玉米赤霉烯酮降解菌的筛选、鉴定及降解酶初步提取

该实验采用富集培养的方法从土壤中筛选出有效降解玉米赤霉烯酮（Zearalenone,ZEN）的菌株。初筛采用ZEN涂布至无机盐培养基作为唯一碳源,复筛是以菌株的发酵液对ZEN的降解率作为评价指标,得到一株菌株A.lwoffi.Haut.1,其发酵液在37 ℃下与5 μg/mL ZEN共培养48 h,降解率高达93.54%。经16S rDNA基因测序分析、生理生化实验和菌落形态观察初步对菌株进行鉴定,并对该菌株的降解活性物质进行初步定位,最终探究丹宁-聚乙二醇法和盐沉法对无细胞上清液进行粗降解酶的提取效果。结果表明,该菌株鉴定判断为鲁氏不动杆菌（Acinetobacter lwoffii）;该菌株的无细胞上清液的降解率最高为82.31%,无细胞上清液经加热处理、蛋白酶K处理和蛋白酶K+SDS处理后,降解率分别为20.10%、41.67%和18.68%,说明降解活性物质主要为胞外酶;当单宁浓度为15 mg/mL,聚乙二醇溶液浓度为10 mg/mL,提出的酶液降解率为64.33%,而盐沉法加入60%硫酸铵提出的粗酶液降解率为20.30%,因此,单宁聚乙二醇法提取降解酶效果更好。该研究为菌株降解ZEN的作用机理提供了研究基础,也为生物降解ZEN提供了新的研究材料。     

玉米赤霉烯酮降解菌的分离鉴定及其降解特性研究

采用富集培养的方法从麦粒中筛选到一株玉米赤霉烯酮(ZEN)降解菌7D3-2,该菌株对ZEN的降解能力高达95%;通过对7D3-2形态、生理生化特性及16S r DNA、gyr A基因系统进化发育地位分析,该菌株被鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。研究了初始p H、培养温度、接种量及ZEN初始质量浓度等环境条件对7D3-2降解ZEN的影响,结果表明:7D3-2降解ZEN的最适温度为30℃,最适p H为7.0;当接种量小于10%时,7D3-2对ZEN的降解率与接种量成正比;ZEN质量浓度在1.0~25.0 mg/L范围内,7D3-2对ZEN的降解差异不显著,当ZEN质量浓度大于25.0 mg/L时,ZEN降解率与ZEN初始质量浓度成反比,但是ZEN被降解的绝对量与其ZEN初始质量浓度呈正相关;降解物质的定域实验结果显示:7D3-2中降解ZEN的物质主要位于细胞外。7D3-2不仅对培养液中的ZEN具有降解效果,也能很好地降解玉米粉中的ZEN。本研究结果不仅可以丰富ZEN降解菌种质资源库,在粮食和饲料的ZEN生物脱毒方面还具有广阔的应用前景。     

玉米赤霉烯酮的分离鉴定及其降解特性研究

采用富集培养的方法从麦粒中筛选到一株玉米赤霉烯酮(ZEN)降解菌7D3-2,该菌株对ZEN的降解能力高达95%;通过对7D3-2形态、生理生化特性及16S r DNA、gyr A基因系统进化发育地位分析,该菌株被鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)。研究了初始p H、培养温度、接种量及ZEN初始质量浓度等环境条件对7D3-2降解ZEN的影响,结果表明:7D3-2降解ZEN的最适温度为30℃,最适p H为7.0;当接种量小于10%时,7D3-2对ZEN的降解率与接种量成正比;ZEN质量浓度在1.0~25.0 mg/L范围内,7D3-2对ZEN的降解差异不显著,当ZEN质量浓度大于25.0 mg/L时,ZEN降解率与ZEN初始质量浓度成反比,但是ZEN被降解的绝对量与其ZEN初始质量浓度呈正相关;降解物质的定域实验结果显示:7D3-2中降解ZEN的物质主要位于细胞外。7D3-2不仅对培养液中的ZEN具有降解效果,也能很好地降解玉米粉中的ZEN。本研究结果不仅可以丰富ZEN降解菌种质资源库,在粮食和饲料的ZEN生物脱毒方面还具有广阔的应用前景。     

1株新型降解玉米赤霉烯酮的沙福芽孢杆菌及其关键酶分析

以玉米赤霉烯酮（zearalenone,ZEN）为主要碳源,采用富集培养法从小麦样品中分离获得1株快速降解ZEN的微生物菌株。通过形态学观察、16S rDNA及促旋酶B亚单位（gyrase B,gyrB）基因序列分析,初步鉴定该菌株为沙福芽孢杆菌（Bacillus safensis）,命名为M7L4,该菌株在基础盐培养基中培养24 h可将10 μg/mL的ZEN完全降解。质谱分析表明,M7L4的活细胞可将ZEN转化为m/z 397.1的新型磷酸化衍生物ZEN-磷酸盐（ZEN-phosphate,ZEN-P）。以菌株M7L4的基因组为模板,扩增出转化ZEN的关键酶磷酸烯醇丙酮酸利用酶（phosphoenolpyruvate-utilizing enzyme,PUE）基因BsaPUE,在大肠杆菌BL21(DE3)中诱导表达并纯化。重组酶Bsa PUE具有磷酸转移酶活性,在有ATP和Mg2+存在的情况下,30 min内可将10 μg/mL ZEN完全转化为ZEN-P。沙福芽孢杆菌及其磷酸转移酶可为生物去除ZEN提供有效的材料资源。     

枯草芽孢杆菌YQ-1降解玉米赤霉烯酮膜蛋白酶的提取及性质分析

玉米赤霉烯酮（Zearalenone,ZEN）是一类常见于发霉谷物、饲料中的真菌毒素,具有生殖、遗传和致癌性等危害,在世界各地的谷物中常超标检出。本课题组前期筛选得到一株高效降解ZEN的枯草芽孢杆菌Bacillus subtilis YQ-1,全细胞催化12 h后ZEN(20μg/mL)的降解率达98.36%。根据降解酶的细胞定位,推测ZEN降解酶定位于细胞膜表面。本实验以ZEN降解率为指标,提取YQ-1细胞膜表面ZEN降解酶蛋白,结合氨基酸序列分析,推测可能的降解酶。结果显示,以辛基-β-葡萄糖苷（OG）、十二烷基-β-D-麦芽糖苷（DDM）、Triton X-100和Triton X-114作为膜蛋白提取剂比Na Cl和尿素提取的蛋白量显著增加,但提取的膜蛋白酶ZEN降解活性较差,以丁醇为提取剂提取得到的水相蛋白粗酶液在12 h内对20μg/mL ZEN的降解率为64.09%,粗酶液蛋白含量为9.86μg/mL,酶活为0.43 U。该降解酶的提取为后续的ZEN降解酶挖掘奠定了实验基础,枯草芽孢杆菌及其ZEN降解酶的研究为霉菌毒素的体外脱毒提供支撑。     

有机磷农药降解酶基因Oph2的克隆及甲基对硫磷降解效果研究

摘要：目的 挖掘可高效表达农残降解酶的基因,并构建含有该表达基因的工程菌,以此提供一种高效去除有机磷农残的新方法。方法 利用基因克隆获得高效表达农残降解酶基因Oph2,采用基因工程手段将其转化至毕赤酵母表达系统,对毕赤酵母的发酵产物进行SDS-PAGE定性研究和酶活定量研究,最后通过与目标底物甲基对硫磷反应,分析工程菌表达降解酶对有机磷农残的去除效果。结果 本研究成功克隆得到了高效表达有机磷降解酶基因Oph2,基因全长1000bp,编码256个氨基酸,酶蛋白分子量为37KD,并利用表达载体pPIC9K成功将其转化至毕赤酵母GS115。构建的工程菌GS115-pPIC9K-Oph2表达有机磷降解酶活性为11.57 U/mL,对6.8 mg/L的甲基对硫磷降解效率为90%以上。结论 本研究得到的工程菌GS115-pPIC9K-Oph2可高效表达有机磷降解酶,该酶活性高,对有机磷农残降解能力强,可有效应用于有机磷农残的降解。     

重组ZEN降解酶Oxa的纯化及特性研究

以表达可高效降解玉米赤霉烯酮(Zearalenone,ZEN,ZEA)的氧化酶Oxa的毕赤酵母工程菌GS115/p PIC9K-Oxa为研究对象,将发酵液经乙醇沉淀、阴离子交换色谱和超滤纯化后,检测重组Oxa酶降解ZEN的活力,分析其二级结构,并对相关酶学特性进行研究。结果表明,氧化酶Oxa的纯化倍数达到20倍以上,纯化后的Oxa针对ZEN的降解率达到80%以上,其二级结构主要由无规卷曲和β-折叠组成,辅以少量的β-转角和α-螺旋,该酶的最适作用温度和pH分别为60℃和9.0,并且在50～70℃和pH9.0～11.0时保持60%以上的ZEN降解率,具有典型的耐高温碱性酶的特征,反应体系中适量的Cu~(2+)、Fe~(2+)和Fe~(3+)的加入能提高其降解活力。本论文为进一步研究氧化酶Oxa的分子结构及作用方式奠定了基础,对ZEN的生物降解具有显著的实际意义。     

两种强启动子在枯草芽孢杆菌中调控表达研究

通过玉米赤霉烯酮(ZEN)降解酶基因ZLHY6的表达水平及降解酶的活性评价,比较了两种组成型强启动子P43与Plap S调控异源基因表达的效果。结果表明,在枯草芽孢杆菌(Bacillus subtilis)Bs 168中,Plap S调控的降解酶基因得到了高效表达,在发酵12 h时降解酶活性达到最高值,酶活为219.02 U/m L。由启动子Plap S介导的ZEN降解酶基因表达载体p WBZ7可以在Bs 168中稳定遗传,为降解酶的高效分泌表达奠定了基础。     

生物质降解菌系中GH11家族耐热木聚糖酶基因的克隆与鉴定

以玉米芯粉为基质富集培养牛粪中生物质降解菌系,提取该降解菌系的宏基因组DNA,并以其为模板,通过简并引物PCR扩增出200bp左右的木聚糖酶基因片段,选择与耐热木聚糖酶相似性最高且丰度最高的基因序列,应用mTAIL-PCR扩增得到1 059bp的基因全长序列(xynHAD4),编码352个氨基酸,经序列比对分析,该木聚糖酶(XynHAD4)属于典型的GH11家族木聚糖酶,与来源于Dictyoglomus thermophilum的xylanase229B相似度为93%。将xynHAD4连接于表达载体pET22b(+),在E.coli BL21中成功得到表达,表达产物经纯化后,测定其分子质量约为36kDa,最适作用温度和pH分别为80℃和6.0,该酶在90℃保温1h,残余酶活为72%,pH作用范围较广,在pH4.0保存1h,残余酶活为82%,可见,XynHAD4属于耐热木聚糖酶,并且在酸性条件下可保持较高稳定性,在食品、饲料等领域具有潜在的应用价值。     

枯草芽孢杆菌纤维素酶基因整合载体的构建

目的:以枯草芽孢杆菌(Bacillus subtilis)为宿主,构建纤维素酶基因整合表达载体,获得能够表达纤维素酶并且降解纤维素的工程菌。方法:通过聚合酶链式反应(polymerase chain reaction,PCR)从B.subtilis LN基因组中克隆同源片段M1、M2基因片段,以质粒pGEM-T为载体,将同源片段M1、M2、启动子P43和葡萄糖苷酶基因CelKg连接在一起构建整合载体pGEM-Kmpgmt,并采用双交换同源重组的方式将其转化进入B.subtilis LN基因组中。结果:通过PCR和双酶切验证整合载体构建完成,并成功整合到野生型B.subtilis LN中,获得重组菌B.subtilis Kpg。刚果红染色结果显示重组菌对羧甲基纤维素钠有降解作用。改良培养基37℃条件下摇瓶培养,重组菌B.subtilis Kpg生长至18 h时上清液中纤维素酶活力比野生型B.subtilis LN提高了115%。     

清除玉米赤霉烯酮菌株的筛选、鉴定及机理初探

通过对我国赤霉病高发区田间取样,筛选具有清除玉米赤霉烯酮(Zearalenone,ZEN)活性的菌株。获得2株清除效果较好的细菌A5、D6,其中D6活性较高。通过生理特征分析、16S r DNA序列比对、GC含量及BIOLOG分析等方法,2株菌都被鉴定为红球菌,DNA-DNA同源性分析表明,这2株菌均为Rhodococcus qingshengii。灭活细胞与ZEN共培养,发现细胞无清除ZEN能力,证明A5、D6不具有吸附ZEN的活性;菌液上清液及细胞内容物与ZEN共培养,证明D6菌株中具有清除ZEN的活性成分为胞内蛋白类物质。同时,通过HPLC荧光检测,发现疑似ZEN代谢产物峰。通过筛选获得了2株可通过胞内酶活降解ZEN的红球菌,为生物法清除真菌毒素提供参考。     

Development and application of analytical methods for the determination of mycotoxins in organic and conventional wheat.

The aim of this study was to develop a multicomponent analytical method for the determination of deoxynivalenol (DON), ochratoxin A (OTA) and zearalenone (ZEN), nivalenol (NIV), 3-acetyl-DON (3-acDON), 15-acetyl-DON (15-acDON), zearalenol (ZOL) and citrinin (CIT) in wheat. It also aimed to survey the presence and amounts of DON, OTA and ZEN in Belgian conventionally and organically produced wheat grain and in wholemeal wheat flours. After solvent extraction, an anion-exchange column (SAX) was used to fix the acidic mycotoxins (OTA, CIT), whilst the neutral mycotoxins flowing through the SAX column were further purified by filtration on a MycoSep cartridge. OTA and CIT were then analysed by high-performance liquid chromatography (HPLC) using an isocratic flow and fluorescence detection, while the neutral mycotoxins were separated by a linear gradient and detected by double-mode (ultraviolet light fluorescence) detection. The average DON, ZEN and OTA recovery rates from spiked blank wheat flour were 92, 83 and 73% (RSDR = 12, 10 and 9%), respectively. Moreover, this method offered the respective detection limits of 50, 1.5 and 0.05 microg kg-1 and good agreement with reference methods and inter-laboratory comparison exercises. Organic and conventional wheat samples harvested in 2002 and 2003 in Belgium were analysed for DON, OTA and ZEN, while wholemeal wheat flour samples were taken from Belgian retail shops and analysed for OTA and DON. Conventional wheat tended to be more frequently contaminated with DON and ZEN than organic samples, the difference being more significant for ZEN in samples harvested in 2002. The mean OTA, DON and ZEA concentrations were 0.067, 675 and 75 microg kg-1 in conventional samples against 0.063, 285 and 19 microg kg-1 in organically produced wheat in 2002, respectively. Wheat samples collected in 2003 were less affected by DON and ZEN than the 2002 harvest. Organic wholemeal wheat flours were more frequently contaminated by OTA than conventional samples (p < 0.10). The opposite pattern was shown for DON, organic samples being more frequently contaminated than conventional flours (p < 0.10).     

Novel oxidative metabolites of the mycoestrogen zearalenone in vitro

The estrogenic mycotoxin zearalenone (ZEN) is known to get metabolized to the alpha-and beta-isomers of zearalenol, but no hydroxylation products of ZEN have yet been reported as metabolites in animals or humans. We have therefore incubated ZEN with microsomes from rat liver in the presence of a nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-regenerating system and analyzed the extracted metabolites with HPLC and GC-MS after trimethylsilylation. A total of 17 in vitro metabolites were observed. The two major metabolites were tentatively identified as monohydroxylated ZEN with the newly introduced hydroxyl group localized in the aliphatic macrocyclic ring. According to the GC-MS analysis, other six monohydroxylation products of ZEN were formed as minor metabolites, together with alpha-and beta-zearalenol and monohydroxylated zearalenols. Thus, ZEN has a considerable propensity for undergoing metabolic hydroxylation reactions in vitro, and the in vivo formation and biological properties of such oxidative metabolites should now be studied.     

Aromatic hydroxylation is a major metabolic pathway of the mycotoxin zearalenone in vitro

Zearalenone (ZEN) is a common mycotoxin, for which only reductive metabolites have been identified so far. We now report that ZEN is extensively monohydroxylated by microsomes from human liver in vitro. Two of the major oxidative metabolites arise through aromatic hydroxylation and are catechols. Their chemical structures have been unambiguously determined by using deuterium‐labeled ZEN and by comparison with authentic reference compounds. Moreover, both catechol metabolites of ZEN were substrates of the enzyme catechol‐O‐methyl transferase. One of the monomethyl ethers represented the major metabolite when ZEN was incubated with rat liver slices, thus demonstrating that catechol formation also takes place under in vivo‐like conditions. Out of ten major human cytochrome P450 (hCYP) isoforms only hCYP1A2 was able to hydroxylate ZEN to its catechols with high activity. Catechol formation represents a novel pathway in the metabolism of ZEN and may be of toxicological relevance.     

Biochemical Characterization of a Low Trypsin Inhibitor Soybean

A low trypsin inhibitor soybean (LTI) was characterized using electrophoresis, enzyme activity measurements, and gel exclusion chromatography. The protein profiles were similar to a control soybean. Gel exclusion chromatography resulted in two peaks of trypsin inhibitor activity in the control. The first peak, absent in LTI, proved to be Kunitz trypsin inhibitor and was electrophoretically isomorphic. The second inhibitor consisted of at least five isotypes and co-eluted with Bowman-Birk trypsin inhibitor. Shorter heating times were required to inactivate both trypsin and chymotrypsin inhibitor activity in LTI compared to control soybeans. The use of LTI may increase the economic viability of soybeans as protein supplements for humans.     

Aerobic and anaerobic in vitro testing of feed additives claiming to detoxify deoxynivalenol and zearalenone

Deoxynivalenol (DON) and zearalenone (ZEN) are mycotoxins produced by fungi of the genus Fusarium which frequently contaminate maize and grain cereals. Mycotoxin-contaminated feed endangers animal health and leads to economic losses in animal production. Several mycotoxin elimination strategies, including the use of commercially available DON and ZEN detoxifying agents, have been developed. However, frequently there is no scientific proof of the efficacy of such adsorbents and degrading products. We therefore tested 20 commercially available products claiming to detoxify DON and/or ZEN either by biodegradation (4 products) or a combination of degradation and adsorption (16 products) under aerobic and anaerobic conditions at approx. pH 7. Under the applied conditions, a complete reduction of DON and consequent formation of the known non-toxic metabolite DOM-1 was exclusively observed in samples taken from the anaerobic degradation experiment of one product. For all other products, incubated under aerobic and anaerobic conditions, a maximum DON reduction of 17% after 72 h of incubation was detected. Aerobic and anaerobic incubation of only one tested product resulted in complete ZEN reduction as well as in the formation of the less-toxic metabolites DHZEN and HZEN. With this product, 68–97% of the toxin was metabolised within 3 h. After 24 h, a ZEN reduction ≥ 60% was obtained with four additional products during aerobic incubation only. Six of the 20 investigated products produced α- and/or β-ZEL, which are metabolites showing similar oestrogenic activity compared to ZEN. Aerobic and anaerobic degradation to unknown metabolites with unidentified toxicity was obtained with 10 and 3 products, respectively. The results of our study demonstrate the importance of in vitro experiments to critically screen agents claiming mycotoxin detoxification.     

Isolation and determination of zearalenone in rice cultures using liquid chromatography with diode array detection.

A rapid method is described for the isolation and determination of zearalenone (ZEN) produced by Fusarium spp., in moist rice culture. Following a simple solvent extraction using acetonitrile:water, the crude extract was defatted with hexane and diluted with methanol. The extract solution containing ZEN was evaporated to dryness, the residue dissolved in acetonitrile and diluted with water. The solution was analysed by liquid chromatography using a UV-diode array detector. The UV spectra and chromatographic data generated from the standard ZEN was stored in a computer and used to identify the toxin in a crude mixture. The purity of the separated peak and the amount of toxin in the crude mixture was determined. The present technique is fast and allows the acquisition of UV spectral information and chromatographic data of ZEN in a single chromatographic operation. Recovery of zearalenone added to the rice was 76-94%.     

Clostridium cellulolyticum D-塔格糖3-差向异构酶基因的克隆、表达及酶活性

D-塔格糖3-差向异构酶是生物法生产新型功能性因子D-阿洛酮糖最为有效的酶。作者克隆到一种新型的D-塔格糖3-差向异构酶基因,来源于微生物Clostridium cellulolyticumH10。以pET-22b(+)为载体质粒,E.coliBL21(DE3)为宿主细胞,构建了基因重组菌,IPTG可诱导目的蛋白质的过量表达;经亲和层析纯化的重组蛋白质样品进行SDS-PAGE分析,在约31 000处出现显著的特征蛋白质条带;活性检测结果表明:该重组酶具有较高的转化活性。     

Stability of zearalenone during extrusion of corn grits

The effects of extrusion cooking on the stability of zearalenone (ZEN) in spiked (4.4 microg/g) food-grade corn grits were investigated using a twin screw extruder. A ground rice culture material containing a high level of ZEN was used to spike the corn grits. The extrusion variables were screw type (mixing and nonmixing), temperature (120, 140, and 160 degrees C), and moisture content (18, 22, and 26%). Both unextruded and extruded samples were analyzed for ZEN by high-performance liquid chromatography. Extrusion cooking of the corn grits resulted in significant reductions of ZEN in grits extruded with either mixing screws or nonmixing screws, but use of mixing screws was somewhat more effective (66 to 83%) overall than nonmixing screws (65 to 77%). Greater reduction of ZEN was observed at either 120 or 140 degrees C than at 160 degrees C. The moisture content of corn grits was not a significant factor affecting reduction of ZEN during extrusion with either mixing or nonmixing screws.