宇佐美曲霉阿魏酸酯酶与木聚糖酶协同作用降解小麦麸皮制备阿魏酸

目的利用宇佐美曲霉(Aspergillus usamii)阿魏酸酯酶与木聚糖酶协同降解小麦麸皮,制备阿魏酸(ferulic acid,FA)。方法将工程酵母菌P.pastoris GS115/AusfaeA和GS115/Ausxyn11A经甲醇诱导发酵,分别获得重组阿魏酸酯酶和木聚糖酶。在pH 5.0、40℃、料液比1∶60(g∶ml)、水解10 h的条件下,探讨木聚糖酶添加量、阿魏酸酯酶与木聚糖酶协同作用对阿魏酸酯酶水解去淀粉麦麸(destarched wheat bran,DSWB),释放FA的影响,并检测酶解产物FA的体外抗氧化活性。结果在0.5g DSWB中单独加入56U阿魏酸酯酶时,FA的释放率为碱提等量麸皮总FA含量的19.48%;在此基础上添加木聚糖酶,能促进阿魏酸酯酶进一步释放FA,当木聚糖酶添加量为300 U时,FA的释放率从19.48%上升至70.10%;木聚糖酶单独作用于DSWB未检测到FA的释放;在反应体系中先添加木聚糖酶,能释放1.23 mg FA,比先添加阿魏酸酯酶提高了0.3 mg;随着FA浓度的增加,FA的还原能力及其对DPPH自由基和羟基自由基的清除能力均呈稳定增长,各浓度FA对羟基自由基的清除能力均高于对DPPH自由基的清除能力。结论阿魏酸酯酶与木聚糖酶之间存在协同作用,双酶共同作用,有利于小麦麸皮的降解,提高FA的释放率。     

重组羧肽酶原B的复性及纯化

目的　建立重组羧肽酶原B复性方法及活性羧肽酶B的纯化方法。方法　重组大肠杆菌发酵后 ,表达的羧肽酶原B包涵体经洗涤、变性 ,采用凝胶过滤的方法对其进行复性及纯化。所得的复性液经Trypsin酶解后 ,采用DEAE FF阴离子交换层析进行羧肽酶B的分离纯化 ,并采用SDS PAGE检测纯度。结果　经凝胶过滤后得到了复性和纯化的羧肽酶原B包涵体 ,在蛋白终浓度相同的情况下 ,复性效率比稀释复性提高了 5 0 % ,经DEAE离子交换柱一步纯化 ,得到了活性羧肽酶B ,纯度达到 90 %以上。以Hippuryl -L arg为底物测活 ,得到的活性酶的比活为 15 0u mg。结论　所建立复性方法及纯化方法为进一步的研究奠定了基础。     

巴斯德梭菌甘油脱水酶基因的克隆表达、纯化及酶学性质的研究

用聚合酶链反应(PCR)技术,从巴斯德梭菌(C lostridium pasteurianum)扩增出甘油脱水酶基因(dhaBCE),在大肠杆菌中高效表达,SDS-PAGE(聚丙烯酰胺凝胶)电泳结果显示,分别有相对分子质量为66、21、16 kD三条特异性蛋白表达条带。用金属镍亲和层析及S-300H凝胶层析将重组蛋白进行分离纯化,所得纯酶的比活为4.26 U/mg。重组酶的最适反应温度42~44℃,最适作用pH=9.10~9.50,它对三个底物结构类似物的米氏常数(Km)值分别是:1,2-丙二醇0.38 mmol/L,甘油0.29 mmol/L,1,2-乙二醇2.0 mmol/L;对辅酶B12的Km值为0.22μmol/L。     

Mg~(2+)和氨基酸对重组大肠杆菌BL21(DE3)生长及羧肽酶原B表达的影响

目的研究Mg2+和氨基酸对重组菌株生长及表达pCPB的影响。方法通过摇瓶和发酵罐培养,观察不同Mg2+浓度和氨基酸对重组菌的生长和羧肽酶原B表达的影响。结果添加1g/L Mg2+能提高质粒的稳定性;添加氨基酸能使羧肽酶原B表达量由18·7g/L提高到24g/L。结论添加适量的Mg2+和氨基酸能促进重组菌的生长和提高目的蛋白的表达量。     

人GINS2基因重组腺病毒表达质粒的构建及鉴定

目的利用AdEasy系统构建携带人GINS2基因的重组腺病毒表达质粒,并进行鉴定。方法以pcDNA3.1-GINS2质粒为模板,PCR扩增GINS2基因,克隆至穿梭质粒pAdTrace-TO4中,构建重组腺病毒穿梭质粒pAdT-GINS2,经双酶切及测序鉴定正确的阳性质粒与骨架质粒pAdEasy-1同时转化感受态大肠杆菌BJ5183,经同源重组获得重组腺病毒质粒pAdE-GINS2,转染HEK293细胞,包装出重组腺病毒Ad-GINS2,大量扩增后,测定病毒滴度,进行PCR鉴定。Western blot法检测重组腺病毒感染的人HL60细胞中GINS2蛋白的表达,MTT法检测重组腺病毒感染对HL60细胞增殖的影响。结果重组腺病毒穿梭质粒pAdT-GINS2经双酶切及测序证明构建正确;经酶切鉴定证明重组腺病毒质粒pAdE-GINS2构建正确;经PCR鉴定证明重组腺病毒Ad-GINS2包装成功,经3轮扩增后病毒滴度可达1.35×1012IU/ml;与空载体感染及未感染的HL60细胞相比,重组腺病毒感染的HL60细胞内GINS2蛋白的表达及细胞增殖水平均明显升高(P<0.05)。结论已成功构建携带人GINS2基因的重组腺病毒表达质粒,感染人HL60细胞后可促进其增殖,为进一步研究该基因在急性髓细胞白血病发生发展中的作用奠定了基础。     

培养基组成对米曲霉产氨基酰化酶的影响

氨基酰化酶是一类能够水解N-酰基-DL-氨基酸上酰胺键的酶。通过单因素实验和正交实验,对米曲霉产氨基酰化酶的培养基组成进行了优化,以提高其产量。确定较优培养基组成为:麸皮2%、尿素1.5%、磷酸二氢钠0.3%、硫酸镁0.35%、Tween-80 0.1%,在此培养基上得到的氨基酰化酶单位酶活可提高到440.51μmol.g-1.h-1。     

稀土和硫酸二乙酯联合诱变米曲霉产氨基酰化酶的研究

为获得高产氨基酰化酶的米曲霉菌株,选用稀土硝酸镧和硫酸二乙酯对米曲霉菌株CICC-2339进行联合诱变,经摇瓶发酵筛选到氨基酰化酶高产菌株SDESLa,其酶活为39.63 μmol·g-1·h-1,较出发菌株提高了39.50%,诱变效果显著.     

人组织激肽释放酶基因的克隆及融合蛋白的表达

目的 开发激肽释放酶基因工程产品,为开展基因治疗高血压奠定基础。方法 采用RT-PCR的方法合成人胰腺 cDNA,从中扩增出Kallikrein(KK)基因。经 XhoI和EcoR I双酶切后,连接到pET-28b(+)载体上,酶切鉴定后,进行核苷酸序列分析和融合蛋白的表达。结果 将IPTG诱导表达的菌体进行SDS-PAGE电泳,与蛋白标准品比较,在相对分子质量31800处可见明显的高表达带。免疫印迹实验表明重组蛋白具有KK的抗原性。结论 已成功克隆并表达了人胰腺组织激肽释放酶基因,为进一步开发基因工程产品及进行基因治疗高血压研究打下了基础。     

旋毛虫与肝癌细胞相关基因CK-1的原核表达及鉴定

目的原核表达旋毛虫与肝癌细胞相关基因CK-1,并进行鉴定。方法以旋毛虫cDNA为模板,PCR扩增CK-1基因,克隆至原核表达载体pET-28a(+)中,构建重组表达质粒pET-28a-CK-1,转化感受态大肠埃希菌(E.coli)BL21(DE3),IPTG分别诱导表达3、4、5 h,表达产物进行12%SDS-PAGE分析。以纯化的旋毛虫肌幼虫免疫小鼠制备鼠抗旋毛虫肌幼虫阳性血清,以H7402细胞免疫小鼠制备鼠抗H7402全细胞蛋白阳性血清,以重组CK-1蛋白免疫小鼠制备鼠抗CK-1蛋白阳性血清,分别以其作为一抗,进行Western blot鉴定。结果重组表达质粒pET-28a(+)-CK-1经双酶切及测序鉴定证明构建正确;IPTG最佳诱导时间为4 h,表达的重组CK-1蛋白以包涵体形式存在,表达量占菌体总蛋白的37.5%;重组CK-1蛋白可被鼠抗旋毛虫肌幼虫阳性血清和鼠抗H7402全细胞蛋白阳性血清识别,鼠抗CK-1蛋白阳性血清可识别旋毛虫肌幼虫蛋白和H7402全细胞蛋白,在相对分子质量38 500处均可见特异性条带,表明重组CK-1蛋白是一个交叉抗原,且具有良好的反应原性。结论原核表达了旋毛虫CK-1基因,为进一步研究旋毛虫的抗肿瘤效应奠定了基础。     

微波辐照选育木聚糖酶高产菌宇佐美曲霉

A high yield xylanase producing strain, A. usamii L336-23, was screened out from its parent strain A.usamii L336 after microwave irradiation. Its productivity of xylanase activity increased by 35.7% from 21000μu·ml-1 to 28500μu·ml-1 and was stable after frequent subcultures and storage for more than two months. The mechanism of microwave mutation was also discussed.     

人源溶菌酶基因在杆状病毒系统中的表达

目的在杆状病毒系统中表达人源溶菌酶(Human lysozyme,hLY)基因,为其临床应用和大规模生产奠定基础。方法将人工合成的含信号肽和不含信号肽的溶菌酶基因克隆至重组杆状病毒表达系统的载体中,构建重组穿梭质粒,转染sf9细胞,SDS-PAGE和Western blot验证蛋白的表达,溶菌试验验证表达产物的溶菌酶活性。结果重组克隆质粒PFastBacHTA-signal-lysozyme和PFastBac HTA-lysozyme的PCR产物分别可见444和393 bp的目的基因条带,测序结果证实重组克隆质粒构建正确。PCR结果显示Lysozyme-1、Lysozyme-2、slysozyme-1、slysozyme-2、slysozyme-3、slysozyme-4号重组穿梭质粒可扩增出目的条带。sf9-PHLY和sf9-PHSLY的表达产物经12%SDS-PAGE分析,分别可见相对分子质量约17 500和19 300的特异蛋白条带;表达的两种重组蛋白均可与鼠抗6×His单抗特异性结合;表达产物具有溶菌酶活性。结论成功在杆状病毒表达系统中表达了hLY基因,为其临床应用和大规模生产奠定了基础。     

The fumitremorgin gene cluster of Aspergillus fumigatus: identification of a gene encoding brevianamide F synthetase

A gene encoding a putative dimodular nonribosomal peptide synthetase (NRPS) was identified within a gene cluster of Aspergillus fumigatus, a species reported to produce fumitremorgins and other prenylated alkaloids. The gene was deleted and overexpressed in the genome reference strain Af293, and was also expressed in the naïve host Aspergillus nidulans, which lacks the equivalent gene cluster. While neither fumitremorgins nor the dipeptide brevianamide F (cyclo‐L ‐Trp‐L ‐Pro), an early intermediate, were detected in wild‐type and deletion strains of A. fumigatus, brevianamide F accumulated in fungal cultures following increased expression of the NRPS gene in both A. fumigatus and A. nidulans. We conclude that the gene Afu8g00170, named ftmA, encodes the NRPS brevianamide synthetase. Brevianamide F is the precursor of a variety of fungal prenylated alkaloids with biological activity, including fumitremorgins A, B and C and tryprostatin B.     

Aspergillus oryzae SAICAR合成酶基因的克隆及反义表达载体的构建

目的 构建Aspergillus oryzae新的红色表现型菌株用于天然色素生产。方法 构建Aspergillus oryzaeRIB40 cDNA文库,应用BLAST网络服务对Aspergillus oryzae RIB40 cDNA文库的EST数据进行同源性比较,从EST克隆扩增Aspergillus oryzae类SAICAR合成酶基因(sh1919f片段),反向插入pUSA真核表达载体。结果 完成反义表达载体的构建。Aspergillus oryzae类SAICAR合成酶氨基酸序列与Pichia jadinii、Saccharomyces cerevisiae和Schizosac-charomyces pombe的SAICAR合成酶氨基酸序列具有高度同源性。结论 类SAICAR合成酶基因反义表达载体的构建为其转染入Aspergillus oryzae菌株筛选红色突变菌株奠定了实验基础。     

Cloning and Expression of Aspergillus tamarii FS132 Lipase Gene in Pichia pastoris

A lipase gene (atl) was cloned from Aspergillus tamarii FS132 for the first time. The gene was found to have an open reading frame of 1024 base pairs (bp), and the coding region of the gene contained two introns (51 bp and 52 bp). Multi-alignment analysis of the deduced amino acid sequence indicated high homology between the enzyme and mono-and diacylglycerol lipases from fungi Aspergillus. The recombinant lipase was expressed in Pichia pastoris GS115 cells. The recombinant lipase was found to have a molecular mass of 36.7 kDa, and it exhibited lipase activity of 20 U/mL in culture supernatant when tributyrin was used as the substrate.     

CdpNPT, an N-prenyltransferase from Aspergillus fumigatus: overproduction, purification and biochemical characterisation

A putative prenyltransferase gene, cdpNPT, was identified in the genome sequence of Aspergillus fumigatus by a homology search by using known prenyltransferases and sequence analysis. CdpNPT consists of 440 amino acids and has a molecular mass of about 50 kDa. The coding sequence of cdpNPT was cloned in pQE60 and overexpressed in E. coli. The soluble His(6)-fusion CdpNPT was purified to near homogeneity and characterised biochemically. The enzyme showed broad substrate specificity towards aromatic substrates and was found to catalyse the prenylation of tryptophan-containing cyclic dipeptides at N1 of the indole moieties in the presence of dimethylallyl diphosphate (DMAPP); geranyl diphosphate was not accepted as prenyl donor. The structures of the enzymatic products were elucidated by NMR and MS analysis. The K(m) value for DMAPP was determined to be 650 microM. Due to substrate inhibition, K(m) values could not be obtained for the aromatic substrates. CdpNPT does not need divalent metal ions for its enzymatic reaction, although Ca(2+) enhances the reaction velocity by up to the threefold. CdpNPT is the first N-prenyltransferase that has been purified and characterised in a homogenous form after heterologous overproduction. Interestingly, it shows significant sequence similarity to other indole prenyltransferases that catalyse the formation of C--C bonds.     

Reverse prenyltransferase in the biosynthesis of fumigaclavine C in Aspergillus fumigatus: gene expression, purification, and characterization of fumigaclavine C synthase FGAPT1

A putative prenyltransferase gene-fgaPT1-has been identified in the biosynthetic gene cluster of fumigaclavines in Aspergillus fumigatus AF293. The gene was cloned and overexpressed in Escherichia coli, and the His6-fusion FgaPT1 was purified to near homogeneity and characterized biochemically. The enzyme was found to convert fumigaclavine A into fumigaclavine C by attaching a dimethylallyl moiety to C-2 of the indole nucleus in a "reverse" manner, that is, by connection of C-3 of the dimethylallyl moiety to an aromatic nucleus. FgaPT1 is a soluble, dimeric protein with a subunit size of 50 kDa. K m(app) values for fumigaclavine A and dimethylallyl diphosphate were determined to be 6 and 13 microM, respectively, while the turnover number was 0.8 s(-1). Metal ions such as Mg2+ and Ca2+ are not essential for the enzymatic activity. FgaPT1 showed relatively strict substrate specificity towards fumigaclavine A, with only dimethylallyl diphosphate being accepted as a donor under our conditions. FgaPT1 is the first reverse prenyltransferase from fungi to have been purified and characterized in homogenous form after heterologous overproduction. Surprisingly, it shows very low sequence similarity to the recently identified prenyltransferase LtxC from cyanobacteria, which also catalyzes the reverse prenylation of an indole nucleus.     

人血管生成抑制素基因的克隆、表达及纯化

目的 构建携带人血管生成抑制素基因的原核表达载体,诱导表达具有活性的血管生成抑制素。方法 用PCR法从人纤溶酶原cDNA中扩增出Kringle 1-3基因,克隆入pET21a(+)载体中,在大肠杆菌BL 21中表达,表达产物经亲和层析纯化。结果 所构建的原核表达载体为高效表达系统,所表达的产物经层析纯化后可获得重组人血管生成抑制素。结论 可获得大量纯化人血管生成抑制素,为进一步临床应用奠定基础。