蜂房哈夫尼菌植酸酶基因appA的克隆、表达及酶学性质研究

从蜂房哈夫尼菌（Hafnia alvei）中克隆获得一个植酸酶编码基因appA, 该基因全长1335bp,编码444个氨基酸,其中前33个氨基酸为信号肽,成熟蛋白的理论分子量为45.2kD。将基因appA 克隆到大肠杆菌E. coli表达载体pET-22b(+),并在大肠杆菌中表达, 表达产物具有植酸酶活性。对表达的酶蛋白进行纯化,并初步研究了该酶的酶学性质,结果表明：酶的作用最适pH值为4.5;在pH 2.0～10.0范围内, 酶活性保留80％以上;酶的作用最适温度为60℃;酶的比活性为356.7U/mg,酶动力学分析表明其K,/i>_m为0.49mmol/L,V_max为238U/mg;该酶对胰蛋白酶和胃蛋白酶有一定的抗性。该研究为哈夫尼菌属来源植酸酶的首次报道。     

蜂房哈夫尼菌植酸酶基因appA的克隆、表达及酶学性质研究

从蜂房哈夫尼菌（Hafnia alvei）中克隆获得一个植酸酶编码基因appA, 该基因全长1335bp,编码444个氨基酸,其中前33个氨基酸为信号肽,成熟蛋白的理论分子量为45.2kD。将基因appA 克隆到大肠杆菌E. coli表达载体pET-22b(+),并在大肠杆菌中表达, 表达产物具有植酸酶活性。对表达的酶蛋白进行纯化,并初步研究了该酶的酶学性质,结果表明：酶的作用最适pH值为4.5;在pH 2.0～10.0范围内, 酶活性保留80％以上;酶的作用最适温度为60℃;酶的比活性为356.7U/mg,酶动力学分析表明其K,/i>_m为0.49mmol/L,V_max为238U/mg;该酶对胰蛋白酶和胃蛋白酶有一定的抗性。该研究为哈夫尼菌属来源植酸酶的首次报道。     

大肠杆菌植酸酶基因appA的克隆与高效表达

从猪粪便中分离并筛选出高效生产酸性植酸酶和磷酸酶双功酶(appA植酸酶)的大肠杆菌菌株。通过PCR方法从该菌株基因组中扩增获得了植酸酶基因appA,测序结果显示该基因编码区全长1,299个核苷酸。将该基因克隆到原核表达载体pET-28a( )上,通过转化的大肠杆菌BL21在试管摇床培养条件下得到了高效表达,其表达量达到692U／mL。酶学特性分析表明其反应的最适pH为4．5,最适温度为60℃。     

大肠杆菌植酸酶基因appA的克隆与高效表达*

从猪粪便中分离并筛选出高效生产酸性植酸酶和磷酸酶双功酶(appA植酸酶)的大肠杆菌菌株。通过PCR方法从该菌株基因组中扩增获得了植酸酶基因appA,测序结果显示该基因编码区全长1,299个核苷酸。将该基因克隆到原核表达载体pET-28a(+)上,通过转化的大肠杆菌BL21在试管摇床培养条件下得到了高效表达,其表达量达到692U/mL。酶学特性分析表明其反应的最适pH为4.5,最适温度为60℃。     

嗜热枯草芽孢杆菌普鲁兰酶基因的表达与重组酶的性质

克隆嗜热枯草芽孢杆菌WY-34普鲁兰酶基因并在大肠杆菌中进行表达,对重组酶进行纯化和酶学性质研究,根据枯草芽孢杆菌的普鲁兰酶蛋白序列,设计PCR引物对WY-34的普鲁兰酶基因进行克隆及异源表达.对表达蛋白的最适pH、pH稳定性及最适温度、温度稳定性等特性进行研究,并测定重组普鲁兰酶的底物特异性.将普鲁兰酶基因pluA克隆及分析序列后,发现基因长度为2.2 kb,编码718个氨基酸,在大肠杆菌中异源表达.通过Ni-IDA亲和层析一步纯化得到比活力为93.2 U/mg的纯酶,SDS-PAGE和凝胶层析测定的分子量分别为76.2 kD和74.3 kD.酶学性质研究表明,该酶的最适温度为40℃,在温度不高于45℃条件下稳定;最适pH为6.0,同一温度下pH 6.0-9.0范围内处理30 min可以保持80％以上的酶活力,此酶对普鲁兰糖有很强的底物特异性.此重组普鲁兰酶的酶学性质表明此酶具有一定的工业化应用价值.     

嗜热子囊菌原变种Thermoascus aurantiacus var.aurantiacus热稳定几丁质酶的克隆表达及活性研究

研究通过RT-PCR和Tail-PCR技术从嗜热子囊菌原变种Thermoascus aurantiacus var.aurantiacus中克隆了一个几丁质酶同源基因。该基因全长1,253bp,包含一个由1,197个碱基构成的开放阅读框,编码398个氨基酸。序列比对分析表明,该基因编码蛋白属于糖苷水解酶18家族的几丁质酶。利用基因重组的方法构建酵母分泌型表达载体,并转化毕赤酵母。在甲醇的诱导下,重组蛋白得到了高效表达,第6天的表达量最高,达到0.433g/L,酶活力为28.96U/mg,同时对表达的几丁质酶进行了纯化,SDS-PAGE检测该蛋白的分子量为43.9kDa。该几丁质酶的最适反应温度为60℃,最适反应pH值为8.0,70℃处理30min仍有45%的相对酶活,具有较好的热稳定性及工业应用价值。     

嗜热毛壳菌热稳定脂肪酶基因(Im)的克隆及其在毕赤酵母中的表达

研究从嗜热毛壳菌Chaetomium thermophilum中克隆了一个新的脂肪酶基因(lm).其中DNA序列包含一个由870个碱基构成的开放阅读框,编码289个氨基酸,含有4个内含子,没有信号肽序列.序列提交GenBank,登录号为GU338248.将该基因在毕赤酵母中表达.在甲醇的诱导下,重组蛋白得到了高效表达,第6天的表达量最高,蛋白达到0.428mg/mL,酶活力为19.77U/mg.SDS-PAGE检测该蛋白的分子量为35kDa.该脂肪酶的最适反应温度为60℃,具有热稳定性,在40-80℃热稳定,80℃处理60min仍有65％的相对酶活.该酶最适反应pH值为10.0,在pH 9.0--12.0酶活相对稳定.该酶具有较好的热稳定性和耐碱性,具有良好的工业应用价值.     

海洋链霉菌Streptomyces olivaceus FXJ7.023来源多功能几丁质酶的克隆、表达及鉴定(英文)

碳水化合物水解酶家族在自然界碳素循环及农业废弃物中几丁质、纤维素等碳水化合物的生物质转化利用中发挥了重要作用。通过PCR技术从海洋链霉菌Streptomyces olivaceus strain FXJ 7.023的fosmid基因组文库中成功克隆得到1个全长885bp的编码295个氨基酸残基的包含1个19个氨基酸残基的N-末端信号肽的壳聚糖酶完全编码区。系统进化分析表明该基因编码蛋白与已报道的Streptomyces sp.SirexAA-E来源壳聚糖酶csnA同源性为71%,与Streptomyces coelicolor A3(2)来源的csn46A同源性为70%。将该编码区重组入原核表达质粒载体pET32a并转化大肠杆菌表达菌株BL21(DE3)plysS,添加IPTG在18℃条件下振荡诱导该蛋白表达,Ni2+-NTA亲和纯化获得分子量为50.3 kDa融合表达蛋白TrxA-SoCsn。该融合重组蛋白在最适反应条件下对底物胶体壳聚糖和羧甲基纤维素的最大酶活分别为3.673U/mg和1.302U/mg,最适反应温度分别为37℃和50℃,最适反应pH分别为pH5.0和pH6.0。TrxA-SoCsn相关的研究结果表明该酶在农业废弃物生物质转化等方面具有一定的应用潜力。     

大肠杆菌腺苷脱氨酶基因的克隆表达

将大肠杆菌K12菌株来源的腺苷脱氨酶基因(add)克隆到载体pET-28a中,并转化至大肠杆菌BL21(DE3)中进行表达。通过IPTG诱导,SDS-PAGE检测和酶活性的测定发现,重组菌表达产生大量腺苷脱氨酶,活性达到51.07U/mg蛋白。通过酶性质的研究,腺苷脱氨酶对腺苷最适pH和温度分别为7.5和40℃,且在40℃下维持稳定。     

来源于Penicillium sp.C1的水产用中性植酸酶基因在毕赤酵母中的表达及性质研究

通过简并引物和TAIL-PCR技术从青霉Penicillium sp. C1菌株的基因组DNA中克隆得到一个新的植酸酶基因,命名为phyC1。其结构基因全长1 477 bp,5’端包含58 bp的内含子序列,该基因编码472个氨基酸和一个终止密码子,前23个氨基酸为信号肽。将PhyC1的成熟蛋白的编码基因在毕赤酵母菌GS115中高效分泌表达,重组蛋白经硫酸铵沉淀和阴离子交换柱和分子筛纯化后达到电泳纯。对其酶学性质分析表明:重组植酸酶PhyC1最适pH为6.5,在pH5.0-10.0的条件下具有较好的稳定性;重组植酸酶PhyC1最适温度为50℃。与现有商品植酸酶相比,PhyC1具有两个显著的特性,如低温条件下具有高活性,尤其是在低于20℃时;在中性pH 7.0条件下仍具有90%以上的相对酶活性,因此,植酸酶PhyC1的优良特性为其在水产行业的应用奠定基础。     

A novel phytase with preferable characteristics from Yersinia intermedia

A Yersinia intermedia strain producing phytase was isolated from glacier soil. The phytase gene, appA, was isolated by degenerate PCR and TAIL-PCR. The full-length fragment contained 2354bp with a 1326-bp open reading frame encoding 441 amino acids. APPA contained the active site RHGXRXP and HD sequence motifs that are typical of histidine acid phosphatases. To our knowledge, this is the first report of the detection of phytase activity and cloning of the relevant gene from Y. intermedia. The gene was overexpressed in Pichia pastoris, and the purified recombinant APPA had a specific activity for sodium phytate of 3960U/mg, which is higher than that of the Citrobacter braakii phytase (previously the highest specific activity known). Recombinant APPA had high activity from pH 2 to 6 (optimum 4.5) and optimal temperature of 55 degrees C; the enzyme was resistant to pepsin and trypsin. These characteristics suggest that APPA may be highly suitable for use in the feed industry.     

Cloning, expression, and characterization of a new phytase from the phytopathogenic bacterium Pectobacterium wasabiae DSMZ 18074

The soft rot bacterium Pectobacterium wasabiae is an economically important pathogen of many crops. A new phytase gene, appA, was cloned from P. wasabiae by degenerate PCR and TAIL-PCR. The open reading frame of appA consisted of 1,302 bp encoding 433 amino acid residues, including 27 residues of a putative signal peptide. The mature protein had a molecular mass of 45 kDa and a theoretical pI of 5.5. The amino acid sequence contained the conserved active site residues RHGXRXP and HDTN of typical histidine acid phosphatases, and showed the highest identity of 48.5% to PhyM from Pseudomonas syringae. The gene fragment encoding the mature phytase was expressed in Escherichia coli BL21 (DE3), and the purified recombinant phytase had a specific activity of 1,072+/-47 U/mg for phytate substrate. The optimum pH and temperature for the purified phytase were pH 5.0 and 50 degrees , respectively. The Km value was 0.17 mM, with a Vmax of 1,714 mmol/min/mg. This is the first report of the identification and isolation of phytase from Pectobacterium.     

来源于柠檬酸杆菌的高比活植酸酶基因在毕赤酵母中的高效表达

柠檬酸杆菌(Citrobacterbraakii)来源的植酸酶是目前报道的比活最高的植酸酶。按照毕赤酵母(Pichiapastoris)对密码子的选择偏向性,对来源于柠檬酸杆菌的高比活植酸酶基因AppA进行了密码子优化改造。改造后的基因AppA(m)按正确的阅读框架融合到毕赤酵母表达载体pPIC9的α-因子信号肽编码序列3′端,通过电击转化得到重组转化子。通过PCR验证,AppA(m)已整合在酵母染色体上。SDS-PAGE分析和表达产物的研究表明,植酸酶得到了高效分泌表达,在5L发酵罐中植酸酶蛋白表达量达到3·2mg/mL发酵液,发酵效价达到每毫升发酵液1·4×107IU以上,高于目前报道的各种植酸酶基因工程菌株的发酵效价。     

Characterization and overproduction of the Escherichia coli appA encoded bifunctional enzyme that exhibits both phytase and acid phosphatase activities

The appA gene that was previously shown to code for an acid phosphatase instead codes for a bifunctional enzyme exhibiting both acid phosphatase and phytase activities. The purified enzyme with a molecular mass of 44,708 Da was further separated by chromatofocusing into two isoforms of identical size with isoelectric points of 6.5 and 6.3. The isoforms had identical pH optima of 4.5 and were stable at pH values from 2 to 10. The temperature optimum for both phytase isoforms was 60 degrees C. When heated at different pH values the enzyme showed the greatest thermal resistance at pH 3. The pH 6.5 isoform exhibited K(m) and Vmax values of 0.79 mM and 3165 U.mg-1 of protein for phytase activity and 5.5 mM and 712 U.mg-1 of protein for acid phosphatase, respectively. The pH 6.3 isoform exhibited slightly lower K(m) and Vmax values. The enzyme exhibited similar properties to the phytase purified by Greiner et al. (1993), except the specific activity of the enzyme was at least 3.5-fold less than that previously reported, and the N-terminal amino acid sequence was different. The Bradford assay, which was used by Greiner et al. (1993) for determination of enzyme concentration was, in our hands, underestimating protein concentration by a factor of 14. Phytase production using the T7 polymerase expression system was enhanced by selection of a mutant able to grow in a chemically defined medium with lactose as the carbon source and inducer. Using this strain in fed-batch fermentation, phytase production was increased to over 600 U.mL-1. The properties of the phytase including the low pH optimum, protease resistance, and high activity, demonstrates that the enzyme is a good candidate for industrial production as a feed enzyme.     

Production of two Highly Active Bacterial Phytases with Broad pH Optima in Germinated Transgenic Rice Seeds

Phytate is the main storage form of phosphorus in many plant seeds, but phosphate bound in this form is not available to monogastric animals. Phytase, an enzyme that hydrolyzes phosphate from phytate, has the potential to enhance phosphorus availability in animal diets when engineered in rice seeds as a feed additive. Two genes, derived from a ruminal bacterium Selenomonas ruminantium (SrPf6) and Escherichia coli (appA), encoding highly active phytases were expressed in germinated transgenic rice seeds. Phytase expression was controlled by a germination inducible alpha-amylase gene (alphaAmy8) promoter, and extracellular phytase secretion directed by an betaAmy8 signal peptide sequence. The two phytases were expressed in germinated transgenic rice seeds transiently and in a temporally controlled and tissue-specific manner. No adverse effect on plant development or seed formation was observed. Up to 0.6 and 1.4 U of phytase activity per mg of total extracted cellular proteins were obtained in germinated transgenic rice seeds expressing appA and SrPf6 phytases, respectively, which represent 46-60 times of phytase activities compared to the non-transformant. The appA and SrPf6 phytases produced in germinated transgenic rice seeds had high activity over broad pH ranges of 3.0-5.5 and 2.0-6.0, respectively. Phytase levels and inheritance of transgenes in one highly expressing plant were stable over four generations. Germinated transgenic rice seeds, which produce a highly active recombinant phytase and are rich in hydrolytic enzymes, nutrients and minerals, could potentially be an ideal feed additive for improving the phytate-phosphorus digestibility in monogastric animals.     

耐热植酸酶突变体的筛选及性质研究

目的:对大肠杆菌Escherichia coli植酸酶基因进行定向进化,获得热稳定性提高的植酸酶突变体。方法:利用易错PCR技术和96微孔板高通量筛选方法获得突变体基因,并对突变酶进行异源表达、纯化及性质研究。结果:通过筛选获得3株热稳定性明显提高的植酸酶突变体APPA1、APPA2、APPA3。酶学性质分析结果表明,3个突变体分子量均约为55kDa,最适pH均为4.5,与野生型无明显差别,热稳定性较野生型均有显著提高,其中突变体APPA3的最适温度为65℃,较野生酶提高5℃,在90℃处理10min后保留50%的酶活。酶的三维结构模拟显示,5个突变位点在植酸酶整体结构上均引入新氢键。结论:通过定向进化获得热稳定性提高的大肠杆菌Escherichia coli植酸酶突变体,对植酸酶的工业应用和研究植酸酶结构与功能关系具有重要意义。     

A Novel Phytase appA from <Emphasis Type="Italic">Citrobacter amalonaticus</Emphasis> CGMCC 1696: Gene Cloning and Overexpression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A novel phytase gene appA, with upstream and downstream sequences from Citrobacter amalonaticus CGMCC 1696, was cloned by degenerate polymerase chain reaction (PCR), and thermal asymmetric interlaced (TAIL) PCR and was overexpressed in Pichia pastoris. Sequence analysis revealed one open reading frame that consisted of 1311 bp encoding a 436–amino-acid protein, which had a deduced molecular mass of 46.3 kDa. The phytase appA belongs to the histidine acid phosphatase family and exhibits the highest identity (70.1%) with C. braakii phytase. The gene was overexpressed in P. pastoris. The secretion yield of recombinant appA protein was accumulated to approximately 4.2 mg·mL⁻¹, and the enzyme activity level reached 15,000 U·mL⁻¹, which is higher than any previous reports. r-appA was glycosylated, as shown by Endo H treatment. r-appA was purified and characterized. The specific activity of r-appA for sodium phytate was 3548 U·mg⁻¹. The optimum pH and temperature for enzyme activity were 4.5 and 55°C, respectively. r-appA was highly resistant to pepsin or trypsin treatment. This enzyme could be an economic and efficient alternative to the phytases currently used in the feed industry.     

Design and expression of a synthetic phyC gene encoding the neutral phytase in Pichia pastoris

The 1074-bp phyCs gene (optimized phyC gene) encoding neutral phytase was designed andsynthesized according to the methylotrophic yeast Pichia pastoris codon usage bias without altering theprotein sequence.The expression vector,pP9K-phyCs,was linearized and transformed in P.pastoris.Theyield of total extracellular phytase activity was 17.6 U/ml induced in Buffered Methanol-complex Medium(BMMY) and 18.5 U/ml in Wheat Bran Extract Induction (WBEI) medium at the flask scale,respectively,improving over 90 folds compared with the wild-type isolate.Purified enzyme showed temperature optimumof 70℃ and pH optimum of 7.5.The enzyme activity retained 97% of the relative activity afterincubation at 80℃ for 5 min.Because of the heavy glycosylation the expressed phytase had a molecularsize of approximately 51 kDa.After deglycosylation by endoglycosylase H (EndoH_f),the enzyme had anapparent molecular size of 42 kDa.Its property and thermostability was affected by the glycosylation.     

来源于Escherichia coli的高比活植酸酶基因的高效表达

高效表达高比活植酸酶是进一步提高植酸酶发酵效价、降低植酸酶生产成本的一个有效途径。对源于Escherichiacoli的高比活植酸酶基因appA ,按照毕赤酵母 (Pichiapastoris)密码子的偏爱进行了密码子优化改造。该改造后的基因appA m按正确的阅读框架融合到毕赤酵母表达载体pPIC9上的α 因子信号肽编码序列 3′端 ,通过电击转化得到重组转化子。对重组毕赤酵母的Southernblotting分析证实植酸酶基因已整合到酵母基因组中 ,并确定了整合基因的拷贝数。Northernblotting分析证实植酸酶基因得到了正常转录。SDS PAGE分析和表达产物的研究表明 ,植酸酶得到了高效分泌表达 ,在 5L发酵罐中植酸酶蛋白表达量达到 2 5mg mL发酵液 ,酶活性 (发酵效价 )达到 7 5× 10 6 IU mL发酵液以上 ,大大高于目前报道的各种植酸酶基因工程菌株的发酵效价。     

A Thermostable phytase from <Emphasis Type="Italic">Neosartorya spinosa</Emphasis> BCC 41923 and its expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

A phytase gene was cloned from Neosartorya spinosa BCC 41923. The gene was 1,455 bp in size, and the mature protein contained a polypeptide of 439 amino acids. The deduced amino acid sequence contains the consensus motif (RHGXRXP) which is conserved among phytases and acid phosphatases. Five possible disulfide bonds and seven potential N-glycosylation sites have been predicted. The gene was expressed in Pichia pastoris KM71 as an extracellular enzyme. The purified enzyme had specific activity of 30.95 U/mg at 37°C and 38.62 U/mg at 42°C. Molecular weight of the deglycosylated recombinant phytase, determined by SDS-PAGE, was approximately 52 kDa. The optimum pH and temperature for activity were pH 5.5 and 50°C. The residual phytase activity remained over 80% of initial activity after the enzyme was stored in pH 3.0 to 7.0 for 1 h, and at 60% of initial activity after heating at 90°C for 20 min. The enzyme exhibited broad substrate specificity, with phytic acid as the most preferred substrate. Its K _m and V _max for sodium phytate were 1.39 mM and 434.78 U/mg, respectively. The enzyme was highly resistant to most metal ions tested, including Fe²⁺, Fe³⁺, and Al³⁺. When incubated with pepsin at a pepsin/phytase ratio of 0.02 (U/U) at 37°C for 2 h, 92% of its initial activity was retained. However, the enzyme was very sensitive to trypsin, as 5% of its initial activity was recovered after treating with trypsin at a trypsin/phytase ratio of 0.01 (U/U).