Engineering of Thermomyces lanuginosus lipase Lip: creation of novel biocatalyst for efficient biosynthesis of chiral intermediate of Pregabalin

Efficient and highly enantioselective hydrolysis of 2-carboxyethyl-3-cyano-5-methylhexanoic acid ethyl ester (CNDE) is the most crucial step in chemoenzymatic synthesis of Pregabalin. By using site-saturation mutagenesis and high-throughput screening techniques, lipase Lip from Thermomyces lanuginosus DSM 10635 was engineered to improve its activity towards CNDE. The triple mutant, S88T/A99N/V116D exhibited a 60-fold improvement in specific activity for CNDE (2.35 U/mg) over the wild-type Lip (0.039 U/mg). Modeling and docking studies demonstrated that the mutant could more effectively stabilize oxygen anions in transition states and the lid of Lip in the open conformation. Additionally, the kinetic resolution of CNDE catalyzed by Escherichia coli cell overexpressing S88T/A99N/V116D mutant afforded (3S)-2-carboxyethyl-3-cyano-5-methylhexanoic acid in 42.4 % conversion and 98 % ee within 20 h with a substrate loading of 1 M (255 g/l). These results demonstrated that a novel and promising biocatalyst was created for efficient chemoenzymatic manufacturing of Pregabalin.     

The Effect of TCM-Induced HAMP on Key Enzymes in the Hydrolysis of AD Model Cells

Neurochemical Research - Alzheimer’s disease (AD) process is characterized classically by two hallmark pathologies: β-amyloid (Aβ) plaque deposition and neurofibrillary tangles of...     

Involvement of Protein Tyrosine Phosphatases BcPtpA and BcPtpB in Regulation of Vegetative Development,Virulence and Multi-Stress Tolerance in Botrytis cinerea

Tyrosine phosphorylation and dephosphorylation have emerged as fundamentally important mechanisms of signal transduction and regulation in eukaryotic cells, governing many processes, but little has been known about their functions in filamentous fungi. In this study, we deleted two putative protein tyrosine phosphatase (PTP) genes (BcPTPA and BcPTPB) in Botrytis cinerea, encoding the orthologs of Saccharomyces cerevisiae Ptp2 and Ptp3, respectively. Although BcPtpA and BcPtpB have opposite functions in conidiation, they are essential for sclerotial formation in B. cinerea. BcPTPA and BcPTPB deletion mutants ΔBcPtpA-10 and ΔBcPtpB-4 showed significantly increased sensitivity to osmotic and oxidative stresses, and to cell wall damaging agents. Inoculation tests showed that both mutants exhibited dramatically decreased virulence on tomato leaves, apples and grapes. In S. cerevisiae, it has been shown that Ptp2 and Ptp3 negatively regulate the high-osmolarity glycerol (HOG) pathway and the cell wall integrity (CWI) pathway. Although both BcPtpA and BcPtpB were able to inactive Hog1 and Mpk1 in S. cerevisiae, in contrast to S. cerevisiae, they positively regulate phosphorylation of BcSak1 (the homologue of Hog1) and BcBmp3 (the homologue of Mpk1) in B. cinerea under stress conditions. These results demonstrated that functions of PTPs in B. cinerea are different from those in S. cerevisiae, and BcPtpA and BcPtpB play important roles in regulation of vegetative development, virulence and in adaptation to oxidative, osmotic and cell-wall damage stresses in B. cinerea.     

NdhO,a Subunit of NADPH Dehydrogenase,Destabilizes Medium Size Complex of the Enzyme in Synechocystis sp. Strain PCC 6803

Two mutants that grew faster than the wild-type (WT) strain under high light conditions were isolated from Synechocystis sp. strain PCC 6803 transformed with a transposon-bearing library. Both mutants had a tag in ssl1690 encoding NdhO. Deletion of ndhO increased the activity of NADPH dehydrogenase (NDH-1)-dependent cyclic electron transport around photosystem I (NDH-CET), while overexpression decreased the activity. Although deletion and overexpression of ndhO did not have significant effects on the amount of other subunits such as NdhH, NdhI, NdhK, and NdhM in the cells, the amount of these subunits in the medium size NDH-1 (NDH-1M) complex was higher in the ndhO-deletion mutant and much lower in the overexpression strain than in the WT. NdhO strongly interacts with NdhI and NdhK but not with other subunits. NdhI interacts with NdhK and the interaction was blocked by NdhO. The blocking may destabilize the NDH-1M complex and repress the NDH-CET activity. When cells were transferred from growth light to high light, the amounts of NdhI and NdhK increased without significant change in the amount of NdhO, thus decreasing the relative amount of NdhO. This might have decreased the blocking, thereby stabilizing the NDH-1M complex and increasing the NDH-CET activity under high light conditions.     

Prevalence of Post-Stroke Cognitive Impairment in China: A Community-Based,Cross-Sectional Study

International hospital-based studies have indicated a high risk of cognitive impairment after stroke, evidence from community-based studies in China is scarce. To determine the prevalence of post-stroke cognitive impairment (PSCI) and its subtypes in stroke survivors residing in selected rural and urban Chinese communities, we conducted a community-based, cross-sectional study in 599 patients accounting for 48% of all stroke survivors registered in the 4 communities, who had suffered confirmed strokes and had undergone cognitive assessments via the Montreal Cognitive Assessment (MoCA), Mini-Mental State Examination (MMSE), and Hachinski Ischemia Scale (HIS). Detection of PSCI was based on scores in these neuropsychological scales. Factors potentially impacting on occurrence of PSCI were explored by comparing demographic characteristics, stroke features, and cardiovascular risk factors between patients with and without PSCI. The overall prevalence of PSCI was 80.97% (95%CI: 77.82%-84.11%), while that of non-dementia PSCI (PSCI-ND) and post-stroke vascular dementia (PSD) was 48.91% (95%CI: 44.91%-52.92%) and 32.05% (95%CI: 28.32%-35.79%), respectively. Prior stroke and complications during the acute phase were independent risk factors for PSCI. The risk of recurrent stroke survivors having PSCI was 2.7 times higher than for first-episode survivors, and it was 3 times higher for those with complications during the acute phase than for those without. The higher prevalence of PSCI in this study compared with previous Chinese studies was possibly due to the combined effects of including rural stroke survivors, a longer period from stroke onset, and different assessment methods. There is an urgent need to recognize and prevent PSCI in stroke patients, especially those with recurrent stroke and complications during the acute phase.