Glucose oxidase (GOD) is an oxygen-consuming dehydrogenase that can catalyze the production of gluconic acid hydrogen peroxide from glucose, and its specific mechanism of action makes it promising for applications, while the low catalytic activity and poor thermostability have become the main factors limiting the industrial application of this enzyme. In this study, we used the glucose oxidase AtGOD reported with the best thermostability as the source sequence for phylogenetic analysis to obtain the GOD with excellent performance. Six genes were screened and successfully synthesized for functional validation. Among them, the glucose oxidase AhGODB derived from Aspergillus heteromorphus was expressed in Pichia pastoris and showed better thermostability and catalytic activity, with an optimal temperature of 40 ℃, a specific activity of 112.2 U/mg, and a relative activity of 47% after 5 min of treatment at 70 ℃. To improve its activity and thermal stability, we constructed several mutants by directed evolution combined with rational design. Compared with the original enzyme, the mutant T72R/A153P showcased the optimum temperature increasing from 40 to 50 ℃, the specific activity increasing from 112.2 U/mg to 166.1 U/mg, and the relative activity after treatment at 70 ℃ for 30 min increasing from 0% to 33%. In conclusion, the glucose oxidase mutants obtained in this study have improved catalytic activity and thermostability, and have potential for application.
Glucose Oxidase/chemistry* ; Enzyme Stability ; Aspergillus/genetics* ; Pichia/metabolism* ; Temperature ; Catalysis ; Fungal Proteins/metabolism* ; Hot Temperature

Objective:To survey the awareness of common primary care clinical guidelines among primary care providers in Qingdao city.Methods:A questionnaire survey on the awareness of common clinical guidelines in primary care was conducted in August 2020 among 659 providers (293 males and 366 females) from 81 primary medical institutions in Qingdao city. The contents of the questionnaire included the general information of primary physicians, the frequency of using the guidelines, the level of understanding of the guidelines, the effect of guidelines in clinical work, the attitudes towards promoting the guidelines, and the interest in learning the guidelines and the way of learning.Results:A total of 659 valid questionnaires were recovered with a recovery rate of 100.0%. The results showed that 14.7% (97/659) practitioners applied the guidelines in most cases, and 31.6% (208/659) frequently used. There were significant differences in use frequency among providers with different practice types, professional titles, education background, practice locations and institutions ( P<0.01). Among participants, 15.2% (100/659) did not know about primary care guidelines, 63.3% (417/659) knew but did not learned guidelines, 21.5% (142/659) knew and studied guidelines carefully. The top five of the learned guidelines (114, 80.3%) were bronchial asthma (113, 79.6%), type 2 diabetes (108, 76.1%), chronic obstructive pulmonary disease (101, 71.1%), and chronic cor pulmonale (83, 58.5%). Most participants who learnt the guidelines viewed the guidelines as quite helpful or very helpful, the score of usefulness for professional knowledge was the highest (4.44±0.60). Most of participants (94.2%, 621/659) thought it was necessary to develop primary care guidelines; and the major factors affecting the promotion were lack of training (79.8%, 526/659), difficulties in access (46.7%, 308/659) and ignorance or neglect (23.2%, 153/659).Most practitioners (93.8%, 618/659) were interested in learning primary care clinical guidelines, and the expected ways of training were online teaching (70.0%, 420/618), training courses(58.3%, 360/618) and special lectures (55.2%, 341/618). Conclusion:The current situation of learning clinical guidelines among primary care providers in Qingdao is not satisfactory, but they are willing to learn the guidelines for improving clinical practice. We should strengthen the training and promotion of primary care guidelines among primary care providers in the future.