Biocatalytic Alkylation Cascades: Recent Advances and Future Opportunities for Late-Stage Functionalization

This Concept article describes the latest developments in the emerging area of late-stage biocatalytic alkylation. Central to these developments is the ability to efficiently prepare S-adenosyl methionine (SAM) cofactor analogues and couple this with enzymatic alkyl transfer. Recent developments in the enzymatic synthesis of SAM cofactor analogues are summarized first, followed by their application as alkyl transfer agents catalyzed by methyltransferases (MTases). Second, innovative methods to regenerate SAM cofactors by enzymatic cascades is reported. Finally, future opportunities towards establishing a generalized platform for late-stage alkylation are described.     

A Methionine Chemical Shift Based Order Parameter Characterizing Global Protein Dynamics

Coupling of side chain dynamics over long distances is an important component of allostery. Methionine side chains show the largest intrinsic flexibility among methyl-containing residues but the actual degree of conformational averaging depends on the proximity and mobility of neighboring residues. The ¹³C NMR chemical shifts of the methyl groups of methionine residues located at long distances in the same protein show a similar scaling with respect to the values predicted from the static X-ray structure by quantum methods. This results in a good linear correlation between calculated and observed chemical shifts. The slope is protein dependent and ranges from zero for the highly flexible calmodulin to 0.7 for the much more rigid calcineurin catalytic domain. The linear correlation is indicative of a similar level of side-chain conformational averaging over long distances, and the slope of the correlation line can be interpreted as an order parameter of the global side-chain flexibility.     

全球蛋氨酸市场现状分析

目前全球蛋氨酸市场由少数几家公司控制 ,其中Novus、Degussa、Adiesso公司分别占有 3 4%、3 0 %和 2 8%的市场份额 ,国内迄今尚未有一套规模型生产装置。而亚洲地区蛋氨酸供应紧张 ,中国作为世界第二大饲料生产国 ,对蛋氨酸的需求更高 ,目前我国主要依靠进口解决需求问题 ,2 0 0 1～ 2 0 0 3年每年进口量均在 5万t/a以上。     

全球氨基酸市场供需与预测

概述了近年来饲料添加剂用氨基酸类的现状和今后发展。着重介绍了氨基酸中主要产品蛋氨酸、赖氨酸和苏氨酸在美国、欧盟和日本的生产和市场发展动向。由于这类食品添加剂需求强劲，目前，该市场正处在回升之中。     

Radiation- and Photo-Induced Oxidation Pathways of Methionine in Model Peptide Backbone under Anoxic Conditions

Within the reactive oxygen species (ROS) generated by cellular metabolisms, hydroxyl radicals (HO^•) play an important role, being the most aggressive towards biomolecules. The reactions of HO^• with methionine residues (Met) in peptides and proteins have been intensively studied, but some fundamental aspects remain unsolved. In the present study we examined the biomimetic model made of Ac-Met-OMe, as the simplest model peptide backbone, and of HO^• generated by ionizing radiation in aqueous solutions under anoxic conditions. We performed the identification and quantification of transient species by pulse radiolysis and of final products by LC-MS and high-resolution MS/MS after γ-radiolysis. By parallel photochemical experiments, using 3-carboxybenzophenone (CB) triplet with the model peptide, we compared the outcomes in terms of short-lived intermediates and stable product identification. The result is a detailed mechanistic scheme of Met oxidation by HO^•, and by CB triplets allowed for assigning transient species to the pathways of products formation.     

Models to predict milk fat concentration and yield of lactating dairy cows: A meta-analysis

Few models have attempted to predict total milk fat because of its high variation among and within herds. The objective of this meta-analysis was to develop models to predict milk fat concentration and yield of lactating dairy cows. Data from 158 studies consisting of 658 treatments from 2,843 animals were used. Data from several feed databases were used to calculate dietary nutrients when dietary nutrient composition was not reported. Digested intake (DI, g/d) of each fatty acid (FA; C12:0, C14:0, C16:0, C16:1, C18:0, C18:1 cis, C18:1 trans C18:2, C18:3) and absorbed amounts (g/d) of each AA (Arg, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Val) were calculated and used as candidate variables in the models. A multi-model inference method was used to fit a large set of mixed models with study as the random effect, and the best models were selected based on Akaike's information criterion corrected for sample size and evaluated further. Observed milk fat concentration (MFC) ranged from 2.26 to 4.78%, and milk fat yield (MFY) ranged from 0.488 to 1.787 kg/d among studies. Dietary levels of forage, starch, and total FA (dry matter basis) averaged 50.8 ± 10.3% (mean ± standard deviation), 27.5 ± 7.0%, and 3.4 ± 1.3%, respectively. The MFC was positively correlated with dietary forage (0.294) and negatively associated with dietary starch (?0.286). The DI of C18:2 (g/d) was more negatively correlated with MFC (?0.313) than that of the other FA. The best variables for predicting MFC were days in milk, FA-free dry matter intake, forage, starch, DI of C18:2, DI of C18:3, and absorbed Met, His, and Trp. The best predictor variables for MFY were FA-free dry matter intake, days in milk, absorbed Met and Ile, and intakes of digested C16:0 and C18:3. This model had a root mean square error of 14.1% and concordance correlation coefficient of 0.81. Surprisingly, DI of C18:3 was positively related to milk fat, and this relationship was consistently observed among models. The models developed can be used as a practical tool for predicting milk fat of dairy cows, while recognizing that additional factors are likely to also affect fat yield.     

Effect of Methionine Sulfoxide on the Synthesis and Purification of Aggregation-Prone Peptides

A two-step synthesis for methionine-containing hydrophobic and/or aggregation-prone peptides is presented that takes advantage of the reversibility of methionine oxidation. The use of polar methionine sulfoxide as a building block in solid-phase peptide synthesis improves the synthesis quality and yields the crude peptide, with significantly improved solubility compared to the reduced species. This facilitates the otherwise often laborious peptide purification by high-performance liquid chromatography. The subsequent reduction proceeds quantitatively. This approach has been optimised with the methionine-rich Tar-DNA-binding protein 43 (307–347), but is also more generally applicable, as demonstrated by the syntheses of human calcitonin and two aggregation-prone peptides from the human prion protein.     

The methionine precursor dl-2-hydroxy-(4-methylthio)butanoic acid protects intestinal epithelial barrier function

dl-2-hydroxy-(4-methylthio)butanoic acid (HMTBA) is a source of dietary methionine (Met) that is widely used in poultry nutrition. We have previously shown that HMTBA is preferentially diverted to the transsulfuration pathway, which gives antioxidant metabolites such as taurine and glutathione. Therefore, here we hypothesize that this Met source can protect epithelial barrier function in an in vitro model of intestinal inflammation of Caco-2 cells. The results show that HMTBA prevents the increase in paracellular permeability induced by H₂O₂ or tumour necrosis factor-α. This effect can be attributed to the increased production of taurine and reduced glutathione. Similar results were obtained for dl-Met, although the protective role of the amino acid was less pronounced than that of the hydroxy analogue. In conclusion, the diversion to the transsulfuration pathway means that this Met precursor is of greater value than previously thought, due to its capacity to improve intestinal homeostasis and the quality of poultry products destined for human consumption.     

Generalized hybrid control synthesis for affine systems using sequential adaptive networks

BACKGROUND: A generalized methodology for the synthesis of a hybrid controller for affine systems using sequential adaptive networks (SAN) is presented. SAN consists of an assembly of neural networks that are ordered in a chronological sequence, with one network assigned to each sampling interval. Using a suitable process model based on oxygen metabolism and an a priori objective function, a hybrid control law is derived that can use online measurements and the states predicted by SAN for computing the desired control action. RESULTS: The performance of the SAN–hybrid controller is tested for simulated fed‐batch production of methionine for three different process conditions. Simulations assume that online measurements of dissolved oxygen (DO) concentration are available. The performance of the SAN–hybrid controller gave an NRMSE of ～10^?4 in the absence of noise, ～10^?3 and ～10^?2 for ± 5% and ± 10% noise in the DO measurement and ～10^?2 for parameter uncertainty when compared with the ideal model prediction. CONCLUSIONS: The observed performance for unmeasured state prediction and control implementation shows that the proposed SAN–hybrid controller can efficiently compute the manipulated variable required to maintain methionine production along the optimized trajectory for different conditions. The test results show that the SAN–hybrid controller can be used for online real‐time implementation in fed‐batch bioprocesses. Copyright © 2009 Society of Chemical Industry