低温脂肪酶的产酶条件优化及其酶学性质

利用单因素筛选和正交试验对Burkholderia sp. SYBC LIP-Y发酵产酶的液体培养基和发酵条件进行了优化,其优化配方为：可溶性淀粉10 g/L、牛肉膏15 g/L、NaNO3 0.252 g/L、橄榄油40ml/L、Triton x-100 10ml/L、初始pH 7.5、接种量10%(V/V),脂肪酶酶活达到85.23U/ml,是优化前的3.63倍。通过对双水相纯化得到的脂肪酶进行酶学性质研究,确定该酶反应的最适pH为10.0,最适温度为30℃,40℃下保温60min酶活性还有80%以上,该脂肪酶为低温脂肪酶,热稳定性好,具有一定的耐醇性,应用前景广阔。     

Melanin Standard Method: Particle Description

Melanin isolated from the ink sac of Sepia officinalis (Sepia melanin) has been proposed as a standard for natural eumelanin. There are no standard methods for the isolation, purification, and storage of melanins. Mild methods designed to preserve the native composition and structure of melanin are needed. The specific aim of the present work, using Sepia melanin, was to develop a mild and generally applicable protocol for the isolation and purification of melanins. It is well established that melanin polymers contain a large number of free carboxylic acid residues. These anionic residues are responsible for the cation exchange properties observed for melanins. Heating melanins with hydrochloric acid at reflux has been demonstrated to lead to extensive decarboxylation. Indeed, heat alone has been shown to cause decarboxylation, and care must be exercised to avoid such conditions. By analogy with cation exchange resins, melanins should be isolated and named according to the associated counterion (e.g., Sepia melanin—K⁺ form). The method reported here avoided extremes in pH and temperature, and was designed to yield melanin in the K⁺ form. Physical disaggregation of particulate melanin using a wet milling step was also found to facilitate removal of significant quantities of adsorbed protein. The following physical parameters were used to monitor the purification and to characterize the resultant melanin: pH, conductance, particle size, and diffuse reflectance spectroscopy.     

Enzymatic degradation of the antibiotic sulfamethazine by using crude extracts of different halophytic plants

Abstract

The biodegradation of the antibiotic sulfamethazine (SMT) by using different crude extracts of halophytes was investigated. For this purpose, crude water extracts of the halophytes Chenopodium quinoa, Sesuvium portulacastrum, and Tripolium pannonicum were prepared. Different amounts of SMT were added to the different extracts (final concentration of 1, 2, and 5?mg L^?1) and incubated at 37?°C. Crude extracts of T. pannonicum were further used to evaluate the degradation rate over time. In order to evaluate the influence of endophytic or naturally plant-associated microorganisms on the biodegradation of SMT, extracts from plants grown in sterile and non-sterile conditions were compared. SMT was analyzed by liquid chromatography coupled to positive ion electrospray mass spectrometry (ESI LC-MS). Based on the findings, crude extracts of T. pannonicum have a high potential to biodegrade SMT with a decrease up to 85.4% (4.27?±?0.10?mg L^?1) from an initial concentration of 5?mg L^?1. The lowest activity was obtained using extracts of C. quinoa with degradation of 4.5%. Extracts of plants cultivated under sterile and non-sterile conditions do not have any significant difference in SMT degradation. Therefore, microorganisms and their enzymatic activities do not seem to play a significant role during this process.     

α-酮戊二酸依赖型双加氧酶催化特性及反应耦联辅因子对其催化羟基化反应的影响

【目的】以重组大肠杆菌表达的枯草芽孢杆菌(Bacillus subtilis)L-异亮氨酸双加氧酶(L-isoleucine dioxygenase,IDO)为研究对象,考察其催化L-异亮氨酸(L-Ile)羟基化反应的影响因素,构建IDO催化合成羟基氨基酸的反应体系。【方法】通过Ni-NTA亲和层析法从重组大肠杆菌(Escherichia coli)BL21/p ET28a-ido中纯化获得重组IDO,以L-Ile为底物,考察重组IDO催化羟基化反应的影响因素,并进一步针对耦联反应优化α-酮戊二酸(α-KG)在重组IDO酶促转化体系中的添加浓度。【结果】基于重组IDO催化L-Ile羟基化的活性测定,计算该酶Km为0.247 mmol/L,kcat为1.260 s-1,kcat/Km为5.101 L/(mmol·s),与其他同源酶动力学参数比较分析表明,重组IDO的底物亲和性及催化效率较高。重组IDO催化反应的最适温度为20°C、最适p H为7.0;在35°C以下较为稳定;反应体系中Fe2+最适浓度为1 mmol/L。重组IDO可催化不同L-氨基酸反应,对L-异亮氨酸、L-正亮氨酸、L-甲硫氨酸的活性较高。通过优化α-KG浓度,反应体系中添加30 mmol/Lα-KG时,可将底物浓度提高至70 mmol/L,产物4-羟基异亮氨酸(4-HIL)的摩尔产率达66.20%,表明α-KG作为反应耦联辅因子,其浓度对重组IDO催化L-Ile羟基化具有显著影响。【结论】重组IDO的底物亲和性、催化效率、最适催化条件、稳定性等基本性质有利于催化L-Ile羟基化反应。在其催化反应体系中,α-KG作为反应耦联辅因子,对酶促转化效果影响显著。研究结果为4-HIL及其他羟基氨基酸的酶促转化提供了研究基础。     

Hydrolysis Rates of Different Small Interfering RNAs (siRNAs) by the RNA Silencing Promoter Complex,C3PO,Determines Their Regulation by Phospholipase Cβ

C3PO plays a key role in promoting RNA-induced gene silencing. C3PO consists of two subunits of the endonuclease translin-associated factor X (TRAX) and six subunits of the nucleotide-binding protein translin. We have found that TRAX binds strongly to phospholipase Cβ (PLCβ), which transmits G protein signals from many hormones and sensory inputs. The association between PLCβ and TRAX is thought to underlie the ability of PLCβ to reverse gene silencing by small interfering RNAs. However, this reversal only occurs for some genes (e.g. GAPDH and LDH) but not others (e.g. Hsp90 and cyclophilin A). To understand this specificity, we carried out studies using fluorescence-based methods. In cells, we find that PLCβ, TRAX, and their complexes are identically distributed through the cytosol suggesting that selectivity is not due to large scale sequestration of either the free or complexed proteins. Using purified proteins, we find that PLCβ binds ∼5-fold more weakly to translin than to TRAX but ∼2-fold more strongly to C3PO. PLCβ does not alter TRAX-translin assembly to C3PO, and brightness studies suggest one PLCβ binds to one C3PO octamer without a change in the number of TRAX/translin molecules suggesting that PLCβ binds to an external site. Functionally, we find that C3PO hydrolyzes siRNA(GAPDH) at a faster rate than siRNA(Hsp90). However, when PLCβ is bound to C3PO, the hydrolysis rate of siRNA(GAPDH) becomes comparable with siRNA(Hsp90). Our results show that the selectivity of PLCβ toward certain genes lies in the rate at which the RNA is hydrolyzed by C3PO.     

Synthesis with good enantiomeric excess of both enantiomers of α-ketols and acetolactates by two thiamin diphosphate-dependent decarboxylases

In addition to the decarboxylation of 2-oxo acids, thiamin diphosphate (ThDP)-dependent decarboxylases/dehydrogenases can also carry out so-called carboligation reactions, where the central ThDP-bound enamine intermediate reacts with electrophilic substrates. For example, the enzyme yeast pyruvate decarboxylase (YPDC, from Saccharomyces cerevisiae) or the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1) can produce acetoin and acetolactate, resulting from the reaction of the central thiamin diphosphate-bound enamine with acetaldehyde and pyruvate, respectively. Earlier, we had shown that some active center variants indeed prefer such a carboligase pathway to the usual one [Sergienko, Jordan, Biochemistry 40 (2001) 7369–7381; Nemeria et al., J. Biol. Chem. 280 (2005) 21,473–21,482]. Herein is reported detailed analysis of the stereoselectivity for forming the carboligase products acetoin, acetolactate, and phenylacetylcarbinol by the E477Q and D28A YPDC, and the E636A and E636Q PDHc-E1 active-center variants. Both pyruvate and β-hydroxypyruvate were used as substrates and the enantiomeric excess was analyzed by a combination of NMR, circular dichroism and chiral-column gas chromatographic methods. Remarkably, the two enzymes produced a high enantiomeric excess of the opposite enantiomer of both acetoin-derived and acetolactate-derived products, strongly suggesting that the facial selectivity for the electrophile in the carboligation is different in the two enzymes. The different stereoselectivities exhibited by the two enzymes could be utilized in the chiral synthesis of important intermediates.     

Determination of platinated purines in oligoribonucleotides by limited digestion with ribonucleases T1 and U2

Platinum complexes which are known to react preferentially with guanine (G) and adenine (A) bases of oligonucleotides can be used as tools to analyze their tertiary structures and eventually to cross-link them. However, this requires efficient methods to allow the identification and quantification of the corresponding adducts which have so far been developed only for oligodeoxyribonucleotides. Maxam-Gilbert type digestions cannot be used for RNAs and HPLC techniques would require too large amounts of expensive material for separation and further characterization. We report a method to determine platination sites on oligoribonucleotides based on the cleavage activity of ribonucleases T1 and U2. To test the method, these enzymes were first used under conditions of limited digestion on 5-mer oligoribonucleotides platinated at a single defined purine. The phosphodiester bond on the 3^′ side of platinated G or A appeared fully resistant to cleavage by ribonuclease T1 or U2, respectively. An inhibitory effect was also observed due to neighboring platinated purines, which decreases with their distance (−2, −1, +1, +2) from the cleavage site and with the enzyme concentration. The method allowed the identification and quantification of the platination sites of a 17-mer oligoribonucleotide, based on the analysis of the mixture of monoplatinated adducts.     

Protein hydrolysate from organic fraction of municipal solid waste compost as nitrogen source to produce lactic acid by Lactobacillus fermentum ATCC 9338 and Lactobacillus plantarum NCIMB 8826

In this work a strategy for obtaining free amino-acids concentrate from an organic fraction of municipal solid waste compost and its use as a nitrogen source for lactic acid production, a compound widely used in different industries, using L. fermentum ATCC 9338 and L. plantarum NCIMB 8826 strains is described. Enzymatic digestion is based on the combined action of endoprotease Alcalase 1.5 MG and exoprotease Flavourzyme 500 MG. The highest degree of hydrolysis obtained under the optimal conditions was 41%. The use of glucanase Viscozyme L prior to protein hydrolysis helped to reduce the viscosity of the solution and promote the action of proteases, increasing its hydrolysis degree by 76%. The hydrolysate contained all 21 amino-acids, making it ideal for lactic acid bacteria growth. During shake flask cultivations the culture media was complemented with glucose as carbon source. Finally, with the hydrolysate, a maximum lactic acid concentration of 9.0 ± 0.2 g·L⁻¹ and 11.1 ± 0.1 g·L⁻¹ for L. fermentum ATCC 9338 and L. plantarum NCIMB 8826 respectively was obtained after 27 h. The innovation of the approach lies in exploiting the overproduction of compost for the production of lactic acid.     

金鱼草胚囊的人工分离

用果胶酶与纤维素酶的混合液离解金鱼草的胚珠,从中分离出完整的胚囊。由固定材料分离的胚囊,经透明染色后可借助干涉差显微装置观察由受精前直至受精后具上百个胚乳细胞时期的胚囊的内部结构。由新鲜胚珠分离胚囊也已初步突破,并对其作了简易的显微化学观察。讨论了活胚囊分离的意义与胚囊离体培养的前景。     

Efficient enzymatic synthesis of 13 C,15N-labeled DNA for NMR studies

The power of heteronuclear NMR spectroscopy to study macromoleculesand their complexes has been amply demonstrated over the last decade. Theobstacle to routinely applying these techniques to the study of DNA has beenthe synthesis of ¹³C,¹⁵N-labeled DNA. Here wepresent a simple and efficient method to generate isotope-labeled DNA forNMR studies that is as easy as that for isotope labeling of RNA. The methodwas used to synthesize a uniformly¹³ C,¹⁵N-labeled 32-nucleotide DNA that binds tohuman basic fibroblast growth factor with high affinity and specificity.Isotope-edited experiments were applied to the¹³ C,¹⁵N-labeled DNA bound to unlabeled protein,and the ¹³ C,¹⁵N-labeled DNA was also examined incomplex with ¹⁵N-labeled protein. The NMR experiments showthat the DNA adopts a well-defined stable structure when bound to theprotein, and illustrate the potential of¹³ C,¹⁵N-labeled DNA for structural studies ofDNA–protein complexes.