【作者】 沈微；

【导师】 诸葛健；

【作者基本信息】 江南大学 ， 发酵工程， 2003， 博士

【摘要】 耐高温α-淀粉酶,能在高温条件下水解淀粉,是目前最重要的工业酶制剂之一,广泛应用于发酵、制糖等工业。高嗜热古细菌Pyrococcus furiosus 产生的胞外α-淀粉酶(PFA)具有优越的耐热性能,较低的最适pH,活性不依赖于钙离子,因此,潜在的应用价值很大。本文主要研究PFA 在真细菌中的分泌表达和在真核生物中的表达,并构建了细菌表达载体以用于上述工作。用PCR 方法从P. furiosus 染色体DNA 中扩增出含信号肽序列的PFA 结构基因,插入大肠杆菌表达载体pET28a 中,构建成重组质粒pET-PFA(s+)。以pET-PFA(s+)为底物,扩增出不含信号肽的PFA 结构基因,插入pET28a。两种重组载体分别转化大肠杆菌BL21 (DE3)。在转化子中,两种结构基因与pET28a 携带的His-tag 融合表达。质粒pET-PFA(s-)的融合表达产物不含信号肽,重组菌在IPTG 诱导下表达出有活性的重组酶。质粒pET-PFA(s+)的表达产物含信号肽,其转化子不能表达出有活性的产物,说明含信号肽的重组酶如果由于融合表达而不能被切除信号肽,则无法形成有活性的结构。质粒pET-PFA(s-)转化子表达的重组酶活力达11.4 U/mL,但大部分的酶活在离心后与细胞碎片一起沉淀。重组酶的最适温度为95℃,最适pH 为5.0,在121℃热处理活性半衰期超过30 min。将表达载体pKK223-3的tac启动子插入载体pET28a多克隆位点,构建成表达载体pEtac。新载体pEtac 为非融合型表达载体,表达产物氨基酸序列与插入基因编码的蛋白完全一致,同时载体自身含lacI 基因能高表达乳糖操纵子阻遏蛋白,因此本底转录低于pKK223-3。将含信号肽的PFA 结构基因插入pEtac 中,构建成重组质粒pEtac-PFA,成功转化大肠杆菌JM109。重组菌在IPTG 诱导下表达出PFA 活性,其中溶解在周质中的酶活达1.4 U/mL,说明PFA 能更多还原

【Abstract】 Thermostable α-amylse, which can catalyze hydrolysis of starch at high temperatures, widely used in sugar and fermentation industry, is one of the most important enzyme products now. Extracellular α-amylase from hyperthermophilic archaeon Pyrococcus furiosus, which is optimally active at about 100℃, is one of the most thermophilic α-amylase found until now. It also shows low optimal pH and the calcium ion independent activity, so it has great potential for industrial use. This research is mainly focused on secreting expression of the PFA in eubacteria and expression of the gene in eukaryotes. The structure gene of P. furiosus extracellular α-amylase (PFA) with signal sequence was amplified from genomic DNA of P. furiosus by using PCR technique. The recombinant vector, pET-PFA(s+), was constructed by inserting the amplified fragment (about 1.4kb) into the expression vector pET-28a. The DNA fragment encoding mature peptide of PFA was amplified from pET-PFA(s+) and was then inserted into pET-28a to construct pET-PFA(s-). Both recombinant plasmids were used to transform Escherichia coli BL21(DE3) respectively. Promoted and controlled by T7 promoter and lac operator, the fusion protein containing His-tag and the peptide of PFA with or without signal peptide could be expressed within the cell of recombinant E. coli. The transformant of pET-PFA(s-) expressed the fusion protein without signal peptide and active recombinant enzyme was tested after the recombinant was induced by IPTG. The transformant of pET-PFA(s+) expressed the fusion protein with signal peptide, and the product was inactive, so if the recombinant enzyme couldn’t be cleaved off it’s signal peptide because the function of the signal peptide is effected by the small peptide, couldn’t form the right structure. The activity level of 11.4 U/mL could be obtained after the transformant of pET-PFA(s-) was induced under standard conditions (in LB medium at 37℃),but most of the enzymatic activity co-precipitated with cell debris after centrifugation. The recombinant enzyme expressed by transformant of pET-PFA(s-) showed similar enzymatic property to the extracelluar α-amylase expressed by P. furiosus. It was optimally active at 100℃and pH 5.0, half life at 121℃was more than 30 min. A new expression vector, pEtac, was constructed by inserting the 243bp fragment between BamH I and EcoR I site of pKK223-3, which contained tac promoter and RBS of pKK223-3, into the multiple cloning site (MCS) of pET-28a. Different to pET-28a, pEtac was not a fusion expression vector, the amino acid sequence expressed was completely correspondent to the gene inserted,meanwhile the vector contained lacI gene which encoded repressor of lac operator, so the basal transcription of pEtac was lower than pKK223-3. Recombinant plasmid pEtac-PFA was constructed by inserting the gene of PFA with signal sequence into MCS of pEtac, and E. coli JM109 was transformed with pEtac-PFA successfully. The recombinant expressed active PFA enzyme after it was induced. The soluble enzymatic activity obtained from periplasmic space of 1 milliliter induced culture was 1.4U, accordingly the PFA with it’s own signal sequence could be secreted into periplasm, and because the proenzyme with it’s signal peptide was inactive, so the signal peptide must be cleaved for active enzyme. By comparison of the activity of recombinant enzymes, it showed that, affected by the His-tag in it’s N-terminal, the fusion protein of PFA and His-tag expressed by transformant of pET-PFA(s-) was not active until it was incubated at 60℃or higher temperature and the recombinant enzyme produced by the transformant of pEtac was active at 40℃even it was not incubated at a high temperature. The DNA fragments used to construct the secreting expression vector of Bacillus subtilis included: 1) replication origin (pAM-α) and tetracycline resistant protein gene (tet) of B. subtilis from pHY300PLK; 2)xylose isomerase promoter of B. megaterium from pWHT02-terminator(Pxyl); 3)signal sequence of α-amylase of B. subtilis 168(sig); 4)replication origin of E. coli and β-lactamase gene (bla) from pUC19. Plasmid pUHxyls, a secreting expression vector of B. subtilis, was constructed by several steps of DNA recombination. It was also a shuttle vector of E. coli and B. subtilis. Because pUHxyls contained the repressor gene of Pxyl, it showed a low level basal transcription when the inducer, xylose, was absent. There was MCS at the downstream of signal sequence, so if the structure gene without signal sequence was inserted into the MCS, it could be expressed and the fusion protein could be secreted into medium. The recombinant plasmid pUHxyls-PFA was constructed by inserting the structure gene without signal sequence into MCS of pUHxyls. B. subtilis WB700, a protease deficiency mutant, was transformed with pUHxyls. The recombinant B. subtilis expressed active PFA enzyme. The expression was strictly controlled by xylose induction. Part of the enzyme was secreted into medium, but about half of the enzyme activity was combined with the cells of the transformant. Preliminary research of the induction conditions showed that high temperature helped to increase the fraction of secreted PFA. Expression of PFA didn’t affect the growth of the transformants. The recombinant plasmid pYX212-PFA was constructed by inserting the gene of PFA withoutsignal sequence into the MCS of a expression vector of Saccharomyces cerevisiae. The recombinant plasmid was used to transform S. cerevisiae W303, the active PFA was expressed within the cell of the recombinant yeast, 0.16 U of enzyme activity could be obtained from per milliliter of culture. The structure gene of PFA without signal sequence was inserted into the site of BamH I of pPIC9K, a secreting expression vector of Pichia pastoris, the site BamH I was at the upstream of α-factor, so the recombinant plasmid pPIC9K-PFA could be used in intracellular expression of the PFA. The plasmid pPIC9K-PFA was linearized with Sal I and used to transform P. pastoris KM71. After induction with methanol, the recombinant P. pastori expressed active PFA enzyme, and the activity was 64 U/mL, and more than 70% of the enzyme activity was in the supernatant after the crude cell exract was centrifuged.更多还原