请帮忙找一篇基因工程相关的外文论文再加上翻译！

Synthesis of optically pure ethyl (S)-4-chloro-3-hydroxybutanoate

by Escherichia coli transformant cells coexpressing

the carbonyl reductase and glucose dehydrogenase genes

由共表达碳酰还原酶和葡萄糖脱氢酶的大肠杆菌转化细胞合成

纯光学（S）-4-氯-3-羟基丁酸乙酯

Abstract The asymmetric reduction of ethyl 4-chloro-3-

oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate

((S)-CHBE) was investigated Escherichia coli cells expressing both the carbonyl reductase (S1) gene from Candida magnoliae and the glucose dehydrogenase (GDH) gene from Bacillus megaterium were used as the

catalyst In an organic-solvent-water two-phase system,(S)-CHBE formed in the organic phase amounted to 258 M (430 g/l), the molar yield being 85% E coli transformant cells coproducing S1 and GDH accumulated 125 M (208 g/l) (S)-CHBE in an aqueous monophase system by continuously feeding on COBE, which is unstable in an aqueous solution In this case, the calculated turnover of NADP+ (the oxidized form of nicotinamide adenine dinucleotide phosphate) to CHBE was 21,600 mol/mol The optical purity of the (S)-CHBE formed was 100% enantiomeric excess in both systems The aqueous system used for the reduction reaction involving E coli HB101 cells carrying a plasmid containing the S1 and GDH genes as a catalyst is simple Furthermore, the system does not require the addition of commercially available GDH or an organic solvent Therefore this system is highly advantageous for the practical synthesis of optically pure (S)-CHBE

本本篇文献研究了利用COBE不对称合成（S）-4-氯-3-羟基丁酸乙酯（CHBE）。大肠杆菌细胞作为催化剂同时表达了来自念珠菌属magnoliae的碳酰还原酶和来自巨大芽孢杆菌的葡萄糖脱氢酶基因。在水/有机溶剂两相体系中，(S)-CHBE在有机相中的浓度可以达到258M（430g/l），摩尔产率达到85%。大肠杆菌的副产物S1和GDH也达到了125M（208g/l），COBE在水相中不稳定，所以(S)-CHBE可以在水单相中不停的生成。在这种情况下，适当的从NADP+到CHBE的转变达到了21,600 mol/mol。所形成的CHBE的旋光度在这种体系中100%对映体过量。在水相中用携带含有S1和GDH基因质粒的E coli HB101作为催化剂不对称还原是比较简单的。并且，这种体系并不额外需要商业GDH或者有机溶剂。因此，这种体系对于实际合成纯光学活性的(S)-CHBE是非常方便的。

Optically active 4-chloro-3-hydroxybutanoic acid esters are useful chiral building blocks for the synthesis of pharmaceuticals The (R)-enantiomer is a precursor of L-carnitine (Zhou et al 1983), and (S)-enantiomer is an important starting material for hydroxymethylglutaryl- CoA (HMG-CoA) reductase inhibitors (Karanewsky et

al 1990) Many studies have described the microbial or enzymatic asymmetric reduction of 4-chloro-3-oxobutanoic acid esters (Aragozzini and Valenti 1992; Bare et al1991; Hallinan et al 1995; Patel et al 1992; Shimizu et al 1990; Wong et al 1985) based on the reduction by baker’s yeast (Zhou et al 1983)We have previously showed that Candida magnoliae AKU4643 cells reduced ethyl 4-chloro-3-oxobutanoate (COBE) to (S)-CHBE with an optical purity of 96% enantiomeric excess (ee) (Yasohara et al 1999) As this yeast has at least three different stereoselective reductases (Wada et al 1998, 1999a, b), the (S)-CHBE produced by this yeast was not optically pure From among these three enzymes, an NADPH-dependent carbonyl reductase, designated as S1, was purified and characterized in some detail (Wada et al 1998) We cloned and sequenced the gene encoding S1 and overexpressed it in Escherichia coli cells This E coli transformant reduced COBE to optically pure (S)-CHBE in the presence of glucose, NADP+, and commercially available glucose dehydrogenase (GDH) as a cofactor generator (Yasohara

et al 2000)

Here, we describe the construction of three E coli transformants coexpressing the S1 from C magnoliae and GDH from Bacillus megaterium genes and analyze the reduction of COBE catalyzed by these strains Previous reports on the enzymatic reduction of COBE to (R)-CHBE with an optical purity of 92% ee (Kataoka et al 1999; Shimizu et al 1990) recommended an organic- solvent two-phase system reaction for an enzymatic or microbial reduction, because the substrate (COBE) is unstable in an aqueous solvent and inactivates enzymes We examined the reduction of COBE to optically pure (S)-CHBE by E coli transformants in a water monophase system reaction and discuss the possible use of this type of reaction system in industrial applications。

具有旋光性的（S）-4-氯-3-羟基丁酸乙酯在药物制剂的合成中是重要的手性化合物。其右旋体是L-卡尼汀的前体，其左旋体是羟甲基戊二酰辅酶A还原酶抑制剂的起始材料。许多研究描述了以面包酵母为基础微生物或者酶的COBE的不对称还原。我们先前已经知道利用来自念珠菌属magnoliae AKU4643 细胞催化COBE生成光学纯度96%的CHBE。这种酵母至少有三种立体选择性的还原酶，这种酵母产生的CHBE并非纯光学的，在这三种酶之中，NADPH-依赖碳酰还原酶，我们克隆并测序编码S1的基因，并在大肠杆菌中过表达。大肠杆菌转化细胞在葡萄糖，NADP+和商业化的葡萄糖脱氢酶作为辅酶因子的启动子催化COBE生成纯光学的CHBE。

我们构建这三种大肠杆菌转化细胞共表达来自的S1和来自巨大芽孢杆菌的GDH，并分析COBE被这几种菌株催化还原的反应机理。先前的报道表明，利用酶催化还原COBE生成CHBE光学纯度可达92%，也提到了因为底物（COBE）在水相中不稳定，并且酶容易钝化，所以利用酶或者微生物在有机溶剂/水两相体系中催化反应。我们研究了在水单相体系中由COBE还原生成纯光学的CHBE，还讨论了这种反应体系在工业应用中可能的用途。

Materials and methods

Bacterial strain and plasmids

The E coli strains used in this study were JM109 and HB101Plasmid pGDA2, in which the GDH gene from B megaterium is inserted into pKK223-3, was kindly provided by Professor I Urabe, Osaka University (Makino et al 1989) Plasmids pSL301 and pTrc99A were purchased from Invitrogen (USA), and Amersham Pharmacia Biotech (UK), respectively Plasmids pUC19 and pSTV28 (Homma et al 1995; Takahashi et al 1995) were purchased from Takara Shuzo (Japan)

材料和方法

菌株和质粒

本次实验中使用的大肠杆菌是JM109 and HB101。来自B megaterium的GDH基因插入到Pkk233-3质粒中，而带有GDH基因片段的pGDA2质粒由到由大阪大学的urabe教授提供。质粒pSL301和 pTrc99A是由美国的Invitrogen公司和英国的公司分别购买的。质粒pUC19和pST28是由日本takara公司购买的。

The recombinant plasmid used in this study was constructed as follows (Fig 1): Plasmid pGDA2 was double-digested with EcoRI and PstI to isolate a DNA fragment of about 09 kilobase pairs (kb) including the GDH gene This fragment was inserted into the EcoRI-PstI site of plasmid pSL301 to construct plasmid pSLG Plasmid pSLG was double-digested with EcoRI and XhoI to isolate a DNA fragment of about 09 kb including the GDH gene

这次实验使用的重组质粒构建如下：质粒pGDA2 被EcoRI 和 PstI双酶切从而分离出一个大小约为09kb的包含有GDH基因的DNA片段。这个片段被插入到质粒Psl301的EcoRI-PstI酶切位点从而构建出质粒pSLG。质粒pSLG被EcoRI和XhoI

To construct plasmid pNTS1G, this 09-kb fragment was inserted into the EcoRI-SalI site of pNTS1, which was constructed to overproduce S1 as described previously (Yasohara et al 2000) To construct plasmid pNTGS1, plasmid pNTG was first generated Two synthetic primers (primer 1, TAGTCCATATGTATAAAGATTTAG,and primer 2 TCTGAGAATTCTTATCCGCGTCCT) were prepared for polymerase chain reaction (PCR) using pGDA2 as the template The PCR-generated fragment was double- digested with NdeI and EcoRI and then inserted into the NdeI EcoRI site of plasmid pUCNT, which was constructed from pUC19 and pTrc99A, as reported (Nanba et al 1999), to obtain pNTG To construct plasmid pNTGS1, two synthetic primers (primer 3, GCCGAATTCTAAGGAGGTTAATAATGGCTAAGAACTTCTCCAACG, and primer 4, GCGGTCGACTTAGGGAAGCGTGTAGCCACCGTC) were prepared using pUCHE, which contains the S1 gene as the template The PCR-generated fragment was double-digested with EcoRI and SalI and then inserted into the EcoRI-SalI site of pNTG to obtain pNTGS1 Plasmid pNTS1G, pNTGS1 or pNTG was transformed into E coli HB101

构建pNTS1是为了过表达前文所提到的S1，这个09kb大小的片段被插入到pNTS1的EcoRI-SalI酶切位点从而构建pNTS1G。为了构建质粒pNTGS1，首先需要构建pNTG。两个合成引物（引物1，TAGTCCATATGTATAAAGATTTAG和引物2，TCTGAGAATTCTTATCCGCGTCCT）和作为模板的pGDA2是PCR反应需要的。PCR得到的片段是由NdeI 和EcoRI双酶切和并插入到质粒pUCNT的NdeI EcoRI酶切位点来得到pNTG。根据报道，pUCNT是由pUC19和 pTrc99A构建而来。为了构建质粒pNTGS1，两个合成引物(引物 3, GCCGAATTCTAAGGAGGTTAATAATGGCTAAGAACTTCTCCAACG, and 引物 4, GCGGTCGACTTAGGGAAGCGTGTAGCCACCGTC)，包括了S1基因作为模板。Pcr产物片段被EcoRI和SalI双酶切然后被插入到pntg的EcoRI-SalI酶切位点得到pntg1质粒pNTS1G, pNTGS1或者 pNTG都是导入大肠杆菌HB101

Plasmid pGDA2 was double-digested with EcoRI and PstI to isolate a DNA fragment of about 09 kb including the GDH gene To construct plasmid pSTVG, this fragment was inserted into the EcoRI-PstI site of plasmid pSTV28 Plasmid pSTVG was transformed into E coli HB101

质粒pGDA2被EcoRI 和 PstI双酶切得到包含GDH基因的09kb大小的DNA片段。为了构建pSTVG质粒，这个片段被插入到pSTV28质粒的EcoRI-PstI的酶切位点。pSTVG质粒被导入到E coli HB101。

Medium and cultivation

The 2×YT medium comprised 16% Bacto-tryptone, 10% yeast

extract, and 05% NaCl, pH 70 E coli HB 101 carrying pNTS1,

pNTG, pNTS1G, or pNTGS1 was inoculated into a test tube containing

2 ml 2×YT medium supplemented with 01 mg/ml ampicillin,

followed by incubation at 37 °C for 15 h with reciprocal shaking

This preculture (05 ml) was transferred to a 500-ml shaking

flask containing 100 ml 2×YT medium The cells were cultivated

at 37 °C for 13 h with reciprocal shaking E coli HB101 carrying

pNTS1 and pSTVG was similarly cultivated in 2×YT medium

supplemented with 01 mg/ml ampicillin and 01 mg/ml chloramphenicol

培养基和培菌

2YT培养基 包含有16%细菌用胰蛋白胨，10%酵母提取物，05% NaCl，pH70

携带有pNTS1,pNTG, pNTS1G, 或 pNTGS1的大肠杆菌HB101被接种到有01mg/ml氨苄青霉素的2ml的2YT培养基，37°C摇床15小时。将05ml菌液接种到100ml2YT培养基的500ml烧瓶中。在37°C摇床培养13小时。携带有pNTS1 和 pSTVG质粒的大肠杆菌HB101在2YT培养基中培养方法相似，只是培养基中要加入01 mg/ml的氨苄青霉素和 01 mg/ml的氯霉素。

Preparation of cell-free extracts and the enzyme assay

Cells were harvested from 100 ml of culture broth by centrifugation, suspended in 50 ml of 100 mM potassium phosphate buffer (pH 65), and then disrupted by ultrasonication The cell debris was removed by centrifugation; the supernatant was recovered as the cell-free extract Carbonyl reductase S1 activity (COBE-reducing activity) was determined spectrophotometically as follows: The assay mixture consisted of 100 mM potassium phosphate buffer (pH 65), 01 mM NADPH, and 1 mM COBE The reactions were incubated at 30 °C and monitored for the decrease in absorbance at 340 nm The assay mixture for GDH activity consisted of 1 M Tris-HCl buffer (pH 80), 100 mM glucose, and 2 mM NADP+ The reactions were incubated at 25 °C and monitored for the increase in absorbance at 340 nm One unit of S1 or GDH was defined as the amount catalyzing the reduction of 1 μmol NADP+ or oxidation of 1 μmol NADPH per minute, respectively Protein concentrations were measured with a protein

assay kit containing Coomassie brilliant blue (Nacalai Tesque, Japan),

using bovine serum albumin as the standard (Bradford 1976)

无细胞抽提液和酶鉴定

将100ml培养液离心收获菌体，用50ml01mol/LpH为65的磷酸缓冲液悬浮，然后超声粉碎。细胞碎片通过离心可以去除，收集上层清液就是无细胞抽提物。碳酰还原酶S1的活性由分光光度计测量如下：测定的混合物包括：01mol/LpH65的磷酸二氢钾缓冲液，01mMNADPH和1mMCOBE。反应在30°C条件下反应，并且随时监测其在340nm处的吸光值。测GDH混合物包括：1M pH 80的Tris-HCl的缓冲液，100mM的葡萄糖，2mM的NADP+。反应在25°C下进行，监测其在340nm处的吸光值。一个单位S1或GDH被定义为每分钟催化还原1μmol NADP+或氧化1 μmol NADPH的量。蛋白质的测定通过含有考马斯亮蓝的蛋白质测定试剂利用牛血清白蛋白作为标准进行测定。

Study of enzyme stability

One milliliter of 100 mM potassium phosphate buffer (pH 65) containing the cell-free extracts of E coli HB101 carrying pNTS1 (S1: 20 U/ml) was mixed with an equal volume of each test organic solvent in a closed vessel After the mixture was shaken at 30 °C for 48 h, the remaining enzyme activities in an aqueous phase were assayed as described above The mixture, containing 100 mM potassium phosphate buffer (pH 65), S1 (20 U/ml), and various concentrations of CHBE, was incubated at 30 °C for 24 h in order to study the enzyme’s stability in the presence of CHBEThe remaining enzyme activities were assayed as described above

酶稳定性的研究

一毫升含有含有pNTS1质粒的E coli HB101的无细胞抽提液的100mM磷酸氢二钾缓冲液（pH65）与等体积的有机溶剂混合。混合物在30 °C震摇48小时后，水相中残留的酶活力即是上述的酶活力。

COBE reduction with E coli cells expressing the S1 gene and E coli cells expressing GDH genes in a two-phase system reaction

The reaction mixture comprised 15 ml culture broth of E coli HB101 carrying pNTG, 17 ml culture broth of E coli HB101 carrying pNTS1, 16 mg NADP+, 4 g glucose, 25 g COBE, 25 ml n-butyl acetate, and about 25 mg Triton X-100 The pH of the reaction mixture was controlled at 65 with 5 M sodium hydroxide At 2 h, 125 g COBE and 25 g glucose were added to the reaction mixture To compare the reaction by E coli transformant coexpressing the GDH and S1 genes, 30 ml culture broth of E coli

HB101 carrying pNTS1G was used instead of culture broth of E coli HB101 carrying pNTG and E coli HB101 carrying pNTS1 Other components and the procedure were the same as described above

表达S1基因和GDH基因的大肠杆菌细胞在两相反应体系中的还原反应

混合物包含有带有pNTG质粒的大肠杆菌HB101的菌液15ml，pNTS1质粒的大肠杆菌HB101的菌液17ml，16 mg NADP+，4 g葡萄糖，25g的COBE，25ml的n-butyl acetate丁酰醋酸盐和大约25mg的聚乙二醇辛基苯基醚Triton X-100。用5M的NaOH溶液将pH控制在65。在反应两小时后，加入125gCOBE和25g葡萄糖到该混合物中。比较大肠杆菌转化细胞共表达GDH和S1基因，携带有pNTS1G质粒的大肠杆菌HB10130ml菌液取代了携带有pNTG和pNTS1质粒的大肠杆菌HB101菌液。其他的成分和步骤和上述的方法相似。

COBE reduction to (S)-CHBE in a two-phase system reaction

The reaction mixture contained 50 ml of culture broth of an E coli HB101 transformant, 32 mg NADP+, 11 g glucose, 10 g COBE, 50 ml n-butyl acetate, and about 50 mg Triton X-100 The reaction mixture was stirred at 30 °C, and the pH was controlled at 65 with 5 M sodium hydroxide Five grams of COBE/55 g glucose and 10 g COBE/11 g glucose were added to the reaction mixture at 3 h and 7 h, respectively; 32 mg NADP+ was added at 26 h

COBE在两相系统中还原生成（S）-CHBE

反应混合物包含50ml E coli HB101转化细胞的培养液，32mgNADP+,11g

葡萄糖，10gCOBE，50ml丁酰醋酸，和大概50mg聚乙二醇辛基苯基醚Triton X-100

在30°C温度下将其混合均匀，并用5M的NaOH溶液将pH控制在65。在第3小时加入5gCOBE和55g葡萄糖或者在第7小时加入10gCOBE和11g葡萄糖，分别在第26小时加入32gNADP+。

COBE reduction to (S)-CHBE in an aqueous system reaction

The reaction mixture was made up of 50 ml of culture broth of an E coli HB101 transformant, 31 mg NADP+, 11 g glucose, and about 50 mg Triton X-100 The reaction mixture was stirred at 30 °C Fifteen grams of COBE was fed continuously by means of a micro-feeding machine at a rate of about 002 g/min for about 12 h The pH of the reaction mixture was controlled at 65 with 5 M sodium hydroxide The reaction mixture was extracted with 100 ml ethyl acetate The organic layer was dried over anhydrous sodium sulfate and then evaporated in vacuo

COBE在水相中还原成(S)-CHBE的反应

反应的体系是由50ml大肠杆菌HB101转化细胞的菌液，31mgNADP+，11g葡萄糖和大约50mg聚乙二醇辛基苯基醚Triton X-100。反应混合物在30°C15mg的COBE通过微量添加机器以002 g/min的速率连续12小时恒定的加入到体系中。用5M的NaOH溶液将pH控制在65。反应混合物用100ml乙酸乙酯萃取。有机层用无水硫酸钠吸干，并在真空中脱水。

Analysis

The organic layer was obtained on centrifugation of the reaction mixture and was assayed for CHBE and COBE by gas chromatography Optical purity of CHBE was analyzed by high-performance liquid chromatography (HPLC), as described previously (Yasohara et al 1999)

Enzymes and chemicals

Restriction enzymes and DNA polymerase were purchased from

Takara Shuzo (Japan) COBE (molecular weight: 16459) was purchased

from Tokyo Kasei Kogyo (Japan) Racemic CHBE (molecular

weight: 16660) was synthesized by reduction of COBE with

NaBH4 All other chemicals used were of analytical grade and

commercially available

分析

离心反应混合物得到的有机层通过气相色谱法测定其CHBE和COBE。COBE的光学纯度如前所述通过高效液相色谱法进行分析。

酶和化学试剂

限制性内切酶和DNA聚合酶由takara公司购得，COBE（分子量：16459）由东京Tokyo Kasei Kogyo公司购得，消旋体CHBE（分子量1666）通过COBE及NaBH4合成。所有其他化学试剂都是分析等级和商业化的试剂。

Construction of E coli transformants overproducing S1 and GDH

To express the carbonyl reductase S1 and GDH genes in the same E coli cells, four expression vectors were constructed (Fig 1) Plasmids pNTS1G and pNTGS1 contain the S1 gene from C magnoliae, the GDH gene from B megaterium, the lac promoter derived from pUC19, and the terminator derived from pTrc99A Plasmid pNTS1 contains the S1 gene, the lac promoter derived from pUC19, and the terminator derived from pTrc99A The enzyme activities in cell-free extracts of the E coli transformants are shown in Table 1 E coli HB101 cells carrying the vector plasmid pUCNT had no detectable S1 or GDH activity E coli HB101 carrying either pNTS1G or pNTGS1 showed S1 and GDH activity without isopropyl-β-D-thiogalactopyranoside (IPTG) induction The S1 activities of these two transformants were lower than the GDH activities To obtain a transformant whose S1 activity was equal to or greater than the level of GDH activity, we used a lower copy vector, pSTV28 (Homma et al 1995; Takahashi et al 1995), to express the GDH gene It may be possible to raise the S1 activity by lowering the GDH activity Plasmid pSTVG contains the GDH gene, the lac promoter, the chloramphenicol resistance gene, and the replicative origin derived from pACYC184 for compatibility with the plasmid pNTS1 In E coli HB101 carrying pNTS1 and pSTVG, the S1 activity was higher than the GDH activity, but this GDH

level may be too low to regenerate in a COBE reduction reaction as described below

过产生S1和GDH的大肠杆菌转化细胞的构建

为了在同一大肠杆菌细胞中表达碳酰还原酶S1和GDH基因，要构建四个表达型载体。质粒pNTS1G 和 pNTGS1包含有来自C magnoliae的S1基因，来自B megaterium的GDH基因，来自pUC19的LAC启动子，从pTrc99A的来的终止子，质粒pNTS1包含有S1基因，来自pUC19的LAC启动子，从pTrc99A的来的终止子。在大肠杆菌转化细胞的无细胞抽提物的酶活力如表一所示。携带有运输质粒pUCNT的大肠杆菌细胞无法检测到其S1和GDH活性。携带有pNTS1G 或 pNTGS1质粒在没有IPTG的诱导下有S1和GDH的活性。在这两个转化菌种中，S1的活力小于GDH的活力。为了得到S1活性等于或者大于GDH的大肠杆菌转化菌株，我们使用低拷贝的载体pSTV28，来表达GDH基因。它可能可以通过降低GDH的活性从而提高S1的活性。质粒pSTVG包含有GDH基因，lac启动子，和氯霉素抗性基因，以及与pNTS1具有相容性的从pACYC184得来的复制起始位点。在携带有pNTS1和pSTVG的大肠杆菌转化细胞中，S1的活性要高于GDH的活性，但是GDH的活性可能会太低而在COBE还原反应中不能再生。

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Logistics and Supply Chain Management（物流及供应链管理）　　学位类型：PGCertPGDipMSc

Management and Marketing（管理与营销）　　学位类型：MSc

Management and International Business（管理和国际商务）　　学位类型：MSc

Management and Finance（管理和财务）　　学位类型：MSc

Master of Business Administration（工商管理硕士）　　学位类型：MBA

Project Management（项目管理）　　学位类型：PGCertPGDipMSc

Quality Management（质量管理）　　学位类型：PGCertPGDipMSc

Real Estate Management（房地产管理）　　学位类型：PGCertPGDipMSc

Risk Management(Block-release or flexible distance learning)（风险管理（块释放或灵活的远程教育））　　学位类型：MSc 共有9个专业

International Business（国际商务）　　学位类型：MBA

Accountancy and Finance(Top-Up)- UK Campus（会计及财务（To- Up课程）-英国校区）　　学位类型：MSc

Accountancy and Finance(CIPathway)（会计及财务（CI分支））　　学位类型：MSc

Accountancy and Finance(Top-Up)- Sri Lanka Delivery（会计及财务（Top-Up课程）-斯里兰卡交换项目）　　学位类型：MSc

Accountancy and Finance(ACCA Pathway)（会计及金融（ACCA分支））　　学位类型：MSc

Business andResearch Degrees（商业固原度）　　学位类型：MPhilPhD

Doctorate in Business Administration(Part-Time)（工商管理博士学位 -（非全日制））　　学位类型：DBA

Finance（金融）　　学位类型：MBA

Management and Finance（管理和财务）　　学位类型：MSc 共有22个专业

Education Secondary Science with Physics（中学科学教育与物理）　　学位类型：PGCert

Education Secondary Science with Chemistry（中学科学教育与化学）　　学位类型：PGCert

Environmental Sustainability（环境的可持续性）　　学位类型：PGCertPGDipMSc

Environmental Sustainability(Design and Construction)（环境的可持续性（设计与施工））　　学位类型：PGCertPGDipMSc

Architecture(RIBA Part 2 Exemption)（建筑协会（RIBA第2条豁免））　　学位类型：MArch

Automotive Calibration and Control（汽车的校准和控制）　　学位类型：PGCertPGDipMSc

Automotive Engineering（汽车工程）　　学位类型：PGCertPGDipMSc

Business Computing（商业电脑）　　学位类型：PGCertPGDipMSc

Computing（计算）　　学位类型：PGCertPGDipMSc

Computer Science（计算机科学）　　学位类型：PGCertPGDipMSc

Construction Project Management（建设工程项目管理）　　学位类型：PGCertPGDipMSc

Data Networks and Security（数据网络和安全）

学位类型：PGCertPGDipMSc

Education Secondary Music（教育中小学音乐）　　学位类型：PGCert

Enterprise Systems Management（企业系统管理）

学位类型：PGCertPGDipMSc

Landscape Architecture（园林）　　学位类型：PGDip

Landscape Architecture（园林）　　学位类型：PGDipMA

Landscape Architecture（园林）　　学位类型：PGDip

Landscape Architecture（园林）　　学位类型：PGDipMA

Mechanical Engineering（机械工程）　　学位类型：PGCertPGDipMSc

Web Technology（网络技术）　　学位类型：PGDipPGDipMSc 共有73个专业

Screen Studies (Distance Learning)（屏幕研究（远程学习））　　学位类型：MA

Social Sciences（社会科学）　　学位类型：MPhilPhD

Future Media: Masterclass（未来媒体：大师）　　学位类型：MA

Future Media: Nano（未来媒体：纳米）　　学位类型：MA

Data Networks and Security + CCNP（数据网络和安全+ CCNP）　　学位类型：MA

Orchestral Performance(Strings)（管弦乐演奏（弦乐））　　学位类型：MMusPGDip

Gamer Camp: Biz, Video Game Enterprise and Production（玩家营：商务，视频游戏的企业和生产）　　学位类型：MSc

Online Journalism(Distance Learning)（新闻在线（远程教育））　　学位类型：MA

Future Media（媒体发展）　　学位类型：MAMSc

Instrumental Performance（器乐演奏）　　学位类型：MMusPGDip

Music Technology（音乐技术）　　学位类型：MMusPGDip

Jazz(Performance or Composition)（爵士（性能或成分））　　学位类型：MMusPGDip

Advanced Healthcare（高级医疗）　　学位类型：MSc

Composition（作曲）　　学位类型：MMusPGDip

Musicology（音乐学）　　学位类型：MMus

Social Media(Distance Learning)（社会化媒体（远程教育））　　学位类型：MA

Conducting(Choral or Orchestral)（指挥（合唱团或管弦乐队））　　学位类型：MMusPGDip

Creative Industries and Cultural Policy(Distance Learning)（创意产业和文化政策（远程教育））　　学位类型：MA

Making (and Saving) Money with Open Data（开放式数据的制作（和保存））　　学位类型：MScMSc

Acting（表演）　　学位类型：MAPGDip

Advanced Practice（高级实践）　　学位类型：PGDipMSc

Acting: The British Tradition（表演：英国传统）　　学位类型：MAPGDip

Arts and Education（艺术教育）　　学位类型：MA

Broadcast Journalism（广播新闻）　　学位类型：PGDip

Chartered Institute of Public Relations Advanced Certificate（学会公共关系高级证书）　　学位类型：PGCert

Creative Industries and Cultural Policy（创意产业与文化政策）　　学位类型：MA

Contemporary Curatorial Practice（当代策展实践）　　学位类型：MA

Criminology（犯罪）　　学位类型：PGCertPGDipMA

Design and Visualisation（设计和可视化）　　学位类型：PGCert

Education（教育）　　学位类型：PGCertPGDipMA

Education Secondary Drama（教育中学戏剧）　　学位类型：PGCert

Education Secondary Mathematics（中学教育数学）　　学位类型：PGCert

English Linguistics(Distance Learning)（英语语言学（远程教育））　　学位类型：PGCertPGDipMA

English（英语）　　学位类型：MPhilPhD

Environmental and Spatial Planning（环境和空间规划）　　学位类型：PGCertPGDipMA

Freelance Photography（自由摄影）　　学位类型：MA

Freelancing and Journalism Enterprise（自由职业者和企业新闻）　　学位类型：MA

Gamer Camp: Pro, Video Games Development（玩家营：临，视频游戏开发）

学位类型：MAMSc

IIA Diploma Internal Audit Practice（IIA文凭内部审计实务）

学位类型：MA

International Broadcast Journalism（国际广播新闻）　　学位类型：MA

International Education（国际教育）　　学位类型：PGCertPGDipMA

International Business Law（国际商法）　　学位类型：LLMPGDipPGDip

International Human Rights（国际人权）　　学位类型：LLMPGDip

Law（法律）　　学位类型：PGDip

Masters in Teaching and Learning(Part-Time)（教学和学习的主人（非全日制））　　学位类型：PGDip

Media and Creative Enterprise（媒体和创意企业）　　学位类型：MA

Music（音乐）　　学位类型：PGCert

Music Radio（音乐电台）　　学位类型：MA

Music Industries（音乐产业）　　学位类型：PGDip

Online Journalism（网络新闻）

学位类型：PGCert

Philosophy（哲学）　　学位类型：MPhilPhD

Performance and Pedagogy（性能和教育学）　　学位类型：MMus

Product Design（产品设计）　　学位类型：PGCertPGDipMA

Primary and Early Years Education（小学和早期教育）　　学位类型：PGCert

Post-Compulsory Education and Training（义务教育后的教育和培训）　　学位类型：PGCert

Psychology（心理学）　　学位类型：PGCertPGDip

Professional Voice Practice（专业的语音实践）　　学位类型：MAPGDip

Public Relations（公共关系）　　学位类型：MA

Radio and Audio Production（收音机和音频制作）　　学位类型：MA

Social Media（社会化媒体）　　学位类型：MA

Research:（研究：）　　学位类型：MPhilPhD

Television Production（电视制作）　　学位类型：PGCertPGDipMA

Visual Communication（视觉传达）　　学位类型：MA

Web Technology（网络技术）　　学位类型：PGCertPGDipMSc

Writing（写作）　　学位类型：MA 共有32个专业

Music Industries(Distance Learning)（音乐产业（远程教育））　　学位类型：MA

Music Technology（音乐技术）　　学位类型：MMusPGDip

Musicology（音乐学）　　学位类型：MMus

Vocal Performance（声乐表演）　　学位类型：MA

Arts and Education（艺术教育）　　学位类型：MA

Art, Health and Well-being（艺术，健康和福祉）　　学位类型：MA

Art and Design（艺术与设计）　　学位类型：MA

Arts Practice and Education（艺术教育与实践）　　学位类型：MA

Arts and Project Management（艺术及项目管理）　　学位类型：MA

Arts and Education（艺术教育）　　学位类型：MA

Digital Arts in Performance（高性能数字艺术）　　学位类型：MA

Education Secondary Art and Design（中学艺术与设计）　　学位类型：PGCert

Education Secondary Instrumental Music（中学器乐）　　学位类型：PGCert

Fashion Styling（时尚造型）　　学位类型：MA

Fashion Promotion（时尚推广）　　学位类型：MA

Fine Art（美术）　　学位类型：MA

Gamer Camp: Pro, Video Games Development（玩家营：临，视频游戏开发）　　学位类型：MAMSc

History of Art and Design（艺术与设计史）　　学位类型：MA

Interior Design（室内设计）　　学位类型：MA

Jewellery, Silversmithing and Related Products（珠宝首饰，银器及相关产品）　　学位类型：MA

Music（音乐）　　学位类型：PGCert

Music Radio（音乐电台）　　学位类型：MA

Music Industries（音乐产业）　　学位类型：MA

Music(Professional Performance)（音乐（专业表现））　　学位类型：PGDip

Music(Specialist Performance)（音乐（专科性能））　　学位类型：PGCert

Product Design（产品设计）　　学位类型：PGCertPGDipMA

Queer Studies in Arts and Culture（艺术和文化的酷儿研究）　　学位类型：MA 共有11个专业

Perioperative Specialist Practice（围手术期专科执业）　　学位类型：PGDip

Health and Social Care(Leadership)（健康与社会关怀（领导））　　学位类型：PGDip

Forensic Psychology（法医心理学）　　学位类型：PGCertPGDipMSc

健康与社会关怀　　学位类型：PGCertPGDipMSc

综合心理治疗　　学位类型：PGCertPGCertPgDipMSc

医学超声　　学位类型：PGCertPGDipMSc

精神健康（高等专科社会工作）纳入AMHP　　学位类型：PGCertPGDipMSc

疼痛管理　　学位类型：PGCertPGDipMSc

公共卫生　　学位类型：PGDipMSc

造影　　学位类型：PGCertPGDipMSc

电信通过远程教育　　学位类型：PGCertPGDipMSc 1个专业

中学设计与科技：食品和纺织品　　学位类型：PGCert

对，确实是declare要删除，你的as已经代表要申明一个变量了

create or replace procedure cpgd_insert(sj in varchar2) as

sl number(10);

begin

select count(1)

into sl

from dba_views

where view_name = 'jbftjl'

and OWNER = 'WINE';

if sl = 1 then

insert into dg_daybb

(epname, gangzhong, ph, guige, rbcp, ybcp, riqi)

select '纵剪机' as epname,

gangzhong,

ph,

guige,

rbcp,

ybcp,

to_date(sj, 'yyyy-mm-dd') as riqi

from jbftjl;

end if;

end;