Mass production of active recombinant Chryseobacterium proteolyticum protein glutaminase in Escherichia coli using a sequential dual expression system and one‐step purification

Abstract

Protein glutaminase (PG) is an enzyme that specifically catalyzes the deamidation of glutamine residues on proteins or peptides, remarkably improving the solubility, emulsification and foaming properties of food proteins and, thereby, conferring great potential in food industry applications. PG is primarily produced from wild strains of Chryseobacterium proteolyticum and the low enzyme production yield restricts large-scale industrial applications. In this context, by evaluating different cleavage site insertions between the pro-region and mature domain of PG as well as different linkers flanking the cleavage site, an E. coli expression and purification protocol has been developed to produce active recombinant PG. To simplify the production workflow, we developed a sequential dual expression system. More than 15 mg of pure and active PG was obtained from 1 L of shaking-flask bacteria culture by one-step nickel affinity chromatography purification. The enzymatic characteristics of the recombinant PG protein were similar to those of native PG. For the deamidation effect of recombinant PG, the deamidation degree (DD) of gliadin reached up to 67% and the solubility increased 84-fold. Thus, this study provides a practical approach to mass producing active PG proteins and investigates its potential applications on food proteins.

Protein-Glutaminase Engineering Based on Isothermal Compressibility Perturbation for Enhanced Modification of Soy Protein Isolate

“…After five rounds of combination iterations of wet experiments, N16M/Q21H/T113E was pronounced as the best-hit triple variant, whose specific activity and catalytic efficiency were 4.28-and 8.12-fold that of the wild type, respectively, which was one of the most excellent performances ever reported to the best of our knowledge (Figure 2D and Figure S2 and Table S3 of the Supporting Information). 18,19 Furthermore, the half-time of N16M/Q21H/T113E was increased by 1.23-fold compared to the wild type (Figure 2E). N16M/N55F/T113E and N16M/N55M/T113E exhibited the highest thermal stability; however, the specific activity increased by only 1.76-and 1.57-fold compared to the wild type.…”

“…4,12,18,20 A twoplasmid system was developed by Lu et al to produce recombinant Pro-PG and human rhinovirus 3C protein to simplify the expression of active PG in E. coli, and the activity of active PG was up to 29 units/mg. 19 Previous studies have also improved the stability, activity, and yield of PG to some extent; however, few studies focused on the modification of PG have been reported. 4,18−21 The PG engineering encountered two major challenges: (1) the lack of efficient directed modification technology to improve the stability, activity, and other traits of PG and (2) an unclear adaptive mechanism of PG under a complex environment (e.g., high shear and high pressure).…”

Enhanced protein glutaminase production from Chryseobacterium proteolyticum combining physico‐chemical mutagenesis and resistance screening and its application to soybean protein isolates

“…established an exogenous expression system in Bacillus glutamicum , and the pro‐PG activities obtained were 0.23 and 26 U mg −1 , respectively. Subsequently, studies have successively established the PG exogenous expression systems of Bacillus licheniformis 10 and Escherichia coli 11 . However, considering the safety of E. coli in food industry, it is not the best choice to serve as the expression platform for PG.…”

“…Subsequently, studies have successively established the PG exogenous expression systems of Bacillus licheniformis 10 and Escherichia coli. 11 However, considering the safety of E. coli in food industry, it is not the best choice to serve as the expression platform for PG. In these expression systems, PG is expressed as a precursor containing signal peptide, and only after cutting off the signal peptide and propeptide can it become a mature PG with biological activity.…”

The characteristics of protein-glutaminase from an isolated Chryseobacterium cucumeris strain and its deamidation application

“…Although Ouyang et al (2021) optimized PG expression in various B. subtilis strains, the maximum PG activity was only 1.10 U/ml. Lu et al (2020) developed an E. coli expression system for the mass production of active PG (15 mg/L). In B. licheniformis CBBD302, GlmU-R-mediated PG expression in active form with a maximum yield of 6.8 U/ml in a 25-L bioreactor ( Niu et al, 2019 ).…”