Catalytic action of L-2-halo acid dehalogenase on long-chain L-2-haloalkanoic acids in organic solvents

A Lyophilized preparation of L-2-halo acid dehalogenase was not only stable but also catalytically active in anhydrous dimethyl sulfoxide, toluene, and other organic solvents. 2-Halo acids with long alkyl (C(5)-C(16)) or aromatic (phenyl and benzyl) side chains were inert in water but dehalogenated effectively in anhydrous dimethyl sulfoxide by the lyophilized enzyme. Long chain 2-haloalkanoic acids such as 2-bromohexadecanoic acids were better as substrate than short-chain halo acids (e.g., 2-chloropropanoic acid). The dehalogenation proceed with inversion of C(2) configuration to produce the corresponding (2R)-2-hydroxy acids in anhydrous dimethyl sulfoxide in the same way as found in water.     

Metabotropic Excitatory Amino Acid Receptor Activation Stimulates Phospholipase D in Hippocampal Slices

Metabotropic excitatory amino acid (EAA) receptors are coupled to effector systems through G proteins. Because various G protein-coupled receptors stimulate the hydrolysis of phosphatidylcholine by phospholipase D (PLD), we examined the possibility that metabotropic EAA receptors exist that are coupled to the activation of PLD. We found that the selective metabotropic glutamate receptor (mGluR) agonists 1S,3R-amino-1,3-cyclopentanedicarboxylic acid (ACPD) and 1S,3S-ACPD, but not the inactive isomer, 1R,3S-ACPD, induce a concentration-dependent increase in PLD activity in hippocampal slices. Selective ionotropic glutamate receptor (iGluR) antagonists did not block 1S,3R-ACPD-induced PLD stimulation. Furthermore, although selective iGluR agonists did not activate this response, the nonselective mGluR-iGluR agonists, ibotenate and quisqualate, caused significant increases in PLD activity (all in the presence of iGluR antagonists). L-2-Amino-3-phosphonopropionic acid, which blocks the mGluR that is coupled to phosphoinositide hydrolysis in various brain regions, activates PLD to the same extent as the active isomers of ACPD. These data suggest that metabotropic EAA receptors exist in hippocampus that are coupled to PLD activation and are pharmacologically distinct from phosphoinositide hydrolysis-coupled mGluRs.     

Regulation by glycophorin of complement activation via the alternative pathway

The effect of glycophorin on complement activation via the alternative pathway was examined by incorporating it into the liposome membrane with trinitrophenylaminocaproyldipalmitoylphosphatidylethanolamine (TNP-Cap-DPPE). Liposomes having incorporated TNP-Cap-DPPE onto the membrane activate the alternative complement pathway of guinea pig as reported previously, and the additional insertion of glycophorin was found to reduce their activating capacity on the alternative complement pathway. This inhibitory effect was cancelled by pretreatment of the glycophorin-containing liposomes with neuraminidase indicating that the sialic acid in glycophorin is playing a role in the regulation of alternative complement pathway-activation on the biological membrane.     

以DL-丝氨酸为原料制备D-丝氨酸的新方法

以DL-丝氨酸为原料经过酯化、拆分和水解制备D-丝氨酸。使L-2,3-二苯甲酰酒石酸(L-DBTA)与DL-丝氨酸甲酯在无水乙醇中于60℃反应形成非对映体盐,冷却到0℃,D-丝氨酸甲酯.L-DBTA二盐析出,过滤后再经水解,得到D-丝氨酸,总收率为74.8%,旋光纯度达到98%以上。     

CHO cell mutants for arginyl-, asparagyl-, glutaminyl-, histidyl- and methionyl-transfer RNA synthetases: identification and initial characterization

A number of conditional lethal mutants of CHO cells that are defective in protein synthesis have been characterized with respect to their biochemical lesions. A defective aminoacyl-tRNA synthetase appears to be the basis of each mutant phenotype. In each strain, the specific activity in vitro of the synthetase cognate for one of the following amino acids was substantially reduced: arginine, asparagine, glutamine, histidine or methionine. One mutant, Arg-1, gave no detectable arginyl-tRNA synthetase activity, suggesting that it contains an altered enzyme that is unstable in vitro. Most of the mutants correspondingly exhibited impaired aminoacylation in vivo under nonpermissive conditions. However, two mutants, Arg-1 and His-1, appeared to have normal levels of acylated tRNA under the nonpermissive conditions which inhibited protein synthesis to approximately 50% and 10%, respectively. The expression of each mutant's phenotype, measured by rates of protein synthesis or growth, was a function of temperature and/or the concentration of amino acid cognate for the synthetase found to be deficient in vitro. The properties of these mutants make them applicable to diverse problems related to translation in mammalian cells.     

Correlation of the O-intermediate rate with the pKa of Asp-75 in the dark, the counterion of the Schiff base of Pharaonis phoborhodopsin (sensory rhodopsin II)

Pharaonis phoborhodopsin (ppR), also called pharaonis sensory rhodopsin II, NpSRII, is a photoreceptor of negative phototaxis in Natronomonas (Natronobacterium) pharaonis. The photocycle rate of ppR is slow compared to that of bacteriorhodopsin, despite the similarity in their x-ray structures. The decreased rate of the photocycle of ppR is a result of the longer lifetime of later photo-intermediates such as M- (ppR(M)) and O-intermediates (ppR(O)). In this study, mutants were prepared in which mutated residues were located on the extracellular surface (P182, P183, and V194) and near the Schiff base (T204) including single, triple (P182S/P183E/V194T), and quadruple mutants. The decay of ppR(O) of the triple mutant was accelerated approximately 20-times from 690 ms for the wild-type to 36 ms. Additional mutation resulting in a triple mutant at the 204th position such as T204C or T204S further decreased the decay half-time to 6.6 or 8 ms, almost equal to that of bacteriorhodopsin. The decay half-times of the ppR(O) of mutants (11 species) and those of the wild-type were well-correlated with the pK(a) value of Asp-75 in the dark for the respective mutants as spectroscopically estimated, although there are some exceptions. The implications of these observations are discussed in detail.     

解磷黑曲霉L-1菌株原生质体诱变育种

以从龙泉山地区酸性红壤中分离的黑曲霉L-1(Aspergillus niger sp.L-1)为出发菌株,通过研究菌株L-1原生质体的形成和再生条件,发现培养30 h的菌丝体在以0.15 mol/L氯化钾为渗透压稳定剂的基本培养基上经过再次培养20 h后,所得菌丝体用0.3%纤维素酶和0.4%蜗牛酶在30 ℃条件下处理...     

臭灵丹草氨基酸成分分析

用氨基酸分析仪分析中药臭灵丹草叶片的氨基酸成分,并比较其水解氨基酸和游离氨基酸含量.结果表明:臭灵丹草中鉴定出18种氨基酸成分,水解氨基酸总量为18.68%,游离氨基酸总量为2.75%.γ-氨基丁酸含量可达0.1%以上.     

三酶级联催化L-苏氨酸生产L-2-氨基丁酸

L-2-氨基丁酸(L-ABA)是一种重要的化工原材料和手性医药中间体,为了实现L-ABA的高效生产,本研究在大肠杆菌EscherichiacoliBL21(DE3)中分别表达大肠杆菌来源的苏氨酸脱氨酶(Threonine deaminase,TD)、苏云金芽孢杆菌来源的亮氨酸脱氢酶(Leucine dehydrogenase,LDH)和博伊丁假丝酵母来源的甲酸脱氢酶(Formatedehydrogenase,FDH),构建体外级联酶催化反应实现L-苏氨酸向L-ABA的转化,体系中TD、LDH和FDH添加最适比例为1∶1∶0.2。为了简化生产工艺,将3种酶在一株菌E. coli 3FT+L中共表达并实现上述配比,在30 L发酵罐中用E. coli 3FT+L全细胞转化12 h,L-ABA的产量为68.5 g/L,底物L-苏氨酸的摩尔转化率达到99.0%。该工艺路线绿色高效,为未来大规模生产L-ABA提供借鉴。