79 results about "Galactosidase activity" patented technology

Methods and compositions are provided for analyzing intracellular events employing fusion proteins comprising the small beta-galactosidase fragment fused to a protein of interest or fragment thereof. By expressing the fusion protein in a cellular host, one can determine the effect of various agents on the activity of the fusion protein, adding the large beta-galactosidase fragment to the fusion protein, either intracellularly or in a lysate and determining the activity of the formed beta-galactosidase using a substrate that forms a detectable product. In this manner, degradation, complex formation, RNAi inhibition or other situation that affects beta-galactosidase activity can be determined.

The invention concerns mutant L. bulgaricus strains bearing a nonsense mutation, in at least one of the sequences coding for the lactose operon, and free from β-galactosidase activity, and lactic starters comprising these strains. The strains and starters can be used to obtain fermented milk products from glucose-added milk.

A method is described for improving the digestibility of a forage diet for ruminant animals. A forage, including alfalfa, Chinese wildrye, corn silage, straw silage, corn stover, ryegrass or TMR, is treated with an enzyme product having cellulase, xylanase, beta-glucanase, pectinase, mannanase and alpha-galactosidase activities.

The invention discloses alpha-galactosidase and an encoding gene and an application thereof. The alpha-galactosidase is as described in the following (a) or (b) or (c): (a) a protein constituted by amino acid residues from 20th site to 739th site of N terminal of sequence 1 in a sequence table; (b) the protein constituted by the amino acid sequence as shown in the sequence 1 in the sequence table; and (c) the protein with the alpha-galactosidase activity, which is derived by replacement and/or deletion and/or addition of one or more amino acid residues in the amino acid sequence of the sequence 1. The alpha-galactosidase can provide technical parameters for establishing a biological reactor with high-efficient expression in the future and be applied in different industrial production fields; furthermore, protein sources are enriched, the protein has low consistency with the known protein amino acid sequence, and new material is provided for taxonomy of the alpha-galactosidase.

The invention discloses an Aspergillus-versicolor-derived alpha-galactosidase Z-GALC, and a coding gene and application thereof. The protein Z-GALC has alpha-galactosidase activity, is derived from Aspergillus versicolor, and is (a) or (b) as follows: (a) protein composed of amino acid sequence disclosed as Sequence 1 in the sequence table; or (b) protein derived from Sequence 1 in the sequence table with alpha-galactosidase activity, which is subjected to substitution and/or deletion and/or addition of one or more amino acid residues of the amino acid sequence disclosed as Sequence 1 in the sequence table. The specific activity of the protein Z-GALC disclosed by the invention as the alpha-galactosidase is 4.07U/mg; the optimum pH value is 7.0; the optimum temperature is 42 DEG C; and the protein Z-GALC has high enzyme activity at the temperature of 37-50 DEG C under the pH value of 6.0-8.0. The invention provides a new way for preparing alpha-galactosidase, and has important application value.

The invention discloses a beta-galactosidase mutant and a related biological material and application thereof.The provided beta-galactosidase mutant is called beta-Gal-2 and shown as A1) or A2), A1) represents protein with the amino acid sequence shown as SEQ ID No.1, and A2) represents protein with beta-galactosidase activity, wherein one or more amino acid residues are substituted and/or deleted and/or added into the amino acid sequence of the protein A1).The mutant has the advantages of being high in adaptability and heat resistance and good in hydrolysis capacity, and influence on enzymatic activity by metal ions is small.The characteristic of heat resistance is extremely prominent, and enzymatic activity of the mutant at the temperature of 60 DEG C can be kept at 80% or more.An extra poisonous inductive agent is not needed when lactobacilli are used as a host to express the mutant, cost is low, expression quantity is large, and the beta-galactosidase mutant has good industrial application prospects.

The invention relates to a method for detecting a quorum sensing quenching bacterial strain. The method comprises the steps of: adding a bacterial strain to be detected in a PIPES (1,4-piperazinediethanesulfonic acid) buffer solution with a pH value of 6.7 to prepare a solution to be detected, wherein AHL (N-acyl Homoserine Lactone) is dissolved in the PIPES buffer solution, then enabling supernatant of the solution to be detected to react with a bacterial solution of A.tumefaciens A136 added with X-gal; after the reaction is ended, detecting OD492 and OD630 (Optical Density) values of a reaction solution, and calculating a standardized beta-galactosidase activity value through a formula of (0.653*OD492-OD630)/(0.267*OD630-OD492), and carrying out different significance analysis through comparing the standardized beta-galactosidase activity value, and finally, determining whether the bacterial strain to be detected is a quorum sensing quenching bacterial strain. The method provided by the invention has the advantages of being simple and rapid, being high in sensitivity, achieving high throughput detection, eliminating most of false positive bacterial strains, and being capable of effectively determining whether the bacterial strain to be detected is the quorum sensing quenching bacterial strain, thereby remarkably improving the accuracy and the pertinence of screening the quorum sensing quenching bacterial strain and saving the time and labor cost.

The invention discloses an alpha-galactosidase, and a coding gene and application thereof. The protein provided by the invention is (a) a protein composed of amino acid residues from 24 site to 751<st> site at the N terminal in Sequence 1 in a sequence table; or (b) a protein composed of amino acid sequences shown in Sequence 1 in the sequence table; or (c) a protein which has alpha-galactosidase activity, is derived from Sequence 1 and is formed by the amino acid sequence of (a) or (b) through the substitution and/or deletion and/or addition of one or more amino acid residues. The protein provided by the invention, serving as alpha-galactosidase, has the following enzymatic properties: the optimum pH value is 4.5, and the protein is stable in the pH value range of 4-10; and the optimum temperature is 60 DEG C, the protein is stable within 55 DEG C, the enzyme activity hardly changes when the protein is kept at 55 DEG C for 30 minutes, and 70% of the activity can be remained when the protein is kept at 60 DEG C for 30 minutes, thus indicating favorable heat stability. The protein provided by the invention has great potentials in industrial application.

The invention discloses a whole-cell biosensor for detecting heavy metal ions in a water-soluble sample and construction and application thereof. The operation object is escherichia coli, no pathogenic risk exists, and the operation is simple and easy to implement. An SRRz cracking gene is used as a report element, so that microbial bacteria are cracked, and the turbidity of bacterial liquid is obviously changed. Detection can be performed through a visible spectrophotometer, and a result can be obtained through direct observation with naked eyes. The response is quick, and only 30-60 minutesare needed from sample contact to result acquisition. X-gal is used for detecting beta-galactosidase released to the outside of the cells, and the operation is simple and convenient. Expensive instruments are not needed in application, so that the detection cost is greatly reduced, and field detection becomes possible. The extracellular beta-galactosidase activity is accurately quantified by usingchromogenic gel containing oNPG, the detection limit is low, and the detection result is accurate. Technical support is provided for daily heavy metal pollution detection and sudden heavy metal pollution detection.

The invention discloses a bacterial strain producing high-temperature-resistant beta-galactosidase and a screening method thereof and belongs to the technical field of biological chemistry and protein engineering. The screening method includes the specific and sequential steps of sample collection, primary screening, secondary screening of culture and beta-galactosidase activity assay. Mainly, soil of hot spring land is collected and boiled, then bacterial colonies are screened, and the technical problem how to produce high-enzyme-activity beta-galactosidase at high temperature is solved through a high-temperature culturing method. The bacterial strain is short in beta-galactosidase production period, high in beta-galactosidase production speed, good in genetic stability, thermal stability and pH stability, high in enzyme catalytic activity and high in production safety, and culturing conditions are simple. The bacterial strain has broad prospects in the fields of food industry and medical and biochemical analysis, in particular to the dairy industry.

The invention discloses an Alpha-galactosidase and a coding gene thereof. The alpha-galactosidase is a protein shown in a) or b) as follows: a) a protein consisting of amino acid residue from the 21-438th position of end N in sequence 1 in a sequence table; b) a protein consisting of the amino acid sequence shown in the sequence 1 in the sequence table; or c) a protein that derives from a) or b),has the activity of Alpha-galactosidase and is obtained from the protein limited by a) or b) through substitution and /or loss and/or addition of one or a plurality of amino acid residues. Applied towider industrial production, the enzyme provides experimental materials for the construction of further industrial high-yield engineering strain and also provides comparative sequences and property research reference for richening the diversity research of the source of Alpha-galactosidase. Therefore the enzyme and the coding gene have extremely favorable commercial application value and potential.

The present invention discloses a method for reaction separation purification of rebaudioside A, and belongs to the technical field of organic chemistry. In the prior art, the stevia sugar crude product mainly contains stevioside, high-quality sweeteners such as rebaudioside A and rebaudioside C, and small amounts of other steviol derivatives, the rebaudioside A and the stevioside have the similar structure, and the dissolubility of the two substances can be increased when the two substances co-exist in a polar solution, such that the multi-stage sectional organic solvent re-crystallization is required to obtain the high-purity rebaudioside A. According to the present invention, the beta-galactosidase having lipase activity and galactosidase activity and derived from sulfolobus solfataricus is utilized to carry out highly specific catalytic hydrolysis on the stevioside, such that the poor-quality stevioside can be subjected to complete catalysis conversion into the water-insoluble steviol, the rebaudioside A and other steviols can not be hydrolyzed so as to remain in the solution and the high-purity rebaudioside A can be obtained through the simple aqueous phase re-crystallization, and the by-produced steviol is the organic synthesis intermediate having the wide use and the added value of higher than the stevioside.

The invention provides a method for raising alpha galactosidase activity, which comprises the following steps: connecting an alpha galactosidase gene (Genbank number: X03102) with a N terminus of a saccharomyces cerevisiae cell wall constituents alpha lectin sequence (Genbank number:M28164), then inserting a C terminus of downstream MFalphal1 signal peptide sequence (Genbank number:M17301) of a saccharomyces cerevisiae expression vector GAL1 promotor (Genbank number:AY428072), constructing an expression cassette, wherein the expression cassette is from the N terminus to the C terminus: the GAL1 promoter, a MF alpha1 signal peptide sequence, the alpha galactosidase gene, the alpha lectin sequence; converting a cerevisiae plasmid containing the expression cassette to the saccharomyces cerevisiae cell. The invention has the advantages that the alpha galactosidase is expressed out of an external surface of the saccharomyces cerevisiae cell wall so that a total contact of enzyme and an extracellular substrate can be realized, therefore the alpha galactosidase activity can be obviously improved.

The present invention relates to methods of releasing galactose from legumes using polypeptides having alpha-galactosidase activity. The invention also relates to polypeptides having alpha-galactosidase activity, polynucleotides encoding the polypeptides, nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of 5 producing the polypeptides. The invention also relates to compositions comprising the polypeptides of the invention and the use of the polypeptides in animal feed.

The vitamin D binding protein (Gc protein) and its small region (approximately 1/5 of the Gc peptide, known as region III) were cloned by means of a baculovirus vector. Cloned Gc protein and cloned domain (Cd) peptide were treated with immobilized β-galactosidase and sialidase to generate macrophage activating factors GcMAFc and CdMAF, respectively. These cloned macrophage activating factors and GcMAF can be used in the treatment of cancer, HIV infection and osteoporosis, and also as adjuvants for immunization and vaccination. Developed methods and antibody-sandwich ELISA kits for detecting serum or plasma α-N-acetylgalactosaminidase activity in cancer and HIV-infected patients.

The invention discloses a construction method and application of a bacterial promoter reporter vector. The method includes the following steps: first constructing a vector containing beta-galactosidase lacZ gene, inserting a target gene promoter into the upstream of the lacZ gene in the vector so as to obtain a reporter vector for detecting the activity of the gene promoter, introducing the reporter vector into to-be-tested strains, and inferring the change of the activity of the target gene promoter by detecting and comparing the activity of the strains and the beta-galactosidase. The reporter vector of the present invention can be used to detect the activity of gene promoters in bacteria and determine the strength of gene expression, and can also be used to detect the presence or absenceof promoter activity of unknown DNA fragments in bacteria, and to determine the location of the gene promoters with low operation cost and stable and reliable result.

The invention discloses a protein with alpha-galactosidase activity and an application of the protein. The protein with alpha-galactosidase activity is any one of A1)-A5) as follows: A1) the protein with the amino acid sequence from the 92th position to the 817th position of a sequence 1 in a sequence table; A2) the protein with the amino acid sequence from the 87th position to the 817th position of the sequence 1 in the sequence table; A3) the protein with the amino acid sequence shown in the sequence 1 in the sequence table; A4) the protein with the amino acid sequence from the 87th position to the 831th position of sequence 5 in the sequence table; A5) the protein with the amino acid sequence shown in sequence 5 in the sequence table. Experiments prove that protein has alpha-galactosidase activity and the protein and a fermentation product of a recombinant microorganism containing protein-encoding nucleic acids can be used for hydrolyzing substances containing alpha-galactoside bonds and preparing alpha-galactosidase preparations.

The invention discloses bacterium beta-galactosidase as well as an encoding gene and an application thereof. A protein provided by the invention is a1) or a2) or a3) or a4), wherein a1) a protein with an amino acid sequence shown as a sequence 2 in a sequence stable; a2) a fused protein obtained by connecting an N end or/and a C end of the protein shown as the sequence 2 in the sequence stable with a label; a3) a protein with an amino acid sequence shown as a sequence 4 in the sequence stable; and a4) a protein with beta-galactosidase activity, which is obtained by carrying out replacement and/or deletion and/or addition of one or more amino acid residues on the protein shown as the a1) or the a2) or the a3). An experiment shows that the protein provided by the invention can be used for efficiently hydrolyzing lactose and can also be used for efficiently synthesizing galacto-oligosaccharides, and has important economic value and practical significance on production of low-lactose milk products and the galacto-oligosaccharides in a food industry.

The invention discloses a preparation method and application of inonotus obliquus polysaccharide. Through water extraction and alcohol precipitation, repeated freeze thawing, deproteinization, dialysis, freeze drying, affinity chromatography and other methods, a polysaccharide component with antioxidant and anti-aging functions is separated and purified from inonotus obliquus; the inonotus obliquus polysaccharide component has strong antioxidant activity; through pretreatment of the inonotus obliquus polysaccharide, etoposide-induced proliferation inhibition and apoptosis of rat aortic smoothmuscle cells can be inhibited, and etoposide-induced cellular senescence beta-galactosidase activity increase, senescence-related heterochromatin aggregation and mitochondrial membrane potential decline can be inhibited; through pretreatment of the inonotus obliquus polysaccharide, etoposide-induced increase in expression levels of p53, p21 and p16 protein and mRNA can be inhibited. The inonotus obliquus polysaccharide components prepared according to the preparation method provided by the invention has good antioxidant and anti-aging activity and has a broad application prospect as a novel anti-aging protective medicine.

The invention provides a lactobacillus plantarum (L.plantarum) CZ401 strain capable of producing exopolysaccharides and beta-galactosidase.The strain is preserved in China Center for Type Culture Collection on March 7, 2016, the address of the China Center for Type Culture Collection is Wuhan University, Wuchang District, Wuhan City of Hubei Province, the preservation number is CCTCC NO:M2016092, and the 16S rDNA sequence of the strain is shown as SEQ ID NO.1.When bacterial colonies are observed on an MRS solid medium, the bacterial colonies are light yellow, smooth in surface and circular, and the centers of the bacterial colonies protrude; it is shown through microscopy observation that the strain is positive in gram staining, rodlike, and free of flagella and spores.The CZ401 strain has higher capacity of producing exopolysaccharides and higher beta-galactosidase activity, can ferment cow's milk to produce lactic acid drinks and has wide application value.

The present invention provides an aqueous composition comprising a protein with enzymatic activity of alpha-galactosidase. The present invention further provides a method of stabilizing an aqueous composition comprising a protein with enzymatic activity of alpha-galactosidase, and a method of preparing a purified aqueous composition comprising the protein with enzymatic activity of alpha-galactosidase.

The invention belongs to the technical field of detection. The invention discloses a method for evaluating the anti-aging efficacy of cosmetics. According to the method, indexes such as a beta-galactosidase activity inhibition effect, a telomerase activity enhancement effect, an oxygen free radical elimination effect, a lipid browning content, a survival rate and the like are evaluated. Accordingto the invention, experimental operation and observation are facilitated, and since chemical substances are directly absorbed into a circulating system, and painful and time-consuming injection or invasive surgery is not needed, so that pressure or tissue damage related to a rodent aging model is avoided. Zebra fish is equivalent to mice for 8-10 days in one day, the experiment period is shorter,and the cost is low; The zebra fish, as a complete living body model, can be used for high-throughput screening of drugs, especially screening of chemical drugs. Embryos and juvenile fishes are transparent, various tissue and organ changes can be directly observed under a microscope, and experiment results are visual and easy to understand.