Acinetobacter sp. Y1的氨氮去除性能及其关键酶活性

【目的】研究Acinetobacter sp.Y1的氨氮(NH_4~+-N)去除性能及其关键酶的提取与酶活性。【方法】以柠檬酸钠为碳源,硫酸铵为氮源,研究菌株Y1的NH_4~+-N去除性能;采用正交实验优化超声波破碎法提取粗酶的条件,SDS-PAGE分析比较渗透压休克法和超声波破碎法获得的粗酶;检测关键酶——羟胺氧化还原酶(HAO)、亚硝酸盐还原酶(NIR)、硝酸盐还原酶(NAR)的酶活性。【结果】24 h内菌株Y1的菌密度(OD600)可达1.280,对NH_4~+-N、总氮(TN)和COD的降解率分别达到98%、94%和92%,硝化过程中羟胺、亚硝酸盐氮、硝酸盐氮不积累,反硝化产生N2;超声波破碎法提取粗酶的最佳工作条件为:破碎功率50 W,工作与间歇时间分别为4 s和7 s,OD600为1.250,总工作时间20 min,关键酶HAO、NIR和NAR的比活力分别为0.011、0.002和0.018 U/mg;渗透压休克法得到的HAO比活力是0.067 U/mg。【结论】Acinetobacter sp.Y1能同时高效去除NH_4~+-N、TN和COD。优化超声波破碎法提取粗酶的条件,检测到HAO、NIR和NAR的酶活性,且渗透压休克法比超声波破碎法更适合用来提取HAO。     

不同细胞破碎方法对无细胞蛋白表达系统细胞抽提物活性的影响

无细胞蛋白表达系统由于能够有效表达膜蛋白等有毒性蛋白,因此近二十年受到了关注,其蛋白表达产率有了显著的提高。细胞抽提物活性的高低是无细胞蛋白表达系统高效运行的关键,若找到简单易行的活性评估方法,将大大降低成本及时间。葡萄糖-6-磷酸脱氢酶 (glucose-6-phosphate dehydrogenase, G-6-PDH)是糖代谢的戊糖磷酸途径中的关键调控酶,该酶可以被用来评估细胞抽提物的活性。以G-6-PDH的活性为指标对无细胞蛋白表达系统中的抽提物活性进行评价,并利用G-6-PDH活性评价体系对机械破碎、高压破碎以及超声破碎三种破碎方法进行了比较,得出了三种破碎方法的最佳破碎条件。机械破碎最佳破碎条件是5 000r/min, 用直径0.1 mm玻璃珠,破碎6次;高压破碎的最佳破碎压力为1 300bar;超声破碎最佳破碎条件是功率强度为总功率的60%,破碎30次。酶活性测定结果显示机械破碎和超声破碎得到的抽提物活性比高压破碎得到的抽提物活性略高。     

超声波萃取烟叶中3-磷酸甘油脱氢酶的研究

将超声波萃取手段引入到烟叶中3-磷酸甘油脱氢酶提取方法中,并通过正交实验确定了超声波萃取烟叶中3-磷酸甘油脱氢酶(GPDH)的最佳条件为：超声波破碎功率为75w,超声波破碎间歇时间为2s,超声波破碎时间为3s,料液比为1:15g/mL;在此最佳条件下测得烟叶中3-磷酸甘油脱氢酶为0.3937U/mg蛋白,均比其他提取手段的酶活要高。     

克雷伯杆菌产1,3-丙二醇关键酶甘油脱水酶的酶活分析方法研究

有氧条件下,建立了克雷伯杆菌破碎方法及其生产1,3-丙二醇代谢途径中关键酶甘油脱水酶的酶活测定方法。甘油脱水酶酶活测定时在超声时间25min,功率为300w的条件下最适破碎频率为破碎时间1s,停息时间4s;甘油脱水酶酶活测定所用磷酸盐缓冲液的最适浓度为0.045mol/L,最适pH值7.2。甘油脱水酶酶活测定反应的最佳温度为37℃,甘油脱水酶酶活测定反应液的最佳吸收波长为290nm。     

Agarivorans albus RZW1-1产琼胶酶的发酵条件优化

为了提高菌株Agarivorans albus RZW1-1的产琼胶酶能力,本研究通过单因素实验探究碳源、氮源、海盐浓度、琼脂浓度、初始pH、装液量、培养时间、培养温度等因素对产酶的影响,得到该菌株的最佳发酵条件。在单因素实验的基础之上,运用正交试验的方法,得到菌株RZW1-1产酶最高的培养基组分。综合单因素实验和正交设计,最终确定了最佳产酶条件为:葡萄糖0.2 g/L、酵母粉9 g/L、海盐浓度4%、琼脂粉浓度0.1%,在装液量25 m L (250 m L三角瓶)、初始pH7.0、28℃、130 r/min条件下培养48 h酶活力达到最高为23.020 U/mL,较基础培养基提高了2.01倍。     

正交试验法确定测定意大利蜜蜂头部乙酰胆碱酯酶反应的最佳条件

用正交试验法研究了酶浓度、底物浓度、反应体系的pH值、反应温度和反应时间5个因素对测定意大利蜜蜂Apis mellifera ligustica Spinola头部乙酰胆碱酯酶(AChE)活性的影响, 并从试验组合中选出最佳条件。蜜蜂AChE活性的测定采用Gorun(1978)改进的Ellman方法, 以碘化硫代乙酰胆碱(ATCI)为底物, 5, 5' 二硫双硝基苯甲酸(DTNB)为显色剂, 测定反应物在412 nm波长下的光密度值, 用考马斯亮蓝G-250法测定蛋白质含量, 经计算得到蜜蜂头部AChE的比活力。对正交试验结果进行极差分析和方差分析, 结果表明各因素对实验结果影响的大小顺序为：温度＞pH值＞时间＞酶浓度＞底物浓度。并得出测定蜜蜂头部AChE活性的最佳条件是：酶终浓度0.2头/mL、底物终浓度0.8 mmol/L、pH值7.5、温度40℃及反应时间5 min。     

糖化酶酶解米渣纯化米蛋白的工艺

研究了糖化酶酶解米渣纯化米蛋白的实验条件:液固比、酶解时间、pH、温度和酶量。通过正交实验优化了酶解主要条件,得到糖化酶水解米渣最佳条件:液固比5:1,时间3 h,pH 4.0,温度65℃和酶量30 U.g-1。在最佳条件下实验,米蛋白的提取率为81.3%,纯度为76.8%。     

对高效石油降解菌株C_(230)酶活的最适pH和温度的探究

以中国甘肃华庆油田附近被石油污染的土壤中分离得到的六株高效石油降解菌A6、L5、L3、M4、B1、B3为菌种材料。通过对六种菌株进行整细胞破碎处理,研究了在不同的pH值和不同温度条件下的各菌株C23O(邻苯二酚2,3-双加氧酶)酶活。实验结果表明:C230酶的最佳反应pH值为7.5;最适反应温度为35℃。     

吡虫啉杀虫剂降解菌Ochrobactum BB -1培养条件的研究

目的:研究培养条件对吡虫啉杀虫剂降解菌苍白杆菌BB -1(Ochrobactum sp.BB -1)生长的影响.方法:采用超声波破碎菌体细胞得到粗酶液;应用单因素试验研究培养条件对吡虫啉杀虫剂降解菌 Ochrobactum BB -1生长的影响.结果:降解酶定域试验表明,BB -1吡虫啉降解酶属于胞内蛋白组分.适合Ochrobactum BB -1生长的最佳碳、氮源分别是甘露醇和酵母膏,浓度分别为30g/L和16g/L;最适培养条件为pH 7.0,250mL三角瓶培养基装液量70mL,培养温度30℃.结论:苍白杆菌BB -1(Ochrobactum BB -1)生长条件要求简单,适于进一步研究其对吡虫啉杀虫剂的降解性能.     

海马总蛋白提取及其酶解条件优化

为了提取海马总蛋白并确定最佳的酶解条件,将海马粉碎后采用水提法提取海马总蛋白。分别选用木瓜蛋白酶、碱性蛋白酶、中性蛋白酶和胰蛋白酶,在不同酶解p H、时间、温度和E/S条件下进行单因素和正交试验法对海马总蛋白的酶解条件进行优化。利用聚丙烯酰胺凝胶电泳和BCA法来分别检测不同条件下的酶解情况及其蛋白含量,根据蛋白水解度来确定其最佳的酶解条件。实验结果表明:从10 g干海马中提取总蛋白为1.056 g,蛋白浓度为0.106 g/m L。海马总蛋白的最佳水解酶为碱性蛋白酶,其最佳酶解条件为:p H 9,E/S为5%,温度为50℃,时间为3 h,获得海马总蛋白的最大水解度为96.9%。研究获得了海马总蛋白及其最佳酶解条件,并为海马多肽生物活性研究奠定了基础。     

Investigation of a Providencia rettgeri strain that has enterobacterial common antigen in the immunogenic state

Antigenic material obtained by phenol-water extraction from Providencia rettgeri strains, Escherichia coli O:14 strains, and mutants of the E. coli O:14 strain were examined by the passive (indirect) hemagglutination technique, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and by immune blotting (lipopolysaccharide (LPS) blotting). Providencia rettgeri 965, like E. coli O:14, was demonstrated to have an enterobacterial common antigen (ECA) in the immunogenic form but, unlike E. coli O:14, it possessed characteristics of a smooth strain. Two populations of molecules were observed to occur in P. rettgeri 965 phenol-water extracts: one consisting of LPS identifiable with specific O antisera and the other of ECA molecules identifiable with E. coli O:14 antiserum or with a monoclonal antibody against ECA.     

An approach for enhancing heterologous production of Providencia rettgeri penicillin acylase in Escherichia coli

Heterologous production of Providencia rettgeri penicillin acylase (PAC) was optimized in Escherichia coli. Several factors, including carbon, temperature, and host effects, were identified to be critical for the enzyme overproduction. The optimum culture conditions for the enzyme production vary for different host/vector systems. With the optimization, both volumetric and specific PAC activities could be significantly improved by more than 50-fold compared to the native expression in P. rettgeri. The heterologous production could be possibly limited by translation or posttranslational steps, depending on the culture temperature and host/vector system. To our knowledge, this is the first evidence demonstrating the limiting step for the production of P. rettgeri PAC and the existence of the P. rettgeri PAC precursor.     

Isolation of insect pathogenic bacteria, Providencia rettgeri, from Heterorhabditis spp.

Nematodes of the genus Heterorhabditis carry bacteria of the genus Photorhabdus into insects including pests of horticultural crops. The bacteria kill the insect and provide conditions which allow for the growth and development of the nematodes. It is reported here that the majority of Heterorhabditis spp. strains tested contained a second bacterial species which was identified as Providencia rettgeri. Injection of the bacteria into waxmoth larvae showed that P. rettgeri was at least as pathogenic as Photorhabdus sp. K122. Both had LD₅₀ values of less than one bacterial cell/larva, but P. rettgeri killed the insects at a considerably faster rate than K122 at both 28°C and 9°C. Since Photorhabdus kills very slowly at low temperatures, it appeared that P. rettgeri might be a better pest control agent under these conditions. However, P. rettgeri was not pathogenic when carried into insect larvae by the nematode, indicating that the nematode suppressed either its release or pathogenicity. It will be necessary to find ways of bypassing or inhibiting this suppression for P. rettgeri to fulfil its potential in pest control.     

A Numerical Taxonomic Study of Proteus-Providence Bacteria

One hundred and six strains from the Proteus-Providence group and 27 other strains from the rest of the Enterobacteriaceae were subjected to 178 morphological. physiological and biochemical tests and the results analysed by computer. Most of the Proteus-Providence strains grouped into six main clusters; (1) Proteus mirabilis , (2) Proteus vulgaris , (3) Proteus morganii , (4) Providencia alcalifaciens, Shigella dysenteriae , (5) Proteus rettgeri , (6) Providencia stuartii . On the basis of these groupings a scheme has been drawn up for distinguishing between the different taxa in the Proteus-Providence group.     

Numerical analysis of electrophoretic protein patterns of Providencia rettgeri strains from human faeces, urine and other specimens

Thirty-one strains of Providencia rettgeri (mainly from humans) were characterized by one-dimensional SDS-PAGE of cellular proteins. The strains came from various countries and comprised 14 from urine, eight from faeces, two from bile (plus one from the liver of a sheep), two from sputum, one from an insect pupa and three the sources of which were unknown. Also included, for reference purposes, were the type strains of the four other Providencia species. The protein patterns, which contained 45-50 discrete bands, were highly reproducible and were used as the basis for two numerical analyses. In the first, which included all the protein bands, the 31 Prov. rettgeri strains formed 13 clusters at the 84% S level. In the second analysis, in which the principal protein bands (in the 33.3-41.3 kD range) were excluded, 29 of the 31 Prov. rettgeri strains formed a single cluster at the 81% S level, whilst the four Providencia reference strains remained unclustered. We conclude that high resolution PAGE combined with computerized analysis of protein patterns provides the basis for typing clinical strains of Prov. rettgeri. Reference strains of each of the 13 PAGE types identified are available from NCTC for inclusion in future studies.     

Numerical analysis of electrophoretic protein patterns of Providencia rettgeri strains from human faeces, urine and other specimens

Thirty-one strains of Providencia rettgeri (mainly from humans) were characterized by one-dimensional SDS-PAGE of cellular proteins. The strains came from various countries and comprised 14 from urine, eight from faeces, two from bile (plus one from the liver of a sheep), two from sputum, one from an insect pupa and three the sources of which were unknown. Also included, for reference purposes, were the type strains of the four other Providencia species. The protein patterns, which contained 45–50 discrete bands, were highly reproducible and were used as the basis for two numerical analyses. In the first, which included all the protein bands, the 31 Prov. rettgeri strains formed 13 clusters at the 84% S level. In the second analysis, in which the principal protein bands (in the 33.3–41.3 kD range) were excluded, 29 of the 31 Prov. rettgeri strains formed a single cluster at the 81% S level, whilst the four Providencia reference strains remained unclustered. We conclude that high resolution PAGE combined with computerized analysis of protein patterns provides the basis for typing clinical strains of Prov. rettgeri. Reference strains of each of the 13 PAGE types identified are available from NCTC for inclusion in future studies.     

雷氏普罗威登斯菌青霉素G酰化酶基因在大肠杆菌中的克隆与表达

利用PCR和分子克隆技术从雷氏普罗威登斯菌（Prouidencia rettgeri)(ATCC29944)的基因组DNA中获得一个青霉素G酰化酶（penicillinGacylase,PGA)基因并将其装入表达质粒pET24a。携带有重组质粒pETPGA的Escherichia coli基因工程菌BL21（DE3）/pETPGA实现了PGA的高效表达,对发酵条件的研究表明基因工程菌在24℃,添加5g/L甘油条件下以1.0mmol/LIPTG诱导1.5h酶活力即达到993.4U/L,比野生菌酶活力（15U/L）提高了66倍。     

Bacterial involvement in apatite genesis

Abstract Bacteria initiated apatite genesis from organic matter in laboratory-controlled conditions. However, the synthesis of apatite, which takes place from inorganic phosphate and aragonite (CaCO₃), appears to be independent of the presence of microorganisms. In vitro experiments using bacterial alkaline phosphatase or calf intestine phosphatase in a definite sterile medium in which the phosphorus source is represented by nucleotides from total yeast RNA, and in vivo experiments with Escherichia coli constitutive alkaline phosphatase mutants, showed the involvement of the alkaline phosphatase with releasing inorganic phosphate in the medium. A strain identified as Providencia rettgeri which in the presence of RNA converts the aragonite to apatite in a few days was isolated from a non-sterile assay. Such a mechanism is proposed to explain the natural formation of apatite in sediments.     

Efficient synthesis of β-lactam antibiotics with very low product hydrolysis by a mutant Providencia rettgeri penicillin G acylase

Applied Microbiology and Biotechnology - Penicillin G acylase (PGA) was isolated from Providencia rettgeri PX04 (PrPGApx04) and utilized for the kinetically controlled synthesis of β-lactam...     

Synthesis and secretion of Providencia rettgeri and Escherichia coli heterodimeric penicillin amidases in Saccharomyces cerevisiae

The Providencia rettgeri and Escherichia coli pac genes encoding heterodimeric penicillin G amidases (PAC) were successfully expressed in Saccharomyces cerevisiae. Furthermore, these recombinant enzymes are secreted from the yeast cell into the medium which is in contrast to bacterial hosts, where the enzymes are retained in the periplasm. Contrary to the P. rettgeri PAC-encoding gene, the E. coli pac is poorly expressed in yeast. The highest yield of P. rettgeri PAC was obtained with a multi-copy plasmid, resulting in of 1500units per liter. This yield is higher by an order of magnitude than that obtained in the best recombinant bacterial expression system. The recombinant P. rettgeri enzyme is only partially and selectively O-glycosylated. Only every sixth or seventh alpha-subunit is glycosylated, while the beta-subunit is not glycosylated at all. N-Glycosylation has not been detected.