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Insufficient catalytic efficiency of flavonoid 6-hydroxylases in the fermentative production of scutellarin leads to the formation of at least about 18% of by-products. Here, the catalytic mechanisms of two flavonoid 6-hydroxylases, CYP82D4 and CYP706X, were investigated by molecular dynamics simulations and quantum chemical calculations. Our results show that CYP82D4 and CYP706X have almost identical energy barriers at the rate-determining step and thus similar reaction rates, while the relatively low substrate binding energy of CYP82D4 may facilitate product release, which is directly responsible for its higher catalytic efficiency. Based on the study of substrate entry and release processes, the catalytic efficiency of the L540A mutation of CYP82D4 increased by 1.37-fold, demonstrating the feasibility of theoretical calculations-guided engineering of flavonoid 6-hydroxylase. Overall, this study reveals the catalytic mechanism of flavonoid 6-hydroxylases, which may facilitate the modification and optimization of flavonoid 6-hydroxylases for efficient fermentative production of scutellarin.
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Sistema Enzimático del Citocromo P-450/metabolismo , Apigenina , GlucuronatosRESUMEN
It is of great significance to use biosynthesis to transform the inorganic substance formaldehyde into organic sugars. Most important in this process was to find a suitable catalyst combination to achieve the dimerization of formaldehyde. In a recent report, an engineered glycolaldehyde synthase was reported to catalyze this reaction. It could be combined with engineered D-fructose-6-phosphate aldolase, a "one-pot enzyme" method, to synthesize L-xylose using formaldehyde and the conversion rate could reach up to 64%. This process also provides a reference for the synthesis of other sugars. With the increasing consumption of non-renewable resources, it was of great significance to convert formaldehyde into sugar by biosynthesis.
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Biocatálisis , Formaldehído , Química , Fructosa-Bifosfato Aldolasa , Metabolismo , XilosaRESUMEN
Objective:To introduce the discovery and nomenclature of the intercondylar foramen of femur (IFF) and foramen of tibial intercondylar eminence (FTIE) and research the close relationship between the high recurrence rate of aggressive tumors around the knee joint and the foramina around the knee joint.Methods:①Radiographic observation and measurement: 3D reconstruction of CT scan of 200 patients in our hospital were used to obverse the common feature、position and measure of Inter-condylar foramen of femur and Foramen of tibial intercondylar eminence. ②Anatomical and histological observation: To proof the existence of IFF and FTIE through the anatomy of 15 cases of car accidents or tumor amputations and 60 cases of autopsy. Then the specific location, the surrounding structure, the proximal coverage, the contents, the apical construction, the wall and the bottom tissues of the IFF and FTIE were studied and analyzed. ③Histological and pathological observation of tumor anatomy: Through the study of the distal femur and tibia malignant tumor tissues(including primary bone tumors and metastatic tumors), we observed the relationship between the foraminal structures and the tumor, judged the situation of concealed transmission and two-way spread through the foramina, and analyzed the relationship between tumor recurrence and foraminal structures. ④The synovial membrane of foramina, especially in cases where the synovium was suspected to be involved by the lesions judged by the radiography was analyzed to observe whether the synovium was infiltrated by the tumor.Results:IFF and FTIE were the inherent physical structure of the human. Their physiological function was the vascular foramina that lead the branches of arteria media genus into the Intercondylar fossa of femur and tibial intercondylar eminence. Their opening was separated with the joint cavity by the synovial tissues, so IFF and FTIE were isolated with joint cavity by the synovial tissues、meniscus and cruciate ligaments. After invading the IFF and FTIE, the aggressive tumors did not break into the joint cavity immediately, but conceal in the foramina and invade the synovium with specific biological behavior with the sequence: reactive edema, hyperplasia, degeneration, calcification, hyaline degeneration (infiltration in some cases), synovial rupture, and then tumor invasion of the articular cavity. Usually, tumors or recurrence has been observed before synovial rupture. We also observed the tendency of tumors to spread along the arteria media genus to the popliteal vessels, peripheral soft tissues and lymphatic vessels with typical radiographic performance like popliteal lymphadenectasis. Color nodules and tumors in other parts could also invade or metastasize into bone through these foramina.Conclusions:IFF and FTIE are foramina nutricium of arteria media genus. They are the inherent physical structure of the human. The foramina play an important role in the spread, concealment and recurrence of peripheralkneeaggressive tumor.
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Objective To introduce the tibial intercondylar eminence hole (TIEH) and study its structure.Explore the connection between TIEH and the pathway how proximal tibial aggressive tumor break into the bony structure from articular cavity.Methods This retrospective study included 200 patient's CT 3-dimensional reconstruction materials from May 2017 to November 2017 in Qilu hospital randomly.There were 115 males and 85 females,the average age was 49 years (ranged from 12 to 90 years).To observe the existence of TIEH and identify its location and measurement with imaging techniques.According to 50 tibial plateau specimen after TKA and 5 specimen after car accident or amputation due to tumor,physical proof the existence of TIEH.The specific location,peripheral structure,coverage,content of TIEH as well as its top,walls and bottom were researched and analysed.Pathological staining was used and 1 cases undertook preoperation contrast agent observation.1 cases of typical cases were reviewed.Results TIEH was ubiquity according to all of the 200 cases.TIEH was located on the depression of tibial plateau,between the attachments of ACL and PCL.The hole was round type,and the diameter was 1.6±0.3 mm,the depth was 9.1±2.1 mm.1-3 Paraforamen (semidiamete≤7 mm) were found around the main TIEH in 53% patients (106/200),the diameter and depth was less than the main hole.The CT value showed the orifice (472.5±30.1 HU) > the pore wall (312.3±22.5 HU) > the pore bottom (202.4±17.3 HU) > the pore (118.3±10.4 HU) > the orifice covering (75.0±11.1 HU).The synovial tissue septum was only 1 mm between the top of hole and the articular cavity.The top of TIEH was surrounded by articular cartilage,the walls and bottom were spongy bone,the content was dense connective tissue that didn't attach to the walls tightly.The peripheral spongy bone was easy to infiltrate by methylene blue.Preoperation radiography showed that TIEH had poor barrier function.Conclusion Tibial intercondylar eminence hole is an intrinsic structure of the human body.The coverage is weak,and it is easy to cause the tumor to hide and recur.The tumor may pass through this hole and bidirectionally enter between the proximal humerus and the joint cavity.
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Xylulose as a metabolic intermediate is the precursor of rare sugars, and its unique pattern of biological activity plays an important role in the fields of food, health, medicine and so on. The aim of this study was to design a new pathway for xylulose synthesis from formaldehyde, which is one of the most simple and basic organic substrate. The pathway was comprised of 3 steps: (1) formaldehyde was converted to glycolaldehyde by benzoylformate decarboxylase mutant BFD-M3 (from Pseudomonas putida); (2) formaldehyde and glycolaldehyde were converted to dihydroxyacetone by BFD-M3 as well; (3) glycolaldehyde and dihydroxyacetone were converted to xylulose by transaldolase mutant TalB-F178Y (from Escherichia coli). By adding formaldehyde (5 g/L), BFD-M3 and TalB-F178Y in one pot, xylulose was produced at a conversion rate of 0.4%. Through optimizing the concentration of formaldehyde, the conversion rate of xylulose was increased to 4.6% (20 g/L formaldehyde), which is 11.5 folds higher than the initial value. In order to further improve the xylulose conversion rate, we employed Scaffold Self-Assembly technique to co-immobilize BFD-M3 and TalB-F178Y. Finally, the xylulose conversion rate reached 14.02%. This study provides a new scheme for the biosynthesis of rare sugars.
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Life system has created rich and colorful genes, to protect the inheritance and prosperity after more than 4 billion years of natural evolution. However, the natural evolution is an extremely slow process, and the origin and evolution of new gene with new function often takes millions of years. Therefore, natural evolution alone cannot meet the rapid development of industrial biotechnological production needs. Using synthetic biology techniques, researchers can design and synthesize new genes based on the known enzyme catalysis mechanism and protein structure according to industrial production requirements, and create various biochemical reactions that cannot be catalyzed by natural living organisms. Although the new gene design technology shows exciting application prospects, there are now still many scientific and technological challenges, such as low success rate of design, low catalytic activity and high synthesis cost. With the rapid development of synthetic biology, the design, transformation, synthesis, screening and other technologies will be integrated into a mature technological process for the new gene design.