技术领域technical field
本发明涉及一种纤维酶体基因的改建方法及通过基因改建获得的新酶，具体涉及一种以热纤梭菌的纤维酶体和它的单体酶为基础的基因改建方法，及基因改建产生的新型木质纤维水解酶，属于基因工程和酶工程领域。The invention relates to a cellulosome gene modification method and a new enzyme obtained through gene modification, in particular to a gene modification method based on the cellulosome of Clostridium thermocellum and its monomeric enzyme, and the gene modification method The produced novel lignocellulosic hydrolyzing enzyme belongs to the fields of genetic engineering and enzyme engineering.
背景技术Background technique
木质纤维是地球植物的支撑组织，主要成分是通过光合作用碳水化合物，是非常丰富的自然资源。同时，自然界很多微生物和植食性动物能够产生一系列酶对木质纤维进行水解，从而构成自然界碳素循环的重要环节。水解木质纤维中纤维素成分的主要酶类包括纤维素酶、纤维素外切酶、纤维二糖酶和b-葡萄糖苷酶等。纤维素酶是b-1,4葡聚糖内切酶的简称；纤维素酶的催化反应是纤维素水解中的限速步骤。Wood fiber is the supporting tissue of plants on the earth, and its main component is carbohydrates through photosynthesis, which is a very rich natural resource. At the same time, many microorganisms and herbivorous animals in nature can produce a series of enzymes to hydrolyze lignocellulose, which constitutes an important part of the carbon cycle in nature. The main enzymes that hydrolyze the cellulose component in lignocellulosics include cellulase, exocellulase, cellobiase and b-glucosidase, etc. Cellulase is short for b-1,4 endoglucanase; the catalyzed reaction of cellulase is the rate-limiting step in cellulose hydrolysis.
纤维素酶在纤维素利用与加工中具有广泛的用途。在造纸工业，纤维素酶在废纸的脱墨处理中是重要的生物催化剂，同时在机械制浆前以纤维素酶预处理可以增加纸浆的强度性能，显著降低机械磨浆时的能量消耗；在纺织工业，利用纤维素酶对棉织物及其混纺织物进行打磨刨光处理，可使织物的硬度适当下降，织物的悬垂度、 回弹性及柔软度获得改善；作为饲料添加剂，纤维素酶能够提高青贮饲料的品质，加强机体对营养物质的吸收，改善瘤胃的发酵功能，从而增加动物体的日增质量，改善产奶量和奶品质，提高经济效益。另外纤维素酶在食品加工和中草药加工等领域也有广泛的用途。但是，植物产生的纤维素结构致密，对纤维素酶的攻击具有很强的抵抗能力；迄今发现的所有天然纤维素酶都只能以有限的速度，缓慢地水解自然的结晶态纤维素。Cellulase has a wide range of applications in cellulose utilization and processing. In the paper industry, cellulase is an important biocatalyst in the deinking treatment of waste paper. At the same time, pretreatment with cellulase before mechanical pulping can increase the strength of pulp and significantly reduce the energy consumption during mechanical refining; In the textile industry, the use of cellulase to polish and plan the cotton fabric and its blended fabrics can reduce the hardness of the fabric appropriately, and improve the drape, resilience and softness of the fabric; as a feed additive, cellulase can Improve the quality of silage, strengthen the body's absorption of nutrients, improve the fermentation function of the rumen, thereby increasing the daily quality of animals, improving milk production and milk quality, and improving economic benefits. In addition, cellulase is also widely used in the fields of food processing and Chinese herbal medicine processing. However, the cellulose produced by plants has a dense structure and is highly resistant to the attack of cellulase; all natural cellulase found so far can only slowly hydrolyze natural crystalline cellulose at a limited speed.
热纤梭菌（Clostridium thermocellum）是1种嗜热厌氧细菌；它能够在细胞表面产生一种水解纤维素和半纤维素酶的多酶聚合体，即纤维酶体（cellulosome）。由于来源于嗜热细菌并且有多种酶的协同作用，纤维酶体的稳定性较好、降解纤维素的表观活性很高。因此几十年来，纤维酶体吸引了大批学者对其进行研究。纤维酶体的结构已经得到充分解析：纤维酶体的中心有1个支架蛋白，它的肽链中有个纤维素结合域（CBD）和多个粘连域（cohesin）；纤维酶体的支架上吸附了多种酶的单体，其中包括纤维素酶、木聚糖酶等；各单体酶的肽链中都具有一段保守的序列，称为锚定域（dockerin），单体酶被分别合成并分泌到细胞表面后以锚定域与支架上的粘连域相结合，从而形成多种酶的聚合体。纤维酶体通过CBD附着到纤维素微丝上对其进行消解。纤维酶体中的多种单体酶附着于同1个支架并共用同1个纤维素结合域（CBD），这种结构不利于通过基因超量表达生产。Clostridium thermocellum is a thermophilic anaerobic bacterium; it can produce a multi-enzyme polymer that hydrolyzes cellulose and hemicellulase, the cellulosome, on the cell surface. Because it is derived from thermophilic bacteria and has the synergistic effect of various enzymes, the cellulosome has good stability and high apparent activity of degrading cellulose. Therefore, for decades, cellulosomes have attracted a large number of scholars to study them. The structure of the cellulosome has been fully resolved: there is a scaffold protein in the center of the cellosome, and its peptide chain has a cellulose-binding domain (CBD) and multiple cohesin domains (cohesin); Monomers that adsorb a variety of enzymes, including cellulase, xylanase, etc.; each monomeric enzyme has a conserved sequence in the peptide chain, called the anchor domain (dockerin), and the monomeric enzymes are separated After being synthesized and secreted to the cell surface, the anchor domain is combined with the adhesion domain on the scaffold to form a polymer of various enzymes. Cellulosomes digest cellulose microfilaments by attaching CBD to them. Multiple monomeric enzymes in the cellosome are attached to the same scaffold and share the same cellulose-binding domain (CBD), a structure that is not conducive to production by gene overexpression.
在分子生物技术高度发展的今天，我们发现纤维酶体的生产有其致命的弱点：（1）纤维酶体产生菌是嗜热厌氧细菌，培养条件苛刻，细胞密度低，发酵产酶的效率非常低；（2）纤维酶体由多个基因编码，总分子质量超过2000 kDa，难以进行体外重组表达；（3）纤维酶体含有近20种不同的单体酶，其中许多单体并不是纤维素水解所必需的，还有些单体酶的活性不及其他来源的同类酶，因而，生产这些单体酶是生物能量的浪费；（4）纤维酶体中各种单体酶的活性有赖于支架上的CBD，离开支架后单体酶的催化活性下降。Today, with the high development of molecular biotechnology, we have found that the production of cellulosomes has its fatal weakness: (1) The cellulosome-producing bacteria are thermophilic anaerobic bacteria with harsh culture conditions, low cell density, and low enzyme production efficiency. Very low; (2) Cellulosomes are encoded by multiple genes with a total molecular mass of more than 2000 kDa, making it difficult to recombine and express in vitro; (3) Cellosomes contain nearly 20 different monomeric enzymes, many of which are not It is necessary for the hydrolysis of cellulose, and the activity of some monomeric enzymes is not as good as that of similar enzymes from other sources. Therefore, the production of these monomeric enzymes is a waste of biological energy; (4) The activity of various monomeric enzymes in cellulosomes depends on For the CBD on the scaffold, the catalytic activity of the monomeric enzyme decreased after leaving the scaffold.
发明内容Contents of the invention
本发明的目的：Purpose of the present invention:
纤维酶体中的各个单体都由独立的基因编码，这些基因都可以通过基因异源高效表达重组进行生产。但是没有支架蛋白的纤维素结合域(CBD)的作用，重组酶的活性不能充分发挥，而此时亚基上锚定域也成为多余。要解决的技术问题是建立一种对纤维酶体中的单体酶进行分子改建的方法，将纤维酶体中依赖于支架蛋白的单体酶构建成独立酶，获得生物活性明显提高的新型木质纤维水解酶，并使新酶得到异源可溶性高效表达，形成高产技术。Each monomer in the cellulosome is encoded by an independent gene, and these genes can be produced through gene heterologous high-efficiency expression recombination. However, without the role of the cellulose-binding domain (CBD) of the scaffold protein, the activity of the recombinase cannot be fully exerted, and the anchor domain on the subunit becomes redundant at this time. The technical problem to be solved is to establish a method for molecular modification of monomeric enzymes in cellulosomes, to construct monomeric enzymes in cellosomes that depend on scaffold proteins into independent enzymes, and to obtain new types of woody enzymes with significantly improved biological activity. Cellulose hydrolyzing enzymes, and the heterologous soluble high-efficiency expression of new enzymes, forming a high-yield technology.
技术方案：Technical solutions:
本发明采用的技术方案是通过基因重组技术，切除纤维酶体的单体酶肽链中的锚定域以减少冗余部件、用1个短链将支架蛋白上的CBD嫁接到单体酶的分子上，获得具有不同分子结构、能够独立结合底物、比单体酶活性高的新型独立酶。本技术方案在本发明中应用于、但是不限于纤维酶体中CelD的分子改建。The technical solution adopted in the present invention is to cut off the anchoring domain in the monomeric enzyme peptide chain of the cellulosome through gene recombination technology to reduce redundant parts, and use a short chain to graft the CBD on the scaffold protein to the monomeric enzyme Molecularly, new independent enzymes with different molecular structures, capable of independently binding substrates, and higher activity than monomeric enzymes were obtained. This technical solution is applied in the present invention, but not limited to the molecular remodeling of CelD in cellulosomes.
纤维酶体中的单体酶CelD（纤维素酶D）是最知名的活性最高的纤维素酶，由基因celD编码。运用本发明的技术方案对celD基因进行改建后产生新型纤维素酶NcelD。NcelD肽链的分子结构特征：不带有与纤维酶体支架蛋白上的粘连域相吸附的锚定域、带有1段链接短肽、有嫁接的CBD；功能特征：是能够结合纤维素的独立酶，不需要也不结合支架蛋白，水解纤维素的活性比自然单体酶CelD提高了66%；生产技术：运用自主发明的基因高效表达载体（美国发明专利US 7,807,460 B2），NcelD的编码基因在大肠杆菌（E. coli）中实现可溶性高效表达。The monomeric enzyme CelD (cellulase D) in the cellulosome is the most well-known and most active cellulase, encoded by the gene celD. The novel cellulase NcelD is produced after rebuilding the celD gene by using the technical scheme of the invention. Molecular structural characteristics of NcelD peptide chain: no anchoring domain adsorbed to the cohesion domain on the cellulosomal scaffold protein, a short chain peptide, and grafted CBD; functional characteristics: it can bind cellulose An independent enzyme that does not require or bind to scaffold proteins, the activity of hydrolyzing cellulose is 66% higher than that of the natural monomeric enzyme CelD; production technology: the use of self-invented high-efficiency gene expression vectors (US patent US 7,807,460 B2), the encoding of NcelD The gene was soluble and highly expressed in Escherichia coli (E. coli).
本发明所需的实验材料及来源：Required experimental materials and sources of the present invention:
热纤梭菌（ATCC 27405）购自美国菌种管理保藏中心，并根据ATCC指南进行细胞培养。细菌DNA的提取、基因的PCR扩增和克隆等根据《分子克隆手册》第三版上的标准方法进行（Sambrook and Russell, 2001, CSHL Press, Cold Spring Harbor, New York）。本实验中所用克隆宿主菌为E. coli DH10B，表达宿主菌为E. coli BL21。从NCBI数据库中获取纤维酶体上支架蛋白基因序列（GenBank accession No. L08665.1）和单体酶celD、celS基因序列（GenBank accession No. X04584.1；L06942.1），并在线分析这些基因的信号肽、锚定域、CBD等序列结构。所采用的表达载体pHsh参见美国发明专利US 7,807,460 B2相关文献，其基因序列参见基因库资料（GenBank accession No. FJ571619）；其中目标基因的表达受sigma 32类型启动子调控。Clostridium thermocellum (ATCC 27405) was purchased from the American Type Control Collection and cell culture was performed according to ATCC guidelines. Bacterial DNA extraction, PCR amplification and cloning of genes were carried out according to the standard methods in the third edition of "Handbook of Molecular Cloning" (Sambrook and Russell, 2001, CSHL Press, Cold Spring Harbor, New York). The cloning host bacteria used in this experiment is E. coli DH10B, and the expression host bacteria is E. coli BL21. Obtain the scaffold protein gene sequence (GenBank accession No. L08665.1) and monomeric enzyme celD and celS gene sequences (GenBank accession No. X04584.1; L06942.1) from the NCBI database, and analyze these genes online The signal peptide, anchor domain, CBD and other sequence structures. For the expression vector pHsh used, please refer to the relevant literature of the US invention patent US 7,807,460 B2, and for the gene sequence, please refer to the GenBank data (GenBank accession No. FJ571619); the expression of the target gene is regulated by the sigma 32 type promoter.
具体操作步骤如下：The specific operation steps are as follows:
（1）目标基因克隆：根据NCBI数据库中热纤梭菌纤维素酶（CelD）基因序列（GenBank accession No. X04584.1，SEQ ID NO.1）设计并合成引物，在上、下游引物中分别引入限制性内切酶识别位点Pst I和XhoI；以热纤梭菌基因组DNA为模板，通过PCR的方法扩增得到目标基因单体酶celD，经Pst I和Xho I双酶切并纯化后，连接到经过同样酶切的表达载体pHsh中；将连接产物通过电击转化到E. coli 细胞中，涂布于含有氨苄青霉素的LB培养基平板，培养出带有目标基因的转化子；从转化子中提取质粒DNA，通过基因序列测定确认目标基因celD克隆成功，celS 的克隆用同样的方法，CelD、CelS等酶的表达质粒被命名为pHsh-celD，pHsh-celS等等。(1) Target gene cloning: According to the Clostridium thermocellum cellulase (CelD) gene sequence (GenBank accession No. X04584.1, SEQ ID NO.1) in the NCBI database, primers were designed and synthesized, and the upstream and downstream primers were respectively Introduce restriction endonuclease recognition sites Pst I and XhoI; using Clostridium thermocellum genomic DNA as a template, the target gene monomer enzyme celD was amplified by PCR method, which was double digested and purified by Pst I and Xho I , connected to the expression vector pHsh that had undergone the same enzyme digestion; the ligated product was transformed into E. coli cells by electric shock, spread on the LB medium plate containing ampicillin, and the transformant with the target gene was cultivated; transformed from The plasmid DNA was extracted from the offspring, and the target gene celD was successfully cloned by gene sequence determination. The same method was used to clone celS. The expression plasmids of enzymes such as CelD and CelS were named pHsh-celD, pHsh-celS and so on.
（2）单体酶中锚定域的删除：根据步骤（1）所得表达质粒pHsh-celD或pHsh-celS的具体测序序列，设计引物以删除单体酶基因celD或celS编码尾部锚定域的核苷酸序列。用产生平末端的高保真性DNA聚合酶如Pyrobest（TaKaRa，大连）进行反向PCR，扩增出包含去除锚定域以外的剩余部分celD-d（SEQ ID NO.2）的质粒片段，为线状DNA，用T4 DNA连接酶对线状DNA进行首尾连接，转化E. coli后得到删去锚定域的单体酶表达质粒。(2) Deletion of the anchor domain in the monozyme: according to the specific sequencing sequence of the expression plasmid pHsh-celD or pHsh-celS obtained in step (1), design primers to delete the anchor domain of the tail of the monozyme gene celD or celS Nucleotide sequence. Use a high-fidelity DNA polymerase that produces blunt ends such as Pyrobest (TaKaRa, Dalian) to perform inverse PCR to amplify a plasmid fragment containing the remaining part of celD-d (SEQ ID NO.2) except for the anchor domain, which is the line The linear DNA was ligated head-to-tail with T4 DNA ligase, and transformed into E. coli to obtain a monomeric enzyme expression plasmid with the anchor domain deleted.
（3）CBD的嫁接：设计1对引物，以热纤梭菌基因组DNA为模板，用产生平末端的高保真性DNA聚合酶如Pyrobest扩增支架蛋白A中编码CBD的DNA片段cbd（SEQ ID NO.3），并且在基因片段cbd上游融合人工设计的链接片段（编码10-60个氨基酸，在酶的主体与纤维素结合域之间起链接作用）后，即得到片段Lcbd（SEQ ID NO.5），将其插入到步骤（2）所得的删除了锚定域的表达质粒pHsh-celD中，形成新酶基因celD-d+Lcbd，我们重新命名为ncelD（SEQID NO.6），转化E. coli后通过筛选和序列测定鉴定出序列正确的表达质粒，命名为pHsh-NcelD。采用同样的方法，我们得到新构建的质粒pHsh-NcelS。(3) CBD grafting: design a pair of primers, use Clostridium thermocellum genomic DNA as a template, and use a high-fidelity DNA polymerase such as Pyrobest to amplify the DNA fragment cbd (SEQ ID NO .3), and after fusing the artificially designed link fragment (encoding 10-60 amino acids, acting as a link between the main body of the enzyme and the cellulose binding domain) upstream of the gene fragment cbd, the fragment Lcbd (SEQ ID NO. 5), insert it into the expression plasmid pHsh-celD with the anchor domain deleted obtained in step (2), to form a new enzyme gene celD-d+Lcbd, which we renamed ncelD (SEQID NO.6), and transform E After coli, the expression plasmid with the correct sequence was identified by screening and sequence determination, and it was named pHsh-NcelD. Using the same method, we obtained the newly constructed plasmid pHsh-NcelS.
单体酶基因celD或celS获得CBD基因后不再附属于原来的纤维酶体，而是独立的新型酶。The single enzyme gene celD or celS is no longer attached to the original cellulosome after acquiring the CBD gene, but an independent new enzyme.
新型酶基因的高效表达：High-efficiency expression of novel enzyme genes:
新型酶的表达质粒pHsh-NcelD和pHsh-NcelS转化E. coli后在30℃进行通气培养3-6小时后，将培养温度提升到40℃-42℃对基因进行诱导表达，继续培养3-6小时后离心收集细胞，以超声波或均质机破碎细胞后获得新酶的粗酶液，进行SDS-PAGE分析。The expression plasmids pHsh-NcelD and pHsh-NcelS of the new enzymes were transformed into E. coli and cultured at 30°C for 3-6 hours, then raised the culture temperature to 40°C-42°C to induce gene expression, and continued to culture for 3-6 hours. The cells were collected by centrifugation after 1 hour, and the crude enzyme solution of the new enzyme was obtained after the cells were disrupted by ultrasonic waves or a homogenizer, and analyzed by SDS-PAGE.
有益效果：Beneficial effect:
（1）本发明的基因改建方法可取得以下有益效果：(1) The gene modification method of the present invention can achieve the following beneficial effects:
纤梭菌产生的纤维酶体上附着有多个单体酶，具有较高的活性和稳定性。单体酶都由独立的基因编码，可以通过基因工程高效表达，但是这些单体酶需要附着于纤维酶体的支架蛋白、借助支架蛋白上的纤维素结合域（CBD）才能充分发挥它们的活性。本发明的方法是切除单体酶上的冗余序列，并且将CBD嫁接到单体酶上，获得不依赖于纤维酶体的新型独立酶，其有益效果是：既具有高活性，又方便基因工程生产。Cellulosomes produced by Clostridium cellulosus are attached with multiple monomeric enzymes, which have high activity and stability. Monomer enzymes are all encoded by independent genes and can be expressed efficiently through genetic engineering, but these monomeric enzymes need to be attached to the scaffold protein of cellulosomes and rely on the cellulose-binding domain (CBD) on the scaffold protein to fully exert their activity . The method of the present invention is to excise the redundant sequence on the monomeric enzyme, and graft the CBD to the monomeric enzyme to obtain a new type of independent enzyme that does not depend on cellosomes. The beneficial effect is that it has high activity and is convenient for gene engineering production.
（2）通过本发明的方法对纤维素酶D进行基因改建取得以下有益效果：(2) Gene modification of cellulase D by the method of the present invention achieves the following beneficial effects:
纤维素酶D（CelD）是纤维酶体中活性最高的单体酶。运用本发明的方法对纤维素酶D进行基因改建后产生新型酶NcelD，其水解可溶性纤维素的活性提高了75%。Cellulase D (CelD) is the most active monomeric enzyme in cellulosomes. The method of the invention is used to genetically modify the cellulase D to produce a novel enzyme NcelD, and its activity of hydrolyzing soluble cellulose is increased by 75%.
（3）用pHsh系统表达新酶取得的有益效果：(3) Beneficial effects obtained by expressing the new enzyme with the pHsh system:
基因高效表达系统pHsh的表达载体是本实验室独家产品，获得美国发明专利（US7,807,460 B2）。新酶NcelD在pHsh载体中得到可溶性超量表达，为酶的工业化生产奠定基础。The expression vector of the high-efficiency gene expression system pHsh is an exclusive product of our laboratory and has obtained a US invention patent (US7,807,460 B2). The new enzyme NcelD was soluble and overexpressed in the pHsh vector, which laid the foundation for the industrial production of the enzyme.
附图说明Description of drawings
图1 新基因的设计和表达质粒结构图。Fig. 1 Design and expression plasmid structure diagram of the new gene.
图2 技术方案示意图。Figure 2 Schematic diagram of the technical solution.
图3 基因表达产生的重组纤维素酶的SDS-PAGE分析图谱。泳道标记：M，蛋白质分子量标准；1，含pHsh载体的菌体细胞中可溶性蛋白图谱；2，含质粒pHsh-CelD菌体细胞中可溶性蛋白图谱；3，含质粒pHsh-NcelD菌体细胞中可溶性蛋白图谱。箭头所指的条带是重组纤维素酶。Figure 3 SDS-PAGE analysis pattern of recombinant cellulase produced by gene expression. Lane markers: M, protein molecular weight standard; 1, soluble protein map in bacterial cells containing pHsh vector; 2, soluble protein map in bacterial cells containing plasmid pHsh-CelD; 3, soluble protein map in bacterial cells containing plasmid pHsh-NcelD protein map. The band indicated by the arrow is the recombinant cellulase.
图4 新酶NcelD与原始单体酶CelD的催化活性的比较。Fig. 4 Comparison of catalytic activity between the new enzyme NcelD and the original monomeric enzyme CelD.
图5 新酶NcelD与原始单体酶CelD的温度稳定性的比较。Figure 5 Comparison of the temperature stability of the new enzyme NcelD and the original monomeric enzyme CelD.
具体实施方式detailed description
下面结合具体实施例对本发明的方法和产品作进一步说明，但不以任何形式限制本发明。The method and product of the present invention will be further described below in conjunction with specific examples, but the present invention is not limited in any form.
实施例1: 从热纤梭菌基因组DNA扩增目标基因Example 1: Target gene amplification from Clostridium thermocellum genomic DNA
引物设计：根据NCBI中报道的热纤梭菌（ATCC 27405）纤维素酶（CelD）基因序列见SEQ ID NO.1，去掉该基因的信号肽序列，设计并合成两条引物序列（5’端分别含有Pst I和Xho I酶切位点）：Primer design: According to the gene sequence of Clostridium thermocellum (ATCC 27405) cellulase (CelD) reported in NCBI, see SEQ ID NO.1, the signal peptide sequence of the gene was removed, and two primer sequences (5' end containing Pst I and Xho I restriction sites respectively):
celD-1：5’ AAAACTGCAGGCAAAAATAACGGAGAATTATC 3’celD-1: 5' AAAACTGCAGGCAAAAATAACGGAGAATTATC 3'
celD-2：5’ CCGCTCGAGTTATATTGGTAATTTCTCGATTAC 3’celD-2: 5' CCGCTCGAGTTATATTGGTAATTTCTCGATTAC 3'
基因组PCR扩增条件：培养热纤梭菌细胞，提取基因组DNA作模板。在50 μL PCR反应体系中加入2 μL （20 ng）模板、2 μL 10 μM引物、4 μL 2.5 μM的dNTP、10 μL 的5×Prime STAR HS DNA 聚合酶缓冲液、31.5 μL 的超纯水和0.5 μL 的Prime STAR HS DNA聚合酶。按温度周期（98℃变性10s，60℃退火15s，72℃延伸2.5min）扩增30个循环，在72℃延伸10分钟。琼脂糖电泳进行检测，发现在1.8 kb附近出现预期特异性扩增带，说明PCR条件合适，产物单一。Genome PCR amplification conditions: Cultivate Clostridium thermocellum cells, extract genomic DNA as a template. Add 2 μL (20 ng) template, 2 μL 10 μM primer, 4 μL 2.5 μM dNTP, 10 μL 5×Prime STAR HS DNA polymerase buffer, 31.5 μL ultrapure water and 50 μL PCR reaction system. 0.5 μL of Prime STAR HS DNA Polymerase. According to the temperature cycle (denaturation at 98°C for 10 s, annealing at 60°C for 15 s, extension at 72°C for 2.5 min), amplify for 30 cycles, and extend at 72°C for 10 minutes. Agarose electrophoresis was performed for detection, and it was found that the expected specific amplification band appeared near 1.8 kb, indicating that the PCR conditions were suitable and the product was single.
实施例2: 热纤梭菌纤维酶体中单体酶基因的表达质粒的构建Embodiment 2: Construction of the expression plasmid of monomeric enzyme gene in Clostridium thermocellum cellulosome
将上述PCR产物割胶回收，进行Pst I和Xho I双酶切，克隆到原核表达载体pHsh质粒上。转化到E. coli DH10B中，并在含有氨苄青霉素（100 μg/mL）的LB培养基中培养转化子。从转化子提取质粒，以Pst I进行酶切，验证插入基因并进行序列测定。结果表明插入基因长度为1827bp，编码含有609个氨基酸残基，序列与数据库中的celD序列一致。表达质粒命名为pHsh-CelD。The above PCR product was recovered by tapping rubber, subjected to Pst I and Xho I double enzyme digestion, and cloned into the prokaryotic expression vector pHsh plasmid. Transform into E. coli DH10B, and culture transformants in LB medium containing ampicillin (100 μg/mL). Plasmids were extracted from transformants, digested with Pst I, and the inserted gene was verified and sequenced. The results showed that the length of the inserted gene was 1827bp, the code contained 609 amino acid residues, and the sequence was consistent with the celD sequence in the database. The expression plasmid was named pHsh-CelD.
实施例3: 基因celD的分子改建Example 3: Molecular reconstruction of gene celD
引物设计：根据NCBI网站数据库中CelD蛋白肽链中的锚定域信息，设计并合成两条反向PCR引物以pHsh-CelD 为模板，PCR扩增以得到包含有去除锚定域编码序列celD-d（SEQ ID NO.2）的质粒片段，自连环化后形成新的质粒pHsh-celD-d；根据NCBI资料中热纤梭菌支架蛋白CipA所含CBD的编码基因cbd（SEQ ID NO.3），同时，设计引物将celD-d与cbd连在一起得到基因序列celD-d+cbd（SEQ ID NO.4），最后设计并合成1对PCR引物扩增带有1个链接片段的Lcbd（SEQ ID NO.5），将celD-d与Lcbd连接后形成新酶基因celD-d+Lcbd，我们重新命名为ncelD（SEQ ID NO.6）。Primer design: According to the anchor domain information in the peptide chain of the CelD protein in the NCBI website database, two reverse PCR primers were designed and synthesized, using pHsh-CelD as a template, and PCR amplified to obtain the anchor domain coding sequence celD- The plasmid fragment of d (SEQ ID NO.2) forms a new plasmid pHsh-celD-d after concatenation; according to the NCBI data, the cbd gene cbd (SEQ ID NO.3 ), at the same time, design primers to link celD-d and cbd together to obtain the gene sequence celD-d+cbd (SEQ ID NO.4), and finally design and synthesize a pair of PCR primers to amplify Lcbd with a linking fragment ( SEQ ID NO.5), the new enzyme gene celD-d+Lcbd is formed after linking celD-d and Lcbd, which we renamed ncelD (SEQ ID NO.6).
dockerin-up: TAACTCGAGCACCACCACCdockerin-up: TAACTCGAGCACCACCACC
dockerin-dn: TTGAGGAGAATTATAGTTGACAdockerin-dn: TTGAGGAGAATTATAGTTGACA
cbd-1: AACGTTGGCAATGCAACACCcbd-1: AACGTTGGCAATGCAACACC
cbd-2: GCTTATTTCAAGGTAGGTGTCcbd-2: GCTTATTTCAAGGTAGGTGTC
以构建好的质粒pHsh-CelD为模板，以dockerin-up和dockerin-dn为引物，进行反向PCR；将PCR产物进行琼脂糖凝胶电泳，并割胶回收得到去除了celD锚定域编码序列的线性DNA片段产物（4036bp）。同时以热纤梭菌基因组为模板，以cbd-1和cbd-2为引物进行PCR扩增基因cbd和链接序列。将PCR产物进行琼脂糖凝胶电泳，并割胶回收得到带有链接序列的Lcbd片段（429bp）。将胶回收产物线性DNA片段和Lcbd片段进行磷酸化连接反应，并电击转化到E. coli DH10B感受态细胞中，在含有氨苄青霉素（100 μg/mL）的LB培养基中培养转化子，提取质粒，进行序列测定，长度为2043 bp的基因序列与预期一致。将改建成功的目标基因的表达产物命名为新型纤维素酶D（NcelD），其表达质粒命名为pHsh-NcelD。Using the constructed plasmid pHsh-CelD as a template and dockerin-up and dockerin-dn as primers, inverse PCR was performed; the PCR product was subjected to agarose gel electrophoresis, and recovered by tapping the gel to obtain the celD anchor domain coding sequence removed. Linear DNA fragment product (4036bp). At the same time, the genome of Clostridium thermocellum was used as a template, and cbd-1 and cbd-2 were used as primers to amplify gene cbd and link sequence by PCR. The PCR product was subjected to agarose gel electrophoresis, and the gel was tapped to recover the Lcbd fragment (429bp) with the linked sequence. The linear DNA fragment and Lcbd fragment of the gel recovery product were phosphorylated and ligated, and transformed into E. coli DH10B competent cells by electric shock, and the transformants were cultured in LB medium containing ampicillin (100 μg/mL) to extract the plasmid , sequence determination was carried out, and the gene sequence with a length of 2043 bp was consistent with the expectation. The expression product of the successfully transformed target gene was named novel cellulase D (NcelD), and its expression plasmid was named pHsh-NcelD.
实施例4: 纤维素酶CelD和新酶NcelD的表达Example 4: Expression of cellulase CelD and new enzyme NcelD
将重组质粒pHsh- CelD和pHsh-NcelD分别转化到E. coli BL21中，获得基因重组菌。重组菌接种在含有氨苄青霉素（100 μg/mL）的LB培养基中，置于30℃下震荡培养3-6小时后，将温度提升到40℃-42℃对基因进行诱导表达，在提高的温度下继续培养3-6小时后，离心收集细胞，以超声波使细胞裂解后离心，上清液为CelD和NcelD的粗酶液，进行SDS-PAGE分析，表明得到了超量表达（图3）。具体诱导表达条件：接种单菌落于含有100 μg/mL的LB培养基中 ，在30℃培养至OD600为0.6-0.8之间，将其迅速转到42℃培养6小时，摇床转速为200 rpm。The recombinant plasmids pHsh-CelD and pHsh-NcelD were transformed into E. coli BL21 respectively to obtain gene recombinant bacteria. The recombinant bacteria were inoculated in LB medium containing ampicillin (100 μg/mL), placed at 30°C for shaking culture for 3-6 hours, and then the temperature was raised to 40°C-42°C to induce gene expression. After continuing to culture at high temperature for 3-6 hours, the cells were collected by centrifugation, and the cells were lysed by ultrasonic waves and then centrifuged. The supernatant was the crude enzyme solution of CelD and NcelD. SDS-PAGE analysis showed that overexpression was obtained (Figure 3) . Specific induction expression conditions: Inoculate a single colony in LB medium containing 100 μg/mL, cultivate at 30°C until the OD600 is between 0.6-0.8, quickly transfer it to 42°C for 6 hours, and the shaker speed is 200 rpm.
实施例5: 重组CelD和NcelD的纯化Embodiment 5: the purification of recombinant CelD and NcelD
培养液5000 rpm，4℃离心10 min收集细胞。用灭菌的去离子水洗涤细胞三次，彻底除去残留的培养基，按照1：2（湿重：体积）的比例重悬于适量的邻苯二甲酸氢钾-咪唑（50mM，pH 6.0）缓冲液中，用高压破碎法破碎细胞后，以16000 rpm， 4℃离心15 min去除细胞碎片，上清液即为胞内粗酶液。将离心得到的粗酶液在60℃水浴锅中进行热处理30 min，以去除大肠杆菌中的杂蛋白。热处理后，4℃下以12000 rpm离心10 min去除热变性的杂蛋白，上清液为通过热处理得到部分纯化的酶液。The culture medium was centrifuged at 5000 rpm for 10 min at 4°C to collect the cells. Wash the cells three times with sterilized deionized water to completely remove the residual medium, and resuspend in an appropriate amount of potassium hydrogen phthalate-imidazole (50mM, pH 6.0) buffer at a ratio of 1:2 (wet weight: volume) After the cells were broken by high-pressure disruption method, they were centrifuged at 16,000 rpm at 4°C for 15 min to remove cell debris, and the supernatant was the crude intracellular enzyme solution. The crude enzyme solution obtained by centrifugation was heat-treated in a 60°C water bath for 30 min to remove impurity proteins in Escherichia coli. After heat treatment, centrifuge at 12,000 rpm for 10 min at 4°C to remove heat-denatured impurity proteins, and the supernatant is partially purified enzyme solution obtained by heat treatment.
必要时用以下方法对酶进行进一步纯化：将热处理后的粗酶液进行DEAE-Sephrose 离子交换层析。将80 mL离子交换树脂装柱后，先用10倍体积的去离子水冲洗用于保存填料的乙醇，再用缓冲液A平衡6～8个柱体积；将部分纯化的酶液泵入离子交换柱，流速为2 mL/min，用缓冲液洗涤2个柱体积将未结合的蛋白彻底洗尽；用400 mL含有0～0.5mol/L NaCl的梯度盐洗脱，收集含目标蛋白的洗脱液，以75%硫酸铵沉淀酶蛋白（4℃过夜），4℃离心20 min后弃上清，加入缓冲液轻轻溶解蛋白并进行透析。所使用的透析缓冲液为Tris-HCl（25mM，pH7.0），在冰上进行透析，每2小时更换一次透析缓冲液（更换3次）获得纯酶。If necessary, use the following method to further purify the enzyme: subject the heat-treated crude enzyme solution to DEAE-Sephrose ion exchange chromatography. After loading 80 mL of ion exchange resin into the column, wash the ethanol used to preserve the filler with 10 times the volume of deionized water, and then use buffer A to balance 6 to 8 column volumes; pump the partially purified enzyme solution into the ion exchange The column, with a flow rate of 2 mL/min, was washed with buffer for 2 column volumes to thoroughly wash away the unbound protein; eluted with 400 mL of gradient salt containing 0-0.5 mol/L NaCl, and collected the eluate containing the target protein. solution, precipitate the enzyme protein with 75% ammonium sulfate (overnight at 4°C), centrifuge at 4°C for 20 min, discard the supernatant, add buffer to gently dissolve the protein and perform dialysis. The dialysis buffer used was Tris-HCl (25 mM, pH 7.0). Dialysis was performed on ice, and the dialysis buffer was changed every 2 hours (3 times) to obtain pure enzyme.
实施例6: 纤维素酶活性测定方法Embodiment 6: cellulase activity assay method
纤维素酶活性一般有两种表述：CMC水解活性、结晶纤维素水解活性。酶活性的测定都是用还原糖分析法，即用酶水解CMC或结晶纤维素底物后，测定反应液中增加的还原糖的量。Cellulase activity generally has two expressions: CMC hydrolysis activity and crystalline cellulose hydrolysis activity. The determination of enzyme activity is all by reducing sugar analysis method, that is, after hydrolyzing CMC or crystalline cellulose substrate with enzyme, the amount of reducing sugar increased in the reaction solution is determined.
反应液的组分：45 µL 50 mM pH 5.0的缓冲液、50 µL 0.5% CMC或结晶纤维素（W/V）、5 µL适当稀释的酶液。Components of the reaction solution: 45 µL 50 mM pH 5.0 buffer, 50 µL 0.5% CMC or crystalline cellulose (W/V), 5 µL appropriately diluted enzyme solution.
反应温度和时间：55℃预热10 min后加入5 µL酶液，如果以CMC为底物在55℃反应5 min；如果以结晶纤维素为底物，在55℃反应30 min。Reaction temperature and time: After preheating at 55°C for 10 minutes, add 5 µL of enzyme solution. If CMC is used as substrate, react at 55°C for 5 minutes; if crystalline cellulose is used as substrate, react at 55°C for 30 minutes.
反应终止与显色：加入300 µL终止剂/显色剂PAHBAH（配方为：4体积的0.5 M NaOH和1体积的溶于0.5 M HCl的5% PAHBAH混合）终止反应；将混合物置于沸水浴中孵育10min，置冰上冷却后，用分光光度计测定在波长410 nm处的吸收值。酶的活性单位定义为每分钟催化产生1 µmol还原糖的酶量。Reaction termination and color development: add 300 µL terminator/chromogen PAHBAH (recipe: 4 volumes of 0.5 M NaOH and 1 volume of 5% PAHBAH dissolved in 0.5 M HCl mixed) to terminate the reaction; place the mixture in a boiling water bath After incubating for 10 min in ice, measure the absorbance at a wavelength of 410 nm with a spectrophotometer. The unit of enzyme activity is defined as the amount of enzyme that catalyzes the production of 1 µmol of reducing sugar per minute.
实施例7: 蛋白质浓度和还原糖浓度定量方法Example 7: Quantitative method for protein concentration and reducing sugar concentration
首先配置Bradford贮存液，其组成为：95 %乙醇10 mL与88 %磷酸20 mL混合，加入35 mg考马斯亮蓝G-250，溶解后室温下避光保存。First prepare Bradford stock solution, its composition is: 10 mL of 95% ethanol mixed with 20 mL of 88% phosphoric acid, add 35 mg of Coomassie Brilliant Blue G-250, dissolve and store in the dark at room temperature.
Bradford工作液配方为：425 mL H2O；15 mL 95 %乙醇；30 mL 85 %磷酸；30 mLBradford贮存液。The formula of Bradford working solution is: 425 mL H2 O; 15 mL 95% ethanol; 30 mL 85% phosphoric acid; 30 mL Bradford stock solution.
配好后，用Whatman 1号滤纸过滤，避光保存于室温，可使用数周，但在使用前要再用滤纸过滤。制备标准曲线：配制标准蛋白溶液 (1 mg/mL牛血清白蛋白)，选择2.5 g～20g的不同蛋白量，加入适量的水，使总体积为100 L，然后加入1 mL的Bradford工作液，上下振荡混匀，放置2 min后在595 nm处测定吸收值，测量应在10 min内完成。线性回归后得到吸光度-牛血清白蛋白量标准曲线为：y = 0.0174x + 0.0051，R2 = 0.995，x表示BSA (g)，y表示吸光度A595。After preparation, filter with Whatman No. 1 filter paper, store at room temperature in the dark, and can be used for several weeks, but filter with filter paper again before use. Preparation of standard curve: Prepare standard protein solution (1 mg/mL bovine serum albumin), select different protein amounts from 2.5 g to 20 g, add appropriate amount of water to make the total volume 100 L, then add 1 mL of Bradford working solution, Shake up and down to mix well, and measure the absorbance at 595 nm after standing for 2 min. The measurement should be completed within 10 min. The standard curve of absorbance-bovine serum albumin amount obtained after linear regression is: y = 0.0174x + 0.0051, R2 = 0.995, x represents BSA (g), y represents absorbance A595 .
样品检测同上法，用适当稀释样品代替牛血清白蛋白，测量待测样品的A595，从BSA标准曲线中确定待测样品的浓度。Sample detection is the same as above method, replace bovine serum albumin with appropriately diluted sample, measure A595 of the sample to be tested, and determine the concentration of the sample to be tested from the BSA standard curve.
葡萄糖的标准曲线的制作：Preparation of standard curve for glucose:
葡萄糖标准曲线制备：配制0.5 mM葡萄糖标准溶液，以不同浓度梯度的葡萄糖与NaOH/对羟基苯甲酸酰肼试剂反应，测定反应液的吸光度A410值。线性回归拟合后得到吸光度-葡萄糖含量标准曲线为：y = 189.23x + 0.03，R2 = 0.999，x表示葡萄糖浓度（µmol），y表示吸光度A410。Glucose standard curve preparation: Prepare 0.5 mM glucose standard solution, react with NaOH/p-hydroxybenzoic acid hydrazide reagent with different concentration gradients of glucose, and measure the absorbance A410 value of the reaction solution. The absorbance-glucose content standard curve obtained after linear regression fitting is: y = 189.23x + 0.03, R2 = 0.999, x represents the glucose concentration (µmol), and y represents the absorbance A410 .
酶学性质的测定：Determination of enzymatic properties:
（1）最适反应温度的测定：取适量稀释的纯化酶液在40～80℃范围内，每隔5℃，分别测定酶活，反应体系为：适量稀释的酶液5 µl，50 mM邻苯二甲酸氢钾-咪唑缓冲液（pH5.5）45 µl，0.5%（W/V）的CMC 50 µl，反应时间为5 min。以最高酶活力为100%，计算相对酶活。(1) Determination of the optimum reaction temperature: take an appropriate amount of diluted purified enzyme solution in the range of 40-80°C, and measure the enzyme activity at intervals of 5°C. The reaction system is: 5 μl of an appropriate amount of diluted enzyme solution, 50 mM Potassium hydrogen phthalate-imidazole buffer (pH5.5) 45 µl, 0.5% (W/V) CMC 50 µl, reaction time 5 min. The relative enzyme activity was calculated with the highest enzyme activity as 100%.
（2）温度稳定性的测定：取适量稀释的纯化酶液分别在45℃、50℃、55℃、60℃、65℃、70℃ pH 5.5下保温60 min，在不同的时间取样测定酶活，以未保温（4℃保存）的酶活力为100%，计算相对酶活，确定酶的温度稳定性。(2) Determination of temperature stability: Take an appropriate amount of diluted purified enzyme solution and incubate at 45°C, 50°C, 55°C, 60°C, 65°C, 70°C at pH 5.5 for 60 minutes, and take samples at different times to determine the enzyme activity , taking the enzyme activity without incubation (stored at 4°C) as 100%, calculate the relative enzyme activity, and determine the temperature stability of the enzyme.
（3）最适反应pH的测定：不同的pH（2.6~9.6）（pH在60℃校正）缓冲液，其中pH 2.6，3.8，4.0，4.2，4.6的是50 mM乙酸-乙酸钠缓冲液，pH 4.5，5.0，5.5，6.0，6.5，7.0，7.5，8.0的是50 mM邻苯二甲酸氢钾-咪唑缓冲液，pH 8.0，8.6，9.2，9.6的是50 mM Tris-甘氨酸缓冲液。于55℃分别测定酶活，反应体系为：5 µl适量稀释的纯化酶液，45µl不同pH值的缓冲液，50 µl 0.5%（W/V） CMC，反应5 min。以酶活力最高为100%，计算相对活性。(3) Determination of the optimum reaction pH: different pH (2.6~9.6) (pH is corrected at 60°C) buffer solutions, among which pH 2.6, 3.8, 4.0, 4.2, 4.6 are 50 mM acetic acid-sodium acetate buffer, pH 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 are 50 mM potassium hydrogen phthalate-imidazole buffer, pH 8.0, 8.6, 9.2, 9.6 are 50 mM Tris-glycine buffer. Enzyme activity was measured at 55°C. The reaction system was: 5 µl of purified enzyme solution diluted in an appropriate amount, 45 µl of buffer solutions with different pH values, 50 µl of 0.5% (W/V) CMC, and reacted for 5 min. The relative activity was calculated with the highest enzyme activity as 100%.
（4）pH稳定性的测定：取5 µl适量稀释的纯化酶液分别加入不同pH值的缓冲液中，在60℃下保温30 min，再加入50 µl 0.5%（W/V） CMC，反应时间5min再分别测定残留酶活性，以未保温（4℃保存）的酶活力为100%，计算相对酶活，比较不同pH条件下酶的稳定性。缓冲液的选择同最适反应pH的测定。反应所用缓冲液为pH 2.6 ~ 9.6的缓冲液。(4) Measurement of pH stability: Add 5 µl of an appropriate amount of diluted purified enzyme solution to buffer solutions with different pH values, incubate at 60°C for 30 min, then add 50 µl of 0.5% (W/V) CMC, and react After 5 minutes, the residual enzyme activity was measured respectively, and the enzyme activity that was not incubated (stored at 4°C) was regarded as 100%, and the relative enzyme activity was calculated to compare the stability of the enzyme under different pH conditions. The choice of buffer is the same as the determination of the optimum reaction pH. The buffer used in the reaction is a buffer with a pH of 2.6-9.6.
试验结果表明，新型酶NcelD的活性和稳定性比自然酶CelD有明显提高（图4、5）。The test results showed that the activity and stability of the new enzyme NcelD were significantly improved compared with the natural enzyme CelD (Figure 4, 5).
SEQUENCE LISTING SEQUENCE LISTING
<110> 江苏大学<110> Jiangsu University
<120> 纤维酶体基因的改建方法及获得的新型纤维素酶<120> Remodeling method of cellulosomal gene and obtained novel cellulase
<160> 6<160> 6
<170> PatentIn version 3.3<170> PatentIn version 3.3
<210> 1<210> 1
<211> 1824<211> 1824
<212> DNA<212>DNA
<213> 热纤梭菌(Clostridium thermoceUum)<213> Thermocellum Clostridium (Clostridium thermoceUum)
<400> 1<400> 1
gcaaaaataa cggagaatta tcaatttgat tcacgaatcc gtttaaactc aataggtttt 60gcaaaaataa cggagaatta tcaatttgat tcacgaatcc gtttaaactc aataggtttt 60
ataccgaacc acagcaaaaa ggcgactata gctgcaaatt gttcaacctt ttatgttgtt 120ataccgaacc acagcaaaaa ggcgactata gctgcaaatt gttcaacctt ttatgttgtt 120
aaagaagacg gaacaatagt gtataccgga acggcaactt caatgtttga caatgataca 180aaagaagacg gaacaatagt gtataccgga acggcaactt caatgtttga caatgataca 180
aaagaaactg tttatattgc tgatttttca tctgttaatg aagaaggaac gtactatctt 240aaagaaactg tttatattgc tgatttttca tctgttaatg aagaaggaac gtactatctt 240
gccgtgccgg gagtaggaaa aagcgtaaac tttaaaattg caatgaatgt atatgaggat 300gccgtgccgg gagtaggaaa aagcgtaaac tttaaaattg caatgaatgt atatgaggat 300
gcttttaaaa cagcaatgct gggaatgtat ttgctgcgct gcggcaccag tgtgtcggcc 360gcttttaaaa cagcaatgct gggaatgtat ttgctgcgct gcggcaccag tgtgtcggcc 360
acatacaacg gaatacacta ttcccatgga ccgtgccata ctaatgatgc atatcttgat 420acatacaacg gaatacacta ttcccatgga ccgtgccata ctaatgatgc atatcttgat 420
tatataaacg gacagcatac taaaaaagac agtacaaaag gctggcatga tgcgggcgac 480tatataaacg gacagcatac taaaaaagac agtacaaaag gctggcatga tgcgggcgac 480
tacaacaaat atgtggtaaa cgccggcata accgttggtt caatgttcct ggcgtgggag 540tacaacaaat atgtggtaaa cgccggcata accgttggtt caatgttcct ggcgtggggag 540
cattttaaag accagttgga gcctgtggca ttggagattc ccgaaaagaa caattcaata 600cattttaaag accacgttgga gcctgtggca ttggagattc ccgaaaagaa caattcaata 600
ccggattttc ttgatgaatt aaaatatgag atagactgga ttcttaccat gcaataccct 660ccggattttc ttgatgaatt aaaatatgag atagactgga ttcttaccat gcaataccct 660
gacgggagcg gaagggtggc tcataaagtt tcgacaagga actttggcgg ctttatcatg 720gacgggagcg gaagggtggc tcataaagtt tcgacaagga actttggcgg ctttatcatg 720
cctgagaacg aacacgacga aagatttttc gtgccctgga gcagtgccgc aacggcagac 780cctgagaacg aacacgacga aagatttttc gtgccctgga gcagtgccgc aacggcagac 780
tttgttgcca tgacggccat ggctgcaaga atattcaggc cttatgatcc tcaatatgct 840tttgttgcca tgacggccat ggctgcaaga atattcaggc cttatgatcc tcaatatgct 840
gaaaaatgta taaatgcggc aaaagtaagc tatgagtttt tgaagaacaa tcctgcgaat 900gaaaaatgta taaatgcggc aaaagtaagc tatgagtttt tgaagaacaa tcctgcgaat 900
gtttttgcaa accagagtgg attctcaaca ggagaatatg ccactgtcag tgatgcagat 960gtttttgcaa accagagtgg attctcaaca ggagaatatg ccactgtcag tgatgcagat 960
gacagattgt gggcggcggc tgaaatgtgg gagaccctgg gagatgaaga ataccttaga 1020gacagattgt gggcggcggc tgaaatgtgg gagaccctgg gagatgaaga ataccttaga 1020
gattttgaaa acagggcggc gcaattctcg aaaaaaatag aagccgattt tgactgggat 1080gattttgaaa acagggcggc gcaattctcg aaaaaaatag aagccgattt tgactgggat 1080
aatgttgcaa acttaggtat gtttacatat cttttgtcag aaagaccggg caagaatcct 1140aatgttgcaa acttaggtat gtttacatat cttttgtcag aaagaccggg caagaatcct 1140
gctttggtgc agtcaataaa ggatagtctc ctttccactg cggattcaat tgtgaggacc 1200gctttggtgc agtcaataaa ggatagtctc ctttccactg cggattcaat tgtgaggacc 1200
agccaaaacc atggctatgg cagaaccctt ggtacaacat attactgggg atgcaacggc 1260agccaaaacc atggctatgg cagaaccctt ggtacaacat attackgggg atgcaacggc 1260
acggttgtaa gacagactat gatacttcag gttgcgaaca agatttcacc caacaatgat 1320acggttgtaa gacagactat gatacttcag gttgcgaaca agatttcacc caacaatgat 1320
tatgtaaatg ctgctctcga tgcgatttca catgtatttg gaagaaacta ttacaacagg 1380tatgtaaatg ctgctctcga tgcgatttca catgtatttg gaagaaacta ttacaacagg 1380
tcttatgtaa caggccttgg tataaatcct cctatgaatc ctcatgacag acgttcaggg 1440tcttatgtaa caggccttgg tataaatcct cctatgaatc ctcatgacag acgttcaggg 1440
gctgacggaa tatgggagcc gtggcccggt taccttgtag gaggaggatg gcccggaccg 1500gctgacggaa tatgggagcc gtggcccggt taccttgtag gaggaggatg gcccggaccg 1500
aaggattggg tggatattca ggacagttat cagaccaatg aaattgctat aaactggaat 1560aaggattggg tggatattca ggacagttat cagaccaatg aaattgctat aaactggaat 1560
gcggcattga tttatgccct tgccggattt gtcaactata attctcctca aaatgaagta 1620gcggcattga tttatgccct tgccggattt gtcaactata attctcctca aaatgaagta 1620
ctgtacggag atgtgaatga tgacggaaaa gtaaactcca ctgacttgac tttgttaaaa 1680ctgtacggag atgtgaatga tgacggaaaa gtaaactcca ctgacttgac tttgttaaaa 1680
agatatgttc ttaaagccgt ctcaactctc ccttcttcca aagctgaaaa gaacgcagat 1740agatatgttc ttaaagccgt ctcaactctc ccttcttcca aagctgaaaa gaacgcagat 1740
gtaaatcgtg acggaagagt taattccagt gatgtcacaa tactttcaag atatttgata 1800gtaaatcgtg acggaagagt taattccagt gatgtcacaa tactttcaag atatttgata 1800
agggtaatcg agaaattacc aata 1824agggtaatcg agaaattacc aata 1824
<210> 2<210> 2
<211> 1611<211> 1611
<212> DNA<212>DNA
<213> 热纤梭菌(Clostridium thermoceUum)<213> Thermocellum Clostridium (Clostridium thermoceUum)
<400> 2<400> 2
gcaaaaataa cggagaatta tcaatttgat tcacgaatcc gtttaaactc aataggtttt 60gcaaaaataa cggagaatta tcaatttgat tcacgaatcc gtttaaactc aataggtttt 60
ataccgaacc acagcaaaaa ggcgactata gctgcaaatt gttcaacctt ttatgttgtt 120ataccgaacc acagcaaaaa ggcgactata gctgcaaatt gttcaacctt ttatgttgtt 120
aaagaagacg gaacaatagt gtataccgga acggcaactt caatgtttga caatgataca 180aaagaagacg gaacaatagt gtataccgga acggcaactt caatgtttga caatgataca 180
aaagaaactg tttatattgc tgatttttca tctgttaatg aagaaggaac gtactatctt 240aaagaaactg tttatattgc tgatttttca tctgttaatg aagaaggaac gtactatctt 240
gccgtgccgg gagtaggaaa aagcgtaaac tttaaaattg caatgaatgt atatgaggat 300gccgtgccgg gagtaggaaa aagcgtaaac tttaaaattg caatgaatgt atatgaggat 300
gcttttaaaa cagcaatgct gggaatgtat ttgctgcgct gcggcaccag tgtgtcggcc 360gcttttaaaa cagcaatgct gggaatgtat ttgctgcgct gcggcaccag tgtgtcggcc 360
acatacaacg gaatacacta ttcccatgga ccgtgccata ctaatgatgc atatcttgat 420acatacaacg gaatacacta ttcccatgga ccgtgccata ctaatgatgc atatcttgat 420
tatataaacg gacagcatac taaaaaagac agtacaaaag gctggcatga tgcgggcgac 480tatataaacg gacagcatac taaaaaagac agtacaaaag gctggcatga tgcgggcgac 480
tacaacaaat atgtggtaaa cgccggcata accgttggtt caatgttcct ggcgtgggag 540tacaacaaat atgtggtaaa cgccggcata accgttggtt caatgttcct ggcgtggggag 540
cattttaaag accagttgga gcctgtggca ttggagattc ccgaaaagaa caattcaata 600cattttaaag accacgttgga gcctgtggca ttggagattc ccgaaaagaa caattcaata 600
ccggattttc ttgatgaatt aaaatatgag atagactgga ttcttaccat gcaataccct 660ccggattttc ttgatgaatt aaaatatgag atagactgga ttcttaccat gcaataccct 660
gacgggagcg gaagggtggc tcataaagtt tcgacaagga actttggcgg ctttatcatg 720gacgggagcg gaagggtggc tcataaagtt tcgacaagga actttggcgg ctttatcatg 720
cctgagaacg aacacgacga aagatttttc gtgccctgga gcagtgccgc aacggcagac 780cctgagaacg aacacgacga aagatttttc gtgccctgga gcagtgccgc aacggcagac 780
tttgttgcca tgacggccat ggctgcaaga atattcaggc cttatgatcc tcaatatgct 840tttgttgcca tgacggccat ggctgcaaga atattcaggc cttatgatcc tcaatatgct 840
gaaaaatgta taaatgcggc aaaagtaagc tatgagtttt tgaagaacaa tcctgcgaat 900gaaaaatgta taaatgcggc aaaagtaagc tatgagtttt tgaagaacaa tcctgcgaat 900
gtttttgcaa accagagtgg attctcaaca ggagaatatg ccactgtcag tgatgcagat 960gtttttgcaa accagagtgg attctcaaca ggagaatatg ccactgtcag tgatgcagat 960
gacagattgt gggcggcggc tgaaatgtgg gagaccctgg gagatgaaga ataccttaga 1020gacagattgt gggcggcggc tgaaatgtgg gagaccctgg gagatgaaga ataccttaga 1020
gattttgaaa acagggcggc gcaattctcg aaaaaaatag aagccgattt tgactgggat 1080gattttgaaa acagggcggc gcaattctcg aaaaaaatag aagccgattt tgactgggat 1080
aatgttgcaa acttaggtat gtttacatat cttttgtcag aaagaccggg caagaatcct 1140aatgttgcaa acttaggtat gtttacatat cttttgtcag aaagaccggg caagaatcct 1140
gctttggtgc agtcaataaa ggatagtctc ctttccactg cggattcaat tgtgaggacc 1200gctttggtgc agtcaataaa ggatagtctc ctttccactg cggattcaat tgtgaggacc 1200
agccaaaacc atggctatgg cagaaccctt ggtacaacat attactgggg atgcaacggc 1260agccaaaacc atggctatgg cagaaccctt ggtacaacat attackgggg atgcaacggc 1260
acggttgtaa gacagactat gatacttcag gttgcgaaca agatttcacc caacaatgat 1320acggttgtaa gacagactat gatacttcag gttgcgaaca agatttcacc caacaatgat 1320
tatgtaaatg ctgctctcga tgcgatttca catgtatttg gaagaaacta ttacaacagg 1380tatgtaaatg ctgctctcga tgcgatttca catgtatttg gaagaaacta ttacaacagg 1380
tcttatgtaa caggccttgg tataaatcct cctatgaatc ctcatgacag acgttcaggg 1440tcttatgtaa caggccttgg tataaatcct cctatgaatc ctcatgacag acgttcaggg 1440
gctgacggaa tatgggagcc gtggcccggt taccttgtag gaggaggatg gcccggaccg 1500gctgacggaa tatgggagcc gtggcccggt taccttgtag gaggaggatg gcccggaccg 1500
aaggattggg tggatattca ggacagttat cagaccaatg aaattgctat aaactggaat 1560aaggattggg tggatattca ggacagttat cagaccaatg aaattgctat aaactggaat 1560
gcggcattga tttatgccct tgccggattt gtcaactata attctcctca a 1611gcggcattga tttatgccct tgccggattt gtcaactata attctcctca a 1611
<210> 3<210> 3
<211> 276<211> 276
<212> DNA<212>DNA
<213> 热纤梭菌(Clostridium thermoceUum)<213> Thermocellum Clostridium (Clostridium thermoceUum)
<400> 3<400> 3
gttgaattct acaacagcaa tccttcagat actactaact caatcaatcc tcagttcaag 60gttgaattct acaacagcaa tccttcagat actactaact caatcaatcc tcagttcaag 60
gttactaata ccggaagcag tgcaattgat ttgtccaaac tcacattgag atattattat 120gttactaata ccggaagcag tgcaattgat ttgtccaaac tcacattgag atattattat 120
acagtagacg gacagaaaga tcagaccttc tggtgtgacc atgctgcaat aatcggcagt 180acagtagacg gacagaaaga tcagaccttc tggtgtgacc atgctgcaat aatcggcagt 180
aacggcagct acaacggaat tacttcaaat gtaaaaggaa catttgtaaa aatgagttcc 240aacggcagct acaacggaat tacttcaaat gtaaaaggaa catttgtaaa aatgagttcc 240
tcaacaaata acgcagacac ctaccttgaa ataagc 276tcaacaaata acgcagacac ctaccttgaa ataagc 276
<210> 4<210> 4
<211> 1887<211> 1887
<212> DNA<212>DNA
<213> 人工序列<213> Artificial sequence
<400> 4<400> 4
gcaaaaataa cggagaatta tcaatttgat tcacgaatcc gtttaaactc aataggtttt 60gcaaaaataa cggagaatta tcaatttgat tcacgaatcc gtttaaactc aataggtttt 60
ataccgaacc acagcaaaaa ggcgactata gctgcaaatt gttcaacctt ttatgttgtt 120ataccgaacc acagcaaaaa ggcgactata gctgcaaatt gttcaacctt ttatgttgtt 120
aaagaagacg gaacaatagt gtataccgga acggcaactt caatgtttga caatgataca 180aaagaagacg gaacaatagt gtataccgga acggcaactt caatgtttga caatgataca 180
aaagaaactg tttatattgc tgatttttca tctgttaatg aagaaggaac gtactatctt 240aaagaaactg tttatattgc tgatttttca tctgttaatg aagaaggaac gtactatctt 240
gccgtgccgg gagtaggaaa aagcgtaaac tttaaaattg caatgaatgt atatgaggat 300gccgtgccgg gagtaggaaa aagcgtaaac tttaaaattg caatgaatgt atatgaggat 300
gcttttaaaa cagcaatgct gggaatgtat ttgctgcgct gcggcaccag tgtgtcggcc 360gcttttaaaa cagcaatgct gggaatgtat ttgctgcgct gcggcaccag tgtgtcggcc 360
acatacaacg gaatacacta ttcccatgga ccgtgccata ctaatgatgc atatcttgat 420acatacaacg gaatacacta ttcccatgga ccgtgccata ctaatgatgc atatcttgat 420
tatataaacg gacagcatac taaaaaagac agtacaaaag gctggcatga tgcgggcgac 480tatataaacg gacagcatac taaaaaagac agtacaaaag gctggcatga tgcgggcgac 480
tacaacaaat atgtggtaaa cgccggcata accgttggtt caatgttcct ggcgtgggag 540tacaacaaat atgtggtaaa cgccggcata accgttggtt caatgttcct ggcgtggggag 540
cattttaaag accagttgga gcctgtggca ttggagattc ccgaaaagaa caattcaata 600cattttaaag accacgttgga gcctgtggca ttggagattc ccgaaaagaa caattcaata 600
ccggattttc ttgatgaatt aaaatatgag atagactgga ttcttaccat gcaataccct 660ccggattttc ttgatgaatt aaaatatgag atagactgga ttcttaccat gcaataccct 660
gacgggagcg gaagggtggc tcataaagtt tcgacaagga actttggcgg ctttatcatg 720gacgggagcg gaagggtggc tcataaagtt tcgacaagga actttggcgg ctttatcatg 720
cctgagaacg aacacgacga aagatttttc gtgccctgga gcagtgccgc aacggcagac 780cctgagaacg aacacgacga aagatttttc gtgccctgga gcagtgccgc aacggcagac 780
tttgttgcca tgacggccat ggctgcaaga atattcaggc cttatgatcc tcaatatgct 840tttgttgcca tgacggccat ggctgcaaga atattcaggc cttatgatcc tcaatatgct 840
gaaaaatgta taaatgcggc aaaagtaagc tatgagtttt tgaagaacaa tcctgcgaat 900gaaaaatgta taaatgcggc aaaagtaagc tatgagtttt tgaagaacaa tcctgcgaat 900
gtttttgcaa accagagtgg attctcaaca ggagaatatg ccactgtcag tgatgcagat 960gtttttgcaa accagagtgg attctcaaca ggagaatatg ccactgtcag tgatgcagat 960
gacagattgt gggcggcggc tgaaatgtgg gagaccctgg gagatgaaga ataccttaga 1020gacagattgt gggcggcggc tgaaatgtgg gagaccctgg gagatgaaga ataccttaga 1020
gattttgaaa acagggcggc gcaattctcg aaaaaaatag aagccgattt tgactgggat 1080gattttgaaa acagggcggc gcaattctcg aaaaaaatag aagccgattt tgactgggat 1080
aatgttgcaa acttaggtat gtttacatat cttttgtcag aaagaccggg caagaatcct 1140aatgttgcaa acttaggtat gtttacatat cttttgtcag aaagaccggg caagaatcct 1140
gctttggtgc agtcaataaa ggatagtctc ctttccactg cggattcaat tgtgaggacc 1200gctttggtgc agtcaataaa ggatagtctc ctttccactg cggattcaat tgtgaggacc 1200
agccaaaacc atggctatgg cagaaccctt ggtacaacat attactgggg atgcaacggc 1260agccaaaacc atggctatgg cagaaccctt ggtacaacat attackgggg atgcaacggc 1260
acggttgtaa gacagactat gatacttcag gttgcgaaca agatttcacc caacaatgat 1320acggttgtaa gacagactat gatacttcag gttgcgaaca agatttcacc caacaatgat 1320
tatgtaaatg ctgctctcga tgcgatttca catgtatttg gaagaaacta ttacaacagg 1380tatgtaaatg ctgctctcga tgcgatttca catgtatttg gaagaaacta ttacaacagg 1380
tcttatgtaa caggccttgg tataaatcct cctatgaatc ctcatgacag acgttcaggg 1440tcttatgtaa caggccttgg tataaatcct cctatgaatc ctcatgacag acgttcaggg 1440
gctgacggaa tatgggagcc gtggcccggt taccttgtag gaggaggatg gcccggaccg 1500gctgacggaa tatgggagcc gtggcccggt taccttgtag gaggaggatg gcccggaccg 1500
aaggattggg tggatattca ggacagttat cagaccaatg aaattgctat aaactggaat 1560aaggattggg tggatattca ggacagttat cagaccaatg aaattgctat aaactggaat 1560
gcggcattga tttatgccct tgccggattt gtcaactata attctcctca agttgaattc 1620gcggcattga tttatgccct tgccggattt gtcaactata attctcctca agttgaattc 1620
tacaacagca atccttcaga tactactaac tcaatcaatc ctcagttcaa ggttactaat 1680tacaacagca atccttcaga tactactaac tcaatcaatc ctcagttcaa ggttactaat 1680
accggaagca gtgcaattga tttgtccaaa ctcacattga gatattatta tacagtagac 1740accggaagca gtgcaattga tttgtccaaa ctcacattga gatattatta tacagtagac 1740
ggacagaaag atcagacctt ctggtgtgac catgctgcaa taatcggcag taacggcagc 1800ggacagaaag atcagacctt ctggtgtgac catgctgcaa taatcggcag taacggcagc 1800
tacaacggaa ttacttcaaa tgtaaaagga acatttgtaa aaatgagttc ctcaacaaat 1860tacaacggaa ttacttcaaa tgtaaaagga aatttgtaa aaatgagttc ctcaacaaat 1860
aacgcagaca cctaccttga aataagc 1887aacgcagaca cctaccttga aataagc 1887
<210> 5<210> 5
<211> 429<211> 429
<212> DNA<212>DNA
<213> 热纤梭菌(Clostridium thermoceUum)<213> Thermocellum Clostridium (Clostridium thermoceUum)
<400> 5<400> 5
aacgttggca atgcaacacc gaccaaggga gcaacaccaa caaatacagc tacgccgaca 60aacgttggca atgcaacacc gaccaaggga gcaaccacaa caaatacagc tacgccgaca 60
aaatcagcta cggctacgcc caccaggcca tcggtaccga caaacacacc gacaaacaca 120aaatcagcta cggctacgcc caccaggcca tcggtaccga caaacacacc gacaaacaca 120
ccggcaaata caccggtatc aggcaatttg aaggttgaat tctacaacag caatccttca 180ccggcaaata caccggtatc aggcaatttg aaggttgaat tctacaacag caatccttca 180
gatactacta actcaatcaa tcctcagttc aaggttacta ataccggaag cagtgcaatt 240gatactacta actcaatcaa tcctcagttc aaggttacta ataccggaag cagtgcaatt 240
gatttgtcca aactcacatt gagatattat tatacagtag acggacagaa agatcagacc 300gatttgtcca aactcacatt gagatattat tatacagtag acggacagaa agatcagacc 300
ttctggtgtg accatgctgc aataatcggc agtaacggca gctacaacgg aattacttca 360ttctggtgtg accatgctgc aataatcggc agtaacggca gctacaacgg aattacttca 360
aatgtaaaag gaacatttgt aaaaatgagt tcctcaacaa ataacgcaga cacctacctt 420aatgtaaaag gaacatttgt aaaaatgagt tcctcaacaa ataacgcaga cacctacctt 420
gaaataagc 429gaaataagc 429
<210> 6<210> 6
<211> 2043<211> 2043
<212> DNA<212>DNA
<213> 人工序列<213> Artificial sequence
<400> 6<400> 6
gcaaaaataa cggagaatta tcaatttgat tcacgaatcc gtttaaactc aataggtttt 60gcaaaaataa cggagaatta tcaatttgat tcacgaatcc gtttaaactc aataggtttt 60
ataccgaacc acagcaaaaa ggcgactata gctgcaaatt gttcaacctt ttatgttgtt 120ataccgaacc acagcaaaaa ggcgactata gctgcaaatt gttcaacctt ttatgttgtt 120
aaagaagacg gaacaatagt gtataccgga acggcaactt caatgtttga caatgataca 180aaagaagacg gaacaatagt gtataccgga acggcaactt caatgtttga caatgataca 180
aaagaaactg tttatattgc tgatttttca tctgttaatg aagaaggaac gtactatctt 240aaagaaactg tttatattgc tgatttttca tctgttaatg aagaaggaac gtactatctt 240
gccgtgccgg gagtaggaaa aagcgtaaac tttaaaattg caatgaatgt atatgaggat 300gccgtgccgg gagtaggaaa aagcgtaaac tttaaaattg caatgaatgt atatgaggat 300
gcttttaaaa cagcaatgct gggaatgtat ttgctgcgct gcggcaccag tgtgtcggcc 360gcttttaaaa cagcaatgct gggaatgtat ttgctgcgct gcggcaccag tgtgtcggcc 360
acatacaacg gaatacacta ttcccatgga ccgtgccata ctaatgatgc atatcttgat 420acatacaacg gaatacacta ttcccatgga ccgtgccata ctaatgatgc atatcttgat 420
tatataaacg gacagcatac taaaaaagac agtacaaaag gctggcatga tgcgggcgac 480tatataaacg gacagcatac taaaaaagac agtacaaaag gctggcatga tgcgggcgac 480
tacaacaaat atgtggtaaa cgccggcata accgttggtt caatgttcct ggcgtgggag 540tacaacaaat atgtggtaaa cgccggcata accgttggtt caatgttcct ggcgtggggag 540
cattttaaag accagttgga gcctgtggca ttggagattc ccgaaaagaa caattcaata 600cattttaaag accacgttgga gcctgtggca ttggagattc ccgaaaagaa caattcaata 600
ccggattttc ttgatgaatt aaaatatgag atagactgga ttcttaccat gcaataccct 660ccggattttc ttgatgaatt aaaatatgag atagactgga ttcttaccat gcaataccct 660
gacgggagcg gaagggtggc tcataaagtt tcgacaagga actttggcgg ctttatcatg 720gacgggagcg gaagggtggc tcataaagtt tcgacaagga actttggcgg ctttatcatg 720
cctgagaacg aacacgacga aagatttttc gtgccctgga gcagtgccgc aacggcagac 780cctgagaacg aacacgacga aagatttttc gtgccctgga gcagtgccgc aacggcagac 780
tttgttgcca tgacggccat ggctgcaaga atattcaggc cttatgatcc tcaatatgct 840tttgttgcca tgacggccat ggctgcaaga atattcaggc cttatgatcc tcaatatgct 840
gaaaaatgta taaatgcggc aaaagtaagc tatgagtttt tgaagaacaa tcctgcgaat 900gaaaaatgta taaatgcggc aaaagtaagc tatgagtttt tgaagaacaa tcctgcgaat 900
gtttttgcaa accagagtgg attctcaaca ggagaatatg ccactgtcag tgatgcagat 960gtttttgcaa accagagtgg attctcaaca ggagaatatg ccactgtcag tgatgcagat 960
gacagattgt gggcggcggc tgaaatgtgg gagaccctgg gagatgaaga ataccttaga 1020gacagattgt gggcggcggc tgaaatgtgg gagaccctgg gagatgaaga ataccttaga 1020
gattttgaaa acagggcggc gcaattctcg aaaaaaatag aagccgattt tgactgggat 1080gattttgaaa acagggcggc gcaattctcg aaaaaaatag aagccgattt tgactgggat 1080
aatgttgcaa acttaggtat gtttacatat cttttgtcag aaagaccggg caagaatcct 1140aatgttgcaa acttaggtat gtttacatat cttttgtcag aaagaccggg caagaatcct 1140
gctttggtgc agtcaataaa ggatagtctc ctttccactg cggattcaat tgtgaggacc 1200gctttggtgc agtcaataaa ggatagtctc ctttccactg cggattcaat tgtgaggacc 1200
agccaaaacc atggctatgg cagaaccctt ggtacaacat attactgggg atgcaacggc 1260agccaaaacc atggctatgg cagaaccctt ggtacaacat attackgggg atgcaacggc 1260
acggttgtaa gacagactat gatacttcag gttgcgaaca agatttcacc caacaatgat 1320acggttgtaa gacagactat gatacttcag gttgcgaaca agatttcacc caacaatgat 1320
tatgtaaatg ctgctctcga tgcgatttca catgtatttg gaagaaacta ttacaacagg 1380tatgtaaatg ctgctctcga tgcgatttca catgtatttg gaagaaacta ttacaacagg 1380
tcttatgtaa caggccttgg tataaatcct cctatgaatc ctcatgacag acgttcaggg 1440tcttatgtaa caggccttgg tataaatcct cctatgaatc ctcatgacag acgttcaggg 1440
gctgacggaa tatgggagcc gtggcccggt taccttgtag gaggaggatg gcccggaccg 1500gctgacggaa tatgggagcc gtggcccggt taccttgtag gaggaggatg gcccggaccg 1500
aaggattggg tggatattca ggacagttat cagaccaatg aaattgctat aaactggaat 1560aaggattggg tggatattca ggacagttat cagaccaatg aaattgctat aaactggaat 1560
gcggcattga tttatgccct tgccggattt gtcaactata attctcctca aaacgttggc 1620gcggcattga tttatgccct tgccggattt gtcaactata attctcctca aaacgttggc 1620
aatgcaacac cgaccaaggg agcaacacca acaaatacag ctacgccgac aaaatcagct 1680aatgcaacac cgaccaaggg agcaacacca acaaatacag ctacgccgac aaaatcagct 1680
acggctacgc ccaccaggcc atcggtaccg acaaacacac cgacaaacac accggcaaat 1740acggctacgc ccaccaggcc atcggtaccg acaaacacac cgacaaacac accggcaaat 1740
acaccggtat caggcaattt gaaggttgaa ttctacaaca gcaatccttc agatactact 1800acaccggtat caggcaattt gaaggttgaa ttctacaaca gcaatccttc agatactact 1800
aactcaatca atcctcagtt caaggttact aataccggaa gcagtgcaat tgatttgtcc 1860aactcaatca atcctcagtt caaggttact aataccggaa gcagtgcaat tgatttgtcc 1860
aaactcacat tgagatatta ttatacagta gacggacaga aagatcagac cttctggtgt 1920aaactcacat tgagatatta ttatacagta gacggacaga aagatcagac cttctggtgt 1920
gaccatgctg caataatcgg cagtaacggc agctacaacg gaattacttc aaatgtaaaa 1980gaccatgctg caataatcgg cagtaacggc agctacaacg gaattacttc aaatgtaaaa 1980
ggaacatttg taaaaatgag ttcctcaaca aataacgcag acacctacct tgaaataagc 2040ggaacatttg taaaaatgag ttcctcaaca aataacgcag acacctacct tgaaataagc 2040
taa 2043taa 2043
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410173567.9ACN103923933B (en) | 2014-04-28 | 2014-04-28 | The reconstruction method of cellulase body gene and the cellulase of acquisition |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410173567.9ACN103923933B (en) | 2014-04-28 | 2014-04-28 | The reconstruction method of cellulase body gene and the cellulase of acquisition |
| Publication Number | Publication Date |
|---|---|
| CN103923933A CN103923933A (en) | 2014-07-16 |
| CN103923933Btrue CN103923933B (en) | 2016-09-14 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410173567.9AExpired - Fee RelatedCN103923933B (en) | 2014-04-28 | 2014-04-28 | The reconstruction method of cellulase body gene and the cellulase of acquisition |
| Country | Link |
|---|---|
| CN (1) | CN103923933B (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106884030A (en)* | 2015-12-16 | 2017-06-23 | 丰益(上海)生物技术研发中心有限公司 | The method for improving the fermentation yield of the fusion protein of containing cellulose binding domain |
| CN107513527B (en)* | 2016-06-16 | 2020-11-27 | 青岛蔚蓝生物集团有限公司 | Cellulase mutant and application thereof |
| CN110734926A (en)* | 2019-10-24 | 2020-01-31 | 江苏大学 | A kind of endoglucanase expression vector and its construction method and application |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FI119325B (en)* | 2005-12-22 | 2008-10-15 | Ab Enzymes Oy | New endoglucanase polypeptides and their preparation and use |
| Title |
|---|
| Accession Number:L08665.1;Gerngross,U.T.等;《GenBank》;19930506;1-2* |
| Accession Number:X04584.1;Joliff,G. 等;《GenBank》;20081023;1-2* |
| 嗜热真菌纤维素酶的CBD与海栖热袍菌的纤维素酶融合表达;乐易林等;《微生物学通报》;20090620;第36卷(第6期);837-841* |
| 梭热杆菌纤维小体研究进展;李爽等;《生物技术通报》;20111231(第5期);31-37* |
| Publication number | Publication date |
|---|---|
| CN103923933A (en) | 2014-07-16 |
| Publication | Publication Date | Title |
|---|---|---|
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