Heteroexpression and biochemical characterization of thermostable citrate synthase from the cyanobacteria Anabaena sp. PCC7120.

The present study recombinantly expressed a citrate synthase from cyanobacteria Anabaena sp. PCC7120 (AnCS) in Escherichia coli and characterized its enzymatic activity. The molecular mass of native AnCS was 88,533.1 Da containing two 44,162.7 Da subunits. Recombinant AnCS revealed the highest activity at pH 9.0 and 25 °C. AnCS displayed high thermal stability with a half-life time (t1/2) of approximately 6.5 h at 60 °C, which was more thermostable than most CS from general organisms, but less than those from hyperthermophilic bacteria. The Km values of oxaloacetate and acetyl-CoA were 138.50 and 18.15 µM respectively, suggesting a higher affinity to acetyl-CoA than oxaloacetate. Our inhibition assays showed that AnCS activity was not severely affected by most metal ions, but was strongly inhibited by Cu2+ and Zn2+. Treatments with ATP, ADP, AMP, NADH, and DTT depressed the AnCS activity. Overall, our results provide information on the enzymatic properties of AnCS, which contributes to the basic knowledge on CS selection for industrial utilizations.

The Influencing Factors of Frailty and Quality of Life in Elderly Patients After Spinal Surgery.

Background: To study the related factors of frailty and quality of life in elderly patients after spinal surgery. Methods: The anxiety, depression, frailty, and quality of life of all patients were assessed by the Anxiety screening scale (GAD-7), Depression screening scale (PHQ-9), Frailty screening scale (FRAIL), and European five-dimensional health scale (EQ-5D-5L) 1 day before surgery (DAY-0). A numeric rating scale (NRS) was used to evaluate patients' pain during activities on the 1st day (POD-1), 3rd day (POD-3), and 30th day (POD-30) after operation. FRAIL scale and EQ-5D-5L were used to evaluate patients' frailty and quality of life on POD-30 and 90th day (POD-90) after the operation. Results: There were significant differences in age, body mass index (BMI), preoperative serum albumin level (ALB), and NRS score on POD-1 between the two groups (P<0.05). Age and PHQ-9 score were positively correlated with EQ-5D-5L score (P<0.05, r Age=0.245, rPHQ-9=0.217), and preoperative ALB level was negatively correlated with EQ-5D-5L score (P<0.05, r ALB=-0.274). Conclusion: The older the age, the larger the BMI and the higher the NRS score on the first day after surgery, the more prone to frailty in elderly patients after spinal surgery; The older age and the lower the preoperative ALB level, the worse the quality of life in elderly patients after spinal surgery.

Association Between Preoperative Sleep Disturbance and Postoperative Delirium in Elderly: A Retrospective Cohort Study.

Purpose: Postoperative sleep disturbance, characterized by diminished postoperative sleep quality, is a risk factor for postoperative delirium (POD); however, the association between pre-existing sleep disturbance and POD remains unclear. This study aimed to evaluate the association between preoperative sleep disturbance and POD in elderly patients after non-cardiac surgery. Patients and methods: This retrospective cohort study was conducted at a single center and enrolled 489 elderly patients who underwent surgery between May 1, 2020, and March 31, 2021. Patients were divided into the sleep disorder (SD) and non-sleep disorder (NSD) groups according to the occurrence of one or more symptoms of insomnia within one month or sleep- Numerical Rating Scale (NRS)≥6 before surgery. The primary outcome was the incidence of POD. Propensity score matching analysis was performed between the two groups. Multiple logistic regression analysis was performed to identify the risk factors for POD. Results: In both the unmatched cohort (16.0% vs 6.7%, P=0.003) and the matched cohort (17.0% vs 6.2%, P=0.023), the incidence of POD was higher in the SD group than in the NSD group. In addition, the postoperative sleep quality and the VAS score at postoperative 24 h were significantly lower in the SD group than in the NSD group. Multivariate logistic regression analysis indicated that age (Odds Ratio, 1.13 [95% CI: 1.04-1.23], P=0.003) and preoperative sleep disturbance (Odds Ratio, 3.03 [95% CI: 1.09-9.52], P=0.034) were independent risk factors for the development of POD. Conclusion: The incidence of POD was higher in patients with pre-existing sleep disturbance than those without it. Whether improving sleep quality for preoperative sleep disturbance may help prevent POD remains to be determined.

Enzymatic Characterization and Coenzyme Specificity Conversion of a Novel Dimeric Malate Dehydrogenase from Bacillus subtilis.

Malate is an important material to various industrials and clinical applications. Bacillus subtilis is a widely used biocatalyst tool for chemical production. However, the specific enzymatic properties of malate dehydrogenase from Bacillus subtilis (BsMDH) remain largely unknown. In the present study, BsMDH was cloned, recombinantly expressed and purified to test its enzymatic properties. The molecular weight of single unit of BsMDH was 34,869.7 Da. Matrix-Assisted Laser-Desorption Ionization-Time-of-Flight Mass Spectrometry and gel filtration analysis indicated that the recombinant BsMDH could form dimers. The kcat/Km values of oxaloacetate and NADH were higher than those of malate and NAD+, respectively, indicating a better catalysis in the direction of malate synthesis than the reverse. Furthermore, six BsMDH mutants were constructed with the substitution of amino acids at the coenzyme binding site. Among them, BsMDH-T7 showed a greatly higher affinity and catalysis efficiency to NADPH than NADH with the degree of alteration of 2039, suggesting the shift of the coenzyme dependence from NADH to NADPH. In addition, BsMDH-T7 showed a relatively lower Km value, but a higher kcat and kcat/Km than NADPH-dependent MDHs from Thermus flavus and Corynebacterium glutamicum. Overall, these results indicated that BsMDH and BsMDH-T7 mutant might be promising enzymes for malate production.

Expression and characterization of a thermostable citrate synthase from Microcystis aeruginosa PCC7806.

Citrate synthase (CS) is an important enzyme in energy conversion and material circulation, participating in many important biochemical processes. In the present study, CS from Microcystis aeruginosa PCC7806 (MaCS) was cloned and expressed in Escherichia coli Rosetta (DE3). The recombinant MaCS was purified and its enzymological properties were characterized. The results showed that MaCS formed dimers in native status. The optimum temperature and pH of MaCS was 30°C and 8.2, respectively. MaCS displayed relative high thermal stability. Treatment at 50°C for 20 min only decreased 11.30% activity of MaCS and the half-life of MaCS was approximately 35 min at 55°C. The kcat and Km of acetyl-CoA and oxaloacetic acid were 17.133 s-1 (kcat) and 11.62 µM (Km), 24.502 s-1 and 103.00 µM, respectively. MaCS activity was not drastically inhibited by monovalent ions and NADH but depressed by divalent ions and some small molecular compounds, especially Mg2+, Zn2+, Co2+ and DTT. Overall, these data contributed to further understanding of energy metabolism in cyanobacteria and also provided basic information for industrial application of CS.