Pgrac

Abstract: The hyperthermophilic α-amylase from Thermococcus sp. HJ21 possesses unique traits (Ca(2+)-independent thermostability and optimal temperature of 95°C) that make it a great potential candidate for use in the food industry. However, this Archaea isolated from a deep-sea thermal vent requires strict control of culture conditions and produces only small amounts of α-amylase. To solve these problems, the α-amylase gene was cloned and expressed in Bacillus subtilis, which is an ideal food-grade host for heterologous protein expression. To express high levels of this α-amylase in B. subtilis, the promoters Pgrac, PxylA, P43, and Phag were used to construct four different expression vectors for testing. The vector containing the PxylA promoter was found to have the highest transcriptional activity and produce the highest amylase activity (19.6 U/ml). To test the secretion efficiency of signal peptides in B. subtilis, three signal peptides were cloned and fused to the α-amylase gene (lacking its native signal peptide). The optimal signal peptide was SamyQ, with a secretion efficiency of approximately 90%. These results indicate that the promoter PxylA and signal peptide SamyQ tested in this study may be useful for the expression and secretion of archaeal proteins in B. subtilis.

Abstract: The present invention relates to a method for the production of hyaluronic acid (HA) in Bacillus subtilis and Escherichia coli through plasmid vectors wherein the gene is under the control of strong promoter Pgrac, and a system for the selection of stable bacterial strains for the production of high levels of hyaluronic acid.

Abstract: The invention discloses recombinant bacillus subtilis producing micromolecular hyaluronic acid and belongs to the technical field of bioengineering. Hyaluronic synthase hasA originated from streptococcus zooepidemicus is integrated into bacillus subtilis to realize production of hyaluronic acid and meanwhile, overexpression on a synthetic route key gene of HA is carried out to realize high yield of HA in bacillus subtilis. On the basis, hyaluronidase originated from leeches is integrated into a bacillus subtilis genome for secretory expression subjected to a promoter Pgrac under induction control of IPTG (isopropyl-beta-d-thiogalactoside). Different micromolecular hyaluronic acid products with different molecular weights are prepared by controlling the secretory expression amount of hyaluronidase and induction starting time. The prepared products are different in ranges of molecular weights and the recombinant bacillus subtilis has important guiding reference meaning for direct production of micromolecular hyaluronic acid in a special particular range by microorganisms. The recombinant bacillus subtilis disclosed by the invention lays a certain foundation for efficiently preparing the micromolecular hyaluronic acid and is suitable for industrial production and application.