appA植酸酶基因与纳豆激酶基因的克隆与表达研究
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摘要
植酸是植物性饲料中磷的主要贮存形式，植酸酶是一种能将植酸水解并释放出无机磷的酶，它广泛存在于微生物与植物中。由于单胃动物如猪、鸡等的消化道中植酸酶活性很低，导致它们无法有效利用植酸磷。同时，植酸也是一种抗营养因子，因为它能螯合许多动物生长所必须的微量元素。
大肠杆菌来源的appA植酸酶是一种新型的植酸酶，与常规的黑曲霉植酸酶相比具有许多优点，如与猪、鸡等畜禽胃的pH环境更加适应，与底物植酸更强的亲合性以及对消化道蛋白酶更强的抵抗性。此外它还是目前所知的植酸酶中水解植酸能力最强的酶。
本实验从猪粪中筛选出一个高产植酸酶的E.coli菌株，根据Dassa等报道的序列设计一对引物通过PCR方法扩增了appA基因。测序结果显示appA基因阅读框架为1299bp，编码432个氨基酸，编码产物理论分子量为47.06kD，同时它也具有其它植酸酶与酸性磷酸酶的活性保守基序RHGxRxP。为了大量表达appA植酸酶，我们将appA基因分别克隆至原核表达载体pET-28a(+)和杆状病毒转移载体pVL-1393中，将其分别置于lac和polyhedrin启动子控制之下。用乳糖诱导使appA基因在大肠杆菌菌株BL21中得到了高效表达。另外，通过共转染和筛选纯化获得了携带appA基因的重组家蚕杆状病毒。感染五龄家蚕后appA植酸酶的表达量达到平均每毫升血淋巴7710U。通过SDS-PAGE发现大肠杆菌与家蚕的表达产物中分别有一条50kD与53kD的特征条带。两个表达系统中表达的重组appA植酸酶的最适温度与最适pH一致，分别为60℃与pH4.5。Ca~(2+)与Mn~(2+)都能提高酶的活性。由appA植酸酶在家蚕生物反应器中的超量表达以及appA植酸酶本身所具有的酶学特性推断，家蚕表达的appA植酸酶适合于作为新一代植酸酶添加剂应用于饲料工业与畜禽养殖业中。
纳豆激酶是一种能够在体内及体外直接分解交联纤维蛋白的新型溶血栓酶。本实验以B.subtilis(natto)基因组DNA为模板扩增了nk基因，测序结果显示与Nakamura报道的纳豆激酶基因高度同源，其氨基酸序列也含有丝氨酸蛋白酶的活性保守中心(serine 221，histidine 64，aspartic acid 32)。将nk基因克隆至转移载体pVL-1393中后用常规方法获得重组病毒，感染家蚕后纳豆激酶获得正确表
达。由于纳豆激酶是一种蛋白酶，我们首先检测并发现表达产物具有蛋白酶活性，
通过纤维蛋白平板检测表达产物具有溶圈活性，表明表达的纳豆激酶具有溶解血
栓的生物学功能。
Phytases are hydrolytic enzymes that initiate the release of phosphate from phytate, the major phosphorus (P) form in animal feeds of plant origin. Phytases are found naturally in microorganisms and plants. Monogastric animals, such as pigs and poultry, are not able to utilize phytate phosphorus efficiently, since they have only low levels of phytase activity in their digestive tracts, phytate also acts as an antinutritional agent in monogastric animals by chelating various microelements needed by the animal.
Escherichia coli phytase appA is an alternative enzyme with performance advantages over the conventional A. niger enzyme. The appA phytase displays several favorable characteristics: an acidic pH optimum close to the physiological pH range of the stomach of pigs and chickens, higher affinity to sodium phytate, greater resistance to pepsin than the commercially available A. niger PhyA, and higher catalytic efficiency for phytate than that of all other known phytases.
In this study, an Escherichia coli strain with high phytase activity was screened from pig excreta. Using primers designed according to the sequence originally described by Dassa, appA gene was amplified by PCR technique. DNA sequencing of the appA gene showed an open reading frame of 1299 bp. The deduced appA phytase composed of 432 amino acids (predicted molecular mass, 47.06 kD) also contained the reserved active-site motif RHGXRXP, which is shared by other phytases and acid phosphatases. To obtain large amounts of appA phytase, the appA gene was subcloned into the prokaryotic expression vector pET-28a(+) and baculovirus transfer vector pVL-1393 under the control of the lac and polyhedrin promoter, respectively. The appA phytase was overexpressed in E. coli strain BL21 induced by lactose. The recombinant baculovirus harboring the appA gene was obtained after co-transfection and several screening rounds. The newly molted 5th instar larva of silkworm Bombyx mori was infected with the recombinant virus. Usin
g a silkworm baculovirus expression vector system (BEVS), a large amount of appA phytase was obtained (up to 7710 per mL hemolymph). SDS-PAGE analysis revealed the molecular mass of
prokaryotic and eukaryotic derived appA phytase was approximately 50 kD and 53 kD, respectively. Both the two phytase had a optimal temperature and pH at 60 癈 and pH 4.5 except the baculovirus derived phytase seemed to be more thermostable. The enzymatic activity of the both prokaryotic and eukaryotic derived appA phytase were increased in the presence of Ca2+ and Mn2+ at a concentration of 1 mM. Due to the high expression efficiency and the enzymatic characteristics, BEVS derived appA phytase seemed to be a valuable candidate for the application of feed supplement.
Nattokinase (NK) is a new fibrinolytic enzyme which cleaves directly cross-linked fibrin in vitro/in vivo. In the present study, the nucleotide sequence of the nattokinase gene was amplified from the genomic DNA of B.subtilis (natto) by PCR. DNA sequence analysis showed that the cloned nk gene was highly homologous to the sequence reported by Nakamura. The deduced amino acid sequence also had the conserved sequences (serine 221, histidine 64, and aspartic acid 32) which was essential for the catalytic center of serine proteases.The nk gene was then cloned into the transfer vector pVL1393. Then Bm cell line was co-transfected with parental Bm-NPV DNA and the transfer plasmid pVL-nk, the recombinant virus was then obtained subsequently with routine procedure. Nattokinase was expressed in silkworm larva by injection with the recombinant virus. As a serine proteases, the expression product was detected with a relatively high protease activity. A similar result was also obtained from fibrin plate assay, it reveal
ed that the expressed NK was provided with fibrinolytic activity.
大肠杆菌来源的appA植酸酶是一种新型的植酸酶，与常规的黑曲霉植酸酶相比具有许多优点，如与猪、鸡等畜禽胃的pH环境更加适应，与底物植酸更强的亲合性以及对消化道蛋白酶更强的抵抗性。此外它还是目前所知的植酸酶中水解植酸能力最强的酶。
本实验从猪粪中筛选出一个高产植酸酶的E.coli菌株，根据Dassa等报道的序列设计一对引物通过PCR方法扩增了appA基因。测序结果显示appA基因阅读框架为1299bp，编码432个氨基酸，编码产物理论分子量为47.06kD，同时它也具有其它植酸酶与酸性磷酸酶的活性保守基序RHGxRxP。为了大量表达appA植酸酶，我们将appA基因分别克隆至原核表达载体pET-28a(+)和杆状病毒转移载体pVL-1393中，将其分别置于lac和polyhedrin启动子控制之下。用乳糖诱导使appA基因在大肠杆菌菌株BL21中得到了高效表达。另外，通过共转染和筛选纯化获得了携带appA基因的重组家蚕杆状病毒。感染五龄家蚕后appA植酸酶的表达量达到平均每毫升血淋巴7710U。通过SDS-PAGE发现大肠杆菌与家蚕的表达产物中分别有一条50kD与53kD的特征条带。两个表达系统中表达的重组appA植酸酶的最适温度与最适pH一致，分别为60℃与pH4.5。Ca~(2+)与Mn~(2+)都能提高酶的活性。由appA植酸酶在家蚕生物反应器中的超量表达以及appA植酸酶本身所具有的酶学特性推断，家蚕表达的appA植酸酶适合于作为新一代植酸酶添加剂应用于饲料工业与畜禽养殖业中。
纳豆激酶是一种能够在体内及体外直接分解交联纤维蛋白的新型溶血栓酶。本实验以B.subtilis(natto)基因组DNA为模板扩增了nk基因，测序结果显示与Nakamura报道的纳豆激酶基因高度同源，其氨基酸序列也含有丝氨酸蛋白酶的活性保守中心(serine 221，histidine 64，aspartic acid 32)。将nk基因克隆至转移载体pVL-1393中后用常规方法获得重组病毒，感染家蚕后纳豆激酶获得正确表
达。由于纳豆激酶是一种蛋白酶，我们首先检测并发现表达产物具有蛋白酶活性，
通过纤维蛋白平板检测表达产物具有溶圈活性，表明表达的纳豆激酶具有溶解血
栓的生物学功能。
Phytases are hydrolytic enzymes that initiate the release of phosphate from phytate, the major phosphorus (P) form in animal feeds of plant origin. Phytases are found naturally in microorganisms and plants. Monogastric animals, such as pigs and poultry, are not able to utilize phytate phosphorus efficiently, since they have only low levels of phytase activity in their digestive tracts, phytate also acts as an antinutritional agent in monogastric animals by chelating various microelements needed by the animal.
Escherichia coli phytase appA is an alternative enzyme with performance advantages over the conventional A. niger enzyme. The appA phytase displays several favorable characteristics: an acidic pH optimum close to the physiological pH range of the stomach of pigs and chickens, higher affinity to sodium phytate, greater resistance to pepsin than the commercially available A. niger PhyA, and higher catalytic efficiency for phytate than that of all other known phytases.
In this study, an Escherichia coli strain with high phytase activity was screened from pig excreta. Using primers designed according to the sequence originally described by Dassa, appA gene was amplified by PCR technique. DNA sequencing of the appA gene showed an open reading frame of 1299 bp. The deduced appA phytase composed of 432 amino acids (predicted molecular mass, 47.06 kD) also contained the reserved active-site motif RHGXRXP, which is shared by other phytases and acid phosphatases. To obtain large amounts of appA phytase, the appA gene was subcloned into the prokaryotic expression vector pET-28a(+) and baculovirus transfer vector pVL-1393 under the control of the lac and polyhedrin promoter, respectively. The appA phytase was overexpressed in E. coli strain BL21 induced by lactose. The recombinant baculovirus harboring the appA gene was obtained after co-transfection and several screening rounds. The newly molted 5th instar larva of silkworm Bombyx mori was infected with the recombinant virus. Usin
g a silkworm baculovirus expression vector system (BEVS), a large amount of appA phytase was obtained (up to 7710 per mL hemolymph). SDS-PAGE analysis revealed the molecular mass of
prokaryotic and eukaryotic derived appA phytase was approximately 50 kD and 53 kD, respectively. Both the two phytase had a optimal temperature and pH at 60 癈 and pH 4.5 except the baculovirus derived phytase seemed to be more thermostable. The enzymatic activity of the both prokaryotic and eukaryotic derived appA phytase were increased in the presence of Ca2+ and Mn2+ at a concentration of 1 mM. Due to the high expression efficiency and the enzymatic characteristics, BEVS derived appA phytase seemed to be a valuable candidate for the application of feed supplement.
Nattokinase (NK) is a new fibrinolytic enzyme which cleaves directly cross-linked fibrin in vitro/in vivo. In the present study, the nucleotide sequence of the nattokinase gene was amplified from the genomic DNA of B.subtilis (natto) by PCR. DNA sequence analysis showed that the cloned nk gene was highly homologous to the sequence reported by Nakamura. The deduced amino acid sequence also had the conserved sequences (serine 221, histidine 64, and aspartic acid 32) which was essential for the catalytic center of serine proteases.The nk gene was then cloned into the transfer vector pVL1393. Then Bm cell line was co-transfected with parental Bm-NPV DNA and the transfer plasmid pVL-nk, the recombinant virus was then obtained subsequently with routine procedure. Nattokinase was expressed in silkworm larva by injection with the recombinant virus. As a serine proteases, the expression product was detected with a relatively high protease activity. A similar result was also obtained from fibrin plate assay, it reveal
ed that the expressed NK was provided with fibrinolytic activity.
引文
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