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1.
从丝状真菌中筛选到一株产α-半乳糖苷酶的菌株F63,对该菌株进行了形态观察和18SrDNA序列分析,该菌株属于青霉属。采用硫酸铵沉淀、阴离子交换层析和分子筛层析等方法分离纯化了该菌株的一种α-半乳糖苷酶。经过聚丙烯酰胺凝胶电泳,此酶蛋白的分子量约为82kDa。该α-半乳糖苷酶反应的最适pH为5.0,最适温度为45℃。此α-半乳糖苷酶的热稳定性在40℃以下,pH稳定性为pH5.0-6.0。与已报道的α-半乳糖苷酶的活性都受到Ag 的强烈抑制不同的是,该α-半乳糖苷酶受Ag 的抑制作用不显著。以pNPG为底物的Km值为1.4mmol/L和Vmax=1.556mmol/L.min-1.mg-1。该酶可以有效降解蜜二糖、棉子糖和水苏糖,但不能降解末端含α-半乳糖苷键的多糖。通过利用质谱技术对纯化的α-半乳糖苷酶进行鉴定以及内肽的N端测序证明该蛋白为一种新的α-半乳糖苷酶。  相似文献   

2.
王剑锋  王璋  李江  饶军 《菌物学报》2012,31(2):251-257
根霉Rhizopus sp.A01发酵豆渣产α-半乳糖苷酶,粗酶液依次经过三相分离、Sephadex G-100凝胶过滤获得了电泳纯的α-半乳糖苷酶,纯化了6.7倍,总酶活回收率达到46%;凝胶过滤和SDS-PAGE显示该酶为相对分子质量为87.6kDa的单体蛋白。该酶水解对硝基苯-α-D-吡喃半乳糖苷的最适pH值为5.0,最适温度为55℃,表观Km、kcat/Km分别为2.56mmol/L、47,400L/mol·s;能微弱水解蜜二糖和棉子糖,水解蜜二糖的速率是水解棉子糖速率的3.4倍;水解活性受多种金属离子的明显抑制,其中Ca2+、Cu2+、Fe3+、Hg+、Mn2+等几乎完全抑制酶活性。该酶活性在pH4.5-9.0保持稳定,在55℃时保温120min,酶活残留48%。  相似文献   

3.
把大肠杆菌β一半乳糖苷酶基因克隆到带有酵母半乳糖可诱导启动子GALl的穿梭表达质粒pYES2中,并把得到的重组质粒分别转化到两种不同遗传性状的宿主菌中,其中一株菌为蛋白酶活性缺失90%以上的pep4-3突变菌株。通过比较两株重组菌产生的β一半乳糖苷酶活性水平发现在所述实验条件下,蛋白酶缺失突变菌株中产生的β一半乳糖苷酶活水平不仅均要高于另一对照菌株,并且pep4-3突变菌株表现出受葡萄糖阻遏的严紧程度高及对诱导反应迅速等特点。此外,带有重组质粒的pep4-3突变菌株在葡萄糖阻遏培养基中最大生长量和重组对照菌株基本相同,但β一半乳糖苷酶在pep4-3突变菌株中的表达对细胞生长的影响明显小于对照菌株。  相似文献   

4.
根据重组工程原理,建立了一种用于构建重组质粒的 “neo/E”(抗生素/单酶切位点)选择与反选择新方法。首先采用 PCR方法扩增出线性打靶分子:然后进行两步体内同源重组,(1)neo/E基因敲入,重组子呈现neo抗性表型;(2)目的基因替换neo/E基因。用限制酶E消化时,发生第二步重组的DNA分子不能被消化,能够转化大肠杆菌受体菌DH5α。应用该方法构建了重组质粒pGL3-Basic PC1900T。PCR及测序鉴定证明:外源片段重组率为20%,所建立的重组工程选择与反选择新技术为质粒构建提供了新的解决方案。  相似文献   

5.
目的:将gfp基因克隆到pGEM3Z-f( )载体。方法:将PCR扩增得到的gfp基因插入到pGEM3Z-f( )质粒的SmaⅠ位点,构建重组质粒转化大肠杆菌DH5α。结果:含重组质粒的大肠杆菌在自然光下呈现黄绿色,而在涂X-gal的培养基上它不仅发出黄绿色荧光,而且还出现蓝色。结论:gfp基因的插入没有引起载体上lacZ基因的失活,而且形成的gfp-lacZ融合基因在大肠杆菌得到了表达。表达产物不仅具有GFP活性,而且保持了β-半乳糖苷酶的生物学活性。  相似文献   

6.
依据同源重组的原理将来源于粟酒裂殖酵母的α-半乳糖苷酶基因m el整合到工业酿酒酵母染色体的甘油合成途径关键酶基因GPD1中,通过G418抗性筛选得到重组子。实验数据表明,重组子S.cerevisiaeMG1利用蜜二糖的能力显著提高,产甘油能力下降。引入外源基因后酵母性状与亲代相比没有显著差异,但生长时具自絮凝能力。MG1分别以玉米粉、小麦淀粉为原料进行浓醪酒精发酵,与亲代工业酿酒酵母比较,发酵液乙醇浓度得到提高,甘油含量降低,蜜二糖消耗殆尽。  相似文献   

7.
蜂毒溶血肽前体蛋白cDNA的克隆及其融合蛋白的表达   总被引:8,自引:0,他引:8  
从蜜蜂毒腺中提取总RNA ,通过RT PCR扩增得到了蜂毒溶血肽前体蛋白的cDNA ,将扩增产物克隆到 pT7Blu T载体上 ,再进一步将插入片段酶切并连接到 pUC1 1 8载体上 ,构建了重组质粒pUMP。DNA序列分析结果表明 ,克隆得到的cDNA序列与所发表序列完全相同 ,且与 β 半乳糖苷酶部分序列构成正确的读码框。含重组质粒 pUMP的大肠杆菌DH5α表达了与β 半乳糖苷酶部分序列融合的蜂毒溶血肽前体蛋白  相似文献   

8.
从嗜热子囊菌(Thermoascus crustaceus)JCM12803克隆α-半乳糖苷酶基因,并对其酶学性质进行系统的研究,旨为获得工业应用性质优良的α-半乳糖苷酶。通过RT-PCR方法从T.crustaceus JCM12803中克隆α-半乳糖苷酶基因序列,并对其酶学性质进行了系统的分析。结果显示,tcgal27A属于糖苷水解酶27家族,基因全长1 918 bp,含有4个内含子,c DNA全长1 419 bp,编码472个氨基酸,预测tcgal27A N端含有24个氨基酸为其可能的信号肽。将TCGal27A在毕赤酵母中成功地进行了表达,所获得的重组TCGal27A具有高比活(336.5 U/mg)及宽泛的底物特异性,对蜜二糖(14.4 U/mg)、棉子糖(9.1 U/mg)、角豆胶(3.6 U/mg)、魔芋粉(1.6 U/mg)、瓜尔豆胶(1.3 U/mg)、水苏糖(0.7 U/mg)都有着不同程度的降解作用。以pNPG为底物时的Km值为1.6mol/m L,Vmax为536.8μmo L/(min·mg)。TCGal27A的最适作用pH为4.5,最适温度为65℃,50℃处理1 h之后酶活力还能保持86.0%。这些结果表明耐高温TCGal27A性质优良,可添加到饲料中,在消化和提高豆粕蛋白能量利用率方面有良好的潜在应用价值。  相似文献   

9.
用PCR方法扩增短芽孢杆菌α—乙酰乳酸脱羧酶基因,引入原核表达载体pBV220中,得到重组质粒pALDl。pALDl中的ALDC基因在大肠杆菌中高效表达,每毫升发酵液产酶80单位以下,比原始菌株提高200余倍。SDS-PAGE蛋白质分析表明,大肠杆菌DH5α(pALDl)表达的ALDC占细胞总蛋白量的40%以上。研究了重组质粒稳定性,大肠杆菌DH5α(PALDl)和HB101(pALDl)分别在无选择压力下30℃连续培养50代以上,41℃诱导不同时间,DH5α(pALDl)在热诱导5h后,未发现质粒不稳定性,HB101(pALDl)在热诱导3h后,质粒基本稳定,但热诱导5h后,丢失质粒的细胞约占70%。  相似文献   

10.
节杆菌BT801基因文库构建及其乙内酰脲酶基因分离与表达   总被引:7,自引:0,他引:7  
L-乙内酰脲酶产生菌节杆菌 BT801的染色体DNA经Sau3A I 部分酶切后分离30kb左右的片段,与经HpaI和PstI酶切的黏粒载体Pkc505进行连接,将连接产物用包装蛋白包装,转染大肠杆菌DH5α得到10 000多个转化子,构建成节杆菌BT801的基因组文库。通过薄层层析等方法筛选得到了1个阳性克隆,通过亚克隆得到了乙内酰脲酶的完整基因,该基因能分别利用自身的启动子和T5启动子在大肠杆菌中进行表达产生有活性的蛋白。  相似文献   

11.
以克雷伯氏菌基因组DNA为模板,扩增得到编码甘油脱氢酶(GDH)的基因dhaD,将其克隆到大肠杆菌表达载体pET-28a(+)上,在E.coliBL21(DE3)中诱导表达,利用表达载体pET-28a(+)上的6·His-Tag标记选用Ni柱亲和层析法纯化表达具有活性的甘油脱氢酶(GDH),纯化后比酶活达到156U/mg,纯化倍数达4.6倍,回收率为67.4%。并初步研究了该酶的酶学性质,酶反应的最适pH为11.0,在pH7.0~12.0范围内稳定;酶反应的最适温度为30℃,稳定范围为25~45℃; 酶动力学参数以甘油为底物的Km为0.54 mmol/L, Vmax为0.49 μmol/(mL·min)。  相似文献   

12.
经过PCR克隆得到硫酸乙酰肝素3-O硫酸基转移酶5(3-OST-5)的基因,将其与大肠杆菌表达载体pET-15b连接后,在大肠杆菌BL21(DE3)中诱导表达,使用镍亲和层析柱纯化得到具有活性的3-OST-5。经测定纯化后的3-OST-5比活达到0.58 U/mg,是纯化前的5.27倍,回收率达80.4%。在此基础上,研究了该酶的酶学性质,酶反应的最适温度为35℃,稳定范围为20-40℃;最适pH为7.0,在pH7.0-9.0范围内稳定。在反应液中加入终浓度为1 mmol/L的K+、Ca2+、Ba2+对酶促反应有一定的促进作用。  相似文献   

13.
右旋糖酐蔗糖酶工程菌株的构建及其培养条件的研究   总被引:3,自引:1,他引:2  
[目的]右旋糖酐蔗糖酶是一种以蔗糖为底物,催化转移D-葡萄糖基生成α-葡聚糖或低聚糖的葡萄糖基转移酶.[方法]利用PCR扩增技术,将已获得的右旋糖酐蔗糖酶基因dexYG亚克隆到表达载体PET28a( )上,转化E.coli BL21(DE3),经过卡那霉素抗性筛选和酶切验证后,得到右旋糖酐蔗糖酶工程菌株BL21(DE3)/pET28-dexYG.[结果]经IPTG诱导该基因在E.coli BL21(DE3)中能有效表达,在诱导过程中菌体生长受到抑制.通过对培养时间、IPTG浓度、培养温度、菌浓(OD600)和pH值等产酶因素的优化考察,得到最佳培养条件为:培养时间5h、IPTG浓度0.5mmol/L、25℃、OD600值1.0和pH6.0.酶活力由最初的5.39U/mL提高到35.62U/mL,其中pH值对产酶活力影响最大,在pH6.0时的最高产酶活力是LB原始pH条件下最高酶活的3.5倍,并且pH值也是导致在诱导后期酶活迅速下降的主要原因之一.[结论]酶的表达和酶活的研究结果表明,构建的工程菌株能够异源高效表达右旋糖酐蔗糖酶,并且表现出较高的酶活力.  相似文献   

14.
高温α-淀粉酶基因突变体在大肠杆菌、毕赤酵母中的表达   总被引:1,自引:0,他引:1  
对地衣芽孢杆菌(Bacillus licheniformis)高温α-淀粉酶(amyE)基因进行改造获得的基因突变体(amyEM),通过PCR扩增,将此基因分别克隆至大肠杆菌表达载体pBV220和毕赤酵母表达载体pPIC9K上,并分别转化大肠杆菌DH5α和毕赤酵母GS115感受态细胞,获得重组大肠杆菌和重组毕赤酵母。通过表达产物的酶活性检测和SDS-PAGE分析,证明突变α-淀粉酶(AmyEM)在大肠杆菌、毕赤酵母中获得有效表达。对重组大肠杆菌产生的α-淀粉酶的粗酶性质分析表明,此酶分子量约为55kDa。其最适反应温度为80℃~90℃,与野生型基因相比,其最适pH均为6.0,但不同的是突变体在pH 5.0~5.5时表现出较高的酶活力;在毕赤酵母细胞的表达产物可分泌至胞外。由于酵母可对蛋白进行糖基化,酶分子量增加到60kDa,最适pH也改变为5.5。此高温α-淀粉酶突变体所具有的在微酸性环境具有较高酶活力的性质,具有重要的潜在工业应用价值。  相似文献   

15.
The DNA fragment encoding Kluyvera citrophila penicillin G acylase (KcPGA) was amplified and cloned into the vector pET28b to obtain a C-terminus His-tagged fusion expression plasmid. The fusion protein KcPGA was successfully overexpressed in Escherichia coli BL21(DE3). The optimal induction concentration of isopropylthio-beta-D-galactoside (IPTG) was found to be 5 microM. The fusion protein was purified in a single step by Ni-IDA affinity chromatograph to a specific activity of 35.3U/mg protein with a final yield of 89% representing a 23-fold purification. The data presented here suggest that the purified fusion protein is stable with respect to pH and temperature. The optimal pH and temperature of recombinant KcPGA are 8.5 and 55 degrees C, respectively. The Km and Vmax are 17.6 microM and 23.8 U/mg, respectively. Therefore, the high yield and high specific activity of recombinant KcPGA produced in E. coli, together with other kinetic parameters, represent an excellent basis for further development of recombinant KcPGA as an immobilized biocatalyst for industrial applications.  相似文献   

16.
High-level production of recombinant glucose isomerase (rGI) is desirable for lactulose synthesis. In this study, the xylA gene encoding glucose isomerase from Actinoplanes missouriensis CICIM B0118(A) was cloned and expressed in E. coli BL21(DE3), and high-level production was performed by optimization of the medium composition. rGI was purified from a recombinant E. coli BL21(DE3) and characterized. The optimum pH value of the purified enzyme was 8.0 and it was relatively stable within the pH range of 7.0-9.0. Its optimum temperature was around 85 degrees C, and it exhibited good thermostability when the temperature was lower than 90 degrees C. The maximum enzyme activity required the presence of both Co2+ and Mg2+, at the concentrations of 200 microM and 8 mM, respectively. With high-level expression and the simple one-step chromatographic purification of the His-tagged recombinant enzyme, this GI could be used in industrial production of lactulose as a potential economic tool.  相似文献   

17.
The dextransucrase gene dsrX from Leuconostoc mesenteroides CGMCC 1.544 was cloned into the vector pET-28a(+) and expressed as a N-terminal His(6)-tag fusion protein of 167.57 kDa in Escherichia coli BL21(DE3). DsrX with the high volumetric activity of 8.8 U ml(-1) culture and the specific activity of 97.37 U mg(-1) crude enzyme extracts was measured in the optimized recombinant expression system. The resultant expression level of the fusion protein amounted to 24.5% of the total cell proteins. The results of affinity chromatography and western blotting indicated that the three sensitive sites of proteolysis existed in the N-terminal catalytic domain of DsrX. Both the recombinant and native enzyme activity were slightly activated by 1 mmol l(-1) Mn(2+) and strongly inhibited by 1 mmol l(-1) Cu(2+) or Al(3+), and their optimum pH values were 5.4. The optimum temperature of the recombinant enzyme for dextran synthesis was 30 degrees C, which was 10 degrees C less than that of the native one. The transglucosylation products of two enzymes were studied by using thin layer chromatography and high-performance anion exchange chromatography. It could be concluded that the better sample-pretreatment temperature in SDS-PAGE was 37 degrees C, which significantly improved the detection of thermal instable enzyme than that of 100 degrees C.  相似文献   

18.
莱鲍迪苷D(Rebaudioside D,RD)是一种稀有具有高甜度的甜菊糖苷类化合物。本文实现了重组大肠杆菌全细胞催化莱鲍迪苷A(Rebaudioside A,RA)合成RD。以水稻c DNA为模板,扩增得到葡萄糖基转移酶基因eugt11,构建了重组菌株E.coli BL21(p ETDuet-eugt11),并成功表达了重组蛋白6His-EUGT11。通过Ni柱亲和层析纯化并在体外酶催化反应表征了其催化活性。将重组菌BL21(p ETDuet-eugt11)应用于催化合成RD研究。探讨了反应体系pH、温度、柠檬酸钠浓度、菌体密度、二价金属离子、二甲苯体积分数、UDPG添加浓度对反应效率的影响。单因素考察结果显示,在菌体密度0.16 g湿细胞/m L反应液,底物RA浓度为1.0 mmol/L,pH 8.0,60 mmol/L柠檬酸钠,1%二甲苯,0.1 mmol/L Zn Cl2,12.0 mmol/L UDPG,反应温度42℃,反应时间24 h的条件下,RD产量为123.6 mg/L(约0.1 mmol/L)。  相似文献   

19.
The gene that encodes thermostable glucose isomerase in Clostridium thermosulfurogenes was cloned by complementation of glucose isomerase activity in a xylA mutant of Escherichia coli. A new assay method for thermostable glucose isomerase activity on agar plates, using a top agar mixture containing fructose, glucose oxidase, peroxidase, and benzidine, was developed. One positive clone, carrying plasmid pCGI38, was isolated from a cosmid library of C. thermosulfurogenes DNA. The plasmid was further subcloned into a Bacillus cloning vector, pTB523, to generate shuttle plasmid pMLG1, which is able to replicate in both E. coli and Bacillus subtilis. Expression of the thermostable glucose isomerase gene in both species was constitutive, whereas synthesis of the enzyme in C. thermosulfurogenes was inducible by D-xylose. B. subtilis and E. coli produced higher levels of thermostable glucose isomerase (1.54 and 0.46 U/mg of protein, respectively) than did C. thermosulfurogenes (0.29 U/mg of protein). The glucose isomerases synthesized in E. coli and B. subtilis were purified to homogeneity and displayed properties (subunit Mr, 50,000; tetrameric molecular structure; thermostability; metal ion requirement; and apparent temperature and pH optima) identical to those of the native enzyme purified from C. thermosulfurogenes. Simple heat treatment of crude extracts from E. coli and B. subtilis cells carrying the recombinant plasmid at 85 degrees C for 15 min generated 80% pure glucose isomerase. The maximum conversion yield of glucose (35%, wt/wt) to fructose with the thermostable glucose isomerase (10.8 U/g of dry substrate) was 52% at pH 7.0 and 70 degrees C.  相似文献   

20.
The gene that encodes thermostable glucose isomerase in Clostridium thermosulfurogenes was cloned by complementation of glucose isomerase activity in a xylA mutant of Escherichia coli. A new assay method for thermostable glucose isomerase activity on agar plates, using a top agar mixture containing fructose, glucose oxidase, peroxidase, and benzidine, was developed. One positive clone, carrying plasmid pCGI38, was isolated from a cosmid library of C. thermosulfurogenes DNA. The plasmid was further subcloned into a Bacillus cloning vector, pTB523, to generate shuttle plasmid pMLG1, which is able to replicate in both E. coli and Bacillus subtilis. Expression of the thermostable glucose isomerase gene in both species was constitutive, whereas synthesis of the enzyme in C. thermosulfurogenes was inducible by D-xylose. B. subtilis and E. coli produced higher levels of thermostable glucose isomerase (1.54 and 0.46 U/mg of protein, respectively) than did C. thermosulfurogenes (0.29 U/mg of protein). The glucose isomerases synthesized in E. coli and B. subtilis were purified to homogeneity and displayed properties (subunit Mr, 50,000; tetrameric molecular structure; thermostability; metal ion requirement; and apparent temperature and pH optima) identical to those of the native enzyme purified from C. thermosulfurogenes. Simple heat treatment of crude extracts from E. coli and B. subtilis cells carrying the recombinant plasmid at 85 degrees C for 15 min generated 80% pure glucose isomerase. The maximum conversion yield of glucose (35%, wt/wt) to fructose with the thermostable glucose isomerase (10.8 U/g of dry substrate) was 52% at pH 7.0 and 70 degrees C.  相似文献   

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