Expression of proteins containing disulfide bonds in an insect cell-free system and confirmation of their arrangements by MALDI-TOF MS

Escherichia coli alkaline phosphatase (AP) and human lysozyme (h-LYZ), which contain two and four disulfide bonds, respectively, were expressed in a cell-free protein synthesis system constructed from Spodoptera frugiperda 21 (Sf21) cells. AP was expressed in a soluble and active form using the insect cell-free system under non-reducing conditions, and h-LYZ was expressed in a soluble and active form under non-reducing conditions after addition of reduced glutathione (GSH), oxidized glutathione (GSSG), and protein disulfide isomerase (PDI). The in vitro synthesized proteins were purified by means of a Strep-tag attached to their C termini. Approximately 41 microg AP and 30 microg h-LYZ were obtained from 1 mL each of the reaction mixture. The efficiency of protein synthesis approached that measured under reducing conditions. Analysis of the disulfide bond arrangements by MALDI-TOF MS showed that disulfide linkages identical to those observed in the wild-type proteins were formed.     

Effect of Water Content on Glass Transition and Protein Aggregation of Whey Protein Powders During Short-Term Storage

The objectives of this study were to investigate the moisture-induced protein aggregation of whey protein powders and to elucidate the relationship of protein stability with respect to water content and glass transition. Three whey protein powder types were studied: whey protein isolate (WPI), whey protein hydrolysates (WPH), and beta-lactoglobulin (BLG). The water sorption isotherms were determined at 23 and 45°C, and they fit the Guggenheim–Andersson–DeBoer (GAB) model well. Glass transition was determined by differential scanning calorimeter (DSC). The heat capacity changes of WPI and BLG during glass transition were small (0.1 to 0.2 Jg⁻¹ °C⁻¹), and the glass transition temperature (T _g) could not be detected for all samples. An increase in water content in the range of 7 to 16% caused a decrease in T _g from 119 down to 75°C for WPI, and a decrease from 93 to 47°C for WPH. Protein aggregation after 2 weeks’ storage was measured by the increase in insoluble aggregates and change in soluble protein fractions. For WPI and BLG, no protein aggregation was observed over the range of 0 to 85% RH, whereas for WPH, ∼50% of proteins became insoluble after storage at 23°C and 85% RH or at 45°C and ≥73% RH, caused mainly by the formation of intermolecular disulfide bonds. This suggests that, at increased water content, a decrease in the T _g of whey protein powders results in a dramatic increase in the mobility of protein molecules, leading to protein aggregation in short-term storage.     

Impact of Surface Active Compounds on Iron Catalyzed Oxidation of Methyl Linolenate in AOT–Water–Hexadecane Systems

Edible oils contain minor surface active components that form micro-heterogeneous environments, such as reverse micelles, which can alter the rate and direction of chemical reactions. However, little is known about the role of these micro-heterogeneous environments on lipid oxidation of bulk oil. Our objective was to evaluate the ability of water, cumene hydroperoxide, oleic acid, and phosphatidylcholine to influence the structure of reverse micelles in a model oil system: sodium bis(2-ethylhexyl) sulfosuccinate (aerosol-OT; AOT) in n-hexadecane. The influence of reverse micelle structure on iron catalyzed lipid oxidation was determined using methyl linolenate as an oxidizable substrate. The size and shape of the reverse micelle were investigated by small-angle x-ray scattering, and water contents was determined by Karl Fischer titrations. Lipid hydroperoxides and thiobarbituric acid reactive substances were used to follow lipid oxidation. Our results showed that AOT formed spherical reverse micelles in hexadecane. The size of the reverse micelles increased with increased water or phosphatidylcholine concentration, but decreased upon addition of cumene hydroperoxide or oleic acid. Iron catalyzed oxidation of methyl linolenate in the reverse micelle system decreased with increasing water concentration. Addition of phosphatidylcholine into the reverse micelle systems decreased methyl linolenate oxidation compared to control and reverse micelles with added oleic acid. These results indicate that water, cumene hydroperoxide, oleic acid, and phosphatidylcholine can alter reverse micelle size and lipid oxidation rates. Understanding how these compounds influence reverse micelle structure and lipid oxidation rates could provide information on how to modify bulk oil systems to increase oxidative stability.     

‘嘎拉’苹果及其杂交后代对炭疽叶枯病的抗性机制分析

为解析苹果对炭疽叶枯病的抗性机制,该研究以‘嘎拉’、‘藤牧1号’苹果及其杂交后代等87个苹果品种(系)为试验材料,进行田间调查及人工接种病菌并检测不同品种(系)中病菌生物量,利用SSR分子标记进行基因型鉴定,分析各品种(系)对炭疽叶枯病的抗性差异,利用荧光定量PCR及酶活检测比较分析水杨酸相关基因、抗性酶基因及酶活性水平差异。结果表明：(1)87个苹果品种(系)对苹果炭疽叶枯病的抗性差异明显,‘嘎拉’、‘2 5’、‘19 19’等品种(系)叶片发病严重,表现出对炭疽叶枯病的高感性,‘藤牧1号’、‘40 9’及‘16 16’等品种(系)叶片无病斑或病斑极少,炭疽叶枯病菌含量显著低于感病性品种(系),其抗性显著。(2)SSR标记S0405127和S0304673的基因型鉴定结果与田间表型调查结果相比,准确率分别为93.10%和91.95%,与人工接种结果相比,准确率分别为91.95%和95.40%。(3)SA相关基因的表达模式结果表明,接种炭疽叶枯病菌4 d后,‘藤牧1号’、‘40 9’及‘16 16’等抗性品种(系)中SA合成相关基因MdEDS1、MdPAD4和MdPAL被强烈诱导表达;同时,SA信号转导相关基因MdNPR1、MdPR1、MdPR5的表达显著高于‘嘎拉’等感病品种(系)。(4)接种炭疽叶枯病菌4 d后,‘藤牧1号’、‘40 9’及‘16 16’等抗性品种(系)的MdSOD、MdPOD酶基因表达水平及酶活性显著高于‘嘎拉’、‘2 5’、‘19 19’等感病品种(系)。研究认为,‘藤牧1号’、‘40 9’及‘16 16’等品种(系)通过调控水杨酸合成和信号转导通路及氧化还原相关反应等提高对炭疽叶枯病的抗性,为挖掘抗性基因以及利用优良种质选育抗病品种奠定了基础。     

Enhancing cofactor recycling in the bioconversion of racemic alcohols to chiral amines with alcohol dehydrogenase and amine dehydrogenase by coupling cells and cell-free system

Alcohol dehydrogenase (ADH) and amine dehydrogenase (AmDH)-catalyzed one-pot cascade conversion of an alcohol to an amine provides a simple preparation of chiral amines. To enhance the cofactor recycling in this reaction, we report a new concept of coupling whole-cells with the cell-free system to enable separated intracellular and extracellular cofactor regeneration and recycling. This was demonstrated by the respective biotransformation of racemic 4-phenyl-2-butanol 1a and 1-phenyl-2-propanol 1b to (R)-4-phenylbutan-2-amine 3a and (R)-1-phenylpropan-2-amine 3b . Escherichia coli cells expressing S-enantioselective CpsADH, R-enantioselective PfODH, and NADH oxidase (NOX) was developed to oxidize racemic alcohols 1a–b to ketones 2a–b with full conversion via intracellular NAD⁺ recycling. AmDH and glucose dehydrogenase (GDH) were used to convert ketones 2a–b to amines (R)- 3a–b with 89–94% conversion and 891–943 times recycling of NADH. Combining the cells and enzymes for the cascade transformation of racemic alcohols 1a–b gave 70% and 48% conversion to the amines (R)- 3a and (R)-3 b in 99% ee, with a total turnover number (TTN) of 350 and 240 for NADH recycling, respectively. Improved results were obtained by using the E. coli cells with immobilized AmDH and GDH: (R)- 3a was produced in 99% ee with 71–84% conversion and a TTN of 1410-1260 for NADH recycling, the highest value so far for the ADH–AmDH-catalyzed cascade conversion of alcohols to amines. The concept might be generally applicable to this type of reactions.     

Synthesis of a new disulfide Fmoc monomer for creating biologically susceptible linkages in peptide nucleic acid oligomers

Peptide nucleic acids (PNA) are one of many synthetic mimics of DNA and RNA that have found applications as biological probes, as nano-scaffold components, and in diagnostics. In an effort to use PNA as constructs for cellular delivery we investigated the possibility of installing a biologically susceptible disulfide bond in the backbone of a PNA oligomer. Here we report the synthesis of a new abasic Fmoc monomer containing a disulfide bond that can be incorporated into a PNA oligomer (DS-PNA) using standard solid phase peptide synthesis. The disulfide bond survives cleavage from the resin and DS-PNA forms duplexes with complementary PNA oligomers. Initial studies aimed at determining if the disulfide bond is cleavable to reducing agents while in a duplex are explored using UV thermal analysis and HPLC.     

Effects of dietary protein contents and habitual endurance exercise on supplemental leucine oxidation in mice

The effects of dietary protein contents and regular exercise on the oxidation of supplemented leucine were examined. In the short-term study, male BALB/cCrSlc mice were fed diets containing 0, 10, 20, 35, and 60% protein: energy ratios for 1 week. In the long-term study, exercised and sedentary mice were fed diets containing 20, 35, and 60% protein ratios for 9 weeks. After the feeding periods, the mice were a bolus administered oral supplements of l-[1-¹³C] leucine. Expired gas was analyzed, and oxidized leucine was expressed as a relative ¹³CO₂/¹²CO₂ ratio. In the short-term study, the peak ¹³CO₂/¹²CO₂ ratio significantly increased with diet protein concentrations. Moreover, the long-term study also showed that the peak ¹³CO₂/¹²CO₂ ratio was significantly increased by high protein diets in both exercised and sedentary mice. Our results indicate that supplemental leucine oxidation is associated with consumption of a high-protein diet, irrespective of exercise status.

Abbreviations: AUC: area under the curve; EX: exercise; RQ: respiratory quotient; SED: sedentary; VO₂/W: oxygen uptake per body weight