多序列比对在克隆表达一株未知芽孢杆菌角蛋白酶基因中的应用

为克隆表达一株未知芽孢杆菌的角蛋白酶基因,本文对芽孢杆菌属来源的13个角蛋白酶基因进行了分类比对,并设计简并引物,成功筛选出一株未知芽孢杆菌的角蛋白酶基因,在大肠杆菌中克隆表达,最后通过SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis)分析和酶活测定验证表达成功,同时还确立重组大肠杆菌BL21(pET-28a-Ker)的最佳诱导温度为20℃,经过0.5 mmol/L IPTG (isopropylthio-galactoside)诱导16 h后,粗酶液中的角蛋白酶酶活达到389.7 U/mL。本文证明了通过多序列比对而设计的角蛋白酶简并引物的有效性,且该方法能够简化角蛋白酶的研究和开发。     

蜡样芽孢杆菌胶原蛋白酶基因的异源表达与活性分析

研究采用降解骨胶原蛋白的蜡样芽孢杆菌MBL13-U作为原菌种,并对蜡样芽孢杆菌MBL13-U进行全基因组测序,克隆获得胶原蛋白酶(ColM13)基因。将目的基因与大肠杆菌的表达载体pET30a进行连接,转入大肠杆菌宿主菌株BL21,获得降解骨胶原蛋白的工程菌pET30a-ColM13/BL21。通过SDS-PAGE测定异源表达的重组蛋白酶ColM13的分子质量,并测定不同诱导时间和诱导浓度对ColM13酶活性的影响,最终确定重组蛋白酶ColM13的酶活最适条件:6‰异丙基硫代半乳糖苷(IPTG,100 mmol/L),37℃诱导6 h,优化后酶活力最高为64.99 U/mL,较优化前提高4.24倍。通过水解圈试验、紫外光谱和扫描电子显微镜等方式验证,表明工程菌构建成功,这为我国畜禽骨骼资源的深度开发和综合利用提供了新的研究思路。     

褐藻胶裂解酶基因在大肠杆菌中的表达及其酶学性质

作者从蜡样芽孢杆菌F1-5-10中提取DNA,通过PCR克隆得到褐藻胶裂解酶基因后,将其构建到pET-30a(+)上并在大肠杆菌BL21菌株中进行高效诱导表达,继而对纯化得到的褐藻胶裂解酶蛋白进行活性检测和酶学特性研究。实验结果表明:PCR扩增出1035bp大小的褐藻胶裂解酶基因algl(GenBank登录号:GU585575),SDS-PAGE电泳结果显示出相对分子质量约为45 000的特异性蛋白质条带。表达条件优化实验结果表明0.4mmol/L浓度的IPTG 32℃诱导4h重组蛋白的表达量最高,约占菌体总蛋白质质量的36%。测定褐藻胶裂解酶酶活性,酶活力为11.7U/mg。酶学性质研究表明:ALGL的酶活最适温度为35℃,热稳定性较差,最适pH值为6.6,Ca2+、Mg2对酶活有激活作用,ALGL催化褐藻胶的Km值为1.89mg/mL,Vmax为15.01U/mL。     

嗜热酸性普鲁兰水解酶Ⅲ的高效分泌表达及其酶学性质

本文探索了嗜热酸性普鲁兰水解酶Ⅲ Tk-PUL在枯草芽孢杆菌表达系统中的高效分泌表达条件,并对重组Tk-PUL的酶学性质进行了初步研究。通过构建Tk-PUL分泌表达信号肽筛选库,并结合高通量筛选方法,确定引导Tk-PUL在枯草芽孢杆菌中高效分泌表达的信号肽。结果表明,在信号肽AmyE的引导下,重组Tk-PUL在枯草芽孢杆菌表达系统中高效分泌表达。Tk-PUL属于单结构域双功能酶,同时具有α-淀粉酶活性和普鲁兰酶活性。重组Tk-PUL的α-淀粉酶活性的最适反应pH为4.5,最适反应温度为100℃,对应的绝对酶活为54.08 U/mg,于100℃的半衰期约为2 h。重组Tk-PUL的普鲁兰酶活性的最适反应pH为4.5,最适反应温度为100℃,对应的绝对酶活为110.39 U/mg,于100℃的半衰期约为2 h。本研究为Tk-PUL在淀粉酶法制糖工业中的应用奠定了基础。     

D-阿洛酮糖 3-差向异构酶基因在枯草芽孢杆菌中的表达

将D-阿洛酮糖3-差向异构酶(DPE)基因利用PCR进行扩增,与枯草芽孢杆菌载体p MA5连接,构建重组质粒p MA5-cbdpe。重组质粒转入枯草芽孢杆菌Bacillus subtilis WB800感受态细胞,利用卡那霉素筛选和PCR鉴定,获得一株DPE重组枯草芽孢杆菌菌株。该重组菌株无需诱导即可产生DPE酶,18 h时酶活可达6.8 U/m L。该酶最适p H为7.0,最适温度为55℃,与大肠杆菌表达的DPE酶酶学性质相似。结果表明,DPE酶可在枯草芽孢杆菌中表达。     

重组枯草芽孢杆菌α-淀粉酶基因工程菌构建与表达

根据GenBank枯草芽孢杆菌α-淀粉酶基因序列设计引物,以枯草芽孢杆菌基因组为模板,PCR克隆α-淀粉酶基因(amy),将α-淀粉酶基因插入穿梭表达载体pP43C,构建重组质粒pP43Camy。随后将重组质粒转化八种蛋白酶缺陷的宿主枯草芽孢杆菌WB800,经筛选获得重组枯草芽孢杆菌α-淀粉酶基因工程菌WB800/pP43Camy1026,工程菌摇瓶发酵酶活力达960U。性质研究表明,重组α-淀粉酶的最适作用温度为70℃,最适反应pH为6.0,具有良好的应用潜力。     

抗辐射不动杆菌碱性脂肪酶基因在毕赤酵母中的表达

本研究将来自抗辐射不动杆菌CMC-1的碱性脂肪酶基因去掉信号肽,经密码子优化及全基因合成后克隆到pPICZαA载体,构建了重组质粒pPICZαA-ARL,质粒线性化后转化毕赤酵母X33,筛选得到分泌表达碱性脂肪酶的重组毕赤酵母X33/pPICZαA-ARL。摇瓶发酵液上清酶活最高达65 U/mL,初步研究了该脂肪酶的酶学性质,其最适作用温度为50℃,最适pH为9.0,最适底物是对硝基苯酚辛酸酯。     

大麦β-淀粉酶在枯草芽孢杆菌中异源表达

该研究拟采用枯草芽孢杆菌异源表达大麦来源β-淀粉酶。选择枯草芽孢杆菌WB800作为宿主,采用同源重组的方法构建表达载体p P4 3NMK-amy B,获得重组枯草芽孢杆菌WB-amy B。重组枯草芽孢杆菌在摇瓶发酵条件下酶活最高可达386 U/m L,纯化后测得其比酶活为613 U/mg。重组酶的最适温度为55℃,最适p H值为5. 0。重组β-淀粉酶水解产麦芽糖能力与大麦β-淀粉酶相当,与普鲁兰酶联用时麦芽糖最大转化率可达81. 8%。重组枯草芽孢杆菌摇瓶发酵水平产酶量高于类似文献报道,重组β-淀粉酶的酶学性质与大麦β-淀粉酶相比几乎相同,完全可以替代大麦β-淀粉酶在工业上的应用。     

耐热耐碱木聚糖酶在大肠杆菌中的高效分泌表达及酶学性质研究

目的研究枯草芽孢杆菌来源的木聚糖酶基因在大肠杆菌BL2l中的高效分泌表达并对其产木聚糖酶进行酶学性质分析。方法全基因合成枯草芽孢杆菌木聚糖酶基因序列并进行密码子优化,经大肠杆菌BL2l表达后获得基因工程菌株,通过SDS-PAGE电泳检测、硫酸铵分级沉淀和AKTA系统分离纯化,分析表达产物的酶学性质。结果重组木聚糖酶为胞外分泌性表达,相对分子质量约43×103;酶促反应最适温度为65℃,最适p H为10.0,最适条件下酶活最高可达1201.5 IU/ml。结论成功获得高效分泌表达重组木聚糖酶的基因工程菌株,该木聚糖酶具较好的耐热性和耐碱性。     

Bacillus stearothermophilus麦芽糖淀粉酶在Bacillus subtilis中的重组表达及发酵优化

为了实现Bacillus stearothermophilus嗜热脂肪芽孢杆菌来源的麦芽糖淀粉酶基因amyM(EC 3.2.1.133)在枯草芽孢杆菌中的重组表达,以opt-amyM/T质粒为模板进行PCR扩增得到目的基因,与表达载体pHY300PLK进行重组连接后转入宿主菌Bacillus subtilis CCTCC M 2016536中进行表达。在TB培养基中发酵培养48 h,麦芽糖淀粉酶酶活达到250.7 U/mL。继续对重组菌氮源种类及复配、氮源质量浓度、碳源质量浓度等摇瓶发酵条件进行优化,确定其最佳产酶条件为:复合氮源为酵母浸膏25 g/L和大豆蛋白胨5 g/L、葡萄糖5 g/L、培养基初始pH 6.5、最适培养温度41℃;在此条件下麦芽糖淀粉酶酶活可达396.7 U/mL,是优化前的1.6倍。在此基础上进一步对麦芽糖淀粉酶进行酶学性质进行测定:麦芽糖淀粉酶最适温度为60℃,最适pH为5.5,半衰期为325 h,Km为0.95 g/L,比活为2646 U/mg。     

Reliable identification of grapevine rootstock varieties using RAPD PCR on woody samples

Since grapevine ( Vitis spp .) rootstock material is being traded increasingly as disbudded woody material a lack of distinctive morphological features on such material necessitates an alternative and reliable means of identification. Methods described here were developed for rapid and efficient extraction of DNA from woody samples rich in phenolic compounds and polysaccharides, and for subsequent identification of varieties by RAPD PCR. Using these methods, and with the application of only one selected RAPD primer, we were able to differentiate sixteen rootstock varieties, including the seven varieties most commonly used in Germany. Problems commonly encountered with reproducibility of RAPD patterns were avoided by choosing primers with a dinucleotide sequence and a high G/C content that allowed a rather high annealing temperature of 45°C. Methods described here should also be useful for other horticultural crops, especially those with woody tissues rich in phenolic compounds and polysaccharides.     

An internet compendium of analytical methods and spectroscopic information for monomers and additives used in food packaging plastics

An internet website (http://cpf.jrc.it/smt/) has been produced as a means of dissemination of methods of analysis and supporting spectroscopic information on monomers and additives used for food contact materials (principally packaging). The site which is aimed primarily at assisting food control laboratories in the European Union contains analytical information on monomers, starting substances and additives used in the manufacture of plastics materials. A searchable index is provided giving PM and CAS numbers for each of 255 substances. For each substance a data sheet gives regulatory information, chemical structures, physico-chemical information and background information on the use of the substance in particular plastics, and the food packaging applications. For monomers and starting substances (155 compounds) the infra-red and mass spectra are provided, and for additives (100 compounds); additionally proton NMR are available for about 50% of the entries. Where analytical methods have been developed for determining these substances as residual amounts in plastics or as trace amounts in food simulants these methods are also on the website. All information is provided in portable document file (PDF) format which means that high quality copies can be readily printed, using freely available Adobe Acrobat Reader software. The website will in future be maintained and up-dated by the European Commission's Joint Research Centre (JRC) as new substances are authorized for use by the European Commission (DG-ENTR formerly DGIII). Where analytical laboratories (food control or other) require reference substances these can be obtained free-ofcharge from a reference collection housed at the JRC and maintained in conjunction with this website compendium.     

Aroma characterization of various apricot varieties using headspace–solid phase microextraction combined with gas chromatography–mass spectrometry and gas chromatography–olfactometry

The characterization of the aromatic profile of several apricot cultivars with molecular tracers in order to obtain objective data concerning the aromatic quality of this fruit was undertaken using headspace–solid phase microextraction (HS–SPME). Six apricot cultivars were selected according to their organoleptic characteristics: Iranien, Orangered, Goldrich, Hargrand, Rouge du Roussillon and A4025. The aromatic intensity of these varieties measured by HS–SPME–Olfactometry were defined and classified according to the presence and the intensity of grassy, fruity and apricot like notes. In the six varieties, 23 common volatile compounds were identified by HS–SPME–GC–MS. Finally, 10 compounds, ethyl acetate, hexyl acetate, limonene, β-cyclocitral, γ-decalactone, 6-methyl-5-hepten-2-one, linalool, β-ionone, menthone and (E)-hexen-2-al were recognized by HS–SPME–GC–O as responsible of the aromatic notes involved in apricot aroma and considered as molecular tracers of apricot aromatic quality which could be utilized to discriminate apricot varieties.     

Tests of potential functional barriers for laminated multilayer food packages. Part I: Low molecular weight permeants

The advent of the functional barrier concept in food packaging has brought with it a requirement for fast tests of permeation through potential barrier materials. In such tests it would be convenient for both foodstuffs and materials below the functional barrier (sub-barrier materials) to be represented by standard simulants. By means of inverse gas chromatography, liquid paraffin spiked with appropriate permeants was considered as a potential simulant of sub-barrier materials based on polypropylene (PP) or similar polyolefins. Experiments were performed to characterize the kinetics of the permeation of low molecular weight model permeants (octene, toluene and isopropanol) from liquid paraffin, through a surrogate potential functional barrier (25 μm-thick oriented PP) into the food simulants olive oil and 3% (w/v) acetic acid. These permeation results were interpreted in terms of three permeation kinetic models regarding the solubility of a particular model permeant in the post-barrier medium (i.e. the food simulant). The results obtained justify the development and evaluation of liquid sub-barrier simulants that would allow flexible yet rigorous testing of new laminated multilayer packaging materials.     

Textural Properties of Cold-set Gels Induced from Heat-denatured Whey Protein Isolates

A 9% whey protein (WP) isolate solution at pH 7.0 was heat-denatured at 80°C for 30 min. Size-exclusion HPLC showed that native WP formed soluble aggregates after heat-treatment. Additions of CaCl2 (10–40 mM), NaCl (50–400 mM) or glucono-delta-lactone (GDL, 0.4–2.0%, w/v) or hydrolysis by a protease from Bacillus licheniformis caused gelation of the denatured solution at 45°C. Textural parameters, hardness, adhesiveness, and cohesiveness of the gels so formed changed markedly with concentration of added salts or pH by added GDL. Maximum gel hardness occurred at 200 mM NaCl or pH 4.7. Increasing CaCl2 concentration continuously increased gel hardness. Generally, GDL-induced gels were harder than salt-induced gels, and much harder than the protease-induced gel.     

Occurrence of bisphenol-F-diglycidyl ether (BFDGE) in fish canned in oil

The levels of bisphenol-F-diglycidyl ether (BFDGE) were quantified as part of a European survey on the migration of residues of epoxy resins into oil from canned fish. The contents of BFDGE in cans, lids and fish collected from all 15 Member States of the European Union and Switzerland were analysed in 382 samples. Cans and lids were separately extracted with acetonitrile. The extraction from fish was carried out with hexane followed by re-extraction with acetonitrile. The analysis was performed by reverse phase HPL C with fluorescence detection. BFDGE could be detected in 12% of the fish, 24% of the cans and 18% of the lids. Only 3% of the fish contained BFDGE in concentrations considerably above 1mg/kg. In addition to the presented data, a comparison was made with the levels of BADGE (bisphenol-A-diglycidyl ether)analysed in the same products in the context of a previous study.     

Analysis of benzo[a]pyrene in spiked fatty foods by second derivative synchronous spectrofluorimetry after microwave-assisted treatment of samples

A simple, rapid and inexpensive method has been developed for the determination of benzo[a     

The development of reference materials for paralytic shellfish poisoning toxins in lyophilized mussel. II: Certification study

This paper describes the second part of a project undertaken to develop certified mussel reference materials for paralytic shellfish poisoning toxins. In the first part two interlaboratory studies were undertaken to investigate the performance of the analytical methodology for several PSP toxins, in particular saxitoxin and decarbamoyl-saxitoxin in lyophilized mussels, and to set criteria for the acceptance of results to be applied during the certification exercise. Fifteen laboratories participated in this certification study and were asked to measure saxitoxin and decarbamoyl-saxitoxin in rehydrated lyophilized mussel material and in a saxitoxin-enriched mussel material. The participants were allowed to use a method of their choice but with an extraction procedure to be strictly followed. The study included extra experiments to verify the detection limits for both saxitoxin and decarbamoyl-saxitoxin. Most participants (13 of 15) were able to meet all the criteria set for the certification study. Results for saxitoxin.2HCl yielded a certified mass fraction of <0.07 mg/kg in the rehydrated lyophilized mussels. Results obtained for decarbamoyl-saxitoxin.2HCl yielded a certified mass fraction of 1.59+/-0.20 mg/kg. The results for saxitoxin.2HCl in enriched blank mussel yielded a certified mass fraction of 0.48 +/- 0.06 mg/kg. These certified reference materials for paralytic shellfish poisoning toxins in lyophilized mussel material are the first available for laboratories to test their method for accuracy and performance.     

Contribution of European research to risk analysis

The European Commission's, Quality of Life Research Programme, Key Action 1—Health, Food & Nutrition is mission-oriented and aims, amongst other things, at providing a healthy, safe and high-quality food supply leading to reinforced consumer confidence in the safety of European food. Its objectives also include the enhancing of the competitiveness of the European food supply. Key Action 1 is currently supporting a number of different types of European collaborative projects in the area of risk analysis. The objectives of these projects range from the development and validation of prevention strategies including the reduction of consumers risks; development and validation of new modelling approaches; harmonization of risk assessment principles, methodologies, and terminology; standardization of methods and systems used for the safety evaluation of transgenic food; providing of tools for the evaluation of human viral contamination of shellfish and quality control; new methodologies for assessing the potential of unintended effects of genetically modified (genetically modified) foods; development of a risk assessment model for Cryptosporidium parvum related to the food and water industries; to the development of a communication platform for genetically modified organism, producers, retailers, regulatory authorities and consumer groups to improve safety assessment procedures, risk management strategies and risk communication; development and validation of new methods for safety testing of transgenic food; evaluation of the safety and efficacy of iron supplementation in pregnant women; evaluation of the potential cancer-preventing activity of pro- and pre-biotic ('synbiotic') combinations in human volunteers. An overview of these projects is presented here.