耐有机溶剂氨基酸脱氢酶基因挖掘与非天然氨基酸的非水相合成

可在非水相体系高效催化不对称还原反应制备手性化合物的氧化还原酶具有重要科学意义与工业应用前景。基于基因挖掘技术,获得了17个耐盐氨基酸脱氢酶的基因,并分析了其进化同源性和蛋白质稳定性热力学参数。选取来源于Natranaerobius thermophilus的苯丙氨酸脱氢酶（PheDH）,进行了基因合成和表达、分离和纯化,获得了耐盐氨基酸脱氢酶,并检测了其有机溶剂耐受性。结果表明,对于催化L-苯丙氨酸的氧化脱氨体系,反应的最适温度为60℃,最适pH为 12。在含有30%的二甲亚砜反应体系中,催化活性是水相体系的1.2倍。而对于催化还原胺化制备L-高苯丙氨酸的体系,最适温度为70℃,最适pH为8.5。在含30%的甲基叔丁基醚和二甲亚砜反应体系中,催化活性分别是原始活性的101.3%和99.2%。研究表明,该耐盐酶具有较好的耐热、耐有机溶剂等抗逆性能。     

Processing of N-terminal unnatural amino acids in recombinant human interferon-beta in Escherichia coli

Incorporation of unnatural amino acids into recombinant proteins represents a powerful tool for protein engineering and protein therapeutic development. While the processing of the N-terminal methionine (Met) residues in proteins is well studied, the processing of unnatural amino acids used for replacing the N-terminal Met remains largely unknown. Here we report the effects of the penultimate residue (the residue after the initiator Met) on the processing of two unnatural amino acids, L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG), at the N terminus of recombinant human interferon-beta in E. coli. We have identified specific amino acids at the penultimate position that can be used to efficiently retain or remove N-terminal AHA or HPG. Retention of N-terminal AHA or HPG can be achieved by choosing amino acids with large side chains (such as Gln, Glu, and His) at the penultimate position, while Ala can be selected for the removal of N-terminal AHA or HPG. Incomplete processing of N-terminal AHA and HPG (in terms of both deformylation and cleavage) was observed with Gly or Ser at the penultimate position.     

Natural and unnatural silks

Natural silk is an important biopolymer with huge potential as it combines superb mechanical properties with environmentally sensitive production methods. Native silk dope taken straight from the gland can easily and without chemical assistance be drawn into strong fibres. Artificial silk fibres, on the other hand, rely on spinning dopes typically ‘reconstituted’ from natural silk fibres by strong chaotropic agents. Such fibres do not form readily, and often require chemical post-spin treatment for stabilisation. In addition these fibres tend to be brittle, and so far have been unable to match native fibres. Here we present novel rheometric data to argue that native and reconstituted silkworm silk dope differ in kind, not just in degree. While native silks behave like typical molten polymers, reconstituted silks do not. We conclude that rheology provides a powerful tool in the quest to learn from the Nature's polymer fibre technology.     

Amino acid oxidase‐catalysed resolution and Pictet–Spengler reaction towards chiral and rigid unnatural amino acids

BACKGROUND: The biosynthesis of structurally complex isoquinoline alkaloids and other natural products occurs via aromatic amino acids such as tyrosine, and chiral and rigid amino acids. These structures are also key building blocks of many active pharmaceutical ingredients. The aim of this work was the exploration of a rapid and straightforward route to chiral 6‐hydroxy‐1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acid. RESULTS: The preparation of (S)‐meta‐tyrosine from racemic meta‐tyro‐ sine with aminoacidoxidase has been developed with ee > 99% and 88% yield. The combination of this resolution with a subsequent Pictet–Spengler reaction enables straightforward and versatile access to chiral (S)‐6‐hydroxy‐1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acid in 30% yield. CONCLUSIONS: This new short chemoenzymatic route to (S)‐6‐hydroxy‐1,2,3,4‐tetrahydroisoquinoline‐3‐carboxylic acid from commercially available DL‐m‐tyrosine is more convenient than other chemical procedures and establishes a new link between the pool of easily accessible racemic aromatic amino acids and the corresponding chiral rigidified amino acids, which are of interest as structural elements of many active pharmaceutical ingredients. These results facilitate synthetic access to a range of active pharmaceutical ingredients and metabolites in chiral form from the oxidation of amino acids. This advances the opportunities to study the molecular interactions with enzymes, receptors and effectors more precisely than with the racemic forms. Copyright © 2007 Society of Chemical Industry     

Hydration free energies calculated using the AMBER ff03 charge model for natural and unnatural amino acids and multiple water models

In this work, we assess calculated hydration free energies of natural and unnatural amino acids compared to experimental hydration free energies of corresponding side-chain analogs using the ff03 charge model. Fine-grid, explicit water thermodynamic integration calculations using two widely used explicit water models (TIP3P and TIP4P-Ew) were performed on 19 natural amino acids and compared with experimental hydration free energies of corresponding side-chain analogs to establish expected accuracy levels for this charge model. Next, parameters previously derived for 17 unnatural amino acids and several new parameters optimized in this work were assessed using the same methodology. We found that the ff03 charge model is correlated with experimental hydration free energies but underestimates the solubilities of several polar natural and unnatural amino acids. Comparisons are presented with other forcefields and water models recently presented in the literature for both natural and modified amino acids.     

Lipase-catalyzed transformations with unnatural acyl acceptors

Lipases are readily available and extremely versatile enzymes. Besides their natural reaction — hydrolysis of lipids — lipases catalyze a large number of other reactions involving a plethora of acyl acceptors such as alcohols, amines, ammonia and hydrogen peroxide. They are also able to accommodate a variety of different structures in the acyl donor. With chiral substrates many of these reactions proceed with a high degree of enantioselectivity. The scope of these lipase-catalyzed transformations with different acyl acceptors is the subject of this review.     

Elucidation of the structure-activity relationships of apelin: influence of unnatural amino acids on binding, signaling, and plasma stability

Apelin is the endogenous ligand of the APJ receptor, a member of the G-protein-coupled receptor family. The apelin-APJ complex has been detected in many tissues and is emerging as a promising target for several pathophysiological conditions. There is currently little information on the structure-activity relationship (SAR) of the apelin hormone. In an effort to better delineate SAR, we synthesized analogues of apelin-13 modified at selected positions with unnatural amino acids, with a particular emphasis on the C-terminal portion. Analogues were then tested in binding and functional assays by evaluating Gi/o-mediated decreases in cAMP levels and by assessing β-arrestin2 recruitment to the APJ receptor. The plasma stability of new compounds was also assessed. Several analogues were found to possess increased binding and higher stability than the parent peptide.     

Phase transition of an unnatural analog of phosphatidylcholine: Phosphatidyl-N-isopropylethanolamine

The phase-transition temperature, of an aqueous dispersion of dipalmitoyl phosphatidyl-N-isopropylethanolamine was determined was determined usingtrans-parinaric acid as a fluorescent probe. Phosphatidyl-N-isopropylethanolamine has a phase-transition temperature that is 3–4 C higher than the corresponding natural analog of dipalmitoyl phosphatidylcholine.     

Engineering the CYP101 system for in vivo oxidation of unnatural substrates

The protein engineering of CYP enzymes for structure–activitystudies and the oxidation of unnatural substrates for biotechnologicalapplications will be greatly facilitated by the availabilityof functional, whole-cell systems for substrate oxidation. Wereport the construction of a tricistronic plasmid that expressesthe CYP101 monooxygenase from Pseudomonas putida, and its physiologicalelectron transfer co-factor proteins putidaredoxin reductaseand putidaredoxin in Escherichia coli, giving a functional invivo catalytic system. Wild-type CYP101 expressed in this systemefficiently transforms camphor to 5-exo-hydroxycamphor withoutfurther oxidation to 5-oxo-camphor until >95% of camphorhas been consumed. CYP101 mutants with increased activity forthe oxidation of diphenylmethane (the Y96F–I395G mutant),styrene and ethylbenzene (the Y96F–V247L mutant) havebeen engineered. In particular, the Y96F–V247L mutantshows coupling efficiency of approximately 60% for styrene andethylbenzene oxidation, with substrate oxidation rates of approximately100/min. Escherichia coli cells transformed with tricistronicplasmids expressing these mutants readily gave 100-mg quantitiesof 4-hydroxydiphenylmethane and 1-phenylethanol in 24–72h. This new in vivo system can be used for preparative scalereactions for product characterization, and will greatly facilitatedirected evolution of the CYP101 enzyme for enhanced activityand selectivity of substrate oxidation.     

Tryptophan fluorescence of calmodulin binding domain peptides interacting with calmodulin containing unnatural methionine analogues

The interactions between the abundant methionine residues ofthe calcium regulatory protein calmodulin (CaM) and severalof its binding targets were probed using fluorescence spectroscopy.Tryptophan steady-state fluorescence from peptides encompassingthe CaM-binding domains of the target proteins myosin lightchain kinase (MLCK), cyclic nucleotide phosphodiesterase (PDE)and caldesmon site A and B (CaD A, CaD B), and the model peptidemelittin showed Ca²⁺-dependent blue-shifts in their maximumemission wavelength when complexed with wild-type CaM. Blue-shiftswere also observed for complexes in which the CaM methionineresidues were replaced by selenomethionine, norleucine and ethionine,and when a quadruple methionine to leucine C-terminal mutantof CaM was studied. Quenching of the tryptophan fluorescenceintensity was observed with selenomethionine, but not with norleucineor ethionine substituted protein. Fluorescence quenching studieswith added potassium iodide (KI) demonstrate that the non-nativeproteins limit the solvent accessibility of the Trp in the MLCKpeptide to levels close to that of the wild-type CaM–MLCKinteraction. Our results show that the methionine residues fromCaM are highly sensitive to the target peptide in question,confirming the importance of their role in binding interactions.In addition, we provide evidence that the nature of bindingin the CaM–CaD B complex is unique compared with the othercomplexes studied, as the Trp residue of this peptide remainspartially solvent exposed upon binding to CaM.     

Application performance and surface morphologies of amino polysiloxanes with different amino values and amino types

Amino polysiloxanes (APSs) with different amino values and amino types were synthesized and applied to cotton fabrics. Softening and smoothening properties of the fabrics treated with APSs were investigated and evaluated by measuring wrinkle recovery angles and friction coefficients, and the morphological features of the APSs adsorbed onto cellulose substrate films were characterized by atomic force microscope (AFM). The results indicate that the amino values and amino types of the APSs have a significant impact on the softening and smoothening properties of the fabrics. APSs with relatively high amino values exhibit superior smoothening property, while APSs with moderate amino values exhibit excellent softening property. Compared to the traditional softener N‐β‐aminoethyl‐γ‐aminopropyl polydimethylsiloxane (APS‐1), the new amino type softeners γ‐piperazinylpropyl polydimethylsiloxane (APS‐2) and N‐γ'‐dimethylaminopropyl‐γ‐aminopropyl polydimethylsiloxane (APS‐3) gave better fabric performance, whereas aminopropyl polydimethylsiloxane (APS‐4) and N‐cyclohexyl‐γ‐aminopropyl polydimethylsiloxane (APS‐5) gave unsatisfactory fabric performance. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Incorporation of an unnatural amino acid in the active site of porcine pancreatic phospholipase A2. Substitution of histidine by l,2,4-triazole-3-alanine yields an enzyme with high activity at acidic pH

The effect of the substitution of the active site histidine48 by the unnatural 1,2,4-triazole-3-alanine (TAA) amino acidanalogue in porcine pancreas phospholipase A₂ (PLA₂) was studied.TAA was introduced biosynthetically using a his-auxotrophicEscherichia coli strain. To study solely the effect of the substitutionof the active site histidine, two nonessential histidines (i.e.His17 and His 115) were replaced by asparagines, resulting ina fully active mutant enzyme (His-PLA₂). In this His-PLA₂ thesingle histidine at position 48 was substituted by TAA withan incorporation efficiency of about 90%, giving a mixture ofHis-PLA₂ and TAA-PLA₂. Based on the charge difference at acidicpH, both forms could be separated by FPLC, allowing for thepurification of TAA-PLA₂ free from His-PLA₂. At pH 6, TAA-PLA₂has a fivefold reduced activity compared with His-PLA₂. Thisreduced activity paralells a reduced rate of covalent modificationwith p-nitrophenacyl bromide of TAA-PLA₂ compared with His-PLA₂.Competitive inhibition gave comparable IC₅₀ values for WT-PLA₂,His-PLA₂ and TAA-PLA₂. These results indicate that the reductionin activity is not caused by a different affinity for the substrate,but more likely results from a reduced k_cat value in TAA-PLA₂.The enzymatic activities for native and mutant PLA₂s were measuredat different pH values. For WT-PLA₂ and His-PLA₂ the activityis optimal at pH 6 and is strongly deminished at acidic pH,with no observable activity at pH 3. In contrast, TAA-PLA₂ isas active at pH 3 as at pH 6. Most likely, the decrease in activityobserved for WT-PLA₂ and His-PLA₂ is caused by the protonationof the active site His48, which is the general base involvedin the activation of the nucleophilic water molecule. In TAA-PLA₂,however, the active site residue TAA48 is unprotonated at bothpH 3 and 6 as a result of the low pK_a of TAA compared with histidine.