【作者】 吴琦；

【导师】 刘世贵；

【作者基本信息】 四川大学 ， 遗传学， 2004， 博士

【摘要】 植酸酶作为一种绿色添加剂，已日益广泛使用于饲料工业中。研究表明，来源于芽孢杆菌的植酸酶属于中性植酸酶不仅具有较好的热稳定性，有助于抵抗饲料制粒或者膨化过程中引起的酶失活，而且其酶促反应的pH有效作用范围在6.5～7.5之间，适合用于消化道呈中性的鲤科鱼类，以及在单胃动物中pH值呈中性的肠道段中表现酶活力。目前，国内外尚无商品化的中性植酸酶投入市场。 本研究从已筛选获得的中性植酸酶产酶菌株枯草芽孢杆菌(Bacillus subtilis) WHNB02出发，根据GenBank上的芽孢杆菌，phyC基因序列设计了一对引物，采用PCR法获得芽孢杆菌植酸酶的全长phyC基因，并将其克隆到pUC18-T载体。序列分析表明，该基因全长1152bp，编码一个383氨基酸的多肽。通过与GenBank中登录的其他6个植酸酶基因的核酸序列和氨基酸序列进行比较，建立了系统进化树，结果表明：所有芽孢杆菌植酸酶具有较高的同源性，来源于7株芽孢杆菌的植酸酶在遗传上分为两大类。利用在线生物信息学数据库及生物软件对本基因进行了信号肽序列、N-糖基化位点、密码子偏爱性等分析，并对其二级结构、疏水区和三维结构进行了预测。B．subtilis WHNB02植酸酶phyC基因序列及其氨基酸序列在GenBank中登录，登录号分别为AF220075和AA043434.1。 根据芽孢杆菌植酸酶phyC基因序列及大肠杆菌表达载体pET-30b(+)多克隆位点序列，采用PCR法获得不含有信号肽序列的植酸酶phyC基因的非融合和融合表达片段。经克隆及序列测定后，构建了带有T7lac启动子的大肠杆菌的植酸酶pET30NFphyC和pET30FphyC表达载体，并转入大肠杆菌BL21(DE3)，实四川大学博士学位论文现了有生物学活性的表达。研究了诱导温度、IPTG诱导浓度和乳糖诱导浓度对植酸酶非融合和融合表达的影响，并对表达目的蛋白的可溶性进行了分析。结果表明，非融合表达在37℃和30℃表达产物以包含体形式存在，无表观酶活，而25℃诱导的表观酶活最高:融合表达载体在37℃表观酶活较低，而30℃诱导的表观酶活最高。表明降低诱导温度有助于实现目的蛋白的可溶性表达。两种表达方式的工PTG最适诱导浓度为0.4mmol/L，乳糖最适诱导浓度为1%，但乳糖诱导表达的时间推迟1一2h，且表达量不及工PTG的诱导。SDS一PAGE分析表明，非融合和融合植酸酶的表达量分别约占菌体总蛋白的13%和15%，分子量分别为40.13KD和43.27KD。 为实现芽抱杆菌植酸酶助尹C基因的分泌表达，以及由酵母翻译后加工可能带来的酶学性质改善，首次进行了该基因在酵母中的表达研究。根据芽饱杆菌植酸酶ph一C基因序列及毕赤巴斯德酵母表达载体pPIC3.SK和pP工CgK多克隆位点序列，采用PCR法获得不含有信号肤序列的植酸酶ph了C基因胞内表达和分泌表达片段。经克隆及序列测定后，构建了重组表达载体pPIc3.SKphyC和ppICgKphyC，经Bgl 11线性化后，电转化毕赤巴斯德酵母宿主菌Gs 115。经MD和MM平板筛选、PCR检测和酶活性测定，获得阳性重组酵母，首次在毕赤巴斯德酵母中实现了有生物学活性芽抱杆菌植酸酶的胞内表达和分泌表达。SDS一PAGE分析表明，胞内植酸酶表达量约占菌体总可溶性蛋白的24%，分子量为42.01KD;而分泌表达的植酸酶为53.SKD和50.gKD两种分子量的蛋白，去糖基化试验证明这是由于产物糖基化程度不同引起的糖蛋白。用麦芽汁半合成培养基对分泌表达重组酵母进行培养，经48h诱导酶活可达到2453U/ml，表达量为出发菌株的10.5倍。 将本研究获得的芽抱杆菌植酸酶的4种基因工程菌表达产物，即大肠杆菌表达的非融合植酸酶和融合植酸酶、酵母胞内表达的植酸酶和分泌表达的植酸酶，通过初步纯化后，对其最适反应温度、热稳定性和最适反应pH值进行了测定。结果表明，4种基因工程植酸酶的最适反应温度分别为55℃，75℃，70℃和65℃，与天然植酸酶相比，分别降低了5℃和增加了巧℃，10℃和5℃。热稳定性测定表明，经75℃处理时，4种基因工程植酸酶分别表现为酶活急剧下降，几乎没有损失，损失较小和几乎没有损失;在90℃水浴smin和10min后四川大学博士学位论文的残留酶活性分别为43.8%和35.5%，94.9%和75.7%，54.6%和42.2%，以及88.0%和78.2%，均比天然植酸酶的有所提高。4种基因工程植酸酶的最适反应pH分别为7.0，7.5，7.5和7.。，催化效率在70%以上的有效反应pH范围分别为pHS.5一8，pHS.5一9，pH6.5一9和pH6一8，比天然植酸酶的扩大了0.5一l个PH。关键词:枯草芽抱杆菌，植酸酶，助一‘基因，大肠杆菌，毕赤巴斯德酵母， 表达尹-更多还原

【Abstract】 As one of the green additives, phytase has been used in feed industry more widely today,. Scientists found Bacillus phytase has the higher thermostablity than Aspergillus phytase. Moreover, its optimum reaction pH is between 6.5 and 7.5, which makes it be used carp with neutral intestinal tract. Up to now, there is no commercial neutral phytase on market yet.According to the Bacillus phytase gene sequences downloaded from GenBank, a pair of primers was designed to amplify the full sequence of phytase phyC gene from B. subtilis WHNB02 by PCR. The PCR product was cloned and sequenced. The sequence analysis results showed the full gene was 1152bp in length, and it coded a 383 amino acid multi-peptide. Compared the nucleotide and amino acid sequences with the other six phytases of Bacillus, their phylogenetic tree was drawn. The results indicated all the seven phytases from Bacillus had the high homology and belonged to two groups genetically. Furthermore, the specialized features of the encoded protein, including signal peptide cleavage site, N-glycosylation sites, codon bias, secondary structure, hydrophobicity and 3D structure, were predicted and analyzed. The gene and its protein sequences were accepted by GenBank with accession numbers AF220075 and AA043434.1.Based on the sequence of AF220075 and multi-clone site of E.coli expression vector pET-30b(+),the non-fusion and fusion expression fragments without signalpeptide sequence were amplified by PCR. The two fragments were cloned, sequenced and inserted into vector pET-30b(+) with lilac promoter. The recombined expression vectors pET30NFphyC and pET30FphyC were screened and transformed into E. coli BL21(DE3). The products with phytase activity were expressed successfully. Induction experiments showed the optimum induction temperature of non-fusion and fusion expression was 25℃ and 30℃ respectively, and the lower induction temperature was helpful to increase soluble protein expression. Their optimum induction concentration of IPTG and lactose was 0.4mmol/L and 1% respectively, but the peak value time of lactose was later about l-2h and lower than that of IPTG. The yield of non-fusion and fusion phytase came to 13% and 15% of total protein and their molecular weights were 40.13KD and 43.27KD, respectively.In order to perform the secretory expression of Bacillus phytase and get better enzymatic characters because of the post-translation modification of yeast, Bacillus phytase phyC gene was expressed in yeast primarily. According to the sequence of AF220075 and multi-clone site of P. pastorisi expression vector pPIC3.5K and pPIC3.5K, the intracellular and secretory expression fragments without signal peptide sequence were amplified by PCR. The products were cloned and sequenced. Then, two recombined expression vectors pPIC3.5KphyC and pPIC9KphyC were constructed. The plasmids were linearized by Bgl II and transformed into Pichia Pastoris GS115 strain by electroporation. Positive strains were screened and purified by culturing on MD plate, MM plate and PCR. Furthermore, they were cultured and induced by methanol. SDS-PAGE analysis showed that phytase was expressed intracellularly, and the yield came to 24% of the total soluble protein and its molecular weight was 42.01KD. However, the phytase expressed secretively was a kind of glycoprotein which had two molecular weight 53.5KD and 50.9KD because of the different glycosylation level. Additionally, in wort semi-synthetic medium, the phytase yield came to 2453U/ml after 48 hours induction, 10.5 times of original B. subtilis strain.All the four kinds of genetic engineering Busillus phytases, non-fusion and fusion phytases expressed in E. coli and intracellular and secretory phytases expressed in P. pastoris, were purified and performed to measure their optimum reaction temperature, thermostability and optimum reaction pH. The results indicated their optimum reaction temperature was 55 ℃, 75 ℃, 70℃ and 65 ℃ respectively. Comparison with the natural phytase, the temperature was lower 5℃ and higher 15℃ , 10℃ and 5更多还原