金属离子对重组N-乙酰鸟氨酸脱乙酰基酶可溶部分酶活力的影响

重组N-乙酰鸟氨酸脱乙酰基酶可在重组菌BL21-pET22b-argE中高效表达,大多以不可溶的包涵体形式存在。文中研究了金属离子浓度及添加时间对该菌生长及可溶表达部分酶活力的影响。结果表明,1.0g/L的Mg2+对生物量和酶活有明显促进作用。Co2+的浓度及添加时间不同,对菌的生长及酶活力的影响结果也不同。培养起始加入Co2+会抑制菌的生长及酶活,而在1.0%乳糖诱导2.5h后加入则可解除生长抑制并促进酶活。Ni2+,Cu2+会抑制菌体的生长及酶活。Mn2+、Mo2+和B3+不影响菌的生长,对酶活有促进作用。添加金属离子后,酶活从16.90U/mL提高到251.91U/mL。     

嗜酸芽孢杆菌普鲁兰酶在大肠杆菌中的表达及发酵优化

为了在3L发酵罐水平上提高重组菌E.coli BL 21(DE3)/pET20b(+)-BapulA的普鲁兰酶的表达量和胞外分泌,将嗜酸芽孢杆菌普鲁兰酶(Bacillus acidopullulyticus)在大肠杆菌中进行重组表达,并在3L发酵罐水平上对重组菌进行发酵优化,考察了发酵条件对重组酶表达的影响。重组菌发酵的最佳条件为:发酵前期菌体生长温度和pH分别为30℃和7.0,当菌体浓度OD_(600)达到50时,发酵温度降低至25℃,此时调节pH为6.2,同时开始流加乳糖[0.4g/(L·h)]进行诱导;在发酵过程中,当菌体浓度OD_(600)依次达到15,45,75时,分别加入浓度为1.5g/L的甘氨酸,当菌体OD600为105时,再加入浓度为3g/L的甘氨酸。发酵结束后普鲁兰酶的胞外酶活达659.0U/mL,最高总酶活达1 910.1U/mL,与摇瓶的初始总酶活(41.1U/mL)和胞外酶活(6.5U/mL)相比,分别提高了45.4倍和100倍。     

β-N-乙酰葡萄糖苷内切酶Endo S异源表达及发酵优化

Endo S(EC 3.2.1.96)是一种来源于化脓链球菌的β-N-乙酰葡萄糖苷内切酶,可以特异性水解免疫球蛋白G(Ig G)可结晶区(Fc片段)N糖链。为实现其高效表达,本研究构建endo S重组表达大肠杆菌E.coli BL21(DE3)/pET28a-endo S,并验证表达蛋白糖苷水解活性,在摇瓶水平单因素优化蛋白质表达,考察了基础培养基种类、碳源、氮源、无机盐、培养基初始pH、发酵温度和时间等因素对产酶的影响,确定最优发酵培养基组成为(g/L):酵母粉10.0,甘油4.0,牛肉浸膏25.4,NaCl 5.0,pH 8.0,最优培养条件为:20℃诱导表达24 h,蛋白表达量为225 mg/L发酵液,是优化前的4.5倍,同时也是目前报道最高表达量的5.6倍。     

脱乙酰基酶在枯草芽孢杆菌中的高效可溶性表达

为提高脱乙酰基酶的可溶性表达含量,对前期挖掘的脱乙酰基酶NAP-Das2. 3基因进行异源表达及发酵优化。将脱乙酰基酶NAP-Das2. 3基因克隆至枯草芽孢杆菌表达载体p P43NMK的npr B信号肽下游,转入B.subtilis WB800构建了重组工程菌B. subtilis WB800/p P43NMK/nap-das2. 3,并对重组菌培养及发酵产酶条件进行了优化。重组菌最适培养和产酶条件分别为甘油6 g/L、牛肉膏30 g/L、Na Cl 10 g/L; p H 7. 5、培养温度37℃、装液量100 m L (500 m L摇瓶)、培养30 h。在优化的条件下,发酵液中脱乙酰基酶酶活达到106. 42 U/L,较出发条件提高了424. 68倍,在5 L发酵罐培养30 h后,脱乙酰基酶酶活达到116. 13 U/L。研究实现了脱乙酰基酶的异源高效可溶性表达,为脱乙酰基酶的应用提供了基础。     

重组大肠杆菌BL21（DE3）-pET28a（＋）-bgl诱导表达β-葡聚糖酶的条件优化

研究自行构建的产β-葡聚糖酶的工程菌E.coli BL21(DE3)-pET28a(+)-bgl在LB培养基中的生长特性,考察种子液的菌龄、培养基起始pH、接种量及诱导起始时发酵液菌浓度等对β-葡聚糖酶产生水平的影响;通过正交试验确定诱导剂IPTG及乳糖添加量、诱导温度及诱导剂作用时间.结果表明:培养基起始pH 7.0,对数生长中期的种子液(OD600为0.35)以接种量(体积分数)10%接入摇瓶发酵培养,37 ℃,200 r/min培养约3 h,菌液OD值达到1.0左右,添加终浓度分别为0.033 6 mmol/L的IPTG及10 mmol/L乳糖,24℃诱导6 h,发酵液清液中酶活达到最高(336.33 U/mL),菌体生长量为1.12 g/L,发酵液中总酶活达到459.32 U/mL,是原始菌株在相同条件下所产酶活的6.62倍.采用优化培养条件及诱导剂作用条件,重组菌在TB培养基中酶活水平进一步提高,诱导剂作用10 h,发酵清液中酶活为1 090.31 U/mL,总酶活1 570.83 U/mL,是原始菌在该条件下酶活的19.73倍,显示出重组菌具有广阔的工业化应用前景.     

冰川细菌Serratia sp.A_(29)-2发酵产低温蛋白酶条件的优化

为提高新疆冰川细菌Serratia sp.A29-2的低温蛋白酶产酶活性,在单因素试验的基础上,通过正交试验和响应面试验,对其发酵培养基和发酵条件进行了优化。研究表明:其最佳培养基组成为蛋白胨15.0 g/L、酵母粉5.0 g/L、麦芽糖10.0 g/L、CaCl20.5 g/L。最优发酵条件是:温度22℃,pH值7.0,时间60 h,NaCl质量分数1.0%。以优化培养基为基础,在最适发酵条件下,菌株Serratia sp.A29-2产低温蛋白酶酶活可达54.25 U/mL,是初始酶活的1.93倍。     

分散泛菌胞内脱乙酰酶在大肠杆菌中重组表达条件优化及酶学特性

目的 对分散泛菌的胞内脱乙酰酶(Pantoea dispersa N-acetylglucosamine deacetylase, Pd-nagA)进行表达条件优化及酶学性质分析。方法 本研究使用聚合酶链式反应(polymerase chain reaction, PCR)技术对分散泛菌全基因组中Pd-nagA基因序列进行全长扩增，将含有Pd-nagA基因的质粒pET-28a(+)转化进入大肠杆菌BL21(DE3)中诱导表达，同时采用单因素实验，优化诱导温度、诱导前菌体生物量、诱导时间以及乳糖类似物异丙基硫代半乳糖苷(isopropyl β-D-1-thiogalactopyranoside, IPTG)的浓度，采用Ni2+-次氮基三乙酸(nitrilotriacetic acid，NTA)进行重组酶的纯化，再对其酶学特性进行测定。结果 以pET-28a(+)质粒作为表达载体，大肠杆菌BL21(DE3)成功表达了可溶形式的Pd-nagA蛋白，分子量约为40 kDa。当诱导温度为28℃，诱导前菌液OD600值为1.2，IPTG浓度为0.6 mmol/L，诱导时间为20 h时，Pd-nagA蛋白得到了大量表达。通过薄层层析法分析表明Pd-nagA重组蛋白能够在体外转化N-乙酰氨基葡萄糖生成氨基葡萄糖，Pd-nagA重组蛋白能够在高盐溶液中保持一半的相对酶活性，该酶储存10 d的相对活性仍然保持在90%以上，具有良好的储存稳定性，催化N-乙酰氨基葡萄糖(N-acetylglucosamine, GlcNAc)生成氨基葡萄糖(glucosamine, GlcN)反应条件简单快捷。结论 本研究成功异源表达了具有催化活性的Pd-nagA酶，并对表达条件进行优化及酶学特性探究，可为工业上单酶催化制备氨基葡萄糖提供一定的理论参考。     

重组融合蛋白MBP-PAI的表达、纯化及酶活测定

为了解决亚油酸异构酶(Linoleic Acid Isomerase,PAI)在大肠杆菌中形成包涵体的问题,选择麦芽糖结合蛋白(Maltose-Binding Protein,MBP)标签和低温诱导表达系统pColdV,构建了大肠杆菌重组表达菌株E.coli BL21(pCold-Mpai),并对其诱导表达条件进行了优化。SDS-PAGE电泳结果显示,融合蛋白MBP-PAI成功表达,重组菌的最佳诱导表达条件为:诱导温度15℃、IPTG添加量0.1 mmol/L、诱导时间12 h,在该诱导条件下,与未融合MBP的PAI相比,MBP-PAI的可溶性表达量为后者的18倍、酶活为后者的1.5倍。经过MBPTrap HP亲和层析柱纯化后,MBP-PAI纯蛋白质的比酶活为1.58 U/mg,能够转化亚油酸形成反10,顺12-共轭亚油酸。     

重组麦芽糖转葡萄糖基酶工程菌发酵条件的优化

对重组麦芽糖转葡萄糖基酶工程菌的发酵条件进行优化,通过单因素试验确定该菌株产麦芽糖转葡萄糖基酶的最佳发酵培养基为：糖蜜0.025g/mL、胰蛋白胨0.015g/mL、MgSO4 ·7H2O与K2HPO4的质量为7:1、FeSO4 ·7H2O 0.5g/L;以葡萄糖氧化酶法为指标测得最佳摇瓶发酵条件为：装液量100mL/250mL、转速200r/min、接种量5%、初始pH 7.0、最佳温度37℃、诱导阶段OD600nm=0.6、诱导温度30℃、诱导时间5h、诱导剂异丙基-β-D-硫代吡喃半乳糖苷(IPTG)浓度1mmol/L。经优化,重组麦芽糖转葡萄糖基酶的酶活力可达950U/mL,比初始条件下提高了近7倍。     

重组N-乙酰鸟氨酸脱乙酰基酶包涵体蛋白的柱上复性

N-乙酰鸟氨酸脱乙酰基酶(NAO)具有广泛的底物选择性,可用于多种活性氨基酸的酶法拆分,具有广阔的工业应用前景。文中采用多种洗涤剂对重组N-乙酰鸟氨酸脱乙酰基酶包涵体进行洗涤去杂,并用DEAE Sepharose Fast Flow层析柱和三缓冲体系作为复性系统对重组N-乙酰鸟氨酸脱乙酰基酶包涵体进行复性实验。结果表明,4mol/L尿素与0.5%Triton X-100联合洗涤可以大量去除杂质;三缓冲体系能有效地实现重组N-乙酰鸟氨酸脱乙酰基酶包涵体的柱上复性。在流速为0.5 mL/min,尿素梯度为21 mL,尿素终浓度为1.8mol/L,蛋白质上样量为0.99 mg的实验条件下,蛋白回收率和复性酶比活分别达到52%和10.27 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.     

Chlorohydrins of bisphenol A diglycidyl ether (BADGE) and of bisphenol F diglycidyl ether (BFDGE) in canned foods and ready-to-drink coffees from the Japanese market

BADGE.2HCl and BFDGE.2HCl were determined in 28 samples of ready-to-drink canned coffee and 18 samples of canned vegetables (10 corn, 5 tomatoes and 3 others), all from the Japanese market. HPLC was used as the principal analytical method and GCMS for confirmation of relevant LC fractions. BADGE.2HCl was found to be present in one canned coffee and five samples of corn, BFDGE.2HCl in four samples of canned tomatoes and in one canned corn. No sample was found which exceeded the 1mg/kg limit of the EU for the BADGE chlorohydrins. However the highest concentration was found for the sum of BFDGE.2HCl and BFDGE.HCl.H₂O at a level of 1.5mg/kg. A Beilstein test confirmed that all cans containing foods contaminated with BADGE.2HCl or BFDGE.2HCl had at lest one part coated with a PVC organosol.     

European priorities for research to support legislation in the area of food contact materials and articles

A strong science base is required to underpin the planning and decision-making process involved in determining future European community legislation on materials and articles in contact with food. Significant progress has been made in the past 5 years in European funded work in this area, with many developments contributing to a much better understanding of the migration process, and better and simpler approaches to food control. In this paper this progress is reviewed against previously identified work-areas (identified in 1994) and conclusions are reached about future requirements for R&D to support legislation on food contact materials and articles over the next 5 or so years.     

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.     

Influence des ions sur le pouvoir hydratant de l'ure e: e tude sur peau de porc ex vivo

This study deals with the influence of ions (NaCl and MgSO4) in a W/O emulsion containing 10% urea. Moisturization kinetics are assessed by corneometry on pig skin ex vivo. The formula's influence on urea penetration is measured by infrared spectrometry with an ATR device and the stripping method. Corneometry and spectroscopy were chosen to record simultaneously the hydratation levels and urea localization into superficial cell layers. Urea crystallization after evaporation of emulsions and aqueous solutions is described. Results show that urea does not hydrate nor penetrate when applied to the skin through an aqueous gel. In a W/O emulsion, sodium chloride increases the ability of urea to moisturize without improving penetration. In vitro urea crystallization is disturbed by sodium chloride or magnesium sulphate for solutions and emulsions. This stabilization by ions is correlated with good moisturization values. The stabilization of urea in the solute state provided by ions increases its water epidermal binding capacity without enhancing penetration.     

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.