14 results about "Substrate concentration" patented technology

This invention discloses a ketoisomerase mutant and its application in the preparation of D-chiral inositol, belonging to the field of genetic engineering technology. The ketoisomerase mutant is obtained by mutating the amino acid sequence shown in SEQ ID NO.1, specifically by mutating amino acid position 62 and / or amino acid position 175 of the amino acid sequence shown in SEQ ID NO.1. After the above-mentioned mutation of the wild-type thermostable ketoisomerase, the resulting ketoisomerase mutant not only retains the advantages of the wild-type thermostable ketoisomerase—its heat resistance and susceptibility to temperature fluctuations—but also exhibits higher enzyme activity. When co-catalyzing the reaction of muscle inositol to D-chiral inositol with inositol dehydrogenase, it can further improve the conversion rate and yield of D-chiral inositol even at high substrate concentrations.

The present application relates to aldose reductase activity analysis technical field, specifically relates to a kind of aldose reductase activity and its inhibitory effect quantitative analysis method.Local substrate concentration regulation is constructed by dividing multiple detection segments, combining bypass sampling channel, electrochemical detection cavity and compensation injection point, and the substrate environment of each detection segment is maintained consistent;Enzyme-free reference channel parallel to microfluidic reaction channel is set, and the total absorbance change amount background is separated, to obtain the real enzymatic absorbance change amount;Based on sliding time window, extract local dynamic enzyme reaction rate, combined with wall adsorption correction to eliminate the influence of enzyme activity attenuation along the way, identify stable reaction interval through local fluctuation coefficient of variation, determine local half response time and local onset time;Local onset time is used as the integration starting point, to calculate comprehensive inhibition intensity, realize inhibitor onset order and inhibition strength spatial quantitative analysis, improve the accuracy, stability and spatial comparability of detection result.

This invention discloses an enzyme preparation and a method for preparing D-chiral inositol, belonging to the field of genetic engineering technology. The enzyme preparation includes inositol dehydrogenase and 2-keto-inositol isomerase. The amino acid sequence of inositol dehydrogenase is shown in SEQ ID NO. 6, and the amino acid sequence of 2-keto-inositol isomerase is shown in SEQ ID NO. 8. Compared with the prior art, the enzyme activities of the inositol dehydrogenase and 2-keto-inositol isomerase with specific amino acid sequences of this invention are further improved. When catalyzing the reaction of muscle inositol to D-chiral inositol, both can further improve the conversion rate and the yield of D-chiral inositol. Furthermore, this inositol dehydrogenase has high tolerance to muscle inositol, and can still efficiently catalyze the reaction to produce D-chiral inositol even at high substrate concentrations, alleviating substrate inhibition.

This invention discloses a sucrose isomerase site mutant, a genetically engineered bacterium, and a method for catalyzing the production of isomaltulose. This invention utilizes PROSS online analysis combined with sequence alignment analysis to screen key amino acid sites related to catalytic activity in sucrose isomerases. Through site-directed mutagenesis, the mutant WT-Gro7-Q474T was obtained, with a maximum specific enzyme activity of 683 U / mg. k cat (s ‑1 The concentration reached 718. By optimizing protein expression conditions, including temperature, IPTG concentration, bacterial concentration, and time, and combining this with the method of co-expression of molecular chaperone proteins, a highly efficient heterologous expression system for sucrose isomerase was established. This invention established an optimal isomaltulose-catalyzed reaction system: in a 2 L reactor, with 1 L of reaction solution and a substrate concentration of 800 g / L sucrose, after 6 hours of reaction, all sucrose was converted, yielding isomaltulose 792.50 g / L, with a conversion rate >99.5%.

This invention belongs to the field of genetic engineering technology and discloses a high-efficiency expression method for recombinant collagenase and its application in the preparation of bovine bone collagen peptides. The collagenase gene derived from Bacillus sp. HUB-I-004 strain (accession number J9CTX3) was optimized using E. coli-preferred codons. A recombinant expression vector was constructed and transformed into E. coli BL21(DE3). After optimization of induction conditions and Ni-NTA affinity purification, a highly active recombinant collagenase with a specific activity of 231.05 U / mg was obtained. Bovine bone collagen was extracted using a high-temperature, high-pressure method. Bovine bone collagen peptides were prepared under optimal conditions of a substrate concentration of 10 mg / mL, an enzyme addition of 20%, and enzymatic hydrolysis at 37℃ for 8 h, significantly improving peptide yield. The obtained product is mainly composed of small molecule peptides and exhibits significant osteoblast proliferation-promoting activity, making it widely applicable in the preparation of health foods and pharmaceuticals.

The application belongs to the technical field of enzyme engineering and genetic engineering, and discloses a ketoreductase mutant, method and application. The mutant comprises the following point mutations: the isoleucine at the 91th position of the amino acid sequence shown in SEQ ID No. 1 is mutated into glycine (I91G), and the alanine at the 139th position is mutated into methionine (A139M). The ketoreductase mutant obtained by the application can produce N-BOC-cis-3-hydroxy-L-proline methyl ester in high selectivity and high yield. Under the conditions of OD 600 =10, 300mM glucose, 2mM NADP + / NAD + , 50mg / ml ls GDH, and a substrate concentration of 200mM, the yield is 96.5% and de>99% after 18h of reaction.

This invention provides the application of β-glucosidase BglE or its mutants in the synthesis of ginsenoside Rg3; the β-glucosidase BglE is derived from *Thermoproteota* archaeon, and its amino acid sequence is shown in SEQ ID NO:2. This invention also provides a β-glucosidase BglE mutant, characterized in that it uses β-glucosidase BglE as the parent, with the following mutations: tyrosine (Y) at position 210 (F) mutated to phenylalanine (F), serine (S) at position 307 (A) mutated to alanine (A) / glycine (G), valine (V) at position 481 (A) mutated to alanine (A) or / and threonine (T) at position 517 (S) mutated to serine (S). The synthesis method of this invention allows for whole-cell feeding under single-enzyme catalysis, eliminating the need for cell disruption and additional hydrogen donors. It achieves a conversion rate exceeding 90% even at high substrate concentrations (40 g / L), enabling efficient one-pot preparation of the target product. The process is simple, offering better cost-effectiveness and promising prospects for industrial application.

This invention belongs to the field of bioengineering and enzyme engineering technology, specifically relating to a method for improving the soluble expression and catalytic activity of chondroitin 4-O-sulfotransferase. Using chondroitin 4-O-sulfotransferase MamC4ST as the research object, this invention constructs various fusion expression systems of soluble tags in prokaryotic hosts, and systematically evaluates its soluble expression behavior and catalytic performance under different host strains and molecular chaperone co-expression conditions. The MBP tag has significant advantages in improving the soluble expression of MamC4ST; under the synergistic effect of the GroEL / GroES molecular chaperone, the recombinant engineered strain MBP-Δ60MamC4ST-Shuttle7-pGro7 exhibits excellent catalytic performance on chondroitin substrates, with conversion and sulfation rates reaching 98.41% and 95.03%, respectively, at a substrate concentration of 2 g / L.

This invention relates to an amino acid ester acyltransferase mutant, its encoding gene, and its application in the synthesis of glutamic-alanine dipeptide. Compared with existing technologies, the amino acid ester acyltransferase mutant disclosed in this invention exhibits high activity, high substrate concentration tolerance, and high selectivity for glutamic-alanine dipeptide synthesis / hydrolysis. In the application of catalyzing the condensation of L-alanine methyl ester and L-glutamine to prepare glutamic-alanine dipeptide, it demonstrates significant advantages in terms of high yield and high space-time yield. Compared with chemical synthesis methods, the synthesis of glutamic-alanine dipeptide using this enzyme does not require group protection and deprotection, the process is simple, the reaction conditions are mild, and the process is environmentally friendly, showing promising application prospects in the industrial production of glutamic-alanine dipeptide.

The present application belongs to the field of food flavor chemistry and sensory evaluation technology, and provides a method for determining the optimal umami substrate concentration for aroma enhancement and a composition thereof. The method first determines the sensory recognition threshold of the umami substrate and the half-maximum intensity concentration of the aroma substance to be tested. Then, the umami enhancement intensity before and after adding a fixed concentration of aroma substance is determined through sensory evaluation. The umami enhancement intensity and substrate concentration data are fitted using a nonlinear single-peak response model to construct a synergistic umami curve. By analyzing the peak center parameter, half-peak full width parameter and asymmetric decay factor of the curve, the optimal umami substrate concentration for the aroma substance to exert the maximum synergistic effect can be accurately locked, and the effective concentration range of the synergistic effect can be quantitatively evaluated. The present application breaks through the cognitive misunderstanding of traditional high-concentration substrate umami enhancement, confirms that the low-concentration threshold region is the optimal synergistic window, and provides a scientific quantitative guidance tool for the food industry to develop low-cost and high-umami products.

This invention discloses an NADPH-dependent alcohol dehydrogenase EcYjgB mutant and its applications. The mutant is based on the wild-type alcohol dehydrogenase EcYjgB with mutations occurring at one or more of the following sites: S46, E51, W52, F54, T92, I108, A114, P116, I118, N240, G262, V264, L265, and A286. The amino acid sequence of the wild-type alcohol dehydrogenase EcYjgB is shown in SEQ ID. 1. This mutant is used to catalyze the selective reduction of high concentrations of 5-hydroxymethylfurfural or furfural to synthesize 2,5-furandiethanol or furfuryl alcohol, respectively. Compared to the wild-type enzyme, this mutant exhibits significantly improved catalytic efficiency and significantly reduced substrate inhibition at high substrate concentrations. It is green and efficient, requires no organic reagents, shortens reaction time, and significantly reduces the difficulty and cost of subsequent separation and purification.

This invention relates to the field of aldose reductase activity analysis technology, specifically to a quantitative analysis method for aldose reductase activity and its inhibitory effect. By dividing the detection into multiple segments and combining a bypass sampling channel, an electrochemical detection chamber, and a compensation injection point to construct a local substrate concentration control system, the substrate environment of each detection segment is maintained consistently. An enzyme-free reference channel parallel to the microfluidic reaction channel is set up to separate the background of the total absorbance change, obtaining the true enzymatic absorbance change. Based on a sliding time window, the local dynamic enzyme reaction rate is extracted, and the influence of enzyme activity decay along the process is eliminated by wall adsorption correction. The stable reaction interval is identified by the local fluctuation coefficient of variation, determining the local half-response time and local onset time. Using the local onset time as the integration starting point, the comprehensive inhibition intensity is calculated, achieving spatial quantitative analysis of the inhibitor's onset order and inhibitory strength, improving the accuracy, stability, and spatial comparability of the detection results.

The application provides a 3-steroid ketone-delta 1 -dehydrogenase, mutants and applications thereof. The 3-steroid ketone-delta 1 -dehydrogenase mutant provided by the application is a mutant of one or more core amino acids of a wild-type 3-steroid ketone-delta 1 -dehydrogenase which are related to catalytic activity of the enzyme. Compared with the wild-type enzyme, the 3-steroid ketone-delta 1 -dehydrogenase mutant of the application significantly improves catalytic efficiency of a C1,2 dehydrogenation reaction. By using a recombinant bacterium expressing the mutant, an intermediate with a methyl substitution at a C6 position of a steroid nucleus can be efficiently converted at a high substrate concentration, a yield of a target product is high, no by-product is generated, and a conversion rate is not less than 95%. The process has a short conversion time, a small amount of biological catalysts, and a simple, mild, and environmentally friendly preparation method, and shows a good industrial application prospect.

This invention discloses an imine reductase mutant and its application in the catalytic synthesis of chiral 1-aminoindenhydrin derivatives. This invention utilizes protein engineering to synthesize an imine reductase mutant derived from *Neosatoria* (…). Neosartorya fumigata imine reductase Nf RedAm underwent molecular modification to obtain a mutant with high activity, high stability, and high substrate tolerance. This mutant can efficiently catalyze the asymmetric reduction of 1-indanone: under optimal conditions, the substrate concentration reaches 10.56 g·L⁻¹. ‑1 Whole-cell catalyst M3 catalyzes the synthesis of products ( R The cumulative concentration of γ-rezagilan reached 11.2 g·L⁻¹. ‑1 The enantioselectivity reaches 98%. Simultaneously, this mutant also exhibits highly efficient catalytic activity and excellent stereoselectivity for 1-indanone derivatives substituted with methyl, halogen, or cyano groups, with product enantioselectivity > 99%. This imine reductase mutant possesses extremely high catalytic activity and substrate tolerance, showing strong industrial application potential in the asymmetric synthesis of chiral 1-aminoindanone derivatives.