化妆品,医药保健品,以及各种生物反应器的应用。
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