磁化水处理菌种在甾体微生物转化过程中的效应*

考察了磁化水处理菌种在甾体微生物羟化转化反应中的效应。磁化处理条件为：磁场强度０．２４～０．２５Ｔ、静置式磁化、作用时间３０ｍｉｎ。研究表明经磁化水处理的绿僵菌菌种,完成１１ａ羟化转化甾体底物１６ａ,１７ａ－环氧黄体酮的能力有明显改善,其效应与添加适量生长调节剂时相当,两者配合使用、提前投入底物时效果加强。这有利于发酵周期的缩短。磁化处理后菌种的优良特性可在传代中保持至第３代。     

Gibberella intermedia C2转化4-雄甾烯-3、17-二酮的研究

甾体化合物具有独特的生理活性,已被广泛应用于抗炎、利尿、免疫、避孕及抗癌等领域。近些年,生物催化与转化在甾体药物中间体合成中发挥的作用日益强大。为了能够合成一些具有潜在价值的新型甾体化合物,以实验室菌种库中保藏的一株Gibberella intermedia C2为研究对象,选取了雄甾烷中一种有广泛用途的化合物4-雄甾烯-3、17-二酮(简称雄烯二酮,AD)为底物进行生物转化。转化液经提取分离,最终获得2个转化产物,经结构鉴定分别为15α-OH-AD和11α,15α-diOH-AD。转化机制研究发现,G.intermedia C2先将底物的15位羟基化生成15α-OHAD,再将其11位羟基化形成双羟基产物。赤霉菌能够特异性、有序地完成对AD的两步羟化反应。此外,通过工艺优化,确定了羟化4AD反应的最适工艺参数如下:发酵培养基的初始pH 6.5,装液量30ml/250ml,底物浓度6.0g/L,转化温度28℃,摇床转速220r/min,转化周期为84h。此时,底物AD的摩尔转化率达到81.5%。     

甾体生物转化技术研究的现状与进展

从半合成原料、菌种选育及改良和生物转化新技术与新工艺(包括底物的物理/化学助溶法,新型转化体系和细胞通透性改良法)等方面对近几年来甾体生物转化进展进行综述。可以预测,在甾体药物的工业化生产过程中,生物转化技术所占比例将大幅度提高。     

拟结晶投料环氧黄体酮犁头霉羟化过程研究

环氧黄体酮羟化产物是多种甾体激素药物的中间体,其11-羟化过程利用犁头霉(Absidiacoerulea)所得转化率远高于其他菌株。采用拟结晶投料方式,将环氧黄体酮颗粒细化后加一定量水,β-环糊精,吐温-80,超声波乳化后投入发酵液中。这种投料方式可避免传统投料中使用有机溶剂毒害细胞的缺点,更利于底物转化。采用多层前传神经网络建立培养基和投料成分配比与转化率关系模型,并将具有全局寻优性能的粒子群优化算法(PSO)应用于培养基和投料成分配比的优化,收敛速度快,效果好。在优化的操作条件下,摇瓶中投料浓度为10g/L时底物转化率达到87.5%,在3.7L发酵罐中投料浓度提高到20g/L时底物转化率仍高达86.6%。     

微生物转化雄甾-4-烯-3，17-双酮的菌种筛选和转化条件的初步研究

雄甾-4-烯-3,17-双酮(简称4AD)是甾体药物的重要中间产物,其11α羟化产物可制成治疗心血管疾病的药物。通过对30株不同种属真菌转化4AD能力的筛选,获得球孢白僵菌(Beauveria bassiana)QY2A对4AD有高效C11α羟化能力,得到目标产物C11α-羟基雄甾-3,17-双酮(简称11α-OH-4AD)。另对该菌株的转化条件进行优化,结果表明:初始pH值6.0,温度28℃,转速180r/min,转化时间60h,助溶剂甲醇终浓度和底物浓度分别为2.5%和2.5g/L时,11α-OH-4AD的转化率为65%,比未优化的转化率提高了51.2%。     

双菌多轮序列协同转化甾体制氢化可的松

建立了蓝色犁头霉AS 3．65和新月弯孢霉AS 3．4381协同多轮转化17α-羟基孕甾-4-烯-3,20-二酮-21-醋酸酯（RSA）割氢化可的松（hydrocortisone,HC）新工艺。在培养好的AS 3．65和AS 3．4381所组成的协同转化体系中,AS 3．65首先将RSA水解为脱氧皮质酮（RS）,这较AS 3．4381单轮批次转化省去了RSA到RS的化学水解工序。在甾体底物RSA平均投料质量浓度为1．3g/L和1g／L的条件下,所选定的协同转化体系可分别被重复利用3轮和6轮,相应的平均产率能维持在较高水平,分别高达81．6％和85％。另外,该工艺明显减少了底物RSA投料浓度对C11位羟化的影响,并有效抑制了AS 3．4381和AS 3．65单独转化过程中出现的14α—OH—RS和11α-OH—RS副产物．     

多胺对裸大麦离体叶片活性氧代谢的影响

裸大麦离体叶片分别在光照和暗诱导下,以腐胺、亚精胺和精胺等３种多胺,分别用２ｍｍｏｌ／Ｌ,０．５ｍｍｏｌ／Ｌ,和０．２ｍｍｏｌ／Ｌ３种浓度处理,均使丙二醛累积减少,延缓过氧化氢酶和ＳＯＤ活性的下降。以ＣａＣｌ２（５ｍｍｏｌ／Ｌ）＋Ｓｐｄ（０．５ｍｍｏｌ／Ｌ）处理,可降低Ｓｐｄ（０．５ｍｍｏｌ／Ｌ）的效应,因此多胺延缓离体叶片衰老与活性氧代谢有关,并且进入细胞时,与Ｃａ发生竞争。     

化学制品在发酵工艺中的利用

把微生物直接作催化剂使用的方法,是由于微生物化学转化甾体的试验取得很大成功,才得到广泛证实的。霉菌、细菌或酵母作用于甾体,催化羟化、脱氢、侧链切断等多种反应。其中有几例已在工业上应用。过去这种方法特别在用化学方法     

离子液体-水双相体系中Gibberella intermedia C1双羟化去氢表雄酮(DHEA)

【目的】考察离子液体-水双相体系中赤霉菌(Gibberella intermedia C1)双羟化甾体类底物去氢表雄酮(DHEA)生成三羟基雄甾烯酮(7α,15α-di OH-DHEA)的生物转化过程。【方法】比较5种不同种类的离子液体([Hmim][PF_6]、[Bmim][PF_6]、[Bmim][BF_4]、[Bmim][NTF2]、[Emim][EtSO_4])对底物转化率和产物得率的影响。优化该双相体系中离子液体的浓度、底物的投料浓度及投料时间等。【结果】选择[Emim][EtSO_4]作为构建该体系的离子液体。摇瓶中最适双相体系转化条件为:菌体生长12 h后,向转化培养基中加入0.8%(体积比)的[Emim][Et SO4],同时投加6 g/L底物DHEA。在5 L发酵罐上,当转化至60 h时,产物浓度高达5.03±0.21 g/L,7α,15α-diOH-DHEA产物摩尔得率达到最高75.5%。【结论】确定了离子液体-水双相转化体系的最适转化条件,并在5 L发酵罐中进行了实验,为该体系的工业化应用奠定了基础。     

梨头霉11α—羟基化制备16β—甲基—11α,17α21—三烃基孕甾—1, …

选育到一株对１６β－甲基－１７α,２１－二羟基孕甾－１,４＝－二烯－３,２０－二酮（Ⅱａ）１１α－羟基化活性强的梨头霉Ａ２８菌株,并发现底物２１－乙酰化（Ⅱｂ）可明显提高１１α－羟工 能力。在适宜的转化条件下,１１ｂ投料浓度０．５％,产物１６β－基１１α,１７α,２１－三羟基孕甾－１,４－二烯－３,２０－二酮（Ⅲ）收率为７３％,结构经波谱分析确认。     

磁化水浸种对玉米生物学效应的初步研究

应用不同磁场强度和不同磁化时间的磁化水浸泡玉米（ZeamaysL．）种子12h,经Knop营养液砂培,对其萌发和幼苗的生长进行了测定。结果表明,磁水处理后的发芽势、发芽率、种子根数、根长、单株叶面积和根苗比显著增加,叶片失水率和低温伤害后的相对电导率显著降低。磁化时间和强度在10-25min和0．10-0．25T（特斯拉）范围内都可促进玉米种子的萌发和幼苗的生长,以10min,0．15T的磁处理方式作用最显著。     

磁处理与超氧化物歧化酶的关系

本文研究了磁处理对番茄早期发育过程中超氧化物歧化酶(Superoxide Dismutase,简称SOD)的影响和关系.结果表明,磁处理不改变SOD同工酸酶带数日,但能提高C带占B,C带活性的比例.经磁处理能提高SOD活性,改变可溶性蛋白含量.试验证明,磁场处理早期效果明显,磁水浇灌显示积累效应,且较稳定.     

Pseudomonas resinovorans SPR1, a newly isolated strain with potential of transforming eugenol to vanillin and vanillic acid

In this study a novel strain was isolated with the capability to grow on eugenol as a source of carbon and energy. This strain was identified as Pseudomonas resinovorans (GenBank accession no. HQ198585) based on phenotypic characterization and phylogenetic analysis of 16S rDNA gene. The intermediates coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and vanillic acid were detected in the culture supernatant during eugenol biotransformation with this strain. The products were confirmed by thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and spectral data achieved from UV-vis, FTIR and mass spectroscopy. Using eugenol as substrate and resting cells of P. resinovorans SPR1, which were harvested at the end of the exponential growth phase, without further optimization 0.24 g/L vanillin (molar yield of 10%) and 1.1g/L vanillic acid (molar yield of 44%) were produced after 30 h and 60 h biotransformation, respectively. The current work gives the first evidence for the eugenol biotransformation by P. resinovorans.     

L(-)-carnitine production using a recombinant Escherichia coli strain

The L(-)-carnitine production by biotransformation using the recombinant strain Escherichia coli pT7-5KE32 has been studied and optimized with crotonobetaine and D(+)-carnitine as substrates. A resting rather than a growing cells system for L(-)-carnitine production was chosen, crotonobetaine being the best substrate. High biocatalytic activity was obtained after growing the cells under anaerobic conditions at 37°C and with crotonobetaine or L(-)-carnitine as inducer. The growth incubation temperature (37°C) was high enough as to activate the heat-inducible λp_L promoter inserted in the plasmid pGP1-2. The best biotransformation conditions were with resting cells, under aerobiosis, with 4 g l⁻¹ and 100 mM biomass and substrate concentrations respectively. Under these conditions the biotransformation time (1 h) was shorter and the L(-)-carnitine yield (70%) higher than previously reported. Consequently productivity value (11.3 g l⁻¹h⁻¹) was highly improved when comparing with other published works. The resting cells could be reused until eight times maintaining product yield levels well over 50% that meant to increase ten times the L(-)-carnitine obtained per gram of biomass.     

Highly efficient conversion of lactate to pyruvate using whole cells of Acinetobacter sp

On an industrial scale, the production of pyruvate at a high concentration from the cheaper lactate substrate is a valuable process. To produce pyruvate from lactate by whole cells, various lactate-utilizing microorganisms were isolated from soil samples. Among them, strain WLIS, identified as Acinetobacter sp., was screened as a pyruvate producer. For the pyruvate preparation from lactate, the preparative conditions were optimized with whole cells of the strain. The cells cultivated in the medium containing 100 mM of l-lactate showed the highest biotransformation efficiency from lactate to pyruvate. The optimized dry-cell concentration, pH, and temperature of reaction were 6 g/L, pH 7.0-7.5, and 30 degrees C, respectively. The influences of ethylenediaminetetraacetic acid (EDTA) and aeration on a biotransformation reaction were carried out under the test conditions. Under the optimized reaction conditions, l-lactate at concentrations of 200 and 500 mM were almost totally stoichiometrically converted into pyruvate in 8 and 12 h, respectively. About 60% of 800 mM of l-lactate was transformed into pyruvate in 24 h. This reduced conversion rate is probably due to the high substrate inhibition in biotransformation.     

Degradation of fluoranthene by a newly isolated strain of <Emphasis Type="Italic">Herbaspirillum chlorophenolicum</Emphasis> from activated sludge

A fluoranthene-degrading bacterial strain FA1 was isolated from activated sludge and identified as Herbaspirillum chlorophenolicum, a newfound bacterial species that can grow well on fluoranthene as sole carbon and energy source. The kinetic characteristic of strain FA1 was tested in the aqueous model system (AMS) and the effects of nonionic surfactants on fluoranthene biodegradation in the AMS were then investigated. Tween 80 exhibited the best solubilization capacity for fluoranthene among three surfactants and its bioavailability decreased with an increase in its concentration and its degradation kinetics fit well with the first-order of power index model. The biotransformation of fluoranthene was greatly improved by Tween 80, and 58.5% fluoranthene degradation was obtained as Tween 80 was 100 mg/l. However, the bioavailability of fluoranthene decreased gradually with the increase of Tween 80 concentration. Bioremediation tests for fluoranthene in soil–water system were designed further to examine the degrading ability of strain FA1 with the presence of indigenous flora or not. The measurements showed that in the presence of indigenous flora, the optimum 30-day fluoranthene degradation in soil–water system reached 77.4%. Evidently, strain FA1 seems both efficient and high-effective and deserves further exploration on the enhanced bioremediation technologies for the treatment of fluoranthene-polluted soil.     

Bioconversion of acrylonitrile to acrylic acid by rhodococcus ruber strain AKSH-84

A new versatile acrylonitrile-bioconverting strain isolated from a petroleum-contaminated sludge sample and identified as Rhodococcus ruber AKSH-84 was used for optimization of medium and biotransformation conditions for nitrilase activity to produce acrylic acid. A simple and rapid HPLC protocol was optimized for quantification of acrylic acid, acrylamide, and acrylonitrile. The optimal medium conditions for nitrilase activity were pH of 7.0, temperature of 30degreesC, agitation of 150 rpm, and inoculum level of 2%. Glycerol as a carbon source and sodium nitrate as the nitrogen source provided good nutritional sources for achieving good biotransformation. Nitrilase activity was constitutive in nature and was in the exponential growth phase after 24 h of incubation under optimal conditions without addition of any inducer. The substrate preference was acrylonitrile and acetonitrile. The present work demonstrates the biotransformation of acrylonitrile to acrylic acid with the new strain, R. ruber AKSH-84, which can be used in green biosynthesis of acrylic acid for biotechnological processes. The nitrilase produced by the isolate was purified and characterized.     

Biotransformation of dl-lactate to pyruvate by a newly isolated Serratia marcescens ZJB-07166

The production of pyruvate using biotransformation from dl-lactate has been recently drawn more and more attentions due to the wide applications of pyruvate in chemicals, drugs, and agrochemicals industries. In the current study, a strain ZJB-07166, which was capable of converting dl-lactate to pyruvate, was newly isolated and characterized and later identified as Serratia marcescens based on the morphology, physiological tests, ATB system and its 16S rDNA sequence. The strain S. marcescens ZJB-07166 was applied in biotransformation of dl-lactate to pyruvate and the detailed time courses for cultivation and biotransformation were investigated. The optimum nitrogen source and carbon source in the microorganism culture for production of lactate dehydrogenase were NH₄Cl and dl-lactate, respectively. The optimum substrate concentration for biotransformation was around 40 mM and EDTA had an obvious stabilizing effect on pyruvate in biotransformation process. The pyruvate production concentration of 210 mM was achieved under the optimum conditions. These results demonstrated that the newly isolated S. marcescens ZJB-07166 was a promising strain for pyruvate production in industrial scale.     

磁化水浸种对西瓜种子萌发及幼苗生理的影响

用不同强度的磁化水(0．05T、0．08T、0．10T、0．15T)浸种西瓜种子研究磁化水对西瓜种子萌发及幼苗生理的影响,结果表明：与对照相比,各种强度磁化水浸种都能提高种子的发芽率、发芽势、发芽指数和幼苗的抗坏血酸含量,其中0．10T磁化水处理后西瓜种子发芽率、发芽势提高幅度最大;对抗坏血酸含量的影响以0．08T处理最明显;经过不同强度磁化水处理后幼苗中的过氧化物酶同工酶酶、酯酶同工酶活性得到不同强度的增强,不同品种表现不同。实验结果说明磁化水处理后能明显提高西瓜种子的萌发能力,并能提高幼苗的各种生理活性。     

Sequential reactors for the removal of endocrine disrupting chemicals by laccase immobilized onto fumed silica microparticles

Abstract

The main objective of this study is the evaluation of the capability of laccase from Myceliophthora thermophila immobilized on fumed silica microparticles (fsMP) for the removal of endocrine disrupting chemicals (EDCs) in two enzymatic reactor configurations. This type of support can also be magnetized to allow the straightforward separation of the biocatalyst under a magnetic field. The support exhibited excellent biocompatibility with the enzyme, superior tolerance to pH and temperature as well as improved stability in comparison with the free enzyme, even in the presence of organic solvents and enzyme inhibitors. The technical feasibility of the removal of EDCs by immobilized laccase was assessed in two types of enzymatic reactors operated in sequential mode: a membrane reactor using fsMP-laccase and a reactor with magnetic separation using magnetized fsMP-laccase. The extent of transformation for the target compounds: bisphenol A (BPA) and 17β-estradiol (E2) was high and comparable to free laccase in both systems (up to 80%). The possibility of reusing the immobilized enzyme, especially for magnetized supports, offers an interesting approach in the development of enzyme based processes for the biotransformation of emerging pollutants.