PG, produced in Chryseobacterium proteolyticum, is a novel food enzyme addition in recent years. PG contains a Cys-His-Asp catalytic triplet that specifically hydrolyzes glutamine residues on proteins or peptide chains and produces glutamate residues and NH4+, without the hydrolytic and cross-linking activity of protease or glutamine aminotransferase(Hashizume, et al., 2011; Yamaguchi, et al., 2001). PG effectively reduces the content of amide groups in proteins and enhances the functional properties of proteins, such as solubility and emulsification of plant proteins. Thus, the potential application values of PG have attracted a lot of attention from the food industry(X. Liu, et al., 2022). It was shown that the solubility of PG-deamidated soy protein was enhanced under acidic and neutral conditions(Suppavorasatit, et al., 2011). The secondary structure of PG deamidated oat protein was more flexible and the protein solution was more homogeneous and stable(Jiang, et al., 2015).
The natural PG-producing strain was discovered in C. proteolyticum 9670T, isolated from the soil in 2000(Yamaguchi & Yokoe, 2000), which was proven to be food safe(Scheuplein, et al., 2007) and certified as a generally recognized as safe (GRAS) strain by US FDA. However, as of present, the recently reported yield of PG in C. proteolyticum is only 2.91 U/mL, which is far below the requirement for industrial applications in the food processing industry(Wang, et al., 2023). Heterologous expression is an alternative strategy to overcome this problem. Currently, The maximum PG enzyme activity of 0.175 U/mL was achieved in E. coli(Lu, et al., 2020). The PG enzyme activity of 7.07 U/mL and 26 U/mg were achieved in Bacillus subtilis(Ouyang, et al., 2021; Yin, et al., 2021) and Corynebacterium glutamicum(Kikuchi, et al., 2008; Qu, et al., 2022; Wang, et al., 2023), respectively. Unfortunately, the expression effect of E. coli, which is the preferred host for superior heterologous gene expression, seems to be not optimistic for PG production.
E. coli, an excellent host for heterologous gene expression, is used in various production and research areas of biology due to its clear background, simple genetic manipulation, broad compatibility, and ability to perform high-density fermentation. Optimization of gene expression using various vectors containing different promoters was widely used to screen for appropriate or high yields of target proteins(Cheng, et al., 2018; Schwaneberg, et al., 2016). The Standard European Vector Architecture (SEVA) creates a set of plasmids with different replication systems for screening suitable hosts and genes(Martinez-Garcia, et al., 2015b). In food application, a natural octapeptide was constructed in E. coli by genetic engineering method to verify the taste of fresh peptide(Zhang, et al., 2017), and an enzyme of alkaline Arxula adeninivorans urate oxidase expressed in E. coli reduced the uric acid level of food(R. Zhang, et al., 2019). In addition, the preparation of monoclonal antibodies against aflatoxin B1(Min, et al., 2011) and the production of d-pantothenic acid(B. Zhang, et al., 2019) were both realized in E. coli, in terms of antibody and metabolic engineering, respectively. All of these cases illustrate that E. coli is an excellent host for heterologous expression.
However, as a Gram-negative bacterium, E. coli produces endotoxins that lead to increased downstream production processes and costs, which is another reason for the difficulty in industrial food production in E. coli. Fortunately, the non-pathogenic probiotic E. coli Nissle 1917 (EcN) of serotype O6:K5:H1, without virulence factors, has been discovered to prevent pathogenic bacteria from attacking the intestinal mucosa in the intestine and has a protective and repairing effect on the intestinal mucosal barrier(Jacobi & Malfertheiner, 2011). Based on the properties of EcN, recent studies have proposed a series of clinical ideas for heterologous gene expression using EcN as a favored chassis for disease treatment, including sustained heterologous expression of 3-hydroxybutyric acid for the treatment of colitis(Yan, et al., 2021), heterologous expression of uric enzyme for the treatment of hyperuricemia(He, et al., 2023), and design of near-infrared nano light for controlling gene expression for the treatment of tumors(Zhu, et al., 2023). EcN has been reviewed recently to be well employed in biomedical engineering for the treatment of infectious diseases, metabolic disorders, and inflammatory bowel diseases (IBDs) as well as cancers(Lynch, et al., 2022; Yu, et al., 2020). However, most of the studies on the heterologous expression of EcN have focused on biomedical engineering and neglected the application value of EcN in the food industry. Due to the probiotic and genetically manipulable properties of EcN, a recent study expressed the sulfotransferase domain of human N-deacetylase/N-sulfotransferase-1 (NDST-1) and the catalytic domain of mouse 3-O-sulfotransferase-1 (3-OST-1) in the engineered strain EcN::T7M with fed-batch fermentation, which brought the yield of NST to 0.21 g/L and the yield of heparosan to 0.85 g/L, respectively(Li, et al., 2021). In addition, EcN was used to engineer a probiotic to produce β-carotene in the gut and metabolize it to vitamin A to treat diarrheal diseases(Miller, et al., 2013). Therefore, the use of non-pathogenic EcN to establish recombinant expression of PG is a very safe and effective strategy in food applications.
Milk is an important part of people's daily dietary structure for nutritional acquisition, especially for its amino acid content. Milk consists of a colloidal particle complex of associated proteins and calcium phosphate with an average size of 150–200 nm(Duerasch, et al., 2018). These conjoined colloids are known as casein micelles(Fox, 2003). Casein is by far the most essential and valuable component of milk, and many dairy products derive their textural, organoleptic, and nutritional properties primarily from casein, which has attracted people to use it for thousands of years. Casein consists of its αs1-casein, αs2-casein, β-casein, and κ-casein and inorganic material, which account for approximately 76–86% of milk(Swaisgood, 2003). However, the solubility of casein is less than 10% compared to natural casein in cow's milk, limiting the application scenarios of casein(Lee, et al., 2010). Chemical modification of casein has been expanded to improve the solubility of casein, including succinylation, acetylation, dephosphorylation, and neutralization(Sarah, et al., 2018; Wu, et al., 2020; Yang, et al., 2016). Sodium caseinate is prepared by fermentation, a process in which non-fat milk or Qula is fermented with lactic acid bacteria (LAB). Subsequently, the solid proteins are separated and adjusted to neutrality with NaOH, and finally, citric acid is added to form sodium caseinate(H. N. Liu, et al., 2013). Sodium caseinate is used in meat processing, bakery, ice cream, and other industries to increase the binder, emulsification, and insufficient nutrition of food products(Garcia, et al., 2004; Sudha, et al., 2014; Voronin, et al., 2021). However, there are some negative effects of chemical methods compared to enzymatic modification, such as loss of nutritional value, decreased emulsification and foaming, and the chemical process still contains a potential consumption threat. The modification of casein by enzymatic methods to improve its functional properties and broaden its application scenarios is still at a limited level of research. The development of a relatively cheap, efficient, and mild soluble casein production process without the intervention of exogenous chemicals is urgent to expand the application of casein in the food industry.
In this study, an expression system for high PG yield was produced in probiotic EcN, which promotes the safe production of PG and simplifies the purifying procedure. The functional properties of casein were significantly improved by PG deamidation which improves its usability as a functional ingredient in the food industry.