(一)技术领域(1) Technical field
本发明涉及生物工程，具体地说是一种表达是疏绵状嗜热丝孢菌Thermomyceslanuginosus AA9家族多糖单加氧酶基因TLAA9-3的毕赤酵母工程菌株Pichia pastorisGS-CT-9-3。The invention relates to bioengineering, in particular to a Pichia pastoris GS-CT-9-3 engineering strain expressing the polysaccharide monooxygenase gene TLAA9-3 of Thermomyceslanuginosus AA9 family.
(二)背景技术(2) Background technology
多糖单加氧酶(英语：Polysaccharide monooxygenases)，又称多糖单氧酶)是一种专门氧化裂解糖苷键，并产生多个纤维寡糖分子的酵素，也是自然界中的常见酵素之一。这类蛋白质在人类的产业上也有所应用，例如用来将纤维素与半纤维素等生物质能降解成可用的小分子。多糖单加氧酶具有多个家族。疏绵状嗜热丝孢菌Thermomyces lanuginosus是一种广泛分布于堆肥中的嗜热真菌。该菌在微晶纤维素为唯一碳源的培养基中50℃条件下可以产生热稳定的纤维素酶和AA9家族多糖单加氧酶。Polysaccharide monooxygenase (English: Polysaccharide monooxygenase, also known as polysaccharide monooxygenase) is an enzyme that oxidatively cleaves glycosidic bonds and produces multiple cellooligosaccharide molecules, and is also one of the common enzymes in nature. This type of protein is also used in human industry, for example, to degrade biomass such as cellulose and hemicellulose into usable small molecules. There are several families of polysaccharide monooxygenases. Thermomyces lanuginosus is a thermophilic fungus widely distributed in compost. The bacterium can produce thermostable cellulase and AA9 family polysaccharide monooxygenase at 50°C in the medium with microcrystalline cellulose as the sole carbon source.
疏绵状嗜热丝孢菌产生的AA9家族多糖单加氧酶TLAA9-3具有氧化裂解纤维素的作用，并且对该菌株产生的内切纤维素酶N24的酶活性具有明显的促进作用,可使其活性提高1.25倍。多糖单加氧酶发挥其活性需要供电子体(抗坏血酸或CDH)的存在，经薄层层析(TLC)检测产生多种纤维寡糖。该酶在缺乏底物的情况下可以产生过氧化氢，并且经飞行质谱鉴定该酶氧化裂解打断纤维素长链既有C1氧化也有C4氧化。The AA9 family polysaccharide monooxygenase TLAA9-3 produced by Thermomyces lanuginosa has the effect of oxidatively cracking cellulose, and can obviously promote the enzymatic activity of endocellulase N24 produced by this strain, which can Make its activity increase 1.25 times. The activity of polysaccharide monooxygenase requires the presence of an electron donor (ascorbic acid or CDH), and various cellooligosaccharides are produced by thin layer chromatography (TLC). The enzyme can produce hydrogen peroxide in the absence of a substrate, and it was identified by flight mass spectrometry that the enzyme oxidatively cleavages and breaks the long chain of cellulose with both C1 oxidation and C4 oxidation.
(三)发明内容(3) Contents of the invention
本发明从疏绵状嗜热丝孢菌Thermomyces lanuginosus获得多糖单加氧酶cDNA序列全长，命名为TLAA9-3，TLAA9-3基因DNA全长1110bp，包括信号肽 序列，起始密码子和终止密码子，无内含子，编码343个氨基酸。具有信号肽序列(1-26aaMAFSNIFSRPARLVLSATVFLSLAQA)及50个O糖基化位点,有一个潜在的N糖基化位点。将TLAA9-3编码的氨基酸序列在国际基因库中进行检索，发现属于多糖单加氧酶第AA9家族。The present invention obtains the full-length polysaccharide monooxygenase cDNA sequence from Thermomyces lanuginosus, which is named TLAA9-3, and the full-length TLAA9-3 gene DNA is 1110bp, including signal peptide sequence, initiation codon and termination Codons, without introns, encode 343 amino acids. It has a signal peptide sequence (1-26aaMAFSNIFSRPARLVLSATVFLSLAQA) and 50 O-glycosylation sites, and one potential N-glycosylation site. The amino acid sequence encoded by TLAA9-3 was searched in the international gene bank, and it was found that it belonged to the AA9 family of polysaccharide monooxygenases.
构建重组表达质粒载体pPICZ α A/TLAA9-3，利用电转仪进行电击转化Pichiapastoris GS115，在Zeocin培养基上筛选，经PCR鉴定筛选阳性转化子，高浓度博来霉素筛选多拷贝转化子，然后进行甲醇诱导表达，获得毕赤酵母工程菌株GS-CT-9-3。该工程菌株接种于含BMGY培养基中，在28℃200rpm/min摇床培养6d后，多糖单加氧酶的表达量为3.35mg/ml，分子量为50kDa，TLAA9-3最适温度为50-55℃，在60℃下是稳定的，处理60min后仍有95％以上的酶活性；70℃处理30min后仍保持75％的活性；80℃处理30min后保持29％的活性；90℃处理30min活性几乎完全丧失。Construct the recombinant expression plasmid vector pPICZ α A/TLAA9-3, transform Pichiapastoris GS115 by electroporation using an electroporator, screen on Zeocin medium, screen positive transformants by PCR identification, and screen multi-copy transformants with high-concentration bleomycin, then The methanol-induced expression was carried out to obtain the Pichia pastoris engineering strain GS-CT-9-3. The engineered strain was inoculated in BMGY-containing medium, and after cultured on a shaker at 28°C 200rpm/min for 6 days, the expression level of polysaccharide monooxygenase was 3.35mg/ml, the molecular weight was 50kDa, and the optimum temperature of TLAA9-3 was 50- It is stable at 55°C and 60°C, and still has more than 95% of the enzyme activity after 60 minutes of treatment; 75% of the activity after 30 minutes of treatment at 70°C; 29% of the activity after 30 minutes of treatment at 80°C; 30 minutes of treatment at 90°C Activity is almost completely lost.
AA9家族多糖单加氧酶TLAA9-3在供电子体(抗坏血酸和CDH)和铜离子存在下能将纤维素长链氧化裂解为不同的纤维寡糖，并且在缺乏底物的情况下可以产生过氧化氢，对内切纤维素酶N24的酶活性具有明显的促进作用，混合酶比原始酶N24的降解纤维素的能力提高1.25倍。多糖单加氧酶第一个组氨酸具有重要作用，前端连有其他氨基酸会影响其活性。不同的多糖单加氧酶氧化裂解纤维素长链发生在不同的连键位置，经飞行质谱鉴定TLAA9-3氧化裂解纤维素长链发生在C1和C4位置。The polysaccharide monooxygenase TLAA9-3 of the AA9 family can oxidatively cleave long-chain cellulose into different cellooligosaccharides in the presence of electron donors (ascorbic acid and CDH) and copper ions, and can produce overexpression in the absence of substrates. Hydrogen peroxide can significantly promote the enzymatic activity of endo-cellulase N24, and the ability of the mixed enzyme to degrade cellulose is 1.25 times higher than that of the original enzyme N24. The first histidine of polysaccharide monooxygenase plays an important role, and other amino acids connected to the front end will affect its activity. Different polysaccharide monooxygenases oxidatively cleaved the long chain of cellulose at different linkage positions, and it was identified by flight mass spectrometry that TLAA9-3 oxidatively cleaved the long chain of cellulose at the C1 and C4 positions.
(四)附图说明：(4) Description of drawings:
图1 AA9家族多糖单加氧酶TLAA9-3的SDS-PAGE分析Fig.1 SDS-PAGE analysis of AA9 family polysaccharide monooxygenase TLAA9-3
泳道1：纯化的AA9家族多糖单加氧酶TLAA9-1Lane 1: Purified AA9 family polysaccharide monooxygenase TLAA9-1
泳道2：低分子量蛋白标准Lane 2: Low molecular weight protein standard
图2 AA9家族多糖单加氧酶TLAA9-3的最适温度Figure 2 Optimum temperature of AA9 family polysaccharide monooxygenase TLAA9-3
图3 AA9家族多糖单加氧酶TLAA9-3对内切纤维素酶N24活性的促进作用Figure 3 The promotion effect of AA9 family polysaccharide monooxygenase TLAA9-3 on the activity of endocellulase N24
图4 AA9家族多糖单加氧酶TLAA9-1氧化裂解纤维素薄层层析检测及供电子体(抗坏血酸)对AA9家族多糖单加氧酶TLAA9-1活性的影响Figure 4 Thin-layer chromatography detection of AA9 family polysaccharide monooxygenase TLAA9-1 oxidatively cleaved cellulose and the effect of electron donor (ascorbic acid) on the activity of AA9 family polysaccharide monooxygenase TLAA9-1
泳道1：添加抗坏血酸的多糖单加氧酶TLAA9-1(E+Vc)Lane 1: Ascorbic acid-added polysaccharide monooxygenase TLAA9-1(E+Vc)
泳道2：未加抗坏血酸的多糖单加氧酶TLAA9-1(E)Lane 2: Polysaccharide monooxygenase TLAA9-1 without ascorbic acid (E)
泳道3：标准纤维寡糖分子(M)Lane 3: Standard cellooligosaccharide molecule (M)
图5 AA9家族多糖单加氧酶TLAA9-1酶解产物飞行质谱分析Figure 5 Flight mass spectrometry analysis of hydrolyzate of AA9 family polysaccharide monooxygenase TLAA9-1
(五)具体实施方式(5) Specific implementation methods
实施例1：疏绵状嗜热丝孢菌Thermomyces lanuginosusermophilum的分离鉴定Example 1: Isolation and identification of lanuginosa thermomyces Thermomyces lanuginosusermophilum
(1)标本采集：从堆肥中采集。(1) Specimen collection: collected from compost.
(2)分离培养：将采集标本取0.5克放置在PDA平板上50℃培养3天后，进行分离纯化。操作步骤参考Cooney and Emerson(1964)文献。(2) Separation and cultivation: 0.5 g of the collected specimens were placed on a PDA plate and cultured at 50° C. for 3 days before separation and purification. The operating steps refer to Cooney and Emerson (1964) literature.
(3)鉴定:参考Cooney and Emerson(1964)和LaTouche(1950)文献。(3) Identification: refer to the documents of Cooney and Emerson (1964) and LaTouche (1950).
实施例2：多糖单加氧酶基因TLAA9-3的克隆Example 2: Cloning of polysaccharide monooxygenase gene TLAA9-3
(1)疏绵状嗜热丝孢菌Thermomyces lanuginosusermophilum总RNA的提取：参照Trizol试剂盒说明。(1) Extraction of total RNA from Thermomyces lanuginosusermophilum: refer to the instructions of the Trizol kit.
(2)cDNA第一条链的合成：按照Takara公司的TaKaRa RNA PCR kit(AMV)Ver3.0试剂盒说明书进行：取1～2μg总RNA，加RNase Free ddH2O至9.5μL，将RNA样品在75℃变性5min，立即在冰浴中冷却5min，然后稍微离心一下，在冰浴中依次加入以下各种成分：10mmol/L dNTP Mixture 2μL，10×RT Buffer(Mg2+)2μL，25mmol/L MgCl24μL，Oligo d(T)-Adaptor Primer 1μL，RNase Inhibiter 0.5μL，AMV Reverse Transcriptase 1μL(FinalVolume 20μL)，将反应液混合后，室温下放10min，然后42℃温育60min，再煮沸5min以灭活反转录酶。加入180μL DEPC处理的ddH2O，稀释至200μL，混匀，稍微离心，保存于-20℃，备用。(2) Synthesis of the first strand of cDNA: according to the instructions of the TaKaRa RNA PCR kit (AMV) Ver3.0 kit from Takara Company: take 1-2 μg of total RNA, add RNase Free ddH2 O to 9.5 μL, and dilute the RNA sample Denature at 75°C for 5 minutes, immediately cool in an ice bath for 5 minutes, then centrifuge slightly, and add the following components in order in the ice bath: 10mmol/L dNTP Mixture 2μL, 10×RT Buffer (Mg2+ ) 2μL, 25mmol/L L MgCl2 4μL, Oligo d(T)-Adaptor Primer 1μL, RNase Inhibiter 0.5μL, AMV Reverse Transcriptase 1μL (FinalVolume 20μL), after mixing the reaction solution, put it at room temperature for 10min, then incubate at 42℃ for 60min, and boil for 5min to Inactivate reverse transcriptase. Add 180 μL of DEPC-treated ddH2 O, dilute to 200 μL, mix well, centrifuge slightly, and store at -20°C for use.
(3)PCR反应(25μL)：cDNA 2μL,10×Buffer 2.5μL,10mmol/L dNTP 2μL,25mmol/LMgCl2 2μL，上、下游引物各2μL，Taq DNA聚合酶0.5μL(5U/μL),ddH2O 12μL。反应条件为94℃5min预变性；94℃40s，56℃40s，72℃1min,共32个循环，72℃延伸10min，4℃保存。(3) PCR reaction (25μL): cDNA 2μL, 10×Buffer 2.5μL, 10mmol/L dNTP 2μL, 25mmol/LMgCl2 2μL, upstream and downstream primers 2μL, Taq DNA polymerase 0.5μL (5U/μL), ddH2O 12μL . The reaction conditions were pre-denaturation at 94°C for 5 minutes; 32 cycles of 94°C for 40 s, 56°C for 40 s, and 72°C for 1 min, extension at 72°C for 10 min, and storage at 4°C.
上游引物：5’-AGGGGTATCTCTCGAGAAAAGACATACCGTGATGACTACACT-3’Upstream primer: 5'-AGGGGTATCTCTCGAGAAAAGACATACCGTGATGACTACACT-3'
下游引物：5’-GAGTTTTTGTTCTAGACCGTAAAAGATTTCAGTTC-3’Downstream primer: 5'-GAGTTTTTGTTCTAGACCGTAAAAGATTTCAGTTC-3'
(4)基因克隆：取0.5μl PCR产物与pMD18-T载体进行连接，操作步骤按照TAKARA公司产品说明书进行。然后连接产物转化大肠杆菌DH5α菌株，在表面涂有氨卞青霉素(100μg/mL)的LB平板上生长过夜。挑取白色菌落，在LB液体培养基中培养过夜。(4) Gene cloning: 0.5 μl of the PCR product was connected to the pMD18-T vector, and the operation steps were carried out according to the product manual of TAKARA company. Then the ligation product was transformed into Escherichia coli DH5α strain and grown overnight on LB plates coated with ampicillin (100 μg/mL). Pick white colonies and culture them overnight in LB liquid medium.
(5)质粒DNA的提取：碱法提取质粒DNA。(5) Extraction of plasmid DNA: extraction of plasmid DNA by alkaline method.
(6)序列测定：DNA双脱氧法测定核苷酸序列，在上海生物工程有限公司进行。序列引物为M13启动子引物。疏绵状嗜热丝孢菌TLAA9-3基因cDNA全长1110bp，包含起始密码子和终止密码子，无内含子，编码343个氨基酸。将此氨基酸序列在国际基因库中进行检索，发现属于多糖单加氧酶第AA9家族。该序列如下：(6) Sequence determination: The nucleotide sequence was determined by the DNA dideoxy method at Shanghai Bioengineering Co., Ltd. The sequence primer is the M13 promoter primer. The full-length cDNA of Thermomyces lanuginosa TLAA9-3 gene is 1110bp, including start codon and stop codon, without intron, encoding 343 amino acids. The amino acid sequence was searched in the international gene bank, and it was found that it belonged to the AA9 family of polysaccharide monooxygenase. The sequence is as follows:
(A)SEQ ID NO 1的信息(A) Information of SEQ ID NO 1
(a)序列特征：*长度：1110碱基对；*类型：核酸；*链型：双链；*拓扑结构：线性(a) Sequence features: *length: 1110 base pairs; *type: nucleic acid; *chain type: double-stranded; *topology: linear
(b)分子类型：DNA(b) Molecule type: DNA
(c)假设：否(c) Assumption: No
(d)反义：否(d) Antisense: No
(e)最初来源：疏绵状嗜热丝孢菌Thermomyces lanuginosus(e) Original source: Thermomyces lanuginosus
(f)序列描述：(f) Sequence description:
(B)SEQ ID NO 2的信息(B) Information of SEQ ID NO 2
(a)序列特征：*长度：343氨基酸；*类型：氨基酸；*链型：单链；*拓扑结构：线性(a) Sequence features: *length: 343 amino acids; *type: amino acid; *chain type: single chain; *topology: linear
(b)分子类型：蛋白质(b) Molecule type: protein
(c)序列描述：(c) Sequence description:
实施方式3：表达载体的构建Embodiment 3: Construction of expression vector
(1)将目的基因CDS序列进行信号肽分析预测，去除信号肽，根据载体信息设计引物，引入XholⅠ和XbaⅠ酶切位点，并补全序列至PpiczαA载体的Kex2蛋白信号切割位点，反向引物3’端去除终止密码子并添加两个碱基以保证序列正常翻译至6×His标签。(1) Analyze and predict the signal peptide of the CDS sequence of the target gene, remove the signal peptide, design primers according to the carrier information, introduce XholI and XbaI restriction sites, and complete the sequence to the Kex2 protein signal cutting site of the PpiczαA vector, reverse The stop codon was removed and two bases were added to the 3' end of the primer to ensure normal translation of the sequence to a 6×His tag.
上游引物：5’-AGGGGTATCTCTCGAGAAAAGACATACCGTGATGACTACACT-3’Upstream primer: 5'-AGGGGTATCTCTCGAGAAAAGACATACCGTGATGACTACACT-3'
下游引物：5’-GAGTTTTTGTTCTAGACCGTAAAAGATTTCAGTTC-3’Downstream primer: 5'-GAGTTTTTGTTCTAGACCGTAAAAGATTTCAGTTC-3'
(2)疏绵状嗜热丝孢菌总RNA的提取:利用Trizol试剂提取。(2) Extraction of total RNA from Thermomyces lanuginosa: extraction with Trizol reagent.
(3)反转录合成cDNA第一条链：取2μg总RNA，加入5×反应缓冲液4μL，10mM dNTP 2μL，核糖核酸酶抑制剂(40－200u/μL)0.5μL引物oligodT(1μg/μL)1μL，反转录酶(10u/μL)2μL，42℃反应60min，然后85℃10min终止反应，稀释至200μL。(3) Synthesize the first strand of cDNA by reverse transcription: Take 2 μg total RNA, add 4 μL of 5× reaction buffer, 2 μL of 10 mM dNTP, 0.5 μL of ribonuclease inhibitor (40-200u/μL) primer oligodT (1 μg/μL ) 1 μL, reverse transcriptase (10u/μL) 2 μL, react at 42°C for 60 minutes, then stop the reaction at 85°C for 10 minutes, and dilute to 200 μL.
(4)PCR反应(25μL)：cDNA 2μL,10×Buffer 2.5μL,10mmol/L dNTP 2μL,25mmol/LMgCl2 2μL，上、下游引物各2μL，Taq DNA聚合酶0.5μL(5U/μL),ddH2O 12μL。反应条件为94℃5min预变性；94℃40s，56℃40s，72℃1min,共32个循环，72℃延伸10min，4℃保存。(4) PCR reaction (25 μL): cDNA 2 μL, 10×Buffer 2.5 μL, 10 mmol/L dNTP 2 μL, 25 mmol/LMgCl2 2 μL, upstream and downstream primers 2 μL, Taq DNA polymerase 0.5 μL (5U/μL), ddH2 O 12 μL. The reaction conditions were pre-denaturation at 94°C for 5 minutes; 32 cycles of 94°C for 40 s, 56°C for 40 s, and 72°C for 1 min, extension at 72°C for 10 min, and storage at 4°C.
(5)基因克隆：取2μL PCR产物与pMD18－T载体进行连接，获得重组质粒pMD18T/TLAA9-3操作步骤按TAKARA公司产品说明书进行。然后连接产物转化大 肠杆菌DH5α，在涂有AMP的LB平板上生长过夜。挑取白色菌落，在LB液体培养基中培养过夜。(5) Gene cloning: Take 2 μL of the PCR product and connect it with the pMD18-T vector to obtain the recombinant plasmid pMD18T/TLAA9-3. The ligation product was then transformed into E. coli DH5α and grown overnight on AMP-coated LB plates. Pick white colonies and culture them overnight in LB liquid medium.
(6)质粒DNA提取：碱法提取质粒DNA。(6) Plasmid DNA extraction: Plasmid DNA was extracted by alkaline method.
(7)用XholI和XbaI对重组质粒pMD18-T/TLAA9-3产物进行双酶切，同时用XholI和XbaI双酶切酵母表达质粒pPICZαA，DNA胶回收试剂盒回收纯化TLAA9-3基因及载体pPICZαA片断。然后进行体外连接，获得重组质粒pPICZαA/TLAA9-3,重组质粒转化大肠杆菌JM109，在含Amp的LB转化平板上挑选单菌落，提取质粒DNA，进行PCR和酶切鉴定，测序确认重组质粒的读码框正确。(7) Use XholI and XbaI to double-enzyme-digest the recombinant plasmid pMD18-T/TLAA9-3 product, and simultaneously use XholI and XbaI to double-enzyme-digest the yeast expression plasmid pPICZαA, and recover and purify the TLAA9-3 gene and vector pPICZαA with the DNA gel recovery kit fragment. Then connect in vitro to obtain the recombinant plasmid pPICZαA/TLAA9-3, transform the recombinant plasmid into Escherichia coli JM109, pick a single colony on the LB transformation plate containing Amp, extract the plasmid DNA, carry out PCR and enzyme digestion identification, and confirm the readout of the recombinant plasmid by sequencing. The code box is correct.
实施例4.表达多糖单加氧酶基因TLAA9-3的酵母工程菌株的构建及筛选Example 4. Construction and screening of yeast engineering strains expressing polysaccharide monooxygenase gene TLAA9-3
(1)重组表达质粒的线性化:将重组表达质粒pPICZ α A/TLAA9-3用限制性内切酶SacⅠ线性化。(1) Linearization of the recombinant expression plasmid: The recombinant expression plasmid pPICZ α A/TLAA9-3 was linearized with restriction endonuclease SacⅠ.
(2)转化：电击转化酵母菌株Pichia pastoris GS115酵母感受态细胞，转化方法参见Invitrogen公司毕赤酵母操作手册。(2) Transformation: Yeast competent cells of the yeast strain Pichia pastoris GS115 were transformed by electric shock. For the transformation method, refer to the Pichia operation manual of Invitrogen Company.
(3)筛选：用灭菌牙签挑取转化子对应点种在Zeocin平板上，28℃培养2－4d,挑取在Zeocin平板上均生长良好的转化子为阳性转化子。挑取阳性转化子分别点种于高浓度博来霉素平板上筛选多拷贝转化子。(3) Screening: Use a sterilized toothpick to pick the corresponding spots of the transformants and inoculate them on the Zeocin plate, culture at 28°C for 2-4 days, and pick the transformants that grow well on the Zeocin plate as positive transformants. Pick positive transformants and spot on high-concentration bleomycin plates to screen for multi-copy transformants.
实施方式5：毕赤酵母Pichia pastoris GS115的诱导表达和活性检测Embodiment 5: Induced expression and activity detection of Pichia pastoris GS115
(1)将阳性转化子接种于含25mL BMGY培养基中，30℃200rpm/min摇床培养24h(OD600值达到1.8)，离心收集菌体，将细胞重悬于适当体积的BMMY培养基中，至OD600值为1.0，28℃200rpm/min继续培养，每24h补充甲醇至终浓度为0.5％，每24h取样，室温10，000g离心5min，取上清进行SDS-PAGE分析。(1) Inoculate positive transformants in 25 mL of BMGY medium, culture at 200 rpm/min at 30°C for 24 hours (OD600 value reaches 1.8), collect the bacteria by centrifugation, and resuspend the cells in an appropriate volume of BMMY medium , to OD600 value of 1.0, 28°C 200rpm/min to continue culturing, supplement methanol every 24h to a final concentration of 0.5%, sample every 24h, centrifuge at room temperature 10,000g for 5min, take the supernatant for SDS-PAGE analysis.
(2)酶活性检测：酶活性测定采用酶解产物薄层层析法，反应体系及反应条件为100μL的酶液，加入200μL的0.5％磷酸膨胀纤维素，100μL的醋 酸缓冲液(0.2mol/L、pH5.0)50℃反应48h，离心后取上清3ul到层析板上，展层后喷洒显色液，85度20min观察结果。蛋白含量测定采用Bradford法。(2) Detection of enzyme activity: Enzyme activity measurement adopts enzymatic hydrolyzate thin-layer chromatography, reaction system and reaction conditions are 100 μ L of enzyme liquid, add 200 μ L of 0.5% phosphoric acid swollen cellulose, 100 μ L of acetate buffer (0.2mol/ L, pH 5.0) reacted at 50°C for 48h, after centrifugation, take 3ul of the supernatant onto the chromatography plate, spray the color developing solution after developing the layer, and observe the result at 85°C for 20min. Protein content was determined by the Bradford method.
(3)重组多糖单加氧酶TLAA9-3的最适反应温度：诱导表达的重组多糖单加氧酶经过GE公司的HisTrap FF crude金属配体亲和层析柱纯化带有组氨酸标记的重组蛋白质，获得电泳均一的纯化蛋白，用纯化的重组多糖单加氧酶测定其性质。在相同PH(PH＝5)不同的温度条件下(30℃，40℃，50℃，60℃，70℃，80℃，90℃)多糖单加氧酶反应36h，然后分别加入纤维素酶，50℃下反应半小时，DNS法测量产生的还原糖的量。数值测量三次求平均值，将最高的酶活力定义为100％。(3) Optimum reaction temperature of recombinant polysaccharide monooxygenase TLAA9-3: the induced recombinant polysaccharide monooxygenase was purified by GE's HisTrap FF crude metal ligand affinity chromatography column with histidine tag Recombinant protein, obtain purified protein with homogeneous electrophoresis, and use purified recombinant polysaccharide monooxygenase to determine its properties. Under the same pH (PH=5) and different temperature conditions (30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C) polysaccharide monooxygenase was reacted for 36 hours, and then cellulase was added respectively, React at 50°C for half an hour, and measure the amount of reducing sugar produced by DNS method. The numerical measurements were averaged three times, and the highest enzyme activity was defined as 100%.
实施方式6：AA9家族多糖单加氧酶TLAA9-3对内切纤维素酶N24活性的促进作用及供电子体的作用Embodiment 6: The promotion effect of AA9 family polysaccharide monooxygenase TLAA9-3 on the activity of endocellulase N24 and the effect of electron donor
(1)AA9家族多糖单加氧酶TLAA9-3对内切纤维素酶N24活性的促进作用(1) The promotion effect of AA9 family polysaccharide monooxygenase TLAA9-3 on the activity of endocellulase N24
采用DNS法，多糖单加氧酶在PH＝5，温度50℃，200RPM的条件下，设置两组多糖单加氧酶与纤维素反应36h，一组加入纤维素酶，一组不加纤维素酶，50℃下反应30min，反应体系为：TLAA9-350μL，N2450μL，200μL的0.5％磷酸膨胀纤维素，100μL的醋酸缓冲液(0.2mol/L、pH5.0)，加入400μL DNS试剂，煮沸10min，在540nm波长下测定光吸收值。以不加多糖单加氧酶TLAA9-3的反应体系中的酶活性定义为100％，分别计算多糖单加氧酶TLAA9-3、内切纤维素酶N24以及混合酶的相对酶活性。重复三次，取平均值。Using the DNS method, polysaccharide monooxygenase was set to react with cellulose for 36 hours under the conditions of pH = 5, temperature 50°C, and 200 RPM. One group added cellulase, and the other group did not add cellulose. Enzyme, react at 50°C for 30min, the reaction system is: TLAA9-350μL, N2450μL, 200μL of 0.5% phosphoric acid swollen cellulose, 100μL of acetate buffer (0.2mol/L, pH5.0), add 400μL of DNS reagent, boil for 10min , Measure the light absorption value at a wavelength of 540nm. The enzyme activity in the reaction system without polysaccharide monooxygenase TLAA9-3 was defined as 100%, and the relative enzyme activities of polysaccharide monooxygenase TLAA9-3, endocellulase N24 and mixed enzymes were calculated respectively. Repeat three times and take the average value.
(2)供电子体(抗坏血酸)对多糖单加氧酶TLAA9-3纤维素酶活性的影响(2) Effect of electron donor (ascorbic acid) on cellulase activity of polysaccharide monooxygenase TLAA9-3
设置两组反应体系，一组加入抗坏血酸，使其终浓度为10mM,另一组不加抗坏血酸,在多糖单加氧酶TLAA9-3最适反应条件下测定其氧化裂解纤维素酶活性，反应48h后，硅胶薄层层析分析产物，测定活性。Two groups of reaction systems were set up, one group was added with ascorbic acid to make the final concentration of 10mM, and the other group was not added with ascorbic acid, and its oxidative cleavage cellulase activity was measured under the optimal reaction conditions of polysaccharide monooxygenase TLAA9-3, and the reaction was 48h Afterwards, the product was analyzed by silica gel thin layer chromatography, and the activity was determined.
实施方式7：AA9家族多糖单加氧酶酶解产物飞行质谱分析Embodiment 7: In-flight mass spectrometry analysis of enzymatic hydrolysis products of AA9 family polysaccharide monooxygenase
将AA9家族多糖单加氧酶TLAA9-2与磷酸膨胀纤维素混合在PH＝5的醋酸安缓冲液中，在最适温度条件下反应48h，取上清做飞行质谱。AA9家族多糖单加氧酶TLAA9-2氧化裂解产生的多糖有多种形式，主要有C1和C4氧化，还可能有C6氧化。C1氧化C6氧化C4水和产物的分子量均增加16，而C4氧化分子量变化为2。飞行质谱测的的分子量减去对应寡糖的分子量再减去氧化减少的碳原子的数量和结合钠离子的数量为氧化后的产物分子量。AA9 family polysaccharide monooxygenase TLAA9-2 and phosphoric acid-swelled cellulose were mixed in acetic acid ammonium buffer solution at pH=5, reacted for 48 hours under optimum temperature conditions, and the supernatant was taken for flight mass spectrometry. The polysaccharides produced by the oxidative cleavage of AA9 family polysaccharide monooxygenase TLAA9-2 have various forms, mainly including C1 and C4 oxidation, and possibly C6 oxidation. The molecular weight of C1 oxidation, C6 oxidation, C4 water and product both increased by 16, while the molecular weight of C4 oxidation changed by 2. The molecular weight measured by flight mass spectrometry minus the molecular weight of the corresponding oligosaccharide minus the number of carbon atoms reduced by oxidation and the number of bound sodium ions is the molecular weight of the oxidized product.
实施方式8：表达TLAA9-3基因的毕赤酵母工程菌株GS-CT-9-3的保藏Embodiment 8: Preservation of Pichia pastoris engineered strain GS-CT-9-3 expressing TLAA9-3 gene
表达TLAA9-3基因的毕赤酵母工程菌株GS-CT-TLAA9-3的保藏单位：中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)；地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所；保藏日期：2016年4月21日；酵母工程菌株编号为：CGMCC No.12385；毕赤酵母工程菌株的分类命名为：巴氏毕赤酵母Pichia pastoris。Deposit unit of Pichia pastoris engineering strain GS-CT-TLAA9-3 expressing TLAA9-3 gene: General Microbiology Center (CGMCC) of China Committee for Culture Collection of Microorganisms; Address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing , Institute of Microbiology, Chinese Academy of Sciences; preservation date: April 21, 2016; yeast engineering strain number: CGMCC No.12385; Pichia pastoris engineering strain classification name: Pichia pastoris.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610366939.9ACN106047736A (en) | 2016-05-26 | 2016-05-26 | Pichia pastoris engineered strain for expressing AA9 family polysaccharide monooxygenase genes TLAA9-3 |
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610366939.9ACN106047736A (en) | 2016-05-26 | 2016-05-26 | Pichia pastoris engineered strain for expressing AA9 family polysaccharide monooxygenase genes TLAA9-3 |
| Publication Number | Publication Date |
|---|---|
| CN106047736Atrue CN106047736A (en) | 2016-10-26 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610366939.9APendingCN106047736A (en) | 2016-05-26 | 2016-05-26 | Pichia pastoris engineered strain for expressing AA9 family polysaccharide monooxygenase genes TLAA9-3 |
| Country | Link |
|---|---|
| CN (1) | CN106047736A (en) |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103384678A (en)* | 2011-02-23 | 2013-11-06 | 诺维信股份有限公司 | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103384678A (en)* | 2011-02-23 | 2013-11-06 | 诺维信股份有限公司 | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
| Title |
|---|
| 张煜等: "里氏木霉纤维二糖水解酶II在毕赤酵母中的高效表达", 《菌物学报》* |
| 耿志刚: "疏绵状嗜热丝孢菌热稳定多糖单加氧酶的克隆、表达和功能研究", 《中国优秀硕士学位论文全文数据库》* |
| Publication | Publication Date | Title |
|---|---|---|
| Borin et al. | Comparative transcriptome analysis reveals different strategies for degradation of steam-exploded sugarcane bagasse by Aspergillus niger and Trichoderma reesei | |
| CN108660145A (en) | The encoding gene and its recombinant expression of heat resistant type Pullulanase and application | |
| CN118652863B (en) | A polysaccharide-cleaving monooxygenase mutant and its gene, engineering bacteria, preparation method and application | |
| CN105950640A (en) | Preparation method of marine bacteria-derived kappa-carrageenase gene and recombinase | |
| US9012186B2 (en) | Hemicellulose-degrading enzymes | |
| CN106085892A (en) | Express the pichia pastoris engineered strain of AA9 family polysaccharide monooxygenase gene TLAA9 4 | |
| Bao et al. | Identification, soluble expression, and characterization of a novel endo-inulinase from Lipomyces starkeyi NRRL Y-11557 | |
| CN107002055B (en) | Fungus-derived high-temperature acidic beta-glucosidase, and coding gene and application thereof | |
| CN109694859B (en) | A kind of thermophilic pectinase and its expression gene and application | |
| CN108165541A (en) | A kind of zymoprotein and its application with betagalactosidase activity | |
| CN101307295A (en) | Engineering strain of Pichia pastoris expressing the gene cbh3 of Chaetomyces thermophila | |
| CN105018444B (en) | The high-temperature acidic beta-glucosidase HiBgl3C and its gene in a kind of humicola lanuginosa source and application | |
| CN106047736A (en) | Pichia pastoris engineered strain for expressing AA9 family polysaccharide monooxygenase genes TLAA9-3 | |
| Ichikawa et al. | The expression of alternative sigma-I7 factor induces the transcription of cellulosomal genes in the cellulolytic bacterium Clostridium thermocellum | |
| CN106047737A (en) | Pichia pastoris engineered strain for expressing AA9 family polysaccharide monooxygenase genes TLAA9-2 | |
| CN105969679A (en) | Pichia engineering strain for expressing AA9-family polysaccharide monooxygenase gene TLAA9-1 | |
| CN105861471B (en) | A kind of neutral low temperature xylosidase CaXyl43A and its gene and application | |
| CN103232949A (en) | Pichia pastoris engineered strain expressing 61-family glycoside hydrolase gene | |
| CN101880682A (en) | Cloning of the endocellulase eg gene of Thermoascus var. photospora 61 family and its encoded protein | |
| CN116064616A (en) | A kind of cellulase gene, cellulase, recombinant vector and application | |
| CN103602604B (en) | Pichia pastoris engineered strain expressing 9-family glycoside hydrolase gene CtAA9d | |
| CN112442488B (en) | Soluble polysaccharide monooxygenase mutant and application thereof | |
| CN101701196A (en) | Engineering strain of Pichia pastoris expressing bgl gene of Chaetomium thermophila | |
| CN104789580A (en) | Novel heat-resistance glucoside hydrolase MtCel3, as well as coding sequence and application thereof | |
| CN101429518A (en) | High temperature resistant xylosidase XynB2, gene encoding the enzyme and uses thereof |
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | Application publication date:20161026 | |
| WD01 | Invention patent application deemed withdrawn after publication |