Abstract: Disclosed is a method for constructing a synthesis and regeneration system based on APS as an active sulfonate donor, belonging to the technical field of biology. The present disclosure provides a new purpose of APS as an active sulfonate donor, and greatly improves the synthesis efficiency of the APS by screening different ATP sulfurylases and adding a pyrophosphatase into a reaction system to eliminate pyrophosphate as a byproduct. Further, the construction of a sulfonation modification system is realized by constructing an APS circulation regeneration system. Compared with a PAPS regeneration system, the APS circulation regeneration system has the advantages of short path and high efficiency, the sulfonation modification efficiency is significantly improved, and the synthesis cost is successfully reduced.

Abstract: The present disclosure discloses a method for efficient catalytic synthesis of PAPS based on constructing an ATP regeneration system, and belongs to the technical field of bioengineering. Efficient production of PAPS is realized through microbial recombination expression and artificial construction of PAPS bifunctional synthetase. On the basis, an ATP regeneration system coupling with polyphosphate kinase from Corynebacterium glutamicum and Mycobacterium tuberculosis can be used for recovering two byproducts: pyrophosphoric acid and ADP at the same time, the equivalent conversion of a substrate and a product is realized, the PAPS generated in a catalysis system has high purity, and the sulfonic acid group donation in most sulfonic acid transfer reactions can be realized.

Abstract: The Corynebacterium glutamicum strain of the present invention is obtained by mutating Corynebacterium glutamicum ATCC 13032, with the mutation sites including: mutating cytosine at site 862902 into thymine; mutating guanine at site 862903 into adenine; mutating cytosine at site 862953 into thymine; mutating adenine at site 862961 into guanine; inserting cytosine and thymine at site 862958; and mutating of guanine at site 862963 by deletion. The Corynebacterium glutamicum strain of the present invention exhibits significantly increased tolerance in high-viscosity environments and growth and metabolism ability under low dissolved oxygen conditions, thereby increasing the yield of mucopolysaccharides, and avoiding the problems where resulting mucopolysaccharides cause the fermentation broth to become viscous and have insufficient dissolved oxygen, which would further limit the metabolism of Corynebacterium glutamicum and ultimately affect the synthesis of mucopolysaccharides.

Abstract: The invention discloses a recombinant Corynebacterium glutamicum for efficient synthesis of highly pure hyaluronic acid and oligosaccharides thereof, belonging to the technical field of bioengineering. The recombinant Corynebacterium glutamicum constructed in the present invention can produce hyaluronic acid with a yield up to 40 g/L, and a crude product purity of 95%. Addition of exogenous hyaluronic acid hydrolase and optimization of the fermentation conditions results in hyaluronic acid oligosaccharides with specific molecular weight, and can further improve the yield of hyaluronic acid to 72 g/L. The invention lays a solid foundation for the efficient synthesis of highly pure hyaluronic acid by microorganisms, and the constructed recombinant Corynebacterium glutamicum is suitable for industrial production and application.

Abstract: Disclosed is a prediction method for tool remaining life of a numerical control machine tool based on a hybrid neural model, including: constructing a hybrid neural network model, specifically including the following steps: constructing sample data according to the sampling frequency of tool data; obtaining a first feature vector representing the tool life by utilizing a convolutional neural network and a long short-term memory network; generating working condition signals of sampling points into a second feature vector representing the tool life by utilizing an NFM neural network; and inputting a current working time of a tool and the acquired feature vectors into a multi-layer perceptron for fusion to predict the tool life.

Abstract: Disclosed is a prediction method for tool remaining life of a numerical control machine tool based on a hybrid neural model, including: constructing a hybrid neural network model, specifically including the following steps: constructing sample data according to the sampling frequency of tool data; obtaining a first feature vector representing the tool life by utilizing a convolutional neural network and a long short-term memory network; generating working condition signals of sampling points into a second feature vector representing the tool life by utilizing an NFM neural network; and inputting a current working time of a tool and the acquired feature vectors into a multi-layer perceptron for fusion to predict the tool life.

Abstract: The present disclosure discloses a method for efficient catalytic synthesis of PAPS based on constructing an ATP regeneration system, and belongs to the technical field of bioengineering. Efficient production of PAPS is realized through microbial recombination expression and artificial construction of PAPS bifunctional synthetase. On the basis, an ATP regeneration system coupling with polyphosphate kinase from Corynebacterium glutamicum and Mycobacterium tuberculosis can be used for recovering two byproducts: pyrophosphoric acid and ADP at the same time, the equivalent conversion of a substrate and a product is realized, the PAPS generated in a catalysis system has high purity, and the sulfonic acid group donation in most sulfonic acid transfer reactions can be realized.

Abstract: A method for predicting a remaining life of a tool of a computer numerical control machine is provided. In the method, indirect measurement indicators of the tool are selected based on monitoring and analyzing a current state of the tool, a prediction model for the remaining life of the tool is established based on data de-noising, feature extraction and a multi-kernel W-LSSVM algorithm. Thereby, a method for predicting a remaining life of a tool of a computer numerical control machine is provided.

Abstract: The invention discloses a recombinant Corynebacterium glutamicum for efficient synthesis of highly pure hyaluronic acid and oligosaccharides thereof, belonging to the technical field of bioengineering. The recombinant Corynebacterium glutamicum constructed in the present invention can produce hyaluronic acid with a yield up to 40g/L, and a crude product purity of 95%. Addition of exogenous hyaluronic acid hydrolase and optimization of the fermentation conditions results in hyaluronic acid oligosaccharides with specific molecular weight, and can further improve the yield of hyaluronic acid to 72 g/L. The invention lays a solid foundation for the efficient synthesis of highly pure hyaluronic acid by microorganisms, and the constructed recombinant Corynebacterium glutamicum is suitable for industrial production and application.

Abstract: A method for predicting a remaining life of a tool of a computer numerical control machine is provided. In the method, indirect measurement indicators of the tool are selected based on monitoring and analyzing a current state of the tool, a prediction model for the remaining life of the tool is established based on data de-noising, feature extraction and a multi-kernel W-LSSVM algorithm. Thereby, a method for predicting a remaining life of a tool of a computer numerical control machine is provided.

Abstract: The disclosure discloses a method for preparing hyaluronan oligosaccharides with odd-numbered degrees of polymerization by hydrolysis, and belongs to the technical field of biological engineering. The disclosure takes macromolecular hyaluronic acid as the substrate, and by controlling the addition amount and reaction time of two hyaluronic acid hydrolases, leech hyaluronidase (LHase) and bovine testicular hyaluronidase (BTH), simultaneously prepares two different structures of hyaluronan oligosaccharides with odd-numbered degrees of polymerization (trisaccharide, pentasaccharide and heptasaccharide). The disclosure provides a new, simple and feasible method for preparing hyaluronan oligosaccharides with odd-numbered degrees of polymerization, thereby laying a foundation for the research on the functions and characteristics of the hyaluronan oligosaccharides with odd-numbered degrees of polymerization.

Abstract: The present invention relates to a method for chondroitin sulfate biosynthesis, belongs to the field of pharmaceuticals. CS was biosynthesized by sulfating the chondroitin with C4ST or C6ST in Tris-HCl buffer assisted with 3?-phosphoadenosine 5?-phophosulfate (PAPS). C4ST and C46ST came from bioengineered Escherichia coli or Pichia pastoris. Chondroitin came from bioengineered Bacillus subtilis 168.

Abstract: The present invention provides a method for scarless in vitro DNA assembly using thermostable exonucleases and ligase, which relates to the field of genetic engineering. The present invention provides a fast method for assembling DNA subfragments with homologous ends, which employs thermostable polymerases and ligase in a thermal cycle of denaturation, annealing, digestion and ligation. After denaturation, DNA subfragments are assembled together via annealing of the homologous end sequences, the unpaired single-stranded overhangs are digested by polymerases, and the resulting nicked gaps are sealed by a ligase. Using this method, 2-6 DNA subfragments were successfully assembled within two hours. This method can be used in conventional DNA recombination and be adapted to high throughput assembly operations. In addition, combinatorial mutations can be easily introduced into the assembled sequence by use of primers with mutated bases.

Abstract: A cyclone filtration assembly unit is described.

Abstract: The present invention relates to a method for chondroitin sulfate biosynthesis, belongs to the field of pharmaceuticals field. CS was biosynthesized by sulfating the chondroitin with C4ST or C6ST in Tris-HCI buffer assisted with 3?-phosphoadenosine 5?-phophosulfate (PAPS). C4ST and C46ST came from bioengineered Escherichia coli or Pichia pastoris. Chondroitin came from bioengineered Bacillus subtilis 168.

Abstract: The present invention relates to the field of biotechnology engineering. It provides a method of constructing a recombinant Bacillus subtilis that can produce specific-molecular-weight hyaluronic acids. By integranted expression of hasA from Streptococcus zooepidemicus and overexpression of genes of HA synthetic pathway, tuaD, glmU and glmS, high yield HA production was achieved in the recombinant strain. Additionally, introduction and functional expression of the leech hyaluronidase in the recombinant strain substantially increased the yield of HA to 19.38 g·L?1. Moreover, HAs with a broad range of molecular weights (103 Da to 106 MDa) were efficiently produced by controlling the expression level of hyaluronidase using RBS mutants with different translational strengths. The method of the present invention can be used to produce low molecular weight HAs at large scale in industrial applications.

Abstract: The present invention provides a method for scarless in vitro DNA assembly using thermostable exonucleases and ligase, which relates to the field of genetic engineering. The present invention provides a fast method for assembling DNA subfragments with homologous ends, which employs thermostable polymerases and ligase in a thermal cycle of denaturation, annealing, digestion and ligation. After denaturation, DNA subfragments are assembled together via annealing of the homologous end sequences, the unpaired single-stranded overhangs are digested by polymerases, and the resulting nicked gaps are sealed by a ligase. Using this method, 2-6 DNA subfragments were successfully assembled within two hours. This method can be used in conventional DNA recombination and be adapted to high throughput assembly operations. In addition, combinatorial mutations can be easily introduced into the assembled sequence by use of primers with mutated bases.

Abstract: The present invention relates to the field of biotechnology engineering. It provides a method of constructing a recombinant Bacillus subtilis that can produce specific-molecular-weight hyaluronic acids. By integranted expression of hasA from Streptococcus zooepidemicus and overexpression of genes of HA synthetic pathway, tuaD, glmU and glmS, high yield HA production was achieved in the recombinant strain. Additionally, introduction and functional expression of the leech hyaluronidase in the recombinant strain substantially increased the yield of HA to 19.38 g·L?1. Moreover, HAs with a broad range of molecular weights (103 Da to 106 MDa) were efficiently produced by controlling the expression level of hyaluronidase using RBS mutants with different translational strengths. The method of the present invention can be used to produce low molecular weight HAs at large scale in industrial applications.

Abstract: The present invention provides a method of rapid directed DNA evolution based on single-stranded combinatorial DNA mutant library. The single- and double-stranded mutant library are constructed either separately or simultaneously using editing primers that contain mutated nucleotides and are targeted to different regions of the parent DNA sequence. The mutant library is then inserted into expression vectors and mutants with desired property are obtained by high throughput screening. Evolution of promoters, enzymes and metabolic pathways were successfully achieved using this method and mutants with excellent properties were obtained. The method of the present invention is simple, rapid, and efficient. It can be used for directed evolution of regulatory sequence such as promoters and ribosome binding sites, and is especially suitable for introducing diverse mutations into protein encoding genes, leading to rapid directed evolution of gene of interest.

Abstract: The present invention provides a novel leech HAase and a method of producing low-molecular-weight HA oligosaccharides using the leech HAase. This invention successfully cloned the first leech HAase gene and provides a method for high-level expression of the leech HAase gene. By controlling the incubation condition, different HA oligosaccharides, particularly HA4, HA6, HA8 and HA10, can be selectively generated using the leech HAase. The large-scale expression of the leech HAase and the enzymatic production of specific HA oligosaccharides are not only useful for the cosmetic, healthcare and the medical industries but also can be a great help to polysaccharides chemical synthesis and cancer research.