Integration of ion exchange resin materials for a downstream-processing approach of an imine reductase-catalyzed reaction

In this study, an ion exchange resin-based downstream-processing concept for imine reductase (IRED)-catalyzed reactions was investigated. As a model reaction, 2-methylpyrroline was converted to its corresponding product (S)-2-methylpyrrolidine with >99% of conversion by the (S)-selective IRED from Paenibacillus elgii B69. Under optimized reaction conditions full conversion was achieved using a substrate concentration of 150 and 500 mmol/L of d -glucose. Seven commercially available cation- and anion-exchange resins were studied with respect to their ability to recover the product from the reaction solution. Without any pretreatment, cation-exchange resins Amberlite IR-120(H), IRN-150, Dowex Monosphere 650C, and Dowex Marathon MSC showed high recovery capacities (up to >90%). A 150-ml preparative scale reaction was performed yielding ~1 g hydrochloride salt product with >99% purity. Any further purification steps, for example, by column chromatography or recrystallization, were not required.     

Genomic and experimental evidence for multiple metabolic functions in the RidA/YjgF/YER057c/UK114 (Rid) protein family

Background

It is now recognized that enzymatic or chemical side-reactions can convert normal metabolites to useless or toxic ones and that a suite of enzymes exists to mitigate such metabolite damage. Examples are the reactive imine/enamine intermediates produced by threonine dehydratase, which damage the pyridoxal 5''-phosphate cofactor of various enzymes causing inactivation. This damage is pre-empted by RidA proteins, which hydrolyze the imines before they do harm. RidA proteins belong to the YjgF/YER057c/UK114 family (here renamed the Rid family). Most other members of this diverse and ubiquitous family lack defined functions.

Results

Phylogenetic analysis divided the Rid family into a widely distributed, apparently archetypal RidA subfamily and seven other subfamilies (Rid1 to Rid7) that are largely confined to bacteria and often co-occur in the same organism with RidA and each other. The Rid1 to Rid3 subfamilies, but not the Rid4 to Rid7 subfamilies, have a conserved arginine residue that, in RidA proteins, is essential for imine-hydrolyzing activity. Analysis of the chromosomal context of bacterial RidA genes revealed clustering with genes for threonine dehydratase and other pyridoxal 5''-phosphate-dependent enzymes, which fits with the known RidA imine hydrolase activity. Clustering was also evident between Rid family genes and genes specifying FAD-dependent amine oxidases or enzymes of carbamoyl phosphate metabolism. Biochemical assays showed that Salmonella enterica RidA and Rid2, but not Rid7, can hydrolyze imines generated by amino acid oxidase. Genetic tests indicated that carbamoyl phosphate overproduction is toxic to S. enterica cells lacking RidA, and metabolomic profiling of Rid knockout strains showed ten-fold accumulation of the carbamoyl phosphate-related metabolite dihydroorotate.

Conclusions

Like the archetypal RidA subfamily, the Rid2, and probably the Rid1 and Rid3 subfamilies, have imine-hydrolyzing activity and can pre-empt damage from imines formed by amine oxidases as well as by pyridoxal 5''-phosphate enzymes. The RidA subfamily has an additional damage pre-emption role in carbamoyl phosphate metabolism that has yet to be biochemically defined. Finally, the Rid4 to Rid7 subfamilies appear not to hydrolyze imines and thus remain mysterious.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1584-3) contains supplementary material, which is available to authorized users.     

Preparation of benzophenone imine complexes of transition metals

Benzophenone imine [M(η¹-NHCPh₂)(CO)_nP_5-n]BPh₄ [M = Mn, Re; n = 2, 3; P = P(OEt)₃, PPh(OEt)₂, PPh₂OEt, PPh₃] complexes were prepared by allowing triflate M(κ¹-OTf)(CO)_nP_5-n compounds to react with an excess of the imine. Hydride-imine [MH(η¹-NHCPh₂)P₄]BPh₄ (M = Ru, Os), triflate-imine [Os(κ¹-OTf)(η¹-NHCPh₂)P₄]BPh₄ and bis(imine) [Ru(η¹-NHCPh₂)₂P₄](BPh₄)₂ [P = P(OEt)₃] derivatives were also prepared. The complexes were characterized spectroscopically (IR, ¹H, ³¹P, ¹³C NMR) and a geometry in solution was also established. Hydride-benzophenone imine [IrHCl(η¹-NHCPh₂)L(PPh₃)₂]BPh₄and [IrHCl(η¹-NHCPh₂)L(AsPh₃)₂]BPh₄ [L = P(OEt)₃ and PPh(OEt)₂] complexes were prepared by reacting hydride IrHCl₂L(PPh₃)₂ and IrHCl₂L(AsPh₃)₂ precursors with an excess of imine. Dihydride IrH₂(η¹-NHCPh₂)(PPh₃)₃ complex was also obtained and a geometry in solution was proposed.     

Cloning and characterization of novel tumor-targeting immunocytokines based on murine IL7

We generated and characterized novel antibody-cytokine fusion proteins (“immunocytokines”) based on murine interleukin-7 (IL7), an immunomodulatory protein which has previously shown anti-cancer activity in preclinical models and whose human counterpart is currently being investigated in clinical trials. The sequential fusion of the clinical-stage antibody fragment scFv(F8), specific to a tumor-associated splice isoform of fibronectin, yielded an immunocytokine (termed “F8-mIL7”) of insufficient pharmaceutical quality and in vivo tumor targeting performance, with a striking dose dependence on tumor targeting selectivity. By contrast, a novel immunocytokine design (termed “F8-mIL7-F8”), in which two scFv moieties were fused at the N- and C-terminus of murine IL7, yielded a protein of excellent pharmaceutical quality and with improved tumor-targeting performance [tumor: blood ratio = 16:1, 24 h after injection]. Both F8-mIL7 and F8-mIL7-F8 could induce tumor growth retardation in immunocompetent mice, but were not able to eradicate F9 tumors. The combination of F8-mIL7-F8 with paclitaxel led to improved therapeutic results, which were significantly better compared to those obtained with saline treatment. The study indicates how the engineering of novel immunocytokine formats may help generate fusion proteins of acceptable pharmaceutical quality, for those immunomodulatory proteins which do not lend themselves to a direct fusion with antibody fragments.     

蛋白质沉淀剂对棉铃虫谷胱甘肽S-转移酶的部分纯化

通过用聚乙烯亚胺（PEI）、硫酸铵、聚乙二醇（PEG）沉淀技术和GSH-Sepharose 4B亲和柱对棉铃虫Helicoverpa armigera (Hübner)幼虫中谷胱甘肽S-转移酶进行了部分纯化研究。结果表明PEG10000和PEG20000的纯化效果优于硫酸铵的沉淀效果。通过PEI沉淀去核酸后,再用硫酸铵沉淀,中肠和脂肪体GST活性分布在70%～75％和60%～65％沉淀段,比活力分别为1 081.49和596.41 nmol/(min·mg),纯化倍数分别为2.53和2.2。在6种PEG中,PEG10000和PEG20000的纯化效果较好。在中肠和脂肪体中PEG10000沉淀的GST活性峰分别在40%～45％和30%～40％,GST比活力分别为795.11和1 080.18 nmol/(min·mg),纯化倍数分别是2.4和3.97。PEG20000沉淀中肠和脂肪体GST的活性峰分别在25%～40％和25%～45％,比活力分别是767.57和945.96 nmol/(min·mg),纯化倍数分别是2.81和3.05。用GSH-Sepharose 4B纯化中肠GST,GST比活力达到5 888.44 nmol/(min·mg),纯化倍数达到107.38。     

S-亚胺还原酶和葡萄糖脱氢酶共表达系统的构建及手性胺的合成

旨在构建S-亚胺还原酶(S-IRED)和葡萄糖脱氢酶(GDH)在大肠杆菌中的一菌双酶共表达系统,实现辅酶NADPH的再生,高效合成手性仲胺。利用无缝克隆的手段设计构建一种单质粒双启动子共表达系统,以全细胞为催化剂催化手性仲胺S-2-甲基吡咯烷(S-2MP)的合成,并研究温度、pH及有机溶剂对双酶反应的影响。成功构建了S-IRED和GDH的重组共表达质粒,实现了S-IRED与GDH在大肠杆菌中的胞内共表达,以亚胺2-甲基吡咯啉(2MPN)为模式底物,以工程菌全细胞催化手性仲胺S-2MP的合成,在低辅酶添加时催化手性胺的产率和光学纯度均高于95%。该双酶共表达体系的最适温度和pH分别为37℃和pH 8,10%以下的甲醇对双酶反应有正向促进作用。大肠杆菌胞内双酶共表达系统的构建实现了辅酶NADPH的原位再生,降低了亚胺还原酶催化合成手性胺的成本,为手性胺的规模制备奠定了基础。     

Rapid detection of a minute amount of lipopolysaccharide from Salmonella in large volumes by poly(ethyleneimine)-cloth enzyme immunoassay

Utilizing the rapidity in capturing Salmonella lipopolysaccharide (LPS) and the macroporous nature of poly(ethyleneimine) immobilized polyester cloth (PEI-cloth), a PEI-cloth disk set in a Swinnex cartridge joined at both ends with 10 ml plastic syringes was repeatedly passed through with an LPS sample in large volumes. The captured LPS on the disk were detected at up to 1 pg, which is equivalent to the amount produced of 16 cells, by cloth enzyme immunoassay.     

An effective route to fluorine containing asymmetric alpha-aminophosphonates using chiral Bronsted acid catalyst

Asymmetric addition of dialkyl phosphites (--CH2CH3, --CH2CH2CH3, --CH(CH3)2, --CH2(CH2)3CH3, --CH2CH2OCH3 and --CH2CH2OC2H5) induced by chiral organocatalyst e.g. (R)- and (S)-3,3'-[3,5-bis(trifluoromethyl)phenyl]2-1,1'-binaphthyl phosphate on fluorinated aldimines derived from cinnamaldehyde has been found effective to give new bioactive alpha-aminophosphonates in good yields (58-73%) and high enantiomeric excess (64.6%-90.6%) under mild conditions.