A Tryptophan 6‐Halogenase and an Amidotransferase Are Involved in Thienodolin Biosynthesis

The biosynthetic gene cluster for the plant growth‐regulating compound thienodolin was identified in and cloned from the producer organism Streptomyces albogriseolus MJ286‐76F7. Sequence analysis of a 27 kb DNA region revealed the presence of 21 ORFs, 14 of which are involved in thienodolin biosynthesis. Three insertional inactivation mutants were generated in the sequenced region to analyze their involvement in thienodolin biosynthesis and to functionally characterize specific genes. The gene inactivation experiments together with enzyme assays with enzymes obtained by heterologous expression and feeding studies showed that the first step in thienodolin biosynthesis is catalyzed by a tryptophan 6‐halogenase and that the last step is the formation of a carboxylic amide group catalyzed by an amidotransferase. The results led to a hypothetical model for thienodolin biosynthesis.     

Predicting the Substrate Scope of the Flavin-Dependent Halogenase BrvH

The recently described flavin-dependent halogenase BrvH is able to catalyse both the bromination and chlorination of indole, but shows significantly higher bromination activity. BrvH was annotated as a tryptophan halogenase, but does not accept tryptophan as a substrate. Its native substrate remains unknown. A predictive model with the data available for BrvH was analysed. A training set of compounds tested in vitro was docked into the active site of a complete protein model based on the X-ray structure of BrvH. The atoms not resolved experimentally were modelled by using molecular mechanics force fields to obtain this protein model. Furthermore, docking poses for the substrates and known non-substrates have been calculated. Parameters like distance, partial charge and hybridization state were analysed to derive rules for predicting activity. With this model for activity of the BrvH, a virtual screening suggested several structures for potential substrates. Some of the compounds preselected in this way were tested in vitro, and several could be verified as convertible substrates. Based on information on halogenated natural products, a new dataset was created to specifically search for natural products as substrates/products, and virtual screening in this database yielded further hits.     

Improving the Stability and Catalyst Lifetime of the Halogenase RebH By Directed Evolution

We previously reported that the halogenase RebH catalyzes selective halogenation of several heterocycles and carbocycles, but product yields were limited by enzyme instability. Here, we use directed evolution to engineer an RebH variant, 3‐LR, with a T_opt over 5 °C higher than that of wild‐type, and 3‐LSR, with a T_m 18 °C higher than that of wild‐type. These enzymes provided significantly improved conversion (up to fourfold) for halogenation of tryptophan and several non‐natural substrates. This initial evolution of RebH not only provides improved enzymes for immediate synthetic applications, but also establishes a robust protocol for further halogenase evolution.     

A Statistical Analysis of the Sequence and Structure of Thermophilic and Non-Thermophilic Proteins

Thermophilic proteins have various practical applications in theoretical research and in industry. In recent years, the demand for thermophilic proteins on an industrial scale has been increasing; therefore, the engineering of thermophilic proteins has become a hot direction in the field of protein engineering. However, the exact mechanism of thermostability of proteins is not yet known, for engineering thermophilic proteins knowing the basis of thermostability is necessary. In order to understand the basis of the thermostability in proteins, we have made a statistical analysis of the sequences, secondary structures, hydrogen bonds, salt bridges, DHA (Donor–Hydrogen–Accepter) angles, and bond lengths of ten pairs of thermophilic proteins and their non-thermophilic orthologous. Our findings suggest that polar amino acids contribute to thermostability in proteins by forming hydrogen bonds and salt bridges which provide resistance against protein denaturation. Short bond length and a wider DHA angle provide greater bond stability in thermophilic proteins. Moreover, the increased frequency of aromatic amino acids in thermophilic proteins contributes to thermal stability by forming more aromatic interactions. Additionally, the coil, helix, and loop in the secondary structure also contribute to thermostability.     

高温菌及其在有机废液处理中的应用

目前食品工业和农业排放的有机废液,未经处理或很少处理就被排放到河流中或环境中,其污染给人造成了巨大的直接和间接的危害.与传统的处理高浓度废水工艺相比,利用高温菌进行的好氧处理系统具有一系列优点:(1)具有更快的生物降解速率,高温下微生物具有更高的活性,代谢速度更快;(2)更高的废水处理效率;(3)残留生物固形物浓度很低;(4)能有效地杀灭病原菌.但是由于高温改变了水体的生理生化性质,使得处理过程更为复杂.阐述了高温菌和高温菌耐热机制,以及在高温环境下处理有机废液的优缺点和动力学方程,列举了部分高温废液处理的实例,对其应用前景进行了初步的探讨.     

Halogenase Engineering for the Generation of New Natural Product Analogues

Halogenases catalyze the incorporation of halogen atoms into organic molecules. Given the importance that halogenation has on the biological activity of small molecules, these enzymes have been subjected to intense engineering efforts to make them more suitable for biotechnology applications. The ability to biohalogenate complex molecules provides, in principle, the opportunity for rapid generation of a series of analogues with new or improved properties. Here we discuss the potential and limitations of using halogenases as biocatalysts, including recent advances in engineering halogenases to generate halogenated natural product analogues.     

嗜热菌在废水处理中的应用及研究进展

介绍了嗜热菌的分类、耐热机制及其在废水处理中的应用.通过实例阐明了利用嗜热菌处理废水的可行性.同时介绍了高温好氧工艺、高温厌氧工艺及其组合工艺的优缺点,并展望了今后的研究方向.     

Detoxification of Synthetic Pyrethroid Insecticides by Thermophilic Microorganisms

Microbially-mediated detoxification of five synthetic pyrethroid insecticides was achieved by a mixed culture growing anaerobically at 75°C. These pyrethroids were hydrolysed to non-insecticidal products after a period of 4–8 weeks' incubation. Of these five permethrin was the most readily transformed and Fastac the most recalcitrant. The mixed culture consisted of a facultative anaerobe and two other obligate anaerobes including a methanogen. This culture was derived from terrestrial sediments from hot-springs in New Zealand. This is the first reported microbial biotransformation of these xenobiotics at a temperature of 75°C. © 1997 SCI.     

More than just a Halogenase: Modification of Fatty Acyl Moieties by a Trifunctional Metal Enzyme

The highly selective oxidative halogenations by non‐heme iron and α‐ketoglutarate‐dependent enzymes are key reactions in the biosynthesis of lipopeptides, and often bestow valuable bioactivity to the metabolites. Here we present the first biochemical characterization of a putative fatty acyl halogenase, HctB, which is found in the hectochlorin biosynthetic pathway of Lyngbya majuscula. Its unprecedented three‐domain structure, which includes an acyl carrier protein domain, allows self‐contained conversion of the covalently tethered hexanoyl substrate. Structural analysis of the native product by ¹³C NMR reveals high regioselectivity but considerable catalytic promiscuity. This challenges the classification of HctB as a primary halogenase: along with the proposed dichlorination, HctB performs oxygenation and an unprecedented introduction of a vinyl‐chloride moiety into the nonactivated carbon chain. The relaxed substrate specificity is discussed with reference to a molecular model of the enzyme–substrate complex. The results suggest that fatty acyl transformation at the metal center of HctB can bring about considerable structural diversity in the biosynthesis of lipopeptides.     

Purification and Characterization of Iso-Ribonucleases from a Novel Thermophilic Fungus

A thermophilic fungus previously isolated from composted horse manure was found to produce extracellular iso-RNases that were purified 127.6-fold using a combination of size exclusion chromatography and a novel affinity membrane purification system. The extent of purification was determined electrophoretically using 4%–15% gradient polyacrylamide gels. RNase activity was dependent on the presence of a metal co-factor with significantly more activity with Zn²⁺ or Mn²⁺ than Mg²⁺. The RNases exhibited maximum activity at both pH 3.0 and pH 7.0 with no activity at pH 2.0 or 10.0. The optimal temperature for the iso-RNase was 70 °C. The molecular weight of the iso-RNase was determined to be 69 kDa using a Sephadex G-75 column.     

多粘类芽孢杆菌极端嗜热多肽的纯化及性质研究

用加热法从多粘类芽孢杆菌(Paenibacillus polymyxa)LM–3菌株的发酵液中纯化得到2个极端嗜热多肽。平板拮抗实验表明,5μL纯化多肽对稻瘟病菌(Magnaporthe grisea)抑菌率达89.6%。SDS–PAGE电泳显示纯化多肽分子量介于6000～7000u之间。多肽复性后,其中之一对稻瘟病菌表现出强的拮抗活性,命名为APPLM3(Antagonism Polypeptide from Paenibacillus polymyxa LM–3);另一个则无此活性,命名为PPLM3(Polypeptide from Paenibacillus polymyxaLM–3)。APPLM3经氨基酸测序,获得了其N–末端5个氨基酸序列(H2N–ANDPR);以该序列为靶序列在NCBI上进行相似性检索,发现其可能与3个假设蛋白(或推导蛋白)相关。APPLM3为首次报道的兼具极端嗜热性和稻瘟病菌拮抗活性的小分子多肽。     

Thermophilic biotrickling filtration of a mixture of isobutyraldehyde and 2‐pentanone

In this study, the possibility of the removal of isobutyraldehyde and 2‐pentanone was investigated in biotrickling filters (BTFs) at higher temperature (52–65 °C). First, the biodegradation of isobutyraldehyde and 2‐pentanone in activated sludge was proven by batch experiments at 52 and 62 °C. In batch experiments isobutyraldehyde was also degraded up to a temperature of 72 °C. Thereafter two bioreactors were operated in parallel, one at ambient temperature (BTF25), and one at 52 °C (BTF52). Maximum elimination capacities of 97 and 139 g m^?3 h^?1 were observed in BTF25 and BTF52, respectively, for isobutyraldehyde. Maximum elimination capacities of 53 and 63 g m^?3 h^?1 were obtained for 2‐pentanone in BTF25 and BTF52, respectively. A significant difference was observed in the operational stability of the two reactors. In the reactor at ambient temperature, operational problems such as foam formation, higher biomass accumulation and organic acid production were observed. In the thermophilic reactor these problems did not occur or were less severe. Copyright © 2007 Society of Chemical Industry     

Effect of Hydraulic Retention Time on Sludge Properties,Cake Layer Structure,and Membrane Fouling in a Thermophilic Submerged Aerobic Membrane Bioreactor

Sludge properties, cake layer structure, and membrane fouling in a membrane bioreactor were studied under various hydraulic retention times (HRT). A decrease in HRT resulted in an increase in extracellular polymeric substance production in bulk sludge and changes in cake layer structure from gel layer to one or two cake layers. Particle size distribution in cake sludge changed with respect to HRT. An evolution in cake layer thickness and porosity was observed with trans-membrane pressure (TMP) jump. The change in cake layer structure might bear more responsibility for the TMP jump than the quantity of cake layer.     

耐热石油降解混合菌群的降解性能研究

针对极端环境下石油污染土壤的生物修复,从克拉玛依油田石油污染土壤中筛选获得一组最适生长温度为45～55℃、最高耐受温度为75℃的耐热石油降解混合菌群KLO-8,该混合菌群由4株单菌组成,16S rDNA序列分析鉴定表明,其中3株为芽孢杆菌(Bacillus sp.)、1株为地芽孢杆菌(Geobacillus sp.)....     

Purification of an Inducible DNase from a Thermophilic Fungus

The ability to induce an extracellular DNase from a novel thermophilic fungus was studied and the DNAse purified using both traditional and innovative purification techniques. The isolate produced sterile hyphae under all attempted growing conditions, with an average diameter of 2 μm and was found to have an optimal temperature of 45 °C and a maximum of 65 °C. Sequencing of the internal transcribed region resulted in a 91% match with Chaetomium sp., suggesting a new species, but further clarification on this point is needed. The optimal temperature for DNase production was found to be 55 °C and was induced by the presence of DNA and/or deoxyribose. Static growth of the organism resulted in significantly higher DNase production than agitated growth. The DNase was purified 145-fold using a novel affinity membrane purification system with 25% of the initial enzyme activity remaining. Electrophoresis of the purified enzyme resulted in a single protein band, indicating DNase homogeneity.     

Identification and Biosynthesis of New Acyloins from the Thermophilic Bacterium Thermosporothrix hazakensis SK20‐1T

Two new acyloin compounds were isolated from the thermophilic bacterium Thermosporothrix hazakensis SK20‐1^T. Genome sequencing of the bacterium and biochemical studies identified the thiamine diphosphate (TPP)‐dependent enzyme Thzk0150, which is involved in the formation of acyloin. Through extensive analysis of the Thzk0150‐catalyzed reaction products, we propose a putative reaction mechanism involving two substrates: 4‐methyl‐2‐oxovalerate as an acyl donor and phenyl pyruvate as an acyl acceptor.     

Hydrogen Production by the Thermophilic Bacterium Thermotoga neapolitana

As the only fuel that is not chemically bound to carbon, hydrogen has gained interest as an energy carrier to face the current environmental issues of greenhouse gas emissions and to substitute the depleting non-renewable reserves. In the last years, there has been a significant increase in the number of publications about the bacterium Thermotoga neapolitana that is responsible for production yields of H₂ that are among the highest achievements reported in the literature. Here we present an extensive overview of the most recent studies on this hyperthermophilic bacterium together with a critical discussion of the potential of fermentative production by this bacterium. The review article is organized into sections focused on biochemical, microbiological and technical issues, including the effect of substrate, reactor type, gas sparging, temperature, pH, hydraulic retention time and organic loading parameters on rate and yield of gas production.     

Stability and Stabilization of Enzymes from Mesophilic and Thermophilic Organisms in Respect to Their Use in FIA-Systems for the Determination of L-Lactate and Acetate

The stabilization effect of ‘bilayer encagement’ on enzymes from mesophilic organisms and their ‘thermophilic’ counterparts was compared. Lactate dehydrogenases from pig heart and from a thermophilic bacterium (Clostridium thermohydrosulfuricum), respectively, showed stabilization factors of 4·5 (at 47°C) and 12·8 (at 70°C), respectively. For ‘thermophilic’ acetate kinase no stabilization effect of encagement was observed. Lactate dehydrogenase and acetate kinase from Clostridium thermohydrosulfuricum were immobilized to controlled porous glass and tested for their long-term stability. The ‘thermophilic’ enzymes showed by far a longer half-life as compared with the corresponding enzymes from pig heart and Escherichia coli, respectively, the half-life time of the flow injection system response with thermophilic lactate dehydrogenase at 35°C attaining 250 h (mesophilic enzyme 89 h), and with thermophilic acetate kinase 79 h (mesophilic enzyme 24 h). © 1997 SCI.