Microbial Oxidation of Hydrocarbons: Properties of a Soluble Methane Monooxygenase from a Facultative Methane-Utilizing Organism, Methylobacterium sp. Strain CRL-26

Methylobacterium sp. strain CRL-26 grown in a fermentor contained methane monooxygenase activity in soluble fractions. Soluble methane monooxygenase catalyzed the epoxidation/hydroxylation of a variety of hydrocarbons, including terminal alkenes, internal alkenes, substituted alkenes, branched-chain alkenes, alkanes (C₁ to C₈), substituted alkanes, branched-chain alkanes, carbon monoxide, ethers, and cyclic and aromatic compounds. The optimum pH and temperature for the epoxidation of propylene by soluble methane monooxygenase were found to be 7.0 and 40°C, respectively. Among various compounds tested, only NADH₂ or NADPH₂ could act as an electron donor. Formate and NAD⁺ (in the presence of formate dehydrogenase contained in the soluble fraction) or 2-butanol in the presence of NAD⁺ and secondary alcohol dehydrogenase generated the NADH₂ required for the methane monooxygenase. Epoxidation of propylene catalyzed by methane monooxygenase was not inhibited by a range of potential inhibitors, including metal-chelating compounds and potassium cyanide. Sulfhydryl agents and acriflavin inhibited monooxygenase activity. Soluble methane monooxygenase was resolved into three components by ion-exchange chromatography. All three compounds are required for the epoxidation and hydroxylation reactions.     

碳源对固定化细胞生产柠檬酸的影响

柠檬酸是利用微生物代谢生产的一种极为重要的有机酸．广泛应用于食品、饮料、化工、冶金、印染等各个领域。在国外,近10年来,利用固定化细胞生产柠檬酸已获得较广泛的研究^〔1－6〕,国内也有学者指出,柠檬酸发酵的趋向是利用固定化细胞进行连续化生产⑺。而国内这方面的研究报道很少〔8,9〕。我们利用海藻酸钙凝胶包埋固定化黑曲霉细胞生产柠檬酸．探讨了碳源种类及其浓度对固定化细胞生产柠檬酸的影响。现将结果报道如下。     

引入非天然氨基酸胶原蛋白表达及交联成键的优化

微生物重组表达胶原蛋白来源清洁,同时具有序列设计灵活和高产量高纯度等优点,作为生物材料在组织工程等领域具有广泛的应用前景。然而如何促进重组胶原分子交联,使其形成更加稳定的空间结构是设计重组胶原纳米材料需要克服的难点。文中通过双质粒系统将非天然氨基酸O-(2-溴乙基)-酪氨酸引入细菌胶原蛋白序列中,并对其发酵条件进行优化,结果表明在25 ℃下,以终浓度为0.5 mmol/L的IPTG和0.06%的阿拉伯糖诱导24 h可以获得高纯度含非天然氨基酸的胶原蛋白。将含非天然氨基酸的胶原蛋白与含半胱氨酸的胶原蛋白在pH为9.0的NH4HCO3缓冲液中进行交联,形成了最大分子粒径可达1 μm的聚集体,为重组胶原蛋白生物材料的设计提供了新思路。     

锌对体外应激海马神经元MTs亚型表达的影响

目的:观察不同锌水平对体外应激海马神经元金属硫蛋白(MTs)亚型表达的影响。方法:取新生1dWis-tar大鼠海马组织进行体外神经元培养,无血清培养24h后,分别向培养液中加入皮质酮、Zn2+特异鳌合剂TPEN,使二者的最终浓度均为1×10-5mol/L,然后加入不同浓度的ZnSO4溶液,使Zn2+的最终浓度分别为1×10-5mol/L、1×10-4mol/L和2×10-4mol/L,作用24h后检测培养液中IL-6和NO含量,以蛋白印迹法检测细胞MTs含量,以RT-PCR检测细胞MT-1mRNA和MT-3mRNA的表达水平。结果:在海马神经元培养液中加入TPEN后,MTs的表达出现明显降低,皮质酮刺激也未见其表达升高。在补锌组,MTs的含量均明显增加,其中以10-4mol/LZn2+组的表达量最高。海马神经元MT-1mRNA和MT-3mRNA的表达水平在皮质酮应激组和补锌组均出现明显升高。另外,锌缺乏和皮质酮刺激可使海马神经元培养上清中的IL-6和NO水平均出现明显升高。结论:不同锌水平对应激海马神经元金属硫蛋白及其亚型mRNA的表达具有调控作用,缺锌可降低金属硫蛋白的表达,而补锌可增加金属硫蛋白的表达。     

Comparative proteome analysis of Aspergillus oryzae 3.042 and A. oryzae 100-8 strains: Towards the production of different soy sauce flavors

Aspergillus oryzae plays a central role in soybean fermentation, particularly in its contribution to the flavor of soy sauce. We present a comparative assessment of the intracellular differences between wild-type strain 3.042 and mutant strain A100-8, at the proteome level. 522 different protein spots were identified by MALDI-TOF MS, with 134 spots being confirmed by MALDI-TOF MS/MS. Of these, 451 were differentially expressed proteins (DEPs). There was at least a two-fold increase for 288 spots, and at least a two-fold decrease for 163 spots, in strain A100-8 when compared to 3.042. Further analysis showed that 63 of the more abundant proteins were involved in glycolysis and the citrate cycle; 43 more abundant proteins and 10 less abundant proteins were related to amino acid biosynthesis and metabolism; two of the more abundant proteins were involved in vitamin biosynthesis; and five of the more abundant proteins and four of the less abundant proteins were related to secondary metabolites. Moreover, quantitative real time PCR showed that the mRNA expression levels of six typical genes we selected were consistent with changes in protein expression. We postulate that there may be a relationship between DEPs and the flavor formation mechanism in A. oryzae.     

A novel carbonyl reductase from Pichia stipitis for the production of ethyl (S)-4-chloro-3-hydroxybutanoate

An NADPH-dependent carbonyl reductase (PsCR) gene from Pichia stipitis was cloned. It contains an open reading frame of 849 bp encoding 283 amino acids whose sequence had less than 60% identity to known reductases that produce ethyl (S)-4-chloro-3-hydroxybutanoates (S-CHBE). When expressed in Escherichia coli, the recombinant PsCR exhibited an activity of 27 U/mg using ethyl 4-chloro-3-oxobutanoate (COBE) as a substrate. Reduction of COBE to (S)-CHBE by transformants in an aqueous mono-phase system for 18 h, gave a molar yield of 94% and an optical purity of the (S)-isomer of more than 99% enantiomeric excess.     

Complete genome sequence of the bacterium Ketogulonicigenium vulgare Y25

Ketogulonicigenium vulgare is characterized by the efficient production of 2KGA from L-sorbose. Ketogulonicigenium vulgare Y25 is known as a 2-keto-L-gulonic acid-producing strain in the vitamin C industry. Here we report the finished, annotated genome sequence of Ketogulonicigenium vulgare Y25.     

Genetic Manipulation of Sex Ratio for the Large-Scale Breeding of YY Super-Male and XY All-Male Yellow Catfish (Pelteobagrus fulvidraco (Richardson))

Yellow catfish has become one of the most important freshwater aquaculture species in China. The mono-sex male yellow catfish has important application value in aquaculture because the male grows generally faster than the sibling females under the same conditions. This study has screened YY super-male and YY physiological female yellow catfish by sex reversal, gynogenesis, and progeny testing, which can help to achieve the large-scale production of YY super-male and XY all-male. From 2008 to 2010, about 123,000 YY super-male were produced, and about 81 million XY all-male fry were produced with 100 % male rate by random sampling. Therefore, these results indicate that YY super-male and YY physiological female yellow catfish can be viable and fertile. We conclude that the mono-sex breeding technique by YY super-male yellow catfish is stable and reliable, which has great potential for application in yellow catfish aquaculture.     

Comparative proteomics analysis of silkworm hemolymph during the stages of metamorphosis via liquid chromatography and mass spectrometry

The silkworm is a lepidopteran insect that has an open circulatory system with hemolymph consisting of blood and lymph fluid. Hemolymph is not only considered as a depository of nutrients and energy, but it also plays a key role in substance transportation, immunity response, and proteolysis. In this study, we used LC‐MS/MS to analyze the hemolymph proteins of four developmental stages during metamorphosis. A total of 728 proteins were identified from the hemolymph of the second day of wandering stage, first day of pupation, ninth day of pupation, and first day as an adult moth. GO annotations and categories showed that silkworm hemolymph proteins were enriched in carbohydrate metabolism, proteolysis, protein binding, and antibacterial humoral response. The levels of nutrient, immunity‐related, and structural proteins changed significantly during development and metamorphosis. Some, such as cuticle, odorant‐binding, and chemosensory proteins, showed stage‐specific expression in the hemolymph. In addition, the expression of several antimicrobial peptides exhibited their highest level of abundance in the hemolymph of the early pupal stage. These findings provide a comprehensive proteomic insight of the silkworm hemolymph and suggest additional molecular targets for studying insect metamorphosis.     

Microbial biosensors: a review

A microbial biosensor is an analytical device which integrates microorganism(s) with a physical transducer to generate a measurable signal proportional to the concentration of analytes. In recent years, a large number of microbial biosensors have been developed for environmental, food, and biomedical applications. Starting with the discussion of various sensing techniques commonly used in microbial biosensing, this review article concentrates on the summarization of the recent progress in the fabrication and application of microbial biosensors based on amperometry, potentiometry, conductometry, voltammetry, microbial fuel cell, fluorescence, bioluminescence, and colorimetry, respectively. Prospective strategies for the design of future microbial biosensors will also be discussed.