ABSTRACT
The synthesis of fine chemicals using multi-enzyme cascade reactions is a recent hot research topic in the field of biocatalysis. The traditional chemical synthesis methods were replaced by constructing in vitro multi-enzyme cascades, then the green synthesis of a variety of bifunctional chemicals can be achieved. This article summarizes the construction strategies of different types of multi-enzyme cascade reactions and their characteristics. In addition, the general methods for recruiting enzymes used in cascade reactions, as well as the regeneration of coenzyme such as NAD(P)H or ATP and their application in multi-enzyme cascade reactions are summarized. Finally, we illustrate the application of multi-enzyme cascades in the synthesis of six bifunctional chemicals, including ω-amino fatty acids, alkyl lactams, α, ω-dicarboxylic acids, α, ω-diamines, α, ω-diols, and ω-amino alcohols.
Subject(s)
Amino Acids , Biocatalysis , Amino Alcohols , Coenzymes/metabolism , DiaminesABSTRACT
Industrial enzymes have become the core "chip" for bio-manufacturing technology. Design and development of novel and efficient enzymes is the key to the development of industrial biotechnology. The scientific basis for the innovative design of industrial catalysts is an in-depth analysis of the structure-activity relationship between enzymes and substrates, as well as their regulatory mechanisms. With the development of bioinformatics and computational technology, the catalytic mechanism of the enzyme can be solved by various calculation methods. Subsequently, the specific regions of the structure can be rationally reconstructed to improve the catalytic performance, which will further promote the industrial application of the target enzyme. Computational simulation and rational design based on the analysis of the structure-activity relationship have become the crucial technology for the preparation of high-efficiency industrial enzymes. This review provides a brief introduction and discussion on various calculation methods and design strategies as well as future trends.
Subject(s)
Biocatalysis , Biotechnology , Enzymes , Chemistry , Metabolism , Metabolic Engineering , Protein Engineering , Structure-Activity RelationshipABSTRACT
Racemases have been applied for the synthesis of enantiomerically pure compounds through the deracemization methods. Mandelate racemase from Pseudomonas putida was the only enzyme that catalyzes the interconversion of mandelate enantiomers. Using genome mining approaches, we identified 9 mandelate racemases (MRs). A novel racemase named ArMR with higher activity and better soluble protein expression, was isolated from Agrobacterium radiobacter. ArMR displayed the optimum catalytic activity at 50 ℃, pH 7.8 in Tris-HCl buffer. The half-life of ArMR at 50, 40 and 30 ℃ was 0.17, 27.2 and 70.7 h, respectively. KM parameter of ArMR towards (R)- and (S)-mandelic acid was 1.44 and 0.81 mmol/L, respectively; the corresponding kcat value was 410 s⁻¹ and 218 s⁻¹. In addition, KM of ArMR towards (R)- and (S)-2-chloro mandelic acid was 6.48 and 6.37 mmol/L, and the corresponding kcat value 0.22 s⁻¹ and 0.23 s⁻¹, respectively. Meanwhile, Mg²⁺ and Mn²⁺ could activate the enzyme whereas Zn²⁺ inactivated the enzyme completely. Discovery of more novel MRs provides supports further research and development of these racemases.
ABSTRACT
Objective To investigate the effect of polyphosphazene(PAGP) microspheres controled release of growth factors on the adhesion and proliferation of bone marrow mesenchymal stem cells (BMSCs).Methods Two kinds of functional poly(alanine ethyl ester-co-glycine ethyl ester) phosphazene microspheres with different ratios of side-substituent groups were synthesized using the emulsion technique.The rate of degradation/hydrolysis of the polymers was carefully tuned to suit the desired application for controlled release.The enzyme-linked immune sorbent assay was utilized to investigate the characterization of the temporal controlled release strategy of microspheres loaded with transforming growth factor-beta 1(TGF-β1) and insulin-like growth factor-l(IGF-1) respectively.The cell adhesion and proliferation stimulated by different growth factors were evaluated by acridine orange staining.Resuits The morphological difference between two PAGP microspheres was identified according to SEM images.The average diameter of two microspheres was 54.22 ± 19.19 μm and 34.11 ± 18.82 μm respectively.The release assay showed that two kinds of microspheres had different release characteristics,with earlier outburst of TGF-β1 and IGF-1 for them to cooperate and later sustained release of TGF-β1 to stimulate the differentiation of stem cells.The result of the acridine orange staining demonstrated that PAGP microspheres supported cell adhesion and growth without obvious cytotoxicity.Meanwhile,the growth factors release strategy significantly improved the proliferation of BMSCs.Conclusion The two polyphosphazene microspheres have a great release-control effect and their controlled release system will have a promising prospect in the future tissue engineering field.
ABSTRACT
Objective To investigate whether TG2 plays an important role in the osteoblast differentiation and mineralization.Methods TG2 mRNA of SaOS-2 cells was knocked down using a lentivirus stably expressing short-hairpin ( sh) RNA targeting TG2.Then the cells were cultured in osteo-inductive medium for 14 d to measure mineralization and for 7 d to measure the levels of osteoblastic differentiation markers including ALP activity and mRNA of collagen I, osteocalcin ( OCN) and BMP-2.The wild-type SaOS-2 cells and scrambled shRNA-transducted SaOS-2 cells served as the controls. Results The controls displayed an increasing trend of the level of ALP activity and mRNA of collagen I, osteocalcin and BMP-2,and notable mineralization at 14 d.When TG2 was knocked down, ALP activity, mRNA of collagen I, osteocalcin and BMP-2 at 7d,and mineralization at 14 d were all significantly lower in comparison with the corresponding values in the controls.Conclusion TG2 is involved in the differentiation and mineralization of osteoblasts in vitro.