Rhodococcus sp.BX2菌对乙腈的降解特性及降解途径研究

对Rhodococcus sp.BX2菌降解乙腈的特性及其降解途径进行了研究.结果显示,在底物浓度为800mg·L-1,接种量为1.0%,培养温度为35℃,环境pH为7.5的条件下,16h时Rhodococcus sp.BX2菌对乙腈的降解率为95.98%;添加葡萄糖可在培养初期加快Rhodococcus sp.BX2菌的生长和对乙腈的降解,蔗糖、乙酰胺和尿素对其影响不大.将BX2菌接种到含有高乙腈浓度(25000mg·L-1)的合成废水中,培养180h后,乙腈降解率可达88.59%.在催化反应60min后,Rhodococcus sp.BX2腈水合酶与腈水解酶的总酶活可达到422.81U·mL-1,对其相关基因序列的分析结果表明,Rhodococcus sp.BX2中同时存在腈水解酶基因和腈水合酶基因,因此,确定乙腈的降解主要由腈水合酶途径完成,可能同时存在腈水解酶的降解途径.     

低剂量全氟辛酸对雄性黑斑蛙生殖毒效应及机理研究

针对新型环境有机污染物全氟辛酸(PFOA)对两栖动物的毒效应问题,以黑斑蛙(Rana nigromaculata)为实验对象,研究了低剂量PFOA暴露对雄性黑斑蛙生殖毒效应及机理.结果表明,在0.001～1mg.L-1低剂量体外暴露20d条件下,PFOA能够显著诱导黑斑蛙精子发生畸形,相对于对照组,精子畸形率呈现明显的剂量-效应关系.酶联免疫试剂盒检测发现,除0.001mg.L-1诱导组外,0.01、0.1和1mg.L-1暴露组的血清雄性激素睾酮和雌性激素雌二醇含量分别显著降低(p<0.01,F=288.7)和上升(p<0.01,F=289.0),说明PFOA作用下黑斑蛙体内性激素水平受到改变.采用荧光定量PCR技术进行进一步的分子生物学检测,发现与性激素水平密切相关的两个基因P450芳香化酶和类固醇激素合成因子(SF-1)均增强表达(0.001mg.L-1诱导组,p<0.05;0.01、0.1和1mg.L-1暴露组,p<0.01,F=140.8).全氟辛酸对两栖类动物的雄性生殖毒效应机理主要是通过介导与性激素分泌相关的两个重要基因P450芳香化酶和类固醇激素合成因子,使其表达上调,从而一方面抑制睾酮的产生,另一方面促进性激素雌二醇的分泌.在0.001mg.L-1的体外暴露剂量下,PFOA就能够明显诱导黑斑蛙精子产生畸形毒性及增强P450芳香化酶和SF-1基因的表达(p<0.05),因此,为保护两栖动物种群,在制订PFOA地表水环境质量标准时应当考虑其下限不能低于0.001mg.L-1.     

堆肥反应器中2种微生物接种剂的堆肥效果研究

在严格控制堆肥条件的情况下,以鸡粪为堆肥基本原料,选用单一微生物巨大芽孢杆菌和复合微生物VT菌为接种剂,通过研究堆肥过程中的温度、氧气浓度、C/N、WSC(水溶性碳)、发芽指数(GI)以及脱氢酶和纤维素酶的活性动态变化来反映这2种微生物接种剂对堆肥效果的影响.结果表明,接种复合微生物菌剂VT的处理在堆肥升温和保持高温效果明显,接种微生物菌剂的2个处理的堆体内氧气浓度都较CK低,其中最低的为接种复合微生物菌剂VT的处理.接种微生物菌剂,C/N下降速度快,WSC浓度相对高,GI值上升速度明显加快,有利于加快堆肥腐熟进程,其中接种复合微生物菌剂VT的效果较单一微生物菌剂巨大芽孢杆菌好.接种微生物菌剂有利于堆肥升温期脱氢酶和纤维素酶活性增加,促进堆肥的氧化还原反应和纤维素的分解,且接种复合微生物VT的效果明显.     

Validation of a conductometric bienzyme biosensor for the detection of proteins as marker of organic matter in river samples

This article describes a conductometric bi-layer based bienzyme biosensor for the detection of proteins as a marker of organic matter in rivers. Proteins were chosen to be used as indicators of urban pollution. The working mechanism of the bienzyme biosensor is based on the enzymatic hydrolysis of proteins into several fractions (peptides and amino acids), which results in a local conductivity change depending of the concentration of proteins. In this work, we began with the optimization of biosensor response using bovine serum albumin (BSA) as standard protein. For this objective seven enzymatic biosensors were prepared: four enzymatic sensors with only one layer of enzyme (proteinase K, trypsin, pronase or protease X) and three other enzymatic sensors with two layers (first layer: membrane containing proteinase K; second layer: one of the three other enzymes: trypsin, pronase or protease X). The biosensors were obtained through the deposition of enzymatic layers and the cross-linking process between enzymes and BSA in saturated glutaraldehyde vapour. The response of the various biosensors, described previously, were compared with the values of total organic carbon (TOC), and those of organic nitrogen (Norg), as determined by the laboratory accredits (CEMAGREF of Lyon) using the traditional method of analysis (NF EN 1484, infrared spectroscopy) and (NF EN 25663, mineralization/colorimetry assay) respectively for each water sample obtained from di erent sites in Lyon (France). The linear correlations obtained with the response of the seven biosensors showed the most important indices of correlations for the biosensor with two enzymatic layers: proteinase K + pronase (pkp). The optimum conditions for the preparation of the pkp biosensor increased the sensitivity and gave a limit of quantification of 0.583 g/L for TOC and 0.218 g/L for Norg in water samples. This sensor shows good reproducibility (2.28%), a capacity to be used at temperatures range 10– 30°C (depending on the season) and moreover a long lifetime (5 weeks).     

Effects of nonionic surfactant Triton X-100 on the laccase-catalyzed conversion of bisphenol A

The laccase-catalyzed conversion of bisphenol A (BPA) in aqueous solutions was studied in the absence and presence of nonionic surfactant Triton X-100. It was found that the addition of Triton X-100 into the reaction system increased the conversion of BPA, especially near the critical micelle concentration of Triton X-100. Also it was found that the stability of laccase was greatly improved in the presence of TritonX-100. Studies on the endogenous fluorescence emission of laccase indicated that there existed an interaction between Triton X-100 and laccase, which was beneficial to folding and stabilizating of laccase. The binding of Triton X-100 to the laccase surface also mitigated the inactivation e ect caused by the free radicals and polymerization products. Under otherwise identical conditions, a lower dosage of laccase was needed for the higher conversion of BPA in the presence of Triton X-100.     

超声协同紫外灭活大肠杆菌实验研究

应用紫外、超声、超声协同紫外对大肠杆菌进行灭活,并从生物学角度对协同机理进行探讨。结果表明:实验条件下,紫外辐照剂量与大肠杆菌灭活率之间具有良好的线性关系,各紫外辐照剂量下的E.coli均发生了光复活现象。超声功率密度对于灭活结果具有显著意义(p0.05),研究中超声灭活大肠杆菌的最大相对功效值对应的工况为7.18W/cm2作用80s。超声的协同作用使紫外线消毒动力学方程K值由0.1增至0.1358,减小了细菌对UV的抗性;与单独紫外作用比较,超声与紫外协同作用的光复活率有所降低。超声与紫外同时作用对细胞形态结构产生严重的破坏,超声作用只能改变细胞的结构,而不能破坏其DNA;利用T4-EndoⅤ对CPDs的特异性识别特性,可发现经各工况处理后,大肠杆菌DNA均被降解,产生大量CPDS,且各工况下ESS含量变化曲线与各工况下大肠杆菌灭活率曲线的相关系数达到0.92。     

消油剂对马粪海胆污染效应的影响

以马粪海胆(Hemicentrotus pulcherrimus)为测试对象,比较了0号柴油分散液(WAFs)与加入消油剂后的乳化液(dis-WAFs)的24、48、72、96 h急性毒性效应,并研究了不同浓度的柴油乳化液对马粪海胆消化腺超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性的影响。实验结果表明:0号柴油分散液对马粪海胆幼胆的半致死浓度分别为18.2、15.5、11.5、9.5 mg/L;0号柴油乳化液对马粪海胆幼胆的24、48、72、96 h半致死浓度分别为11.7、9.1、7.4、5.1 mg/L。暴露相同时间时,0号柴油乳化液的毒性大于0号柴油分散液。亚致死浓度的dis-WAFs污染对抗氧化酶活性影响存在剂量-效应关系。同一剂量组随着污染时间的延长,SOD和CAT的活力表现为先诱导后抑制的趋势,受污染个体在污染解除之后,其SOD和CAT酶活性得到恢复。SOD和CAT可以作为海洋底栖环境石油烃污染监测的潜在的生物标志物之一。     

Mn(Ⅱ)氧化细菌的微生物学研究进展

Mn(Ⅱ)氧化细菌广泛存在于自然界中,对其生理特性、氧化机制和功能等的研究,对于进行含锰水处理过程中生物除锰机理的探究具有重大意义.本文就国内外对Mn(Ⅱ)氧化细菌的氧化机理、酶学研究等进展进行了总结.普遍认为,Mn(Ⅱ)的氧化机理可分为:(1)间接氧化,即微生物通过新陈代谢作用改变自身微环境,如pH、Eh,从而实现Mn(Ⅱ)的化学氧化;(2)直接氧化,即锰氧化酶的直接催化氧化或通过特定的键合作用实现氧化.目前酶学研究中所涉及到的酶有:木质素过氧化酶、锰过氧化物酶、漆酶、木质素降解酶等,所涉及到的微生物有:生盘纤发菌、杆状菌、土微菌属、假单胞菌、真菌等.本文同时也提出了Mn(Ⅱ)氧化细菌的微生物学研究中存在的问题,对进一步的研究作了展望.表1参45     

产1,3-丙二醇温控重组大肠杆菌JM109(pBV220-yqhD-dhaB)的构建

1,3-丙二醇(1,3-PD)是一种重要的化工原料,其最重要的用途是作为合成聚酯PTT的单体.由于微生物发酵法生产1,3-PD具有操作简单,不易产生有毒副产物等特点,已得到广泛关注.本研究在前期工作的基础上,分别获得了来源于肺炎克雷伯氏菌的甘油脱水酶编码基因dhaB和来源于大肠杆菌的1,3-PD氧化还原酶同工酶编码基因yqhD,利用温控表达载体pBV220串联构建了重组质粒pBV220-yqhD-dhaB,将其转化大肠杆菌得到产1,3-丙二醇温控重组大肠杆菌JM109(pBV220-yqhD-dhaB).该重组菌在LB培养基中,30℃好氧培养12 h至对数生长中期,再经42℃好氧诱导发酵4 h,测得胞内甘油脱水酶和1,3-丙二醇氧化还原酶同工酶的酶活力分别达到260 U/mg蛋白和140U/mg蛋白;在含甘油40 g/L的发酵培养基中,30℃好氧培养12 h至对数生长中期,再经42℃好氧诱导发酵4 h,测得发酵液中1,3-PD含量为8.5 g/L.这将为进一步构建基因工程菌生产1,3-PD打下坚实的基础.图6表1参18     

一种新的中温酸性α-淀粉酶的分离纯化及酶学性质

从Bacillus sp.中分离纯化了一种新的中温酸性α-淀粉酶,并对其酶学性质进行了研究.粗酶经硫酸铵沉淀、 DEAE-Sepharose Fast Flow阴离子层析、 Sephadex G-75凝胶过滤层析等步骤后获得电泳均一的α-淀粉酶,其分子量(Mr)约为56×103.该酶最适反应pH为5.0,在pH 5.0～11.0范围内稳定,具有较好的耐酸特性.该酶最适反应温度为40～50 ℃,在40～60 ℃范围内稳定.在60 ℃、 pH 4.5时,该酶能快速有效水解可溶性淀粉;在pH 5.0时该酶对多种生淀粉底物也具有良好的水解作用.因此,该酶有助于解决中温条件淀粉加工行业中,液化酶和糖化酶由于适用pH值的差异而无法联用的难题,是一种具有研究价值和应用前景的中温酸性α-淀粉酶.经LC-MASS-MASS分析得到了酶蛋白中两个肽段的氨基酸序列,通过比对发现,该酶与NCBI中已报道的α-淀粉酶序列具有一定的同源性,同时,在氨基酸水平上也存在着差异.