Modification of protease-producing strains
Protease is a class of enzyme that catalyzes the hydrolysis of peptide bonds in proteins, which is widely found in animals, plants and microorganisms, and it is one of the most used enzyme preparations in industrial enzyme species, accounting for about 60% of the total amount of enzymes. Microbial sources of proteases are abundant, diverse and easy to produce, becoming the main source of proteases on the market. The commercial production of protease can be traced back to the beginning of the last century, in 1908 the Germans have begun to use pancreatic enzyme tanning leather, in 1911, the United States Wallerstein company produced papain as a clarifying agent for beer. in the early sixties, the Netherlands produced detergents with the addition of protease. In addition, also combined with practical to carry out heat-resistant, acid-resistant, salt-resistant protease production bacteria selection and breeding, as well as petroleum as raw material fermentation production of alkaline protease research.
There are many kinds of proteases, and their molecular weight size, spatial structure and function are very different, but each family of proteases still maintains a highly conserved structural domain. At present, more than 100 types of proteases have been crystallized or highly purified, and many of them have been elucidated in terms of their primary and stereo structures (tertiary structure).
2 Modification of protease strains
The construction of engineering bacteria for efficient expression of protease and the optimization of its enzymatic properties have been the hotspot of domestic and foreign scholars, and the means usually used are: mutation breeding, protoplasmic fusion, construction of genetically engineered bacteria, and in vitro directional evolution technology.
2.1 Mutagenesis breeding and protoplasmic fusion
Mutagenesis breeding is mainly through radiation mutagenesis or some chemical reagents to induce mutations in the genetic material of the bacterium to obtain mutant strains with excellent traits. For example, after UV mutagenesis of enzyme-producing strains by Cai Wanling et al, their enzyme activity increased by 16% compared with the original strain. Yanli Li et al. screened the neutral protease-producing enzyme activity of Aspergillus oryzae ZW-06 up to 15,000 U/g of dry curd by Co60-directed mutagenesis, which was 74% higher than that before mutagenesis. Huang Hongying et al. combined low-energy N+ ion implantation technology on the wild-type Bacillus licheniformis for four times with different physicochemical means of mutagenesis, to obtain a high-yielding strain, the crude enzyme activity of 12425. 9 U/mL, 17.1 times higher than the departure strain.
Protoplasmic fusion technology is a process in which the cells of a biparental strain are de-walled for protoplasmic fusion, so as to exchange and recombine their genomes to obtain stable recombinants. For example, Pan Yanyun et al. used the protoplasmic fusion method to fuse Bacillus licheniformis 2709 with Bacillus subtilis BD105, which contains alkaline protease gene cloning vector pDW2, to obtain a high-yielding strain A16, with fermentation enzyme production 50%-100% higher than that of 2709, and enzyme production of up to 30,000 U/mL in the shake flask test.
2.2 Construction of genetic engineering bacteria
The construction of engineering bacteria usually requires suitable expression hosts and expression vectors or expression elements. Firstly, the target gene is cloned into the expression vector to obtain the recombinant vector, then transformed into the expression host, through the screening process, and finally obtain the high-yield strain process. It is also possible to integrate the exogenous target gene fragments directly into the genome of the host to obtain high-yielding strains.
[October 18-20] 2024 The 2nd Conference on Advanced Enzyme Engineering and Enzyme Technology Applications
Min Zhang et al. ligated the promoter-signal peptide sequence ( sacR ) of the sacB gene with the Bacillus subtilis neutral protease gene and cloned it into the vector pHP13, constructed the inducible expression secretion vector pHP13SN containing the neutral protease gene, and then transformed it into Bacillus subtilis DB104, which showed 48% increase in enzyme viability as compared with that of the departure strain. wangHui et al. cloned Banpr, a neutral protease from Bacillus amyloliquefaciens K11, and constructed a recombinant expression plasmid, pUB110-Banpr, with Bacillus amyloliquefaciens K11 as the expression host, and the enzyme activity in shake flask fermentation reached 8995 ± 250 U/mL, and 28084 ± 1282 U/mL in 15 L fermentation system, which was much more than that of the industrial strain Bacillus subtilis AS.1398 (8000-10000 U/mL).
2.3 Improvement of protease properties
With the development of in vitro directed evolution techniques, proteins with improved or novel functions can be obtained by random mutation of coding genes, DNAShuffling techniques and directed screening without the knowledge of the three-dimensional structural information and mechanism of action of the target protein.
Enzyme stability is a key factor in the success of commercialization. Enzyme stability can be improved by introducing salt bridges, disulfide bonds, aromatic interactions and increasing the affinity of the enzyme for calcium ions thereby increasing the stiffness of the molecule through targeted mutagenesis. Replacement of Gly61 or Ser98 of the protease BPN’ with Cys, and of both, resulted in an increase in the thermal stability of the enzyme without a change in catalytic efficiency.
By replacing Val72 with Ile, Ala92 with Thr, and Gly131 with Asp of B. subtilis protease, the mutant enzyme can catalyze substrate activity 2 times higher than that of the wild enzyme at 10°C. In 2005, the Danish company Novozymes introduced a low-temperature protease “Polarzyme”, which can be added to detergent. By adding it into detergent, the washing temperature decreased from 60°C and 40°C to 30°C and 20°C, saving 60% of electric power, and the sales volume of detergent “carecoldwash” with low-temperature protease increased by seven times.
3 Outlook
Proteases have been used in a variety of fields that are closely related to our daily lives, and this environment places high demands on the production and characterization of proteases. Therefore, in-depth understanding of the catalytic mechanism of protease, the relationship between spatial structure and catalytic function, and directional modification of enzyme-producing strains to meet the requirements of industrial production are still the development trend of enzyme engineering in the future. In addition, besides the selection and breeding of good strains, efforts should be made to try to construct new expression systems or multiple protease gene co-expression systems, to construct more expression vectors for proteases, so as to realize the effective expression of different components and further improve the expression amount, enzyme activity and enzyme stability.
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