Optimizing Protein-Glutaminase Expression in Bacillus subtilis

Ouyang, Xiaoying; Liu, Yingjie; Qu, Ruidan; Tian, Mingyi; Yang, Ting; Zhu, Rui; Gao, Hongliang; Jin, Mei; Huang, Jing

doi:10.1007/s00284-021-02404-0

Cited by 17 publications

(14 citation statements)

References 19 publications

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“…The enzyme activity unit was defined as the amount of enzyme required to produce 1 μmol of ammonia per minute. 20 Determination of Apparent Melting Temperatures. The purified enzyme sample (20 μL) was mixed with 5 μL of 100× SYPRO Orange dye in the 96-well plate and centrifuged at 4 °C for 5 min.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…4,12,18−20 Moreover, genetic engineering has been applied to enhance PG expression, such as signal peptide optimization of the protein secretion pathway. 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.…”

Section: ■ Introductionmentioning

confidence: 99%

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

Zheng

Long

Zhang

et al. 2022

J. Agric. Food Chem.

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Protein-glutaminase plays a significant role in future food (e.g., plant-based meat) processing as a result of its ability to improve the solubility, foaming, emulsifying, and gel properties of plant-based proteins. However, poor stability, activity, high pressure, and high shear processing environments hinder its application. Therefore, we developed an application-oriented method isothermal compressibility perturbation engineering strategy to improve enzyme performance by simulating the high-pressure environment. The best variant with remarkable improvement in specific activity and half-time, N16M/Q21H/T113E, exhibited a 4.28-fold increase compared to the wild type in specific activity (117.18 units/mg) and a 1.23-fold increase in half-time (472 min), as one of the highest comprehensive performances ever reported. The solubility of the soy protein isolate deaminated by the N16M/Q21H/T113E mutant was 55.74% higher than that deaminated by the wild type, with a tinier particle size and coarser texture. Overall, this strategy has the potential to improve the functional performance of enzymes under complex food processing conditions.

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Section: ■ Materials and Methodsmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

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

Zheng

Long

Zhang

et al. 2022

J. Agric. Food Chem.

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“…The indophenol method was used to determine the concentration of free ammonia after PG hydrolyzed aminoacyl groups on glutamine residues in proteins ( Ouyang et al, 2021 ; Yin et al, 2021 ). The PG activity was determined using Cbz-Gln-Gly or casein as a substrate, and the detection procedures were according to the method of Ouyang et al (2021) . One unit of the enzyme was defined as the amount that released 1 μmol of ammonia per min under the reaction conditions.…”

Section: Methodsmentioning

confidence: 99%

“…E. coli , Bacillus subtilis , Bacillus licheniformis , and C. glutamicum have been used for PG over-expression ( Zhang et al, 2021 ). 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).…”

Section: Introductionmentioning

confidence: 99%

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

Dai

et al. 2022

Front. Microbiol.

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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.

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“…Bacillus have high secretory capacity and become the main producer of the enzyme in industrial production ( Westers et al, 2004 ). However, the yield and activity of alkaline protease in Bacillus is usually low ( Zhang et al, 2020 ; Ouyang et al, 2021 ; Zhang et al, 2021 ; Espoui et al, 2022 ). To achieve high-efficiency expression of target proteins, researchers had reformed expression elements, such as signal peptides, transcription factors, and molecular chaperones ( Liu et al, 2019 ; Suberu et al, 2019 ; Gong et al, 2020 ; Neef et al, 2021 ; Su et al, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

Genetic engineering for enhanced production of a novel alkaline protease BSP-1 in Bacillus amyloliquefaciens

Jiang

Liu

et al. 2022

Front. Bioeng. Biotechnol.

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Alkaline protease has been widely applied in food, medicine, environmental protection and other industrial fields. However, the current activity and yield of alkaline protease cannot meet the demand. Therefore, it is important to identify new alkaline proteases with high activity. In this study, we cloned a potential alkaline protease gene bsp-1 from a Bacillus subtilis strain isolated in our laboratory. BSP-1 shows the highest sequence similarity to subtilisin NAT (S51909) from B. subtilis natto. Then, we expressed BSP-1 in Bacillus amyloliquefaciens BAX-9 and analyzed the protein expression level under a collection of promoters. The results show that the P43 promoter resulted in the highest transcription level, protein level and enzyme activity. Finally, we obtained a maximum activity of 524.12 U/mL using the P43 promoter after fermentation medium optimization. In conclusion, this study identified an alkaline protease gene bsp-1 from B. subtilis and provided a new method for high-efficiency alkaline protease expression in B. amyloliquefaciens.

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Optimizing Protein-Glutaminase Expression in Bacillus subtilis

Cited by 17 publications

References 19 publications

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

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

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

Genetic engineering for enhanced production of a novel alkaline protease BSP-1 in Bacillus amyloliquefaciens

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