Creatinine levels in biological fluids are important indicators for the clinical evaluation of renal function. Creatinase (CRE, EC3.5.3.3) is one of the key enzymes in the enzymatic measurement of creatinine concentration, and it is also the rate-limiting enzyme in the whole enzymatic cascade system. The poor catalytic activity of CRE severely limits its clinical and industrial applications. To address this issue, a semi-rational design is applied to increase the activity of a creatinase from Alcaligenes sp. KS-85 (Al-CRE). By high-throughput screen of saturation mutagenesis libraries on the selected hotspot mutations, multiple variant enzymes with increased activity are obtained. The five-point best variant enzyme (I304L/F395V/K351V/Y63S/Q88A) were further obtained by recombine the improved mutations sites that to showed a 2.18-fold increased specific activity. Additionally, structure analysis is conducted to understand the mechanism of the activity change. This study paves the way for a better practical application of creatinase and may help further understand its catalytic mechanism.
Creatinine ; Mutagenesis, Site-Directed ; Ureohydrolases/genetics* ; Catalysis

Background Since the outbreak of the COVID-19 epidemic, staff of the centers for disease control and prevention (CDC) have been burdened with heavy epidemic prevention control, and excessive occupational stress can cause depression and other psychological problems. Objective To explore the status of occupational stress, resilience, and depression of CDC staff and potential relationships between them. Methods From December 2020 to April 2021, a survey was conducted at provincial and municipal levels, and the stratified cluster sampling method was used at county (district) level to select a total of 3514 samples. Their occupational stress, resilience, and depression status were evaluated using the Chinese Effort-Reward Imbalance Scale (ERI), the Chinese Connor-Davidson Resilience Scale (CD-RISC), and the Patient Health Questionnaire (PHQ-9). Independent sample t test, analysis of variance, χ² test, Pearson correlation analysis, and mediation test (structural equation model) were conducted. Results The positive rate of occupational stress was 34.29% in the CDC staff, the resilience score was 66.28±15.32, and the positive rate of depression was 48.58%. Significant differences were found in the positive rates of occupational stress among different groups of gender, age, education background, marital status, administrative duty, weekly exercise frequency, chronic disease prevalence, and participation in epidemic control (P<0.05); in the resilience scores among different groups of gender, age, administrative duty, weekly exercise frequency, chronic disease prevalence, and participation in epidemic control (P<0.05); in the positive rates of depression among different groups of gender, age, educational background, personal monthly income, weekly exercise frequency, chronic disease prevalence, and participation in epidemic control (P<0.05). Occupational stress was negatively correlated with resilience (r=−0.165, P<0.01). Resilience was negatively correlated with depression (r=−0.383, P<0.01). Occupational stress was positively correlated with depression (r=0.343, P<0.01). The structural equation embracing a partial mediating effect of resilience on the relationship occupational stress and depression was established, and the partial mediating effect was 0.039, accounting for 10.46% of the total effect. Conclusion High positive rates of occupational stress, reduced resilience, and depression are shown among CDC staff in the context of the COVID-19 epidemic, and resilience partially mediates the effect of occupational stress on depression. The study findings suggest that improving resilience may reduce occupational stress and depression in CDC staff.