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Walnut kernels, selected as the raw material, underwent hydrolysis with protein-glutaminase (PG) following pulping. This process laid the foundation for producing set-type walnut yogurt, involving steps such as blending, homogenization, sterilization, and fermentation. The study investigated the impact of protein-glutaminase and five distinct starter cultures labeled I~V on the quality of walnut yogurt. This was evaluated by examining the yogurt's fermentation characteristics, physicochemical indicators, and microstructure. Furthermore, walnut yogurt developed under these optimal conditions was compared to traditional milk yogurt to assess its quality and characteristics. The findings revealed a significant improvement in the fermentation characteristics and textural parameters of yogurt fermented by walnut milk following enzymatic hydrolysis compared to non-enzymatic walnut milk. The optimal conditions were identified as 0.2% enzyme concentration and a 40-minute reaction time, with the pH, acidity, hardness, and consistency of walnut yogurt being 4.3, 55°T, 85.30 g, and 583.96 g·s, respectively. Walnut yogurt developed using starter culture I exhibited the highest acidity level, maintaining minimal fluctuation over a 21-day storage period. In contrast, yogurts prepared with alternative starter cultures demonstrated varying degrees of stratification and sedimentation. The yogurt fermented with culture I showed superior viscoelastic properties, presenting a denser gel network. Conversely, yogurts fermented with cultures IV and V displayed reduced apparent viscosity, lower values of G' (storage modulus), and G'' (loss modulus), along with a looser gel structure. Additionally, walnut yogurt produced with starter culture I maintained bacterial counts exceeding 106 CFU/mL throughout the storage duration. Despite walnut yogurt requiring further refinement in aspects such as consistency, water-holding capacity, and whey release rate compared to milk yogurt, the overall impact of PG was positive, enhancing the gelation quality of walnut yogurt. The yogurt prepared with starter culture I (Lactobacillus bulgaricus and Streptococcus thermophilus) was notably improved, receiving favorable overall assessments.

BACKGROUND: Protein glutaminase (PG) is a novel protein modification biotechnology that is increasingly being used in the food industry. However, the current level of fermentation of PG‐producing strains still does not meet the requirements of industrial production. To obtain the mutant strains with high PG production, the atmospheric and room temperature plasma (ARTP) combined with LiCl chemical mutagen were used in mutagenesis of a PG producing Chryseobacterium proteolyticum 1003. RESULTS: A mutant strain (WG15) was successfully obtained based on malonic acid resistance screening after compound mutagenesis of the starting strain C. proteolyticum 1003 using ARTP with LiCl, and it was confirmed to be genetically stable in PG synthesis after 15 generations. The protein glutaminase production of WG15 was 2.91 U mL−1 after optimization of fermentation conditions, which is 48.69% higher than the original strain C. proteolyticum 1003. The PG obtained from fermentation showed good activities in deamidation of soy protein isolate. The solubility and foaming properties of the PG‐treated soy protein isolate were significantly increased by 36.50% and 10.03%, respectively, when PG was added at the amount of 100 U mL−1. In addition, the emulsifying activity and emulsion stability of the treated soy protein isolate were improved by 12.44% and 10.34%, respectively, on the addition of 10 U mL−1 PG. The secondary structure of the soy protein isolate changed after PG treatment, with an increased proportion of glutamate. CONCLUSION: The results of the present study indicate that the PG produced by this mutant strain could improve the functional properties of soybean protein isolate and the C. proteolyticum mutant WG15 has great potential in food industry. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

BACKGROUND: Novel thermo‐reversible hydrogels that undergo gelation in feedback to external stimuli have numerous applications in the food, biomedical, and functional materials fields. Muscle myofibrillar protein (MP) has long been known for thermally irreversible gelation. Once the reversible gelation of MP is achieved, its scope for research and application will expand. RESULTS: The work reported here achieved, for the first time, a thermo‐reversible MP gelation by elaborate deamidation using protein glutaminase (PG). The protein concentration and PG reaction time within windows of 1.0–2.5% and 8 h or 12 h were observed to be vital for creating thermo‐reversible gels. The gel strength increased with protein concentration. The gel displayed a perforated lamellar microstructure, which resulted in a high water‐holding capacity. The rheological results revealed the thermo‐reversibility of the gel was robust for up to five cycles of heating and cooling. The thermally reversible gelation is closely related to the reversible assembly between individual α‐helix and helical coiled coil. Hydrophobic interactions proved to be predominantly involved in the formation and stabilization of the gel network structure. CONCLUSION: This work increases the scope of research into the thermo‐responsive behavior of MP‐based gel. It can foster advances in research into the applications of muscle proteins and into the use of PG as a novel ingredient in the food industry. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

In this study, we successfully obtained a novel source protein glutaminase PG5 with specific activity of 10.4 U/mg, good tolerance and broad substrate profile through big data retrieval. Structural analysis and site-directed mutagenesis revealed that the catalytic pocket of Mature-PG5 contained a large number of aromatic amino acids and hydrophobic amino acids, and that Ser72 greatly affects the properties of the catalytic pocket and the affinity of PG5 for the substrate. In addition, molecular dynamics analysis revealed that the opening and closing between amino acid residues Gly65 and Thr66 with Cys164 at the catalytic cleft could affect substrate binding and product release. In addition, PG5 effectively improved the solubility of fish myofibrillar proteins under low-salt conditions while enhancing their foaming and emulsification properties. This study offers valuable insights into the catalytic mechanism of PG5, which will contribute to its future directed evolution and application in the food industry. [Display omitted] • A novel protein glutaminase PG5 was obtained through big data mining. • Residue Ser72 in catalytic pocket affect the affinity of PG5 toward substrate. • The opening and closing between Gly65 and Thr66 with Cys164 affect substrate hydrolysis. • PG5 enhanced the solubility and functional properties of fish MPs in low-salt condition. [ABSTRACT FROM AUTHOR]

Plant protein-based hydrogels have enormous potential applications, and their great functional properties and sustainability are considered the best alternatives to reduce the environmental burden of animal protein resources. However, heat-induced plant protein-based hydrogels often exhibit a rough and opaque network structure, limiting their application in various scenarios where gel transparency is crucial. In this study, transparent hydrogels were successfully prepared using deamidated soy protein isolate (SPI) modified by protein-glutaminase (PG). Compared with the non-transparent SPI hydrogel, the deamidated SPI hydrogel possessed a soft and intricate chain-like network, exhibiting excellent light transmittance and certain mechanical strength. The results indicated that deamidation treatment unfolded the structure of SPI, shifting the protein conformation from ordered to disordered, and leading to the transformation of large insoluble protein aggregates into small soluble aggregates. In addition, deamidation treatment effectively inhibited the thermal aggregation rate and aggregation counts of SPI. This was attributed to the conversion of glutamine to glutamic acid, which enhanced the electrostatic repulsion between protein molecules. The present work offers a promising approach for preparing highly transparent plant protein-based hydrogels, which are conducive to developing the next generation of plant protein-based functional materials to replace existing petroleum-based materials. [Display omitted] • Transparent soy protein hydrogel is successfully prepared without the addition of any gelling agents. • The deamidation reaction depolymerizes the insoluble soy protein aggregates. • Deamidation treatment inhibits the thermal aggregation of soybean protein. • Deamidated soy protein exhibits a more flexible gel network structure. [ABSTRACT FROM AUTHOR]

Thermo-reversible gel of myofibrillar protein (MP) can be made by tactics of elaborate deamidation using protein-glutaminase (PG), and this work aimed to disclose the link between thermally reversible gelation of MP and the coiled-coil (CC). Enzymatic deamidation fragmented myofibril filaments and triggered structural reassembly to create small-sized aggregates. The coiling and dissociation of CC structure in the myosin tails is the fundamental structural basis of the PG deamidated MP (DMP) in the dynamic evolution of reversible gelation. After specific inhibition of CC assembly by trifluoroethanol (TFE), the thermo-reversible gel ability of DMP was impaired, which confirmed that the dynamic assembly of CC with temperature response played a key role in the thermo-reversible gelation of DMP. The findings may broaden the molecular basis of natural CC reversible gelation and foster advances for the development of new muscle protein products.

Protein glutaminases (PG; EC = 3.5.1.44) are enzymes known for enhancing protein functionality. In this study, we cloned and expressed the gene chryb 3 encoding protein glutaminase PG3, exhibiting 39.4 U/mg specific activity. Mature-PG3 featured a substrate channel surrounded by aromatic and hydrophobic amino acids at positions 38-45 and 78-84, with Val81 playing a pivotal role in substrate affinity. The dynamic opening and closing motions between Gly65, Thr66, and Cys164 at the catalytic cleft greatly influence substrate binding and product release. Redesigning catalytic pocket and cocatalytic region produced combinatorial mutant MT6 showing a 2.69-fold increase in specific activity and a 2.99-fold increase at t 65 °C 1/2 . Furthermore, MT6 boosted fish myofibrillar protein (MP) solubility without NaCl. Key residues such as Thr3, Asn54, Val81, Tyr82, Asn107, and Ser108 were vital for PG3-myosin interaction, particularly Asn54 and Asn107. This study sheds light on the catalytic mechanism of PG3 and guided its rational engineering and utilization in low-salt fish MP product production.

Protein glutaminase (PG; EC 3.5.1.44) is a class of food-grade enzyme with the potential to significantly improve protein functionality. However, its low catalytic activity and stability greatly hindered industrial application. In this study, we employed structural-based engineering and computational-aided design strategies to target the engineering of protein glutaminase PG5, which led to the development of a combinatorial mutant, MT8, exhibiting a specific activity of 31.1 U/mg and a half-life of 216.2 min at 55 °C. The results indicated that the flexible region in MT8 shifted from the C-terminus to the N-terminus, with increased N-terminal flexibility positively correlating with its catalytic activity. Additionally, MT8 notably boosted fish myofibrillar proteins (MPs) solubility under the absence of NaCl conditions and enhanced their foaming and emulsifying properties. Key residues like Asp31, Ser72, Asn121, Asp471, and Glu485 were crucial for maintaining PG5-myosin interaction, with Ser72 and Asn121 making significant energy contributions. [Display omitted] • Structure-based and computational-aided strategies were utilized for PG5 rational design. • Catalytic activity and t 1/2 55°C of combinatorial mutant MT8 improved by 3-fold and 3.57-fold. • The solubility of deamidated myofibrillar proteins (DMPs) by MT8 were enhanced. • Key amino acids involved in the interaction at the PG5-myosin complex were identified. [ABSTRACT FROM AUTHOR]

Protein glutaminase (PG; EC 3.5.1.44) is a novel deamidase that helps to improve functional properties of food proteins. Currently, the highest activated PG enzyme activity was 26 U/mg when recombinantly expressed via the twin-arginine translocation (Tat) pathway in Corynebacterium glutamicum. In this study, superfolder green fluorescent protein (sfGFP) was used to replace traditional signal peptides to facilitate efficient heterologous expression and secretion of Propeptide-Protein glutaminase (PP) in Bacillus subtilis. The fusion protein, sfGFP-PP, was secreted from 12 h of fermentation and reached its highest extracellular expression at 28 h, with a secretion efficiency of about 93 %. Moreover, when fusing sfGFP with PP at the N-terminus, it significantly enhances PG expression up to 26 U/mL by approximately 2.2-fold compared to conventional signal-peptides- guided PP with 11.9 U/mL. Finally, the PG enzyme activity increased from 26 U/mL to 36.9 U/mL after promoter and RBS optimization. This strategy not only provides a new approach to increase PG production as well as extracellular secretion but also offers sfGFP as an effective N-terminal tag for increased secreted production of difficult-to-express proteins. [ABSTRACT FROM AUTHOR]

Protein glutaminase (PG), originating from Chryseobacterium proteolyticum, can catalyze the deamidation of glutamine residues in plant proteins into glutamic acid, thus enhancing its functional properties. However, the low yield of PG limits its industrial production. In this study, the yield of PG in C. proteolyticum TM1040 increased by 121 %, up to 7.30 U/mL in a 15 L fermenter after medium optimization. Subsequently, purified PG was obtained by cation exchange chromatography (CEX) coupled with hydrophobic interaction chromatography (HIC). The degree of deamidation (DD) of wheat gluten after purified PG deamidation was 87.11 %, which is superior to chemical deamidation in safety and DD. The emulsifying and foaming properties of deamidated wheat gluten were 2.67 and 18.86 times higher, and the water- and oil-holding properties were 4.23 and 18.77 times higher, respectively. The deamidated wheat gluten with enhanced functional properties was used to improve the flavor and texture in baking cakes., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier Ltd.)

Deamidation is a promising tool to improve solubility and other functional properties of food proteins. One possibility of protein deamidation is the use of a protein glutaminase (PG; EC 3.5.1.44), an enzyme that catalyzes the deamidation of internal glutamine residues in proteins to glutamic acid residues. The PG from Chryseobacterium proteolyticum is the only one described in literature to date and is commercially available (Amano Enzyme Inc., Japan; PGA). Based on a similarity search, we discovered a predicted, uncharacterized protein from Bacteroides helcogenes and this protein was verified as a PG. After recombinant production and purification, the novel PG (BH-PG) was biochemically characterized and compared with PGA. Some advantageous characteristics for potential application of BH-PG compared with PGA were the higher temperature stability (residual activity after 24 h of incubation at 50 °C was 87% for BH-PG and 2% for PGA), an optimum pH value at acidic conditions (pH 5.5) and less product inhibition by ammonia that is released during the deamidation of proteins (residual activity after adding 40 mM ammonia was 77% for BH-PG and 27% for PGA). Finally, the applicability of BH-PG and PGA was compared by gluten deamidation experiments. Consequently, the final solubility of the nearly insoluble food protein gluten was 94% after BH-PG treatment, whereas the solubility was around 66% when using PGA. [ABSTRACT FROM AUTHOR]

Background: Protein glutaminase specifically deamidates glutamine residue in protein and therefore significantly improves protein solubility and colloidal stability of protein solution. In order to improve its preparation efficiency, we exploited the possibility for its secretory expression mediated by twin-arginine translocation (Tat) pathway in Bacillus licheniformis. Results: The B. licheniformis genome-wide twin-arginine signal peptides were analyzed. Of which, eleven candidates were cloned for construction of expression vectors to mediate the expression of Chryseobacterium proteolyticum protein glutaminase (PGA). The signal peptide of GlmU was confirmed that it significantly mediated PGA secretion into media with the maximum activity of 0.16 U/ml in Bacillus subtilis WB600. A mutant GlmU-R, being replaced the third residue aspartic acid of GlmU twin-arginine signal peptide with arginine by site-directed mutagenesis, mediated the improved secretion of PGA with about 40% increased (0.23 U/ml). In B. licheniformis CBBD302, GlmU-R mediated PGA expression in active form with the maximum yield of 6.8 U/ml in a 25-l bioreactor. Conclusions: PGA can be produced and secreted efficiently in active form via Tat pathway of B. licheniformis, an alternative expression system for the industrial-scale production of PGA. [ABSTRACT FROM AUTHOR]

Thermo-reversible gel of myofibrillar protein (MP) can be made by tactics of elaborate deamidation using protein-glutaminase (PG), and this work aimed to disclose the link between thermally reversible gelation of MP and the coiled-coil (CC). Enzymatic deamidation fragmented myofibril filaments and triggered structural reassembly to create small-sized aggregates. The coiling and dissociation of CC structure in the myosin tails is the fundamental structural basis of the PG deamidated MP (DMP) in the dynamic evolution of reversible gelation. After specific inhibition of CC assembly by trifluoroethanol (TFE), the thermo-reversible gel ability of DMP was impaired, which confirmed that the dynamic assembly of CC with temperature response played a key role in the thermo-reversible gelation of DMP. The findings may broaden the molecular basis of natural CC reversible gelation and foster advances for the development of new muscle protein products., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

In the present study, effect of deamidation by protein glutaminase (PG; EC 3.5.1.44) on structure of wheat gluten (WG) and its possible to construct high internal phase emulsions (HIPEs) system were investigated. The deamidation reaction was performed for different durations (0–48 h). The deamidation degree (DD, 0–70.5%) of WG was increased with an increase of reaction time (0–48 h). After the specific deamidation by PG, the solubility of WG was significantly improved at neutral pH while maintaining its molecular weight. Unlike native WG, deamidated WG (DWG) had the ability to form stable HIPEs. Moreover, HIPEs prepared using DWGs with a moderate DD (28.7–40.5%) exhibited remarkable characteristics in terms of their network structure, rheological behavior and friction coefficient. This study demonstrated the application of PG-modified WG for HIPE preparation and characterized their structural and functional relationship, providing a promising approach for the production of stabilized emulsions in food systems. • Deamidation by PG improved solubility and emulsifying ability of wheat gluten. • Unlike native wheat gluten, HIPEs could be stabilized by deamidated wheat gluten. • Deamidation degree was critical to rheological and tribological behaviors. [ABSTRACT FROM AUTHOR]

• Chicken breast myofibrillar protein (CMP) treated by protein-glutaminase (PG). • PG tailored CMP net charge by deamidating glutamine residue to glutamic acid. • Enhanced net charge led to the dissociation of myofibril and release of myosin. • Viscosity increased with PG deamidation for 8 h, but it decreased for16 h. • PG can boost the solubility of CMP in water and tune the colloidal state. Inspired by the salt-in effect, the potential use of protein-glutaminase (PG) to increase the intrinsic charges of chicken breast myofibrillar proteins (CMPs) for enhanced water solubility was tested. The degree of deamidation (DD) and solubility of CMPs increased with PG reaction time. Over 60% of CMPs were soluble in water under a DD of 6.5% due to specific conversion of glutamine to glutamic acid. PG deamidation could remarkably increase the net charge of CMPs with a merit in maintaining most of the amino acid and protein subunit compositions. Such a high electrostatic repulsion exerted a transformation of β-sheet into α-helix, unfolded the structure to expose hydrophobic residues, and allowed the dissociation of myofibril and release of subunits (myosin, actin or their oligomers), leading to a stable colloidal state. This work may foster the engineering advances of protein micro-modification in the tailor manufacture of muscle protein-based beverages. [ABSTRACT FROM AUTHOR]

The effect of enzymatic deamidation by protein-glutaminase (PG) on protein solubility and flavor binding potential of soymilk was studied. Treatment of soymilk with PG for 2 h (temperature of 44 °C and enzyme:substrate ratio (E/S) of 40 U/g protein) resulted in high degree of protein deamidation (66.4% DD) and relatively low degree of protein hydrolysis (4.25% DH). Deamidated (DSM) and control soymilks (CSM) did not differ with respect to aroma, but differed in taste characteristics by sensory evaluation. Protein solubility in DSM was enhanced at weakly acidic conditions (pH 5.0), but did not differ from non-deamidated soymilk at pH values of 3.0 and 7.0. Odor detection thresholds for the flavor compounds vanillin and maltol were approximately 5 and 3 fold lower, respectively, in DSM than in CSM. Dose-response curves (Fechner's law plots and n exponents from Stevens's power law) further demonstrated that DSM had a lower flavor binding potential than CSM. PG deamidation has the potential to reduce flavor binding problems encountered in high protein-containing foods and beverages. Practical Application: The findings of this study can help lead to the development of technology to produce protein-containing foods with improved functional properties, especially protein solubility, and potentially decreased flavor fade problems associated with flavor-protein interactions, especially with carbonyl containing flavor compounds. [ABSTRACT FROM AUTHOR]

The protein glutaminase (PG) secreted by the Gram-negative bacterium Chryseobacterium proteolyticum can deamidate glutaminyl residues in several substrate proteins, including insoluble wheat glutens. This enzyme therefore has potential application in the food industry. We assessed the possibility to produce PG containing a pro-domain in Corynebacterium glutamicum which we have successfully used for production of several kinds of proteins at industrial-scale. When it was targeted to the general protein secretion pathway (Sec) via its own signal sequence, the protein glutaminase was not secreted in this strain. In contrast, we showed that pro-PG could be efficiently produced using the recently discovered twin-arginine translocation (Tat) pathway when the typical Sec-dependent signal peptide was replaced by a Tat-dependent signal sequence from various bacteria. The accumulation of pro-PG in C. glutamicum ATCC13869 reached 183 mg/l, and the pro-PG was converted to an active form as the native one by SAM-P45, a subtilisin-like serine protease derived from Streptomyces albogriseolus. The successful secretion of PG via this approach confirms that the Tat pathway of C. glutamicum is an efficient alternative for the industrial-scale production of proteins that are not efficiently secreted by other systems. [ABSTRACT FROM AUTHOR]