Purified protein glutaminase from Chryseobacterium proteolyticum enhances the properties of wheat gluten.

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.

A novel method for high level production of protein glutaminase by sfGFP tag in Bacillus subtilis.

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.

Effects of Flammulina velutipes polysaccharide with ice recrystallization inhibition activity on the quality of beef patties during freeze-thaw cycles: An emphasis on water status and distribution.

The antifreeze activity of Flammulina velutipes polysaccharide (FVP) autoclave-extracted with dilute alkaline and effects of FVP on moisture status, size of ice crystals, physical and chemical characteristics of beef patties during repeated freeze-thaw (F-T) cycles were investigated. Results showed that FVP exhibited ice recrystallization inhibition activity and was able to alter the onset freezing/melting temperature of beef patties. 0.01% FVP significantly alleviated (P < 0.05) the decrement in water holding capacity by inhibiting water migration, restraining the mobility of water, and reducing the size of ice crystals of beef patties during the repeated F-T cycles. In addition, FVP could effectively inhibited oxidation reaction and protein aggregation of beef patties with significant decreases in TBARS value, protein turbidity, contents of total sulfhydryl and carbonyl of myofibrillar protein, and an increase in protein solubility during the repeated cycles. These results suggest FVP could be developed to be a promising cryoprotectant in frozen patties.

Effect of protein-glutaminase on the texture, rheology, microstructure and sensory properties of skimmed set-type yoghurt.

The effects of enzymatic deamidation by protein-glutaminase (PG) on the texture, rheology, microstructure, and sensory properties of skimmed set-type yoghurt were studied. The proportion of small-particle size milk protein micelles (10-50 nm) increased significantly from 0 to 99.39% after PG deamidation. Cryo-SEM results revealed that PG-treated yoghurt had a denser and less open 3D structure. PG was effective at inhibiting post-acidification during storage at 4 ℃. The water holding capacity of PG-treated yoghurt (0.12 U·mL-1) increased by more than 15%. The fluidity and viscosity of yoghurt were significantly improved with increasing PG dose. Sensory evaluation revealed that PG (0.06 U·mL-1) significantly improved the smoothness and creaminess of skimmed set-type yoghurt, which corresponded to the pastiness in texture. In summary, PG can effectively address the problems of post-acidification, gel fracture, and flavors change in skimmed set-type yoghurt, providing new applications for PG in the food industry.

Biochemical characteristics and potential application of a thermostable starch branching enzyme from Bacillus licheniformis.

Slowly digestible starch (SDS) has attracted increasing attention for its function of preventing metabolic diseases. Based on transglycosylation, starch branching enzymes (1,4-α-glucan branching enzymes, GBEs, EC 2.4.1.18) can be used to regulate the digestibility of starch. In this study, a GBE gene from Bacillus licheniformis (bl-GBE) was cloned, expressed, purified, and characterized. Sequence analysis and structural modeling showed that bl-GBE belong to the glycoside hydrolase 13 (GH13) family, with which its active site residues were conserved. The bl-GBE was highly active at 80 °C and a pH range of 7.5-9.0, and retained 90% of enzyme activity at 70 °C for 16 h. bl-GBE also showed high substrate specificity (80.88 U/mg) on potato starch. The stability and the changes of the secondary structure of bl-GBE at different temperature were determined by circular dichroism (CD) spectroscopy. The CD data showed a loss of 20% of the enzyme activity at high temperatures (80 °C), due to the decreased content of the α -helix in the secondary structure. Furthermore, potato starch treated with bl-GBE (300 U/g starch) showed remarkable increase in stability, solubility, and significant reduction viscosity. Meanwhile, the slowly digestible starch content of bl-GBE modified potato starch increased by 53.03% compared with native potato starch. Our results demonstrated the potential applications of thermophilic bl-GBE in food industries.

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

Protein-glutaminase (PG), a deamidation enzyme commercially derived from Chryseobacterium proteolyticum, is used to improve the solubility and other functional properties of food proteins. In this study, a new PG-producing strain, Chryseobacterium cucumeris ZYF120413-7, was isolated from soil, and it had a high PG yield and a short culture time. It gave the maximum PG activity with 0.557 U/ml on Cbz-Gln-Gly after 12 h of culture, indicating that it was more suitable for PG production. The enzyme activity recovery and purification fold were 32.95% and 161.95-fold, respectively, with a specific activity of 27.37 U/mg. The PG was a pre-pro-protein with a 16 amino acids putative signal peptide, a pro-PG of 118 amino acids, and a mature PG of 185 amino acids. The amino acid sequence identity of PG from strain ZYF120413-7 was 74 and 45%, respectively, to that of PG from C. proteolyticum 9670T and BH-PG. The optimum reaction pH and temperature of PG was 6 and 60°C, respectively. Enzyme activity was inhibited by Cu2+. The optimum PG substrate was Cbz-Gln-Gly, and the Km and Vmax values were 1.68 mM and 1.41 µM mg protein-1 min-1, respectively. Degree of deamidation (DD) of soy protein isolate (SPI) treated by purified PG was 40.75% within the first 2 h and 52.35% after 18 h. These results demonstrated that the PG from C. cucumeris ZYF120413-7 was a promising protein-deamidating enzyme for improving the functionality of food proteins.

Methionine biosynthesis pathway genes affect curdlan biosynthesis of Agrobacterium sp. CGMCC 11546 via energy regeneration.

Curdlan is a water-insoluble exopolysaccharide produced by Agrobacterium species under nitrogen starvation. The curdlan production in the ΔmdeA, ΔmetA, ΔmetH, and ΔmetZ mutants of methionine biosynthesis pathway of Agrobacterium sp. CGMCC 11546 were significantly impaired. Fermentation profiles of four mutants showed that the consumption of ammonia and sucrose was impaired. Transcriptome analysis of the ΔmetH and ΔmetZ mutants showed that numerous differentially expressed genes involved in the electron transfer chain (ETC) were significantly down-regulated, suggesting that methionine biosynthesis pathway affected the production of energy ATP during the curdlan biosynthesis. Furthermore, metabolomics analysis of the ΔmetH and ΔmetZ mutants showed that ADP and FAD were significantly accumulated, while acetyl-CoA was diminished, suggesting that the impaired curdlan production in the ΔmetH and ΔmetZ mutants might be caused by the insufficient supply of energy ATP. Finally, the addition of both dibasic sodium succinate as a substrate of FAD recycling and methionine significantly restored the curdlan production of four mutants. In conclusion, methionine biosynthesis pathway plays an important role in curdlan biosynthesis in Agrobacterium sp. CGMCC 11546, which affected the sufficient supply of energy ATP from the ETC during the curdlan biosynthesis.

Characterization and rheological properties analysis of the succinoglycan produced by a high-yield mutant of Rhizobium radiobacter ATCC 19358.

Succinoglycan is an industrially important exopolysaccharide biosynthesized by bacteria. In this study, mutant strain 18052 N-11 was obtained from the wild type strain Rhizobium radiobacter ATCC 19358 by NTG mutagenesis. It has a high yield succinoglycan of 32.5 g/L cultured in a 15 L-fementer for 72 h. Succinoglycan SG-A from the wild type strain has two components, and the molecular weights were 1.55 × 107 Da and 1.26 × 106 Da, respectively. While, succinoglycan SG-N from the mutant strain was a homogeneous polysaccharide, and the molecular weight was 1.01 × 107 Da. The molecular weight of both succinoglycan was higher than those reported in literatures. DSC thermogram of SG-A showed a higher endothermic peak than that of SG-N due to the higher crystallinity of SG-A. The dynamic frequency sweep test of SG-A and SG-N showed that the elastic modulus G' and viscosity modulus G" curves intersected at 65 °C, indicating the thermally induced order-disorder conformation. The results of effect of concentrations (2.5-15%) and temperatures (25-75 °C) on apparent viscosity of SG-A and SG-N showed that the succinoglycan solutions exhibited non-Newtonian, shear-thinning behavior. Both SG-A and SG-N showed an excellent emulsification activity. The characterizations and rheological properties make SG-A and SG-N prominent candidates in food, cosmetics, pharmaceutical and petroleum industries.

Glutamine synthetase gene glnA plays a vital role in curdlan biosynthesis of Agrobacterium sp. CGMCC 11546.

Curdlan is a neutral linear exopolysaccharide produced by Agrobacterium spp. under nitrogen-limiting conditions. In this study, we explored the role of glnA in curdlan biosynthesis in Agrobacterium sp. CGMCC 11546. The curdlan production of the ΔglnA strain was impaired, decreasing by 93% compared with that of the wild-type strain after 96 h fermentation. Analysis of fermentation profiles revealed that cell growth and utilization of carbon and nitrogen sources were impaired in the ΔglnA strain. Transcriptome analysis indicated that various of genes involved in curdlan biosynthesis were downregulated after 24 h fermentation in the ΔglnA strain, particularly genes involved in heme synthesis and the electron transport chain, which are essential for energy generation. Metabolomics analysis revealed flavin adenine dinucleotide (FAD) and adenosine diphosphate (ADP) accumulation in the ΔglnA strain, suggesting insufficient energy supply. Furthermore, glnA overexpression led to an 18% increase in the curdlan yield of the ΔglnA mutant compared with that of the wild-type strain after 96 h fermentation. Taken together, the findings demonstrate that glnA plays a vital role in curdlan biosynthesis by supplying ATP via regulating the expression of genes involved in heme synthesis and the electron transport chain.

Characterization and improvement of curdlan produced by a high-yield mutant of Agrobacterium sp. ATCC 31749 based on whole-genome analysis.

Curdlan is a bacterial, water-insoluble, linear homopolysaccharide that has been widely used in the food industry. In this study, genome information of strain CGMCC 11546, a UV-induced high-yield mutant of the model curdlan-producing strain Agrobacterium sp. ATCC 31749, was used to investigate the molecular mechanism of curdlan biosynthesis. The maximum curdlan yield of 47.97 ± 0.57 g/L was obtained from strain CGMCC 11546 by using optimal media containing 60 g/L sucrose, 6 g/L yeast, 2 g/L KH2PO4, 0.4 g/L MgSO4·7H2O, 2 g/L CaCO3, 0.1 g/L FeSO4·7H2O, 0.04 g/L MnSO4, and 0.02 g/L ZnCl2 at 30 °C and 280 rpm after 96 h of fermentation. The gel strength of curdlan was improved by 41 % by knocking out the ß-1,3-glucanase genes exoK and exsH of strain CGMCC 11546. Furthermore, the application of curdlan from the ΔexoK-exsH strain in noodles significantly improved the eating quality of both raw and cooked noodles.

Characterization and optimization of production of bacterial cellulose from strain CGMCC 17276 based on whole-genome analysis.

Bacterial cellulose (BC) has received considerable attention as an environment-friendly, biodegradable nanomaterial. In this study, the strain Komagataeibacter sp. nov. CGMCC 17276, which showed rapid cell growth and high BC-production ability, was isolated and classified into a novel species in the Komagataeibacter genus. Four BC synthase operons were annotated using whole-genome analysis, partially explaining the high BC yield of strain CGMCC 17276. Operons bcs Ⅱ and bcs Ⅲ showed high transcriptional levels under static and agitated culture conditions, indicating their importance in BC synthesis. Of the eight suitable carbon sources identified by whole-genome analysis, the highest BC production was achieved using glycerol as a single carbon source. Finally, waste glycerol was successfully used as an eco-friendly and sustainable strategy for BC production. This study provides valuable insights into the mechanism of BC synthesis, genetic structure of BC-producing strains, and industrialization of BC production using an eco-friendly and low-cost strategy.

Comparison of bacterial nanocellulose produced by different strains under static and agitated culture conditions.

Bacterial nanocellulose (BNC) has many advantages over plant cellulose, which make it widely used in many fields, especially in the food industry. In this study, three strains including BCA263, BCC529, and P1 were selected for characteristics analysis of BNCs under static and agitated culture conditions. The BNCs produced under static culture condition were in the shape of uniform membrane, while BNCs produced under agitated culture were in form of small agglomerates and fragments. BCA263 and BCC529 strains were more suitable for static culture, while P1 strain was more suitable for agitated culture. BNCs produced under static culture condition exhibited higher crystallinity, stronger tensile strength, denser network structure, higher temperature resistance and good flame retardancy; while BNCs produced under agitated culture condition exhibited larger porous and lower crystallinity. Furthermore, BNCs produced under agitated culture condition were more suitable as a stabilizer of coffee milk beverage.

Separation of two microbial transglutaminase isomers from Streptomyces mobaraensis using pH-mediated cation exchange chromatography and their characterization.

Microbial transglutaminase (MTGase) derived from Streptomyces mobaraensis has been widely used in the food, biotechnology and medicine fields. The lot-to-lot consistency and product stability of MTGase must be ensured. The structure and charge variants of MTGase can influence its bioactivity. In this study, MTGase isomers (MTG I1 and MTG I2) were found during the separation of MTGase by pH-mediated cation-exchange chromatography. MTG I1 and MTG I2 had the same molecular weight and N-terminal amino acid sequences, but they showed charge heterogeneity. The affinity of MTG I2 for substrates was higher than that of MTG I1, and the thermal stability and the acid-base tolerance of MTG I1 were significantly higher than that of MTG I2. Therefore, the ratio of MTG I1/MTG I2 was positively correlated with the stability of MTGase. The buffer pH and the ionic strength of the eluent had significant effects on the separation of MTG I1 and MTG I2, and the elution gradient steepness and column load showed little effect on the separation of the MTG I1 and MTG I2 peaks. We built a stable and repeatable separation method for MTG I1 and MTG I2. MTG I1 could transform into MTG I2, but MTG I2 was unable to transform into MTG I1, making the transformation of MTG I1 to MTG I2 was irreversible. When MTG I2 was removed from the MTGase, a portion of the MTG I1 could transform into MTG I2. Therefore, one way to increase the stability of MTGase was to reduce the transformation of MTG I1 to MTG I2.