Potential of fermentation profiling via rapid measurement of amino acid metabolism by liquid chromatography-tandem mass spectrometry

Monitoring amino acid metabolism during fermentation has significant potential from the standpoint of strain selection, optimizing growth and production in host strains, and profiling microbial metabolism and growth state. A method has been developed based on rapid quantification of underivatized amino acids using liquid chromatography-electrospray tandem mass spectrometry (LC-MS-MS) to monitor the metabolism of 20 amino acids during microbial fermentation. The use of a teicoplanin-based chiral stationary phase coupled with electrospray tandem mass spectrometry allows complete amino acid analyses in less than 4 min. Quantification is accomplished using five isotopically labeled amino acids as internal standards. Because comprehensive chromatographic separation and derivatization are not required, analysis time is significantly less than traditional reversed- or normal-phase LC-based amino acid assays. Intra-sample precisions for amino acid measurements in fermentation supernatants using this method average 4.9% (R.S.D.). Inter-day (inter-fermentation) precisions for individual amino acid measurements range from 4.2 to 129% (R.S.D.). Calibration curves are linear over the range 0-300 microg/ml, and detection limits are estimated at 50-450 ng/ml. Data visualization techniques for constructing semi-quantitative fermentation profiles of nitrogen source utilization have also been developed and implemented, and demonstrate that amino acid profiles generally correlate with observed growth profiles. Further, cellular growth events, such as lag-time and cell lysis can be detected using this methodology. Correlation coefficients for the time profiles of each amino acid measured illustrate that while several amino acids are differentially metabolized in similar fermentations, a select group of amino acids display strong correlations in these samples, indicating a sub-population of analytes that may be most useful for fermentation profiling.     

Transport of amino acids in intact 3T3 and SV3T3 cells. Binding activity for leucine in membrane preparations of ehrlich ascites tumor cells.

Transport of amino acids into 3T3 and SV3T3 (SV40 virus-transformed 3T3) cells was measured on glass cover slips. The 3T3 and SV3T3 cells contain both A (alanine preferring) and L (leucine prefferring) systems for neutral amino acid transport. Initial rates of uptake of amino acids are about twofold higher in SV3T3 than in 3T3 cells. Other parameters measured, however, do not indicate marked differences in the transport of amino acids by the two cell types. L-system amino acids, such as leucine, are subject to trans-stimulation in both cell lines, whereas A-system amino acids, such as alanine and glycine, are not. Leucine was transported to higher levels in confluent cells than in nonconfluent cells. Glycine, however, shows distinctly less transport activity as the cells become confluent. Ehrlich ascites cell plasma membranes were prepared and assayed for amino acid-binding activity. Leucine-binding activity was detected by equilibrium dialysis in Triton X-100-treated membrane preparations.     

Effects of the feed solution concentration on the separation degree in Donnan dialysis for binary systems of amino acids

The separations of amino acids by Donnan dialysis using an ion-exchange membrane were studied. Donnan dialytic experiments were carried out using an anion-exchange membrane, glutamic acid–phenylalanine or glutamic acid–alanine mixed solutions as the feeds, and sodium hydroxide solutions as the stripping ones. The initial concentrations of the two kinds of amino acids in the feed solutions were equal and in the range of 0.5–50 mol m⁻³. The amino acid fluxes were measured for each feed solution. Above the feed concentration of 10 mol m⁻³, the glutamic acid flux was over 100 times greater than that of the other amino acid, and it was found that the Donnan dialysis was applicable to the separation of the amino acids. On the other hand, below 10 mol m⁻³, the amino acid fluxes varied in a complicated manner with the concentration, and below 1 mol m⁻³ there was little difference between the fluxes of the two amino acids.Furthermore, after soaking the membrane in solutions having the same concentrations as the feed in the Donnan dialysis, uptake of the amino acids into the membrane was also measured. By comparing the experimental results of both the flux and uptake of the amino acids, the reason why the flux varied in a complicated manner with the concentration was discussed.     

Dialytic transport of carboxylic acids through an anion exchange membrane

The transport behavior of five carboxylic acids of relevance in biotechnology (acetic, propionic, lactic, oxalic, citric) in diffusion dialysis and neutralization dialysis through an anion exchange membrane is investigated. The dependence of acid anion flux on base concentration in neutralization dialysis is analyzed in terms of two limiting situations (boundary layer control and membrane control) by an empirical two-parameter flux equation in formal analogy to a Langmuir function. When coupled to a life fermenter, neutralization dialysis is a means to control the pH of the fermentation medium. By removing biotoxic acids, it improves microbial productivity, as exemplified with the Propioni system producing vitamin B₁₂ and propionic acid.     

Fractionation of casein hydrolysates using polysulfone ultrafiltration hollow fiber membranes

The objective of this study was to characterize the fractionation profile of casein hydrolysates obtained with polysulfone hollow fiber ultrafiltration membranes. The two-step ultrafiltration process developed by Turgeon and Gauthier [J. Food Sci., 55 (1990) 106] was used: a caseinate solution was submitted to proteolysis with chymotrypsin or trypsin, and the reaction mixture (RM) was subsequently ultrafiltered using a 30 kDa (MWCO) hollow-fiber polysulfone membrane. The total hydrolysate permeating from this first step was further fractionated using a 1 kDa (MWCO) membrane, producing the mixture of polypeptides (retentate) and the amino acid fraction (permeate). The effect of enzyme specificity and of membrane retentivitiy on the total composition (total nitrogen, fat, lactose, minerals) and amino acid profile of the fractions was studied. The overall composition of the fractions was not significantly affected by the nature of the enzyme but the degree of hydrolysis and the molecular weight distribution profile analyses showed a marked effect of the enzyme specificity, with trypsin giving a larger proportion of small peptides (< 200 Da) in the mixture of polypeptides. Amino acid profile analyses provided useful information on the phenomena governing the fractionation of amino acids with a polysulfone membrane: (1) the target amino acids of the enzyme are concentrated in the permeate as a result of their presence in all peptides produced by hydrolysis, (2) polar amino acids are retained by the membrane, (3) non-polar amino acids are not selectively rejected by the membrane. Our results suggest that the charge/hydrophobicity balance of the peptides produced is the predominant factor determining the fractionation of casein hydrolysates.     

Keratinolytic enzyme-mediated biodegradation of recalcitrant feather by a newly isolated Xanthomonas sp. P5

We isolated and characterized a novel feather-degrading Xanthomonas sp. P5 with keratinolytic activity. In improved medium containing 0.1% (w/v) feather, maximal keratinolytic activity was observed at 5 days (69.0 ± 0.6 U/mL). This value was 7.1-fold higher than the yield in basal feather medium. The strain P5 degraded feather completely after 7 days. Feather degradation resulted in free thiol group, soluble protein and amino acids formation, indicating that sulfitolysis and proteolysis may be responsible for feather degradation by the strain P5. Total free amino acid concentration in the cell-free supernatant was around 188.6 μM. Asparagine, methionine, histidine and threonine were the major amino acids released in the culture. Xanthomonas sp. P5 exhibited multiple plant growth-promoting attributes such as siderophore, indoleacetic acid, ammonia, hydrolytic enzyme and antifungal activity. Our results indicate that Xanthomonas sp. P5 could be not only used to upgrade the nutritional value of recalcitrant feather waste but is also a potential candidate for the development of biofertilizer or biocontrol agent applicable to crop plant soil.     

Replacing 32 proline residues by a noncanonical amino acid results in a highly active DNA polymerase

Protein engineering may be achieved by rational design, directed evolution-based methods, or computational protein design. Mostly these methods make recourse to the restricted pool of the 20 natural amino acids. With the ability to introduce different new kinds of functionalities into proteins, the use of noncanonical amino acids became a promising new method in protein engineering. Here, we report on the generation of a multifluorinated DNA polymerase. DNA polymerases are highly dynamic enzymes that catalyze DNA synthesis in a template-dependent manner, thereby passing several conformational states during the catalytic cycle. Here, we globally replaced 32 proline residues by the noncanonical imino acid (4R)-fluoroproline in a DNA polymerase of 540 amino acids (KlenTaq DNA polymerase). Interestingly, the substitution level of the proline residues was very efficient (92%). Nonetheless, the introduction of (4R)-fluoroproline into the DNA polymerase resulted in a highly active fluorinated enzyme, which was investigated in primer extension and PCR assays to analyze activity, selectivity, and stability in comparison to the parental enzyme. The DNA polymerase retained fidelity, activity, and sensitivity as the parental wild-type enzyme accompanied by some loss in thermostability. These results demonstrate that a noncanonical amino acid can be used for substitutions of natural counterparts in a highly dynamic enzyme with high molecular weight without effecting crucial enzyme properties. Furthermore, the employed DNA polymerase represents a promising starting point for directed DNA polymerase evolution with noncanonical amino acids.     

Flow injection on-line dialysis coupled to high performance liquid chromatography for the determination of some organic acids in wine

A combined system of flow injection on-line dialysis sample pretreatment and high performance liquid chromatographic separation/detection (FID-HPLC) was developed for simultaneous determination of six organic acids (tartaric, malic, lactic, acetic, citric and succinic acids). A sample or mixed standard solution (400 μL) was injected into a donor stream (water) of FID system and was pushed further through a dialysis cell, while an acceptor solution (water) was held in the opposite side of the dialysis membrane. The dialysate containing organic acids in the acceptor solution was then flowed to an injection loop of the HPLC valve, where it was further injected into the HPLC system and analysed under normal HPLC conditions, using a reversed-phase (C₁₈) analytical column and UV detection (210 nm). The order of elution was tartaric, malic, lactic, acetic, citric and succinic acids with the analysis time of 8 min. The FID system could be operated in parallel with HPLC separation, providing sample throughput of 7.5 h⁻¹. Dialysis efficiencies of six organic acids were in range of 4.6-9.5%. Calibration graphs for all the mentioned organic acids were linear over the range of 250-7500 mg L⁻¹. Precisions for all the organic acids were within 5.4%. The proposed system was successfully applied for analysis of some Thai wines. By spiking wine samples with mixed acid standard solutions, the percentage recoveries in range of 84-104 were found. This system has advantages of fast and high degrees of automation for dialysis sample pretreatment, on-line sample separation and dilution, good clean-up for prolongation of life-time of the HPLC column and low consumption of chemicals and materials.     

Adaptive Evolution of Saccharomyces cerevisiae with Enhanced Ethanol Tolerance for Chinese Rice Wine Fermentation

High tolerance towards ethanol is a desirable property for the Saccharomyces cerevisiae strains used in the alcoholic beverage industry. To improve the ethanol tolerance of an industrial Chinese rice wine yeast, a sequential batch fermentation strategy was used to adaptively evolve a chemically mutagenized Chinese rice wine G85 strain. The high level of ethanol produced under Chinese rice wine-like fermentation conditions was used as the selective pressure. After adaptive evolution of approximately 200 generations, mutant G85X-8 was isolated and shown to have markedly increased ethanol tolerance. The evolved strain also showed higher osmotic and temperature tolerances than the parental strain. Laboratory Chinese rice wine fermentation showed that the evolved G85X-8 strain was able to catabolize sugars more completely than the parental G85 strain. A higher level of yeast cell activity was found in the fermentation mash produced by the evolved strain, but the aroma profiles were similar between the evolved and parental strains. The improved ethanol tolerance in the evolved strain might be ascribed to the altered fatty acids composition of the cell membrane and higher intracellular trehalose concentrations. These results suggest that adaptive evolution is an efficient approach for the non-recombinant modification of industrial yeast strains.     

氨基酸分子改性的SiO2杂化材料用于胰蛋白酶固定化

为改善二氧化硅载体材料本身的生物相容性和疏水性,维持包埋生物分子的活性,本文对水解前驱体3-氨基丙基三甲氧基硅烷进行氨基酸分子改性。具体过程包括N-Fmoc-L-缬氨酸和氯化亚砜反应生成N-Fmoc-L-缬氨酰氯,再和3-氨基丙基三甲氧基硅烷反应生成N-（3-三甲氧基硅基）丙基-N′-Fmoc-L-缬氨酰胺后。然后去除Fmoc,得到N-（3-三甲氧基硅基）丙基-L-缬氨酰胺作为氨基酸修饰的硅源前驱体。通过IR、MS、1H-NMR等分析测试手段对合成得到的各个化合物的结构进行了表征。利用正硅酸甲酯（TMOS）和N-（3-三甲氧基硅基）丙基-L-缬氨酰胺为复合硅源,经过溶胶-凝胶过程来包埋了胰蛋白酶,研究得到最适的固定化条件为,N-（3-三甲氧基硅基）丙基-L-缬氨酰胺的含量为15mol%。在该条件下,固定化胰蛋白酶活力的绝对值是199U,游离酶的酶活力的绝对值是103U, 四甲氧基硅烷直接包埋的固定化酶活力的活性是38 U。在该条件下,杂化硅源得到的固定化酶的活性是以四甲氧基硅烷水解前驱体的固定化酶活性的5倍,杂化硅源固定化胰蛋白酶的最相比游离酶,酶的最高活力提高的几乎2倍。这些结果表明氨基酸分子对水解前驱体修饰以后,水解产生的固定化载体具有良好的生物相容性。通过改性载体制备的固定化酶,对甲醇变性剂的稳定性,对酸碱的抵抗性及热稳定性也有明显地提高。     

黑曲霉酸性蛋白酶在食醋酿造中的催化效应

从麸醋母曲中分离出黑曲霉Asp 5609并以此为诱变出发菌株,采用紫外线照射结合亚硝基胍诱变剂处理,筛选出酸性蛋白酶高产变异株Asp 5609—7。用盐溶加温提取法从Asp 5609-7固体发酵物料中提取酸性蛋白酶粗酶液,经DE-30型中空纤维柱超滤浓缩,用于食醋酿造物料蛋白的酶解催化,可大幅度提高醋液中氨基酸含量。     

Effect of the activity levels of the added proteolytic enzyme mixture on free amino acids in ripening Ossau-Iraty cheese

A proteolytic enzymatic preparation (using one of three enzyme concentrations and, hence, one of three different enzymatic activity levels) was added (before clotting) to the milk used to manufacture Ossau-Iraty ewes'-milk cheese. The free amino acids were analysed by reversed-phase high-performance liquid chromatography and the sulphosalicylic acid-soluble N fraction was quantified by the trinitrobenzenesulphonic acid method for use as an index of proteolysis during ripening. Sensory analysis of the cheeses began after two months of ripening. Use of the enzymatic preparation increased the rate of release of amino acids in an amount proportional to the enzyme concentration employed. The effect of the preparation was more pronounced in the early months of ripening, with the differences in the free amino acid contents of the various batches decreasing as ripening progressed. Levels of certain free amino acids, such as taurine, tyrosine and valine, were virtually unaffected by the addition of the enzymatic preparation, whereas levels of such amino acids as serine, glycine, arginine and proline were reduced. Texture defects in the cheeses were observed, namely, reduced elasticity and creaminess and increased brittleness. Similarly, enzymatic treatment also gave rise to bitter flavours that were not characteristic of the normal taste and aftertaste of Ossau-Iraty cheese and these changes were proportional to the quantity of enzyme added.     

The Thermostability of Two Kinds of Recombinant ∆6-Fatty Acid Desaturase with Different N-Terminal Sequence Lengths in Low Temperature

Two recombinant Rhizopus stolonifer ?6-fatty acid desaturase enzymes with different-length N-termini were cloned and expressed in Saccharomyces cerevisiae strain INVScl: LRsD6D begins with the sequence of the N-terminal of the R. stolonifer ?6-fatty acid desaturase native, encoding a deduced polypeptide of 459 amino acids (M-S-T-L-D-R-Q-S-I-F-T-I-K-E-L-E-S-I-S-Q-R-I-H-D-G-D-E-E-A-M-K-F), whereas SRsD6D begins with the amino acid sequence of the predicted ORF, encoding a deduced polypeptide of 430 amino acids (M-K-F) and LRsD6D is longer than SRsD6D by 29 amino acids (M-S-T-L-D-R-Q-S-I-F-T-I-K-E-L-E-S-I-S-Q-R-I-H-D-G-D-E-E-A). Bioinformatic analysis characterized the two recombinant ?6-fatty acid desaturase enzymes with different-length N-termini, including three conserved histidine-rich motifs, hydropathy profile, and a cytochrome b5-like domain in the N-terminus. When the coding sequence was expressed in S. cerevisiae strain INVScl, the coding produced ?6-fatty acid desaturase activity exhibited by RsD6D, leading to a novel peak corresponding to γ-linolenic acid methyl ester standards, which was detected with the same retention time. The residual activity of LRsD6D was 74 % at 15 °C for 4 h and that of SRsD6D was 43 %. Purified recombinant LRsD6D was more stable than SRsD6D, indicating that the N-terminal extension, containing mostly hydrophobic residues, affected the overall stability of recombinant LRsD6D.     

Esterification Synthesis of Ethyl Oleate in Solvent-Free System Catalyzed by Lipase Membrane from Fermentation Broth

In this study, the immobilized lipase was prepared by fabric membrane adsorption in fermentation broth. The lipase immobilization method in fermentation broth was optimized on broth activity units and pH adjustments. The viscose fermentation broth can be used with a certain percentage of dilution based on the original broth activity units. The fermentation broth can be processed directly without pH adjustment. In addition, the oleic acid ethyl ester production in solvent-free system catalyzed by the immobilized lipase was optimized. The molar ratio of ethanol to oil acid, the enzyme amount, the molecular amount, and the temperature were 1:1, 12% (w/w), 9% (w/w)(based the total amount of reaction mixture), and 30 °C, respectively. Finally, the optimal condition afforded at least 19 reuse numbers with esterification rate above 80% under stepwise addition of ethanol. Due to simple lipase immobilization preparation, acceptable esterification result during long-time batch reactions and lower cost; the whole process was suitable for industrial ethyl oleate production.     

Amperometric L-glutamate sensor using a novel L-glutamate oxidase from Streptomyces platensis NTU 3304

Streptomyces platensis NTU 3304, isolated from soil samples, produces extracellular L-glutamate oxidase in liquid culture. Strains of this species have never been reported to be able to produce this enzyme. The purified enzyme was immobilized onto a cellulose triacetate membrane which was held at an oxygen electrode. The sensor was specific to L-glutamate in accordance with the properties of the novel L-glutamate oxidase. The time required for each assay in batch operation was less than 3 min. A linear relationship is observed between the decrease in dissolved oxygen and the concentration of L-glutamate between 20 and 140 mg l^?1 (ca. 0.12 and 0.84 mM). The sensor retained 95% of its original activity after 400 assays over a period of 3 weeks. The sensor was applied to determine the concentration of L-glutamate in broth samples during L-glutamic acid fermentation. Good correlations were achieved between results obtained with the sensor and by enzymatic analysis using glutamate dehydrogenase.     

Optical resolution of various amino acids using a supported liquid membrane encapsulating a surfactant-protease complex

We have encapsulated a surfactant-protease complex (the main protease used being alpha-chymotrypsin) in an organic phase of a supported liquid membrane (SLM) for the optical resolution of various amino acids. L-Isomers of amino acids were enantioselectively permeated through the SLM. The mechanism of the amino acid permeation through the SLM was considered to be as follows; an L-amino acid was enantioselectively esterified with ethanol by a surfactant-protease complex encapsulated in the SLM, and the resulting L-amino acid ethyl ester dissolved into the organic phase of the SLM and diffused across the SLM. Another surfactant-alpha-chymotrypsin complex in the receiving phase catalyzed ester hydrolysis to produce the initial L-amino acid and ethanol, which are water-soluble. Thus, the L-amino acid was selectively transported to the receiving phase through the SLM on the basis of the molecular recognition of the surfactant-protease complex in the SLM. It was found that the catalytic activity and enantioselectivity of the surfactant-protease complex governed the permeate flux of amino acids and the enantiomeric excess in the membrane separation.     

Functionalization of cellulose dialysis membranes for chiral separation using beta-cyclodextrin immobilization

A membrane-based chiral separation system for the separation of racemic tryptophan solutions is developed by the covalently binding beta-cyclodextrin onto the surface of commercial cellulose membranes. The immobilization process is monitored by XPS. AFM demonstrates the evolutionary transition of membrane surface morphology before and after the CD immobilization. Due to their different complexation with immobilized CD, dialysis transport experiments show d-tryptophan preferential permeability through the immobilized CD membranes, and the enantioselectivity is 1.10. A model based on the existence of a thin chiral solution layer of amino acid at the interface between the feed solution and the membrane has been proposed. This chiral separation model has been verified using the chiral separation results of racemic amino acids and binding constants of amino acids with CD. The effect of membrane's pore size on enantioselectivity has also been investigated. The immobilized CD membrane, having MWCO 1000, exhibits the highest enantioselectivity to the racemic tryptophan solution.     

淀粉酶产色链霉菌TUST2中ε-聚赖氨酸降解酶的纯化和性质

分离得到产抗菌聚氨基酸--ε-聚赖氨酸菌株淀粉酶产色链霉菌TUST2,从中纯化了ε-聚赖氨酸降解酶,并对其性质进行了研究.结果表明,该酶为膜结合蛋白.为提取该降解酶,先收集菌体细胞并用超声波破碎,细胞膜部分用1.0 moL/L NaSCN溶液溶解.将粗酶液进行Sephadex G100凝胶柱层析分离.用100mmol/L磷酸缓冲液洗脱,收集活性部分.纯化后的样品用SDS-PAGE检测,酶亚基分子量约为54700.酶活力在pH=6.0～9.0间稳定,最适宜pH=7.0.酶的最适温度为30℃,在10～50℃水浴30 min酶活力未见明显下降.研究了不同金属离子对酶活力的影响,结果表明,Zn~(2+),Cu~(2+)和Fe_(3+)可分别提高酶活力29.72%,15.85%和15.08%;但Ag~(+),Hg~(2+),Co~(2+)和Mn~(2+)对酶活力有强烈的抑制作用.Ca2~(2+),K~+和Ba~(2+)对酶活力没有影响.添加4%Tween-80能提高酶活力10%,但EDTA能强烈抑制酶活力.研究结果表明,此降解酶的性质与白色链霉菌产生的ε-聚赖氨酸降解酶的性质相似.     

Controlling E. coli adhesion on high-k dielectric bioceramics films using poly(amino acid) multilayers

The influence of high-k dielectric bioceramics with poly(amino acid) multilayer coatings on the adhesion behavior of Escherichia coli (E. coli) was studied by evaluating the density of bacteria coverage on the surfaces of these materials. A biofilm forming K-12 strain (PHL628), a wild-type strain (JM109), and an engineered strain (XL1-Blue) of E. coli were examined for their adherence to zirconium oxide (ZrO(2)) and tantalum oxide (Ta(2)O(5)) surfaces functionalized with single and multiple layers of poly(amino acid) polyelectrolytes made by the layer-by-layer (LBL) deposition. Two poly(amino acids), poly(l-arginine) (PARG) and poly(l-aspartic acid) (PASP), were chosen for the functionalization schemes. All three strains were found to grow and preferentially adhere to bare bioceramic film surfaces over bare glass slides. The bioceramic and glass surfaces functionalized with positively charged poly(amino acid) top layers were observed to enhance the adhesion of these bacteria by up to 4-fold in terms of bacteria surface coverage. Minimal bacteria coverage was detected on surfaces functionalized with negatively charged poly(amino acid) top layers. The effect of different poly(amino acid) coatings to promote or minimize bacterial adhesion was observed to be drastically enhanced with the bioceramic substrates than with glass. Such observed enhancements were postulated to be attributed to the formation of higher density of poly(amino acids) coatings enabled by the high dielectric strength (k) of these bioceramics. The multilayer poly(amino acid) functionalization scheme was successfully applied to utilize this finding for micropatterning E. coli on bioceramic thin films.