14 results about "Specific substrate" patented technology

The invention discloses a glycosyl transferase mutant and an application of the glycosyl transferase mutant in synthesis of rebaudioside. According to the invention, single-point mutation and multi-point mutation are carried out on the basis of a glycosyl transferase amino acid sequence as shown in SEQ ID NO: 1, and a mutant with improved catalytic activity is obtained. The catalytic activity, the substrate specificity and / or the substrate specificity of the glycosyl transferase mutant are / is changed, and the catalytic activity of enzyme to a specific substrate can be remarkably improved by mutation at a specific site. And carrying out induced expression and protein purification on the obtained mutation sequence to obtain the mutant enzyme. The mutant enzyme is used as a catalyst, and UDPG is used as a glycosyl donor, so that the catalytic reaction efficiency of substrates such as stevioside ST, rebaudioside A (RebA) and rebaudioside D (RebD) can be obviously improved. According to the glycosyl transferase UGT76G1 mutant constructed by the research, the catalytic activity of the glycosyl transferase UGT76G1 mutant is improved, and efficient production of rebaudioside M is realized by optimizing a reaction system.

This invention discloses a quantitative detection kit for arginine peptidase, a qualitative detection kit for arginine peptidase, and their usage methods. The quantitative detection kit utilizes the specific substrate Nα-benzoyl-DL-arginine-4-ylbenzamide hydrochloride, which can be hydrolyzed by arginine peptidase to 4-ylbenzamide. 4-ylbenzamide has a maximum absorption peak at 405 nm, and the specific arginine peptidase activity can be rapidly detected at room temperature using spectrophotometry. The qualitative kit utilizes the specific substrate Nα-benzoyl-DL-arginine-β-naphthamide hydrochloride, which can be hydrolyzed by arginine peptidase to p-β-naphthamide. β-naphthamide combines with 4-(dimethylamino)cinnamaldehyde to form a compound that is visible to the naked eye as a pink color. The disulfide bond cleaving agent cuts the mucus disulfide bonds, exposing bacterial enzyme sites, and the synergistic effect of the enzyme activator enhances the reaction rate of arginine peptidase hydrolysis of the substrate, significantly improving the detection sensitivity of arginine peptidase in tongue coating samples.

This invention discloses a glycosyltransferase mutant and its application in the synthesis of rebaudioside. Based on the amino acid sequence of the glycosyltransferase shown in SEQ ID NO:1, this invention performs single-point and multi-point mutations to obtain mutants with enhanced catalytic activity. The catalytic activity, substrate specificity, and / or substrate specificity of the glycosyltransferase mutant are altered; mutations at specific sites can significantly improve the enzyme's catalytic activity for specific substrates. The mutant enzyme is obtained by inducing expression and purifying the protein from the obtained mutant sequence. Using the mutant enzyme as a catalyst and UDPG as a glycosyl donor, the catalytic reaction efficiency for the substrates steviol glycoside ST, rebaudioside A (RebA), and rebaudioside D (RebD) can be significantly improved. The glycosyltransferase UGT76G1 mutant constructed in this study improves its catalytic activity, and the efficient production of rebaudioside M is achieved through optimization of the reaction system.

The invention relates to the technical field of enzyme activity detection, in particular to a standardized Glu-C incision enzyme activity spectrum detection method. The method provided by the invention realizes rapid, accurate and low-cost enzyme activity detection by using a specific substrate Z-Phe-Leu-Gl-4-pNA and optimized reaction conditions, and specifically comprises the following steps: diluting a Glu-C endonuclease sample, mixing the diluted Glu-C endonuclease sample with a substrate solution, putting the mixture into a preheated Tris-HCl buffer solution, monitoring absorbance change at the wavelength of 405nm, and calculating the enzyme activity through a specific formula. A standardized Glu-C incision enzyme activity detection system is established, the detection time is short, the cost is low, the detection method has a wide linear range, a low detection limit and high precision, and a technical basis is provided for establishing an industrial unified standard.

The present application relates to the field of bioengineering, and particularly relates to genome, expression module, recombinant strain and application thereof in preparation of rhodiolin. The present application provides a genome comprising one or more of expression gene, knockout gene, overexpression gene and inhibition gene. The present application constructs a strain capable of utilizing sugarcane molasses as the only carbon source to generate rhodiolin, thereby reducing the cost of materials; then the metabolic network of the microorganism is restructured through metabolic engineering, the metabolic flow is optimized, the metabolic flux among different metabolic modules is reasonably distributed, and the efficient synthesis of the target product is promoted; finally, the mining and screening of enzymes are crucial, the enzymes with higher activity and higher specificity for specific substrates are mined and screened based on the combination of the trained large language model and the machine learning model and the structural information of the substrates, which significantly improves the titer and conversion rate of rhodiolin generation, and at the same time, reduces the generation of by-products and the accumulation of precursor substances, and will promote the large-scale production and application of rhodiolin.

The present application belongs to the technical field of biotechnology and enzyme engineering, and relates to a molecular modification method for improving substrate specificity of aldehyde ketone reductase, which is achieved by constructing a substrate library of aldehyde ketone reductase AKR13B3 and analyzing kinetic parameters, determining a mutation site by performing loop structure analysis and amino acid sequence alignment on a complex structure of aldehyde ketone reductase AKR13B3, coenzyme NADPH and substrate 3-keto-DON, and screening a mutant with increased substrate specificity, the present application designs the geometric shape of the entrance loop region of the substrate binding pocket of AKR13B3 to change substrate preference and realize the increase of catalytic activity of the enzyme on specific substrates, in addition, the present application provides a feasible scheme for designing aldehyde ketone reductase with good substrate specificity, and provides great hope for developing high-efficiency enzyme preparations.

The application provides a glucose dehydrogenase mutant, a preparation method, application and freeze-dried powder thereof, and relates to the technical field of biotechnology.The glucose dehydrogenase mutant provided by the application has an amino acid sequence as shown in SEQ ID NO.1.The glucose dehydrogenase mutant has better stability compared with a wild type, and has more specific substrate specificity, and can be used for preparing a blood glucose detection product.

The invention belongs to the technical field of protein research engineering, and discloses a transporter screening method based on an artificial intelligence technology, and the method is based on the artificial intelligence technology, according to protein sequence characteristics, whether protein belongs to transporter is predicted, and the transporter is predicted to be outward transporter, inward transporter or bidirectional transporter. And predicting whether the transporter can transport a specific substrate or not, and screening out the transporter with a specific function by integrating calculation results of the three steps. Compared with a conventional single-scale universal machine learning model, the method provided by the invention provides a more comprehensive prediction solution for screening of transport proteins, and the cost consumed by a wet experiment is greatly reduced.

Provided is a method of controlling a substrate transfer system transferring substrates by controlling a first transfer device and a second transfer device comprising: a process of acquiring process module-specific substrate transfer positions for each of the plurality of process modules, wherein the process module-specific substrate transfer positions are substrate transfer positions of the mounting parts of the first transfer device for allowing the second transfer device to place substrates at centers of the stages, the second transfer device transferring substrates from the mounting parts of the load lock module to the stages of the process modules.

The present application belongs to the field of biomedical detection technology, and particularly relates to a reaction solution for detecting trypsin-like transpeptidase activity and application thereof. The present application uses BAPNA as a specific substrate, which can produce a detectable signal change in the transpeptidation reaction, and a high-concentration amino group-glycine amide is added as a receptor molecule to enhance the transpeptidation reaction efficiency. Therefore, the present application solves the technical problem that the enzyme activity cannot be detected by the immunization method, has high reaction efficiency, stable and reliable detection process, shortened operation time (within 15 minutes), simple operation, strong practicability, can be used for evaluating the severity of acute allergic reaction, providing scientific guidance for the diagnosis of mast cell-related airway inflammation and mast cell activation syndrome, monitoring the effect of allergic treatment, and has clinical value in airway inflammation typing, differential diagnosis and efficacy monitoring through induced sputum or BAL fluid trypsin activity determination.

The invention discloses taxus chinensis-sourced dioxygenase as well as a preparation method and application thereof, and belongs to the technical field of natural product biosynthesis. The dioxygenase TcOGD1 and the dioxygenase TcOGD2 are identified and obtained from taxus chinensis var mairei, and both the dioxygenase TcOGD1 and the dioxygenase TcOGD2 can participate in oxidation modification and skeleton rearrangement reaction of taxane natural products. The TcOGD1 shows strong catalytic activity and wider substrate adaptability, in-vitro synthesis of the 5 / 7 / 6 type taxane taxupine J with the potential of reversing multidrug resistance of tumors is realized, and a new class of 5 / 7 / 6 type taxane compound is obtained. The TcOGD2 can produce selective oxidation products for specific substrates. The dioxygenase can efficiently catalyze conversion of various taxane structures in vitro, the framework type and structural diversity of taxane natural products are expanded, a key tool is provided for synthesis of taxane drug lead compounds and new drug development, and the dioxygenase has good application prospects.

The invention relates to improved limonene synthases for biosynthetic production of limonene. engineered limonene synthases with improved characteristics compared to reference limonene synthases are provided. Some engineered limonene synthases have improved limonene production yield. Other engineered limonene synthases have switched enantioselectivity. Still other engineered limonene synthases have improved yield with specific substrates. Some engineered limonene synthases have improvements in a combination of one or more of limonene production yield, enantioselectivity and substrate specificity. Also provided herein is a method of screening limonene synthase, e.g., for characterization of limonene synthases. Further provided is a method of producing limonene using the engineered limonene synthases provided herein.

The invention discloses a human trophoblast organ-like model and a construction method and application thereof. The method comprises the following steps: firstly, digesting and separating trophoblast cells from human placenta villus, inoculating the trophoblast cells into matrigel, and adding an ADMEM / F12 culture medium containing specific growth factors for culturing to form trophoblast-like organs; then morphological observation is carried out, and expression of P-gp, BCRP and MRP1 / 2 is detected from mRNA and protein level to carry out model verification; a co-incubation method is adopted, a plurality of ABC transporter specific substrates are adopted, and the influence of verapamil, KO143 and probenecid on the transportation of the ABC transporter specific substrates is researched; and finally, the method is applied to evaluation of substrate transport inhibition of a clinical drug on the ABC transporter protein. The human trophoblast organ model is constructed for the first time and is applied to ABC transporter mediated drug transport research, the operation method is efficient and rapid, and the organ model can be widely applied to in-vitro high-throughput screening of clinical drugs on ABC transporter inhibitors. The method has a wide application prospect.

The invention belongs to the technical field of biomedical detection, and particularly relates to reaction liquid for detecting trypsin transpeptidase activity and application of the reaction liquid. BAPNA is adopted as a specific substrate, detectable signal change can be generated in the transpeptidation reaction, then high-concentration amino group-glycinamide is added as an acceptor molecule, and the transpeptidation reaction efficiency is enhanced. Therefore, the technical problem that the enzyme activity cannot be detected by an immunoassay is solved, the reaction efficiency is high, the detection process is stable and reliable, the operation time is shortened to be within 15 minutes, the operation is simple and convenient, and the practicability is high; the kit can be used for evaluating the severity of acute anaphylaxis, provides scientific guidance for diagnosis of mast cell related airway inflammation and mast cell activation syndrome, monitors the anaphylaxis treatment effect, and has clinical value in the aspects of airway inflammation typing, differential diagnosis and curative effect monitoring by inducing phlegm or BAL liquid trypsin activity determination.