992 resultados para carbohydrate analysis


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Several serological methods have been used for the characterization and identification of soybean bradyrhizobia. However, some problem were non-reactivity of certain strains and cross-reactivity among others. Since lipopolysaccharide (LPS) can often be used in strain identification, the objective was to investigate the antigenic properties and polyacrylamide gel electrophoretic pattern of 12 Brazilian strains of Bradyrhizobium japonicum that nodulate soybean and to compare them to standard strains. The close correlation between the LPS patterns obtained by SDS-PAGE and the serological analysis permitted us to assign the strains to nine groups different or the same as the standard strains.

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组特殊自养氨氧化混合种群,表现:无机环境种群生长迅速、生物量高;在一个完全无机的自养生长环境中,不仅保持高氨氧化速率,并出现丰富的异养微生物种群;该种群置于异养、厌氧环境中,迅速表现出产氢特征。对于这样一个特殊的生态体系,研究其共生机理,以及联接这些种群之间的碳源和能源问题,将具有非常重要意义。我们拟从种群特征、细胞表面分泌产物、游离体系产物多糖、蛋白和脂肪酸方面开展研究。 第一部分,自养氨氧化混合种群的基本特征。采用氨氧化培养基,进行种群氨氧化特征研究;采用扫描电镜观察自养混合种群的微观特征;沉降、离心去除微生物种群,分析水相中的总有机碳、糖类等物质;利用LB培养基进行种群的分离、纯化,并采用DGGE手段对微生物种群结构进行分析。结果表明,接入菌种后(2/5000(V/V)),培养液中氨(200mg/L)在3-5天内快速降解;亚硝酸盐与氨氮变化呈负相关趋势,仅有少量硝酸盐含量(< 30mg/L)。氨氧化种群的生物量增长与氨氧化趋势一致,初始生物量7.75 mg/L(蛋白含量),3-5天后生物量快速增长,并达到最高63.06 mg/L(蛋白含量)。电镜图片显示,种群外包裹一层粘液。离心除去菌体后,检测培养液总有机碳和糖的含量,同样表现出与生物量增长相似的特征,分别由初始的3.73、2.35 mg/L,3-5内天迅速增加,并分别达到最大值35.19、27.45 mg/L。经初步分离、纯化并对纯化菌株进行测序,获得了10株异养微生物分别为布鲁氏菌科苍白杆菌属、纤维单孢菌、类芽孢菌属、黄杆菌属、无色杆菌、鞘脂单胞菌、嗜麦芽寡养单胞菌、噬氢菌属、硫红球菌、假单胞菌;DGGE显示,约有20分条离带,我们对其中的两条优势条带进行切割回收测序,鉴定为欧洲亚硝化单胞菌(Nitrosomonas eur)。 第二部分:混合种群自养-异养菌共生的可能机制。在对微生物种群特征初步分析基础上,针对胞外糖类组分可能被微生物代谢分解,我们重点对微生物细胞蛋白质与糖类进行分析。采用超声结合RIPA裂解液裂解,SDS-PAGE电泳分析混合种群总蛋白种类,并通过氨基酸分析仪及红外光谱法分析氨基酸组成及蛋白红外特征。采用超声破碎结合反复冻融对细胞样品进行处理,提取液采用醇沉、Sevage脱氮白,凝胶过滤方法脱盐和分级分离。对提取物的糖分析包括:紫外扫描,红外光谱,核磁共振,单糖组成分析;扫描电镜观察菌群破裂现象。SDS-PAGE分析结果表明:氨氧化种群不同生长阶段都显示出42kD蛋白表达量很高,d4时42kD蛋白表达已经很强,4-7d内一直持续这种过量表达,直到d8后表达开始减弱。说明42kD蛋白可能与氨氧化密切相关。红外光谱分析显示:细胞提取物的特征峰分布在3427.42cm-1、1718.18 cm-1和1681.72 cm-1、1160.07和1086.74 cm-1,分别对应为OH、 C=O、C-O-C基团,表明具有蛋白的典型特征;氨基酸分析显示蛋白中的Gly,Asp,Ala,Glu含量相对较高。 提取物中胞外多糖分离谱图得到不均一组分,共得到6个收集峰;紫外扫描在201-213 nm处有多糖吸收峰,同样表明多糖成分不均一性;多糖红外光谱特征峰主要分别在3400.49 cm-1、2920.28 cm-1、1154.54和1087.52 cm-1,对应OH、-CH2- or CH 、C-O-H or C-O-C等多糖特征基团;多糖提取物核磁共振1H d4.3~5.9之间出现强吸收峰,这是1H中,多糖存在的明显证据,1H NMR中,其中O-乙酰基的甲基上的氢信号为d1.1~1.3之间。糖肟全苯甲酸酯衍生物的HPLC测定中,得到单一的单糖峰,由于时间问题,还未进行更深入的试验;电镜图片显示,种群中的细胞有大量的破裂现象。 实验表明,自养氨氧化混合种群显示出快速的氨氧化速率,氨氧化过程生物量和有机质的增加明显。微生物种群包裹粘液层,并分离纯化出大量的异养菌;去除菌体后的游离培养液中存在有机质(包括多糖)说明无机自养生长体系中存在异养菌生长、繁殖的二次碳源;细胞提取物中蛋白条带数目多、种类丰富;细胞多糖提取物具有明显的多糖特征,以及单糖的存在。结合种群的显微特征和游离体系中的有机质的检测结果,我们认为,无机自养生长体系中,种群细胞生长过程中发生的破裂现象可能是导致大量的蛋白、多糖释放到游离胞外,并成为其他异养菌生长的碳源和氮源。这可能是自养体系中,大量异养菌共生的可能机制,至于是什么原因引起种群生长过程中产生的破裂现象,还有待下一步深入研究。 A group of mixed autotrophic ammonia oxidizing populations, having much biological characteristic tested by concerned personnel for pilot test: Performed rapid population growth and obtained high biomass in inorganic environment; Not only maintained a high rate of ammoxidation, promoted a wealth of heterotrophic microbial populations growth in a totally inorganic and autotrophic growth environment; Placed in heterotrophic and anaerobic environment,had the performance characteristics that could rapidly produce hydrogen.For such a special ecological system, Study its symbiotic mechanism and the connection between these populations of carbon and energy issues, will have a very important significance. We intended from the characteristics of the population, the secretion product of cell surface, free substance in the liquid medium like polysaccharide, protein and fatty acids carrying out research. Part I: The basic features of mixed autotrophic ammonia oxidizing populations . Use inorganic liquid medium, processed study for ammonia oxidation characteristics of the population; we used scanning electron microscopy to get micro-features of autotrophic ammonia oxidizing populations .The medium was carried out settlement and centrifugal then removed the microbial populations, after all of that we analysis the water phase for total organic carbon(TOC), carbohydrate and other substances; Solid ammonia oxidizing medium was adopted to separation and purification of population, DGGE means was for structure analysis of microbial population. The results showed that after the inoculum of bacteria (2 / 5000 (V / V)), ammonia in the culture medium (200 mg / L) was rapid degradation in 3-5 days; ammonia and nitrite have the negative correlation between changes in the trend, then only a small amount of nitrate content (<30mg / L). The biomass growth of ammoxidation population in line with the trend of ammonia oxidation, the initial volume of it was 7.75 mg / L (protein content), in 3-5 days upto 63.06 mg / L (protein content). Electron microscope image showed, the populations were wrapped in a layer of mucus, including the a large number ruptted micorbe , Centrifuge to remove bacteria, then detected the medium for total organic carbon and sugar content, result took on the same characteristics with biomass growth, that were from the initial 3.73、2.35 mg / L respectively, in 3-6 days achieved rapid increase in the maximum to 35.19、27.45 mg / L respectively. After initial separation、 purification ,then processed sequencing to strains purified and got the result that there were 10 heterotrophic microorganisms : Brucella Branch pale bacillus, Cellu lomonas, Bacillus species category, a Flavobacterium, colorless Bacteria, Aeromonas sheath fat, little support maltophilia Aeromonas, macrophages species hydrogen, sulphur-MI, Pseudomonas bacteria spores; DGGE display, there were 20 separation bands approximately. Part II: Mixed populations that autotrophic - heterotrophic bacteria symbiotic mechanism. On the basis of preliminary analysis of microbial population characteristics, aiming at extracellular carbohydrate components might be decomposition by microbial, we focused on microbial cell protein and carbohydrate analysis. Using ultrasound combined with RIPA lysis cracking the cells, SDS-PAGE electrophoresis analysis the total protein species of the population, and through the amino acid analyzer studied the compositions of amino acid and infrared spectroscopy analysis of a protein infrared characteristics. Using ultrasound combined with repeatedly freezing and thawing to treated the cell sample, then took the means that alcohol precipitation, deproteinization by Sevage, gel filtration aimed at desalination and grade separation to deal with the lysates . The extraction of sugar analysis included: UV scanning, IR, NMR, single-sugar composition analysis. SDS-PAGE analysis showed that: 42 kD protein expression was very high at different growth stages of mixed autotrophic ammonia oxidizing populations , on the fourth day, 42 kD protein expression had been very strong, 4-7d, it had continued this excessive expression, then started to weaken after 7 days. 42 kD protein that might be closely associated with ammonia oxidation. Infrared spectral analysis showed that: cell extracts with the characteristic that the peak distribution in 3427.42 cm-1、1718.18 cm-1 and 1681.72 cm-1、1160.07 cm-1 and 1086.74 cm-1 corresponding to OH、C = O、C-O-C Groups which had the typical characteristics of protein; and analysis showed that amino acids including Gly, Asp, Ala, Glu ,the content in the protein is relatively high. Exopolysaccharide in the extracts had the separation map that it was uneven, received a total of six collection peaks by the detection mode of phenol-sulphruic acid method ; ultraviolet scan in the 201-213 nm department had polysaccharide absorbing peak, the same ingredients that polysaccharide heterogeneity; infrared polysaccharide spectral characteristics of the main peak at 3400.49 cm-1, 2920.28 cm-1, 1154.54 and 1087.52 cm-1, corresponding OH,-CH2-or CH, C-O-H or C-O-C;and other characteristics of polysaccharide group; 1H NMR of polysaccharide extract appeared absorption peak between d4.3 ~5.9, which is the apparent evidence of polysaccharide, In 1H NMR, the hydrogen signal of one of O-acetyl was between 1.1 to 1.3. The determination of Sugar oxime whole benzoate derivatives by HPLC, there was a single-sugar peak, as a matter of time, yet more in-depth test. Summary: Mixed autotrophic ammonia oxidizing populations show us that it had the ability in ammonia oxidizing and it was great, organic matter and biomass increased significantly in the process of ammonia oxidation. Microbial populations was wrapped up slime layer, the phenomenon of cell breakdown obviously, and there were a lot of separation and purification of the heterotrophic bacteria; a lot of organic matter (including polysaccharides)remined in the medium that removal of cell indicated the inorganic system existed secondary carbon sources that could be used by the heterotrophic bacteria ; there were a large number proteins bands of cell extract, rich variety; cell extracts of polysaccharide had obvious characteristics of polysaccharide, and the existence evidence of single-sugar. Combined population of microscopic characteristics and free of organic matter in the test results, we believe that the health of inorganic system, population growth occurred in the course of the breakdown of the phenomenon is likely to lead to a lot of protein and polysaccharide released into the extracellular free, And other heterotrophic bacteria use them to the growth as carbon and nitrogen. This may be autotrophic system, the large number of heterotrophic bacteria symbiotic mechanism.

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A procedure was developed to extract polyols and trehalose (protectants against stress) from fungal conidia. Conidia were sonicated (120 s) and immersed in a boiling water bath (5.5 min) to optimize extraction of polyols and trehalose, respectively. A rapid method was developed to separate and detect low-molecular-weight polyols and trehalose using high-performance liquid chromatography (HPLC). An ion exchange column designed for standard carbohydrate analysis was used in preference to one designed for sugar alcohol separation. This resulted in rapid elution (less than 5 min), without sacrificing peak resolution. The use of a pulsed electrochemical detector (gold electrode) resulted in limits of reliable quantification as low as 1.6 μg ml-1 for polyols and 2.8 μg ml-1 for trehalose. This is very sensitive and rapid method by which these protectants can be analysed. It avoids polyol derivatization that characterizes analysis by gas chromatography and the long run times (up to 45 min) that typify HPLC analysis using sugar alcohol columns.

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Cell surface proteins obtained by alkaline extraction from isolated cell walls of Mortierella pusilla and M. candelabrum, host and nonhost, respectively, to the mycoparasite, Piptocephalis virginiana, were tested for their ability to agglutinate mycoparasite spores. The host cell wall protein extract had a high agglutinating activity (788 a.u. mg- t ) as compared with the nonhost extract (21 a.li. mg- t ). SDS-polyacrylamide gel electrophoresis of the cell wall proteins revealed four protein bands, a, b, c, and d (Mr 117, 100, 85 and 64 kd, respectively) at the host surface, but not at the nonhost surface, except for the faint band c. Deletion of proteins b or c from the host cell wall protein extract significantly reduced its agglutinating activity. Proteins band c, obtained as purified preparations by a series of procedures, were shown to be two glycoproteins. Carbohydrate analysis by gas chromatography demonstrated that glucose and Nacetylglucosamine were the major carbohydrate components of the glycoproteins. It was further shown that the agglutinating activity of the pure preparation containing both band c was 500-850 times that of the single glycoproteins, suggesting the involvement of both glycoproteins in agglutination. The results suggest that the glycoproteins band c are the two subunits of agglutinin present at the host cell surface. The two glycoproteins band c purified from the host cell wall protein extract were further examined after various treatments for their possible role in agglutination, attachment and appressorium formation by the mycoparasite. Results obtained by agglutination and attachment tests showed: (1) the two glycoprotein-s are not only an agglutinin responsible for the mycoparasite spore agglutination, but may also serve as a receptor for the specific recognition, attachment and appressorium formation by the mycoparasite; (2) treatment of the rnycoparasite spores with various sugars revealed that arabinose, glucose and N-acetylglucosamine inhibited the agglutination and attachment activity of the glycoproteins, however, the relative percentage of appressorium formation was not affected by the above sugars; (3) the two glycoproteins are relatively stable with respect to their agglutinin and receptor functions. The present results suggest that the agglutination and attachment may be mediated directly by certain sugars present at the host and mycoparasite cell surfaces while the appressorlum formation may be the response of complementary combinations of both sugar and protein, the two parts of the glycoproteins at the interacting surfaces of two fungi.

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Lipids constitute a significant portion of the biomass of earth and lipolytic enzymes play a very important role in lipid turn over. Apart from their biological significance, lipolytic enzymes are also very important in the fields of nutrition, food technology, medicine and preparative and analytical lipid biochemistry. Recent developments in the study of proteins and enzymes have largely benefited the study of lipolytic enzymes, that some of these enzymes were isolated in pure form. Even today there is a continuous search for new and potent sources of these lipolytic enzymes. The zest for elucidating the structure and mechanism of action of the enzymes obtained in pure form for biochemist still remains unabated. The literature shows no record of such an effort for the study of lipases from marine sources. The fact that many fishes like oil sardine, mackerel, cat fish, seer etc. contains large amounts of lipid shows the possibility of the existence of lipases in significant amounts necessitating their exhaustive study. Such a study will, not only provide alternate sources for lipase but also will provide methods to curb lipolysis and the resultant rancidity and off flavor development in fish and fishery products.

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Tannases have attracted wider attention because of their biotechnological potential, especially enzymes from filamentous fungi and other microorganisms. However, the biodiversity of these microorganisms has been poorly explored, and few strains were identified for tannase production and characterization. This article describes the production, purification and characterization of a glucose- and solvent-tolerant extracellular tannase from Aspergillus phoenicis. High enzymatic levels were obtained in Khanna medium containing tannic acid up to 72 h at 30 °C under 100 rpm. The purified enzyme with 65% of carbohydrate content had an apparent native molecular mass of 218 kDa with subunits of 120 kDa and 93 kDa and was stable at 50 °C for 1 h. Optima of temperature and pH were 60 °C and 5.0-6.5, respectively. The enzyme was not affected significantly by most ions, detergents and organic solvents. While glucose did not affect the tannase activity, the addition of a high concentration of gallic acid did. The Km values were 1.7 mM (tannic acid), 14.3 mM (methyl-gallate) and 0.6 mM (propyl-gallate). The enzyme was able to catalyze the transesterification reaction to produce propyl-gallate. All biochemical properties suggest the biotechnological potential of the glucose- and solvent-tolerant tannase from A. phoenicis. © 2012 Elsevier B.V. All rights reserved.

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Pós-graduação em Agronomia (Horticultura) - FCA

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In recent years, growing attention has been devoted to the use of lignocellulosic biomass as a feedstock to produce renewable carbohydrates as a source of energy products, including liquid alternatives to fossil fuels. The benefits of developing woody biomass to ethanol technology are to increase the long-term national energy security, reduce fossil energy consumption, lower greenhouse gas emissions, use renewable rather than depletable resources, and create local jobs. Currently, research is driven by the need to reduce the cost of biomass-ethanol production. One of the preferred methods is to thermochemically pretreat the biomass material and subsequently, enzymatically hydrolyze the pretreated material to fermentable sugars that can then be converted to ethanol using specialized microorganisms. The goals of pretreatment are to remove the hemicellulose fraction from other biomass components, reduce bioconversion time, enhance enzymatic conversion of the cellulose fraction, and, hopefully, obtain a higher ethanol yield. The primary goal of this research is to obtain kinetic detailed data for dilute acid hydrolysis for several timber species from the Upper Peninsula of Michigan and switchgrass. These results will be used to identify optimum reaction conditions to maximize production of fermentable sugars and minimize production of non-fermentable byproducts. The structural carbohydrate analysis of the biomass species used in this project was performed using the procedure proposed by National Renewable Energy Laboratory (NREL). Subsequently, dilute acid-catalyzed hydrolysis of biomass, including aspen, basswood, balsam, red maple, and switchgrass, was studied at various temperatures, acid concentrations, and particle sizes in a 1-L well-mixed batch reactor (Parr Instruments, ii Model 4571). 25 g of biomass and 500 mL of diluted acid solution were added into a 1-L glass liner, and then put into the reactor. During the experiment, 5 mL samples were taken starting at 100°C at 3 min intervals until reaching the targeted temperature (160, 175, or 190°C), followed by 4 samples after achieving the desired temperature. The collected samples were then cooled in an ice bath immediately to stop the reaction. The cooled samples were filtered using 0.2 μm MILLIPORE membrane filter to remove suspended solids. The filtered samples were then analyzed using High Performance Liquid Chromatography (HPLC) with a Bio-Rad Aminex HPX-87P column, and refractive index detection to measure monomeric and polymeric sugars plus degradation byproducts. A first order reaction model was assumed and the kinetic parameters such as activation energy and pre-exponential factor from Arrhenius equation were obtained from a match between the model and experimental data. The reaction temperature increases linearly after 40 minutes during experiments. Xylose and other sugars were formed from hemicellulose hydrolysis over this heat up period until a maximum concentration was reached at the time near when the targeted temperature was reached. However, negligible amount of xylose byproducts and small concentrations of other soluble sugars, such as mannose, arabinose, and galactose were detected during this initial heat up period. Very little cellulose hydrolysis yielding glucose was observed during the initial heat up period. On the other hand, later in the reaction during the constant temperature period xylose was degraded to furfural. Glucose production from cellulose was increased during this constant temperature period at later time points in the reaction. The kinetic coefficient governing the generation of xylose from hemicellulose and the generation of furfural from xylose presented a coherent dependence on both temperature and acid concentration. However, no effect was observed in the particle size. There were three types of biomass used in this project; hardwood (aspen, basswood, and red maple), softwood (balsam), and a herbaceous crop (switchgrass). The activation energies and the pre-exponential factors of the timber species and switchgrass were in a range of 49 - 180 kJ/mol and from 7.5x104 - 2.6x1020 min-1, respectively, for the xylose formation model. In addition, for xylose degradation, the activation energies and the preexponential factors ranged from 130 - 170 kJ/mol and from 6.8x1013 - 3.7x1017 min-1, respectively. The results compare favorably with the literature values given by Ranganathan et al, 1985. Overall, up to 92 % of the xylose was able to generate from the dilute acid hydrolysis in this project.

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The polysaccharides extracted from Claviclonium ovatum were studied by a combination of compositional assays, reductive partial hydrolysis, linkage analysis, Fourier Transform infrared (FTIR) spectroscopy, and C-13, H-1, and C-13/H-1 heteronuclear multiple quantum correlation (HMQC) two-dimensional nuclear magnetic resonance (NMR) spectroscopy. The chemical and spectroscopic data showed that the alkali-modified C. ovatum polysaccharides are composed of a nearly idealized repeating unit of 6'-O-methylcarrabiose 2,4'-disulfate (the repeating unit of 6-O-methylated iota-earrageenan), although some minor components were also present. The C. ovatum galactans are the most highly methylated carrageenans reported. (C) 2004 Elsevier Ltd. All rights reserved.

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A basic lectin (pI approximately 10.0) was purified to homogeneity from the seeds of winged bean (Psophocarpus tetragonolobus) by affinity chromatography on Sepharose 6-aminocaproyl-D-galactosamine. The lectin agglutinated trypsinized rabbit erythrocytes and had a relative molecular mass of 58,000 consisting of two subunits of Mr 29,000. The lectin binds to N-dansylgalactosamine, leading to a 15-fold increase in dansyl fluorescence with a concomitant 25-nm blue shift in the emission maximum. The lectin has two binding sites/dimer for this sugar and an association constant of 4.17 X 10(5) M-1 at 25 degrees C. The strong binding to N-dansylgalactosamine is due to a relatively positive entropic contribution as revealed by the thermodynamic parameters: delta H = -33.62 kJ mol-1 and delta S0 = -5.24 J mol-1 K-1. Binding of this sugar to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of D-galactose. Studies with other sugars indicate that a hydrophobic substituent in alpha- conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are critical for sugar binding to this lectin. Lectin difference absorption spectra in the presence of N-acetylgalactosamine indicate perturbation of tryptophan residues on sugar binding. The results of stopped flow kinetics with N- dansylgalactosamine and the lectin are consistent with a simple one- step mechanism for which k+1 = 1.33 X 10(4) M-1 s-1 and k-1 = 3.2 X 10(- 2) s-1 at 25 degrees C. This k-1 is slower than any reported for a lectin-monosaccharide complex so far. The activation parameters indicate an enthalpically controlled association process.

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Endogenous prion proteins (PrP) play the central role in the pathogenesis of transmissible spongiform encephalopathies. The carbohydrate N-acetylgalactosamine 4-O sulfotransferase 8 (CHST8) promotes the conversion of the cellular PrP(C) into the pathogenic PrP(d). Six sequence variants within the CHST8 gene were identified by comparative sequencing and genotyped for a sample of 623 animals comprising bovine spongiform encephalopathy (BSE)-affected and healthy control cows representing German Fleckvieh (German Simmental), German Holstein (Holstein-Friesian) and Brown Swiss. Significant differences in the allele, genotype and haplotype frequencies between BSE-affected and healthy cows indicate an association of sequence variant g.37254017G>T with the development of the disease in Brown Swiss cattle.

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ABSTRACT : BACKGROUND : Diets that restrict carbohydrate (CHO) have proven to be a successful dietary treatment of obesity for many people, but the degree of weight loss varies across individuals. The extent to which genetic factors associate with the magnitude of weight loss induced by CHO restriction is unknown. We examined associations among polymorphisms in candidate genes and weight loss in order to understand the physiological factors influencing body weight responses to CHO restriction. METHODS : We screened for genetic associations with weight loss in 86 healthy adults who were instructed to restrict CHO to a level that induced a small level of ketosis (CHO ~10% of total energy). A total of 27 single nucleotide polymorphisms (SNPs) were selected from 15 candidate genes involved in fat digestion/metabolism, intracellular glucose metabolism, lipoprotein remodeling, and appetite regulation. Multiple linear regression was used to rank the SNPs according to probability of association, and the most significant associations were analyzed in greater detail. RESULTS : Mean weight loss was 6.4 kg. SNPs in the gastric lipase (LIPF), hepatic glycogen synthase (GYS2), cholesteryl ester transfer protein (CETP) and galanin (GAL) genes were significantly associated with weight loss. CONCLUSION : A strong association between weight loss induced by dietary CHO restriction and variability in genes regulating fat digestion, hepatic glucose metabolism, intravascular lipoprotein remodeling, and appetite were detected. These discoveries could provide clues to important physiologic adaptations underlying the body mass response to CHO restriction.