Aspergillus niger genetically engineered bacterium used for producing Rhizopus chinensis lipase, and preparation method thereof

Abstract

The invention discloses a Rhizopus chinensis lipase expression cassette, an expression carrier containing the Rhizopus chinensis lipase expression cassette, and Aspergillus niger genetically engineered bacterium containing the Rhizopus chinensis lipase expression cassette and a preparation method thereof. The preparation method of the Aspergillus niger genetically engineered bacterium comprises following steps: coding gene RCL of Rhizopus chinensis lipase, aspergillus nidulans tryptophan synthetase C terminator TtrpC, and promoter Pgpdp used for extension of Penicillium glyceraldehyde-3-phosphate dehydrogenase gene are obtained; RCL super expression cassette Pgpdp-RCL-TtrpC is constructed, transformation of agrobacterium tumefaciens is realized with a T-DNA binary vector, and transformation of Aspergillus niger is carried out. The Aspergillus niger genetically engineered bacterium is capable of secreting food-grade Rhizopus chinensis lipase with high efficiency; lipase activity is as high as 263IU / ml; no fungaltoxin or antibiotic is generated under enzyme-producing conditions; and the Aspergillus niger genetically engineered bacterium is especially suitable for large-scale production of food-grade Rhizopus chinensis lipase.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an Aspergillus niger genetically engineered bacterium for producing Rhizopus chinensis lipase and a preparation method thereof, and an application of the Aspergillus niger genetically engineered bacterium in producing Rhizopus sinophila lipase. Background technique [0002] Aspergillus niger (Aspergillus niger) is an important industrial microorganism, which is widely used in industrial enzyme preparations, and can produce more than 30 kinds of enzyme preparations such as lipase, amylase, acid protease, cellulase, and pectinase; It has a history of hundreds of years in food brewing and processing, so it is recognized by the US FDA as a GRAS (Generally recognized as safe) strain, which can be used in the production of food and pharmaceutical grade enzyme preparations. Because Aspergillus niger has excellent protein secretion ability, high safety, mature fermentation an...

AI Technical Summary

Problems solved by technology

However, the natural Rhizopus chinensis has limited ability to produce lipase, and for food safety considerations, the lipase secreted by it cannot be directly used as a food additive

With the development of molecular biology, the gene of RCL was cloned, and the researchers realized the high expression of RCL in Pichia pastoris (Chinese patent, publication number: CN102911888A), but methanol needs to be added during the fermentation process of Pichia pastoris, and methanol A highly toxic chemical substance, a small amount of methanol will cause central nervous system damage, eye damage and metabolic acidosis, so RCL produced with methanol as an inducer still cannot be used as a food additive

The inventors found in the research that if RCL is fused with other commonly used promoters in fungal expression research (such as Aspergillus nidulans tryptophan synthase promoter PtrpC, Aspergillus niger glucoamylase promoter PglaA, etc.), RCL cannot be effectively achieved. In addition, if the mature peptide of RCL is fused with other secretion signals, secretory expression cannot be efficiently achieved in Aspergillus niger

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