TGase Production Research Articles

LexA, an SOS response repressor, activates TGase synthesis in Streptomyces mobaraensis

Transglutaminase (EC 2.3.2.13, TGase), an enzyme that catalyzes the formation of covalent cross-links between protein or peptide molecules, plays a critical role in commercial food processing, medicine, and textiles. TGase from Streptomyces is the sole commercial enzyme preparation for cross-linking proteins. In this study, we revealed that the SOS response repressor protein LexA in Streptomyces mobaraensis not only triggers morphological development but also enhances TGase synthesis. The absence of lexA significantly diminished TGase production and sporulation. Although LexA does not bind directly to the promoter region of the TGase gene, it indirectly stimulates transcription of the tga gene, which encodes TGase. Furthermore, LexA directly enhances the expression of genes associated with protein synthesis and transcription factors, thus favorably influencing TGase synthesis at both the transcriptional and posttranscriptional levels. Moreover, LexA activates four crucial genes involved in morphological differentiation, promoting spore maturation. Overall, our findings suggest that LexA plays a dual role as a master regulator of the SOS response and a significant contributor to TGase regulation and certain aspects of secondary metabolism, offering insights into the cellular functions of LexA and facilitating the strategic engineering of TGase overproducers.

Identification of multiple regulatory genes involved in TGase production in Streptomyces mobaraensis DSM 40587

Microbial transglutaminase (TGase) is a protein that is secreted in a mature form and finds wide applications in meat products, tissue scaffold crosslinking, and textile engineering. Streptomyces mobaraensis is the only licensed producer of TGase. However, increasing the production of TGase using metabolic engineering and heterologous expression approaches has encountered challenges in meeting industrial demands. Therefore, it is necessary to identify the regulatory networks involved in TGase biosynthesis to establish a stable and highly efficient TGase cell factory. In this study, we employed a DNA-affinity capture assay and mass spectrometry analysis to discover several transcription factors. Among the candidates, eight were selected and found to impact TGase biosynthesis. Notably, SMDS_4150, an AdpA-family regulator, exhibited a significant influence and was hence named AdpASm. Through electrophoretic mobility shift assays, we determined that AdpASm regulates TGase biosynthesis by directly repressing the transcription of tg and indirectly inhibiting the transcription of SMDS_3961. The latter gene encodes a LytR-family positive regulator of TGase biosynthesis. Additionally, AdpASm exhibited negative regulation of its own transcription. To further enhance TGase production, we combined the overexpression of SMDS_3961 with the repression of SMDS_4150, resulting in a remarkable improvement in TGase titer from 28.67 to 52.0 U/mL, representing an 81.37% increase. This study establishes AdpA as a versatile regulator involved in coordinating enzyme biosynthesis in Streptomyces species. Furthermore, we elucidated a cascaded regulatory network governing TGase production.

Improved Productivity of Streptomyces mobaraensis Transglutaminase by Regulating Zymogen Activation.

Streptomyces mobaraensis transglutaminase (TGase) is extracellularly expressed as a zymogen and then activated by TGase-activating protease (TAP). In this study, we reported the strategy for improving TGase production via the regulation of TAP activity in S. mobaraensis. First, we analyzed the effects of three inorganic nitrogen sources on TGase production. With 30 mM nitrogen content, the time to the peak of TGase activity induced by (NH4)2SO4 or NH4Cl was 72 h, 12 h earlier than that of the fermentation without adding NH4 +. SDS-PAGE analysis indicated that NH4 + accelerated the TGase activation in S. mobaraensis. Then, we examined the effect of NH4 + on TAP biosynthesis using a TGase-deficient S. mobaraensis strain. It showed that NH4 + enhanced the TAP activity at the early stage of the fermentation, which was dependent on the concentration and time of NH4 + addition. Last, the yield and productivity of S. mobaraensis TGase were increased by 1.18-fold and 2.1-fold, respectively, when optimal NH4 + addition (60 mM and 12 h) was used. The fermentation period was shortened from 84 to 48 h. The NH4 + addition also increased the storage stability of crude enzyme at room temperature. These findings will benefit the TGase production and its activation mechanism in S. mobaraensis.

Transglutaminase from UV Mutated Bacillus cereus NRC215: Production, Purification, and Characterization

Transglutaminase (EC 2.3.2.13, TGase) recorded the highest activity (0.101 U/ml) in bacterial isolate NRC215. 16S rRNA sequencing revealed that NRC215 was identified as Bacillus cereus NRC215 under accession number MT229271 in the NCBI database. UV irradiation was employed to improve TGase production. Five rifampin (RIF) resistant mutants were only isolated from UV-treated Bacillus cereus NRC215 for three minutes. The best mutant, BCrif5, exhibiting induced rifampin resistance, gave TGase with higher activity (0.148 U/ml). The ISSR PCR technique was employed to detect these new rearrangements resulting from UV mutagenesis between the wild-type strain and its mutants. Moreover, TGase has been purified by three-step procedures resulting in a recovery of 28 and 34.63% for wild and BCrif5 strains, respectively. The optimal purified TGase activity was exhibited at pH 7 for wild strain while the mutant BCrif5 at pH 5.0 and 40 °C for both wild and BCrif5 strains. Bacillus cereus NRC215 TGase was activated by Ba+2 (102.50 and 107.06%), while it was inhibited by Cu+2 (30% and 22.35%) for wild and BCrif5 strains, respectively. It could be concluded that Bacillus cereus NRC215 is a promising strain for TGase production, which is beneficial as a food additive.

Enhanced Production of Transglutaminase in Streptomyces mobaraensis through Random Mutagenesis and Site-Directed Genetic Modification.

Streptomyces transglutaminase (TGase) is widely used to improve food texture properties. In this study, random mutagenesis and site-directed genetic modification were used to improve the production of TGase in Streptomyces mobaraensis. First, S. mobaraensis DSM40587 (smWT) was subjected to atmospheric and room-temperature plasma mutagenesis, and then a mutant (smY2019) with a 5.5-fold increase in TGase yield was screened from approximately 3000 × 25 (round) mutants. Compared to smWT, smY2019 exhibits a 3.2-fold higher TGase mRNA level and two site mutations within the -10 region of the TGase promoter. The recombinant expression analysis in the TGase-deficient S. mobaraensis suggests that the mutated TGase promoter is more robust than the wild-type one. Finally, we integrated two additional TGase expression cassettes into the smY2019 genome, yielding the recombinant strain smY2019-3C with a 103% increase in TGase production compared to smY2019. The smY2019-3C strain with 40 U/mL of TGase yield could be a suitable candidate for the industrial production of TGase.

Utilization of agro-industrial waste for production of Transglutaminase from Streptomyces mobaraensis

This work studied the production of Transglutaminase (TGase) using wheat bran as carbon source. The medium components and culture conditions were optimized by statistical Box-Behnken response surface methodology. The release of active Transglutaminase was enhanced by adding (i) protease to remove pro-region to make inactive enzyme to active form, (ii) Cetyl trimethyl ammonium bromide (CTAB) which facilitated more secretion. Under finally optimized conditions viz. 5 g wheat bran, protease: 39.14 U, magnesium chloride (MgCl2): 0.10 M, CTAB: 0.08% and inoculation size: 2% led to 4-fold (12.949 ± 0.061 IU/g) increased TGase production over that of un-optimized conditions. The application of TGase was shown to be useful in effective casein cross-linking.

Comparative proteome analysis of Streptomyces mobaraensis under MgCl2 stress shows proteins modulating differentiation and transglutaminase biosynthesis

Much effort has been devoted to studying the production of Streptomyces transglutaminase (TGase). However, more exploration into the mechanism of TGase biosynthesis is necessary to enhance its production further. The effect of excessive metal stress on Streptomyces mobaraensis's TGase activity, growth rate, and mycelium differentiation were evaluated. To elucidate the regulatory mechanism of TGase production and cell differentiations, a proteomic analysis and qRT-PCR of S. mobaraensis was performed. This study showed that the TGase biosynthesis was enhanced while the cell growth was inhibited under MgCl2 stress at the earlier stage of incubation. Furthermore, MgCl2 stress resulted in early cell differentiation compared to the control group. The proteomic analysis indicated that both the nucleotide metabolism and primary metabolism were repressed at the onset of TGase production, explaining the observed decrease in cell growth rate. Several enriched enzymes in the nitrogen metabolic pathways confirmed that the metabolic fluxes for the syntheses of glycine and serine were increased. Furthermore, some stress or stress-related proteins were expressed at a low level in the strain cultivated in normal medium but were highly expressed at the onset of TGase production.

Enhancing transglutaminase production of Streptomyces mobaraensis by iterative mutagenesis breeding with atmospheric and room-temperature plasma (ARTP)

ObjectivesTo improve the fermentation production of transglutaminase (TGase) from Streptomyces mobaraensis for applications in the food industry, the atmospheric and room-temperature plasma (ARTP) mutagenesis was applied to breed S. mobaraensis mutants with increased TGase production.ResultsAfter eight rounds of iterative ARTP mutagenesis, four genetically stable mutants, Sm5-V1, Sm6-V13, Sm2-V10, and Sm7-V12, were identified, which showed increased TGase production by 27, 24, 24, and 19%, respectively. The best mutant Sm5-V1 exhibited a maximum TGase activity of 5.85 U/mL during flask fermentation. Compared to the wild-type strain, the transcription levels of the zymogen TGase genes in the mutants increased significantly as indicated by quantitative real-time PCR, while the gene nucleotide sequences of the mutants did not change at all. It was shown that the overexpression of TGase zymogen gene in the mutants contributes to the increase in TGase production.ConclusionsARTP is a potentially efficient tool for microbial mutation breeding to bring some significant changes required for the industrial applications.

Improving the active expression of transglutaminase in Streptomyces lividans by promoter engineering and codon optimization.

BackgroundTransglutaminases (TGase), which are synthesized as a zymogen (pro-TGase) in Streptomyces sp., are important enzymes in the food industry. Because this pro-peptide is essential for the correct folding of Streptomyces TGase, TGase is usually expressed in an inactive pro-TGase form, which is then converted to active TGase by the addition of activating proteases in vitro. In this study, Streptomyces hygroscopicus TGase was actively produced by Streptomyces lividans through promoter engineering and codon optimization.ResultsA gene fragment (tg1, 2.6 kb) that encoded the pro-TGase and its endogenous promoter region, signal peptide and terminator was amplified from S. hygroscopicus WSH03-13 and cloned into plasmid pIJ86, which resulted in pIJ86/tg1. After fermentation for 2 days, S. lividans TK24 that harbored pIJ86/tg1 produced 1.8 U/mL of TGase, and a clear TGase band (38 kDa) was detected in the culture supernatant. These results indicated that the pro-TGase was successfully expressed and correctly processed into active TGase in S. lividans TK24 by using the TGase promoter. Based on deletion analysis, the complete sequence of the TGase promoter is restricted to the region from −693 to −48. We also identified a negative element (−198 to −148) in the TGase promoter, and the deletion of this element increased the TGase production by 81.3 %, in contrast to the method by which S. lividans expresses pIJ86/tg1. Combining the deletion of the negative element of the promoter and optimization of the gene codons, the yield and productivity of TGase reached 5.73 U/mL and 0.14 U/mL/h in the recombinant S. lividans, respectively.ConclusionsWe constructed an active TGase-producing strain that had a high yield and productivity, and the optimized TGase promoter could be a good candidate promoter for the expression of other proteins in Streptomyces.

Improvement of transglutaminase production by extending differentiation phase of Streptomyces hygroscopicus: mechanism and application

Streptomyces transglutaminase (TGase) is an important industrial enzyme that catalyzes cross-linking of proteins. It is secreted as a zymogene and then is activated by proteases under physiological conditions. Although the activation process of TGase has been well investigated, the physiological function of TGase in Streptomyces has not been revealed. In this study, physiological function of TGase from Streptomyces hygroscopicus was found to be involved in differentiation by construction of a TGase gene interruption mutation strain (Δtg). The mutant Δtg showed an absence of differentiation compared with the parent strain. Furthermore, the production of TGase was found to be increased with the extending growth arrest phase of mycelium in submerged cultures. Thus, to enhance yield of TGase, the mycelium differentiation of Streptomyces was regulated via low temperature stress in a 3-L stirred-tank fermenter. The production of TGase increased by 39% through extending the growth arrest phase for 4h. This study found that TGase is involved in Streptomyces differentiation and proposed an approach to improve TGase production by regulation of mycelium differentiation in submerged cultures.

Enhancement of transglutaminase production in Streptomyces mobaraensis DSM 40587 by non-nutritional stress conditions: Effects of heat shock, alcohols, and salt treatments

Stress-mediated bioprocess is a strategy designed to enhance biological target productivity. In this study, an attempt was made to enhance transglutaminase (TGase) production by Streptomyces mobaraensis by using different stress conditions including heat shock, alcohols and salt stress. Results showed that the effects of stress on TGase production depended on the type applied. For heat shock, TGase production (1.32 U/ml) was recorded maximum in the culture treated at 48 h post inoculation in water bath at 60 °C for 1 min. For alcohols treatment, the maximum activity of TGase (1.77 and 1.75 U/ml) was obtained when 3% methanol was added into the medium at 0 or 24 h of fermentation. However, a 3.5-fold increased production of TGase (3.8 U/ml) was observed in the medium supplemented with 0.2 mol/l MgCl2 compared with the basic medium at the beginning of fermentation. In conclusion, TGase production from S. mobaraensis was improved by heat shock, methanol and salt stress treatments, MgCl2 stress was the most effective.

Enhancement of transglutaminase production in Streptomyces mobaraensis as achieved by treatment with excessive MgCl2

In this study, we first tested the capacity for eight different salts as stress-mediated bioprocesses in the production of transglutaminase (TGase). A significant effect on the cell growth and TGase production was obtained with the highest yield of TGase being observed at 96 h of incubation (4.3 U/ml) when the basic medium was supplemented 0.10 M MgCl(2), as opposed to that observed with the basic medium control (2.1 U/ml at 120 h). Data from Western blot assays showed that transformation of pro-TGase to its mature enzyme occurred more rapidly in MgCl(2) medium. Furthermore, total protease, metalloprotease, and serine protease were also synthesized at a faster rate in the medium containing MgCl(2). The results demonstrate that MgCl(2) enhanced the production of key proteases involved in the activation of TGase biosynthesis. To explore the mechanism, viability assay was performed. The results show that MgCl(2) induced the mycelia differentiation, decreased cell growth rate, and stimulated cell death. We argue that TGase production was promoted by the stimulation of mycelium differentiation induced by MgCl(2) stress.

Transglutaminase를 생산하는 Streptomyces platensis의 분자생물학적인 연구를 위한 접합 전달법 확립

식품단백질의 물성과 기능성을 개선시켜 산업적인 가치가 매우 높은 TGase를 생산하는 S. platensis YK-2의 분자 유전학적인 연구를 위해 형질전환방법을 확립하였으며 본 연구를 위해 도입된 방법은 대장균을 plasmid DNA의 공여체로, S. platensis의 포자를 수용체로 사용하는 접합전달법이다. S. platensis를 위한 최적 접합전달조건은 50 mM의 <TEX>$MgCl_2$</TEX>를 첨가한 MS배지에 열처리를 하지 않은 포자와 <TEX>$5{\times}10^7$</TEX>의 plasmid DNA 공여체인 E. coli를 사용하는 것이다. 또 본 연구를 통해 얻어진 접합전달체의 attB site에 대한 분석을 통해 S. platensis chromosome의 pirin 상동체를 코드하는 ORF내에 attB site가 단일위치로 존재하고 있으며 이미 밝혀진 다른 방선균유래 attB site의 염기서열에 대해 77.8%~96.3%의 상동성을 나타냈다. Streptomyces platensis YK-2, newly isolated from forest soil, produces transglutaminase (TGase), which catalyses an acyl transfer reaction between the primary grade amine and protein or <TEX>$\gamma$</TEX>-carboxyamide group of peptide bound glutamine residues. For a molecular genetic study of S. platensis, an effective transformation method was established by using a conjugal transfer of DNA from Escherichia coli to spores of actinomycetes. The highest transconjugation frequency of S. platensis was obtained on an MS medium containing 50 mM <TEX>$MgCl_2$</TEX>, using <TEX>$5{\times}10^7\;E$</TEX>. coli as a DNA donor and <TEX>$1{\times}10^8$</TEX> spores without heat treatment as a host. We also identified that S. platensis contains a single attB site within an ORF encoding a pirin-homolog, and that its attB site sequence shows high homology to that of S. logisporoflavus. In addition, it was confirmed by phenotypic analyses of exconjugants that the introduction of heterologous DNA into the attB site of the S. platensis chromosome does not affect its morphological differentiation and TGase production.

Effects of oxygen volumetric mass transfer coefficient on transglutaminase production by Bacillus circulans BL32

The effects of oxygenation in cultures of Bacillus circulans BL32 on transglutaminase (TGase) production and cell sporulation were studied by varying the agitation speed and the volume of aeration. Kinetics of cultivations has been studied in batch systems using a 2 L bioreactor, and the efficiency of agitation and aeration was evaluated through the oxygen volumetric mass transfer coefficient (kLa). It was adopted a two-stage aeration rate control strategy: first stage to induce biomass formation, followed by a second stage, in which cell sporulation was stimulated. A correlation of TGase production, spores formation, and oxygen concentration was established. Under the best conditions (500 rpm; 2 vvm air flow, followed by no air supply during stationary phase; kLa of 33.7 h−1), TGase production reached a volumetric production of 589 U/L after 50 h of cultivation and the enzyme yield was 906 U/g cells. These values are 61% higher than that obtained in shaker cultures and TGase productivity increased 82%, when kLa varied from 4.4 to 33.7 h−1. The maximal cell concentration increased four times in relation to shaker cultures and the cultivation time for the highest TGase activity was reduced from 192 h to just 50 h. These results show the importance of bioprocess design for the production of microbial TGase, especially concerning the oxygen supply of cultures and the induction of cell sporulation.

Environmental Effects on Transglutaminase Production and Cell Sporulation in Submerged Cultivation of Bacillus circulans

In this research, the effects of pH, temperature, and oxygen on growth kinetics of a newly isolated strain of Bacillus circulans from the Amazon and their correlations with transglutaminase (TGase) production and cell sporulation were investigated. Statistical experimental methods were used to optimize these parameters, while induction of sporulation was achieved by oxygen culture control. Full factorial composite experimental design and response surface methodology were experimentally tested. The model showed that temperature has a positive and significant effect on TGase production (P < 0.05) while pH and temperature, associated with anoxic conditions, have a marked effect on cell sporulation which is consistently linked with TGase production. The contour plot of results showed that the best culture conditions for TGase production of B. circulans were 30 degrees C, initial pH 8.5, and the highest production was obtained in late-stationary culture phase with maximal specific enzyme activity of 655 U g(-1) of cells (0.37 U/mL). A correlation between enzyme production and cell sporulation, as mediated by oxygen culture conditions, was also demonstrated and, although demonstrated only for B. subtilis, it corroborates the molecular mechanisms involved in this process. It can be suggested that B. circulans BL32 is a strong biological system for the industrial production of TGases.

Solid state bioreactor production of transglutaminase by Amazonian Bacillus circulans BL32 strain

In this work, we investigated the production of transglutaminase (TGase) by an Amazonian isolated strain of Bacillus circulans by solid-state cultivation (SSC). Several agro-industrial residues, such as untreated corn grits, milled brewers rice, industrial fibrous soy residue, soy hull, and malt bagasse, were used as substrates for microbial growth and enzyme production. Growth on industrial fibrous soy residue, which is rich in protein and hemicellulose, produced the highest TGase activity (0.74 U g(-1) of dried substrate after 48 h of incubation). A 2(3) central composite design was applied to determine the optimal conditions of aeration, cultivation temperature and inoculum cell concentration to TGase production. The best culture conditions were determined as being 0.6 L air min(-1), 33 degrees C and 10 log (10) CFU g(-1) of dried substrate, respectively. Under the proposed optimized conditions, the model predicted an enzyme production of 1.16 U g(-1) of dried substrate, closely matching the experimental activity of 1.25 U g(-1). Results presented in this work point to the use of this newly isolated B. circulans strain as a potential alternative of microbial source for TGase production by SSC, using inexpensive culture media.

Production of transglutaminase by Streptomyces isolates in solid-state fermentation.

To screen Streptomyces isolates for transglutaminase (TGase) production in solid-state fermentation (SSF) on various substrates. Streptomyces mobaraensis NRRL B-3729, Streptomyces paucisporogenes ATCC 12596 and Streptomyces platensis NRRL 2364 strains were screened for extracellular TGase production in SSF on different substrates. High-protein-content beans, peas and lentils proved to be the best substrates. Good TGase production was obtained on liver kidney beans and green mung beans in a 4- to 6-day SSF. Temperature optima of the enzymes varied between 45 to 50 degrees C. Molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) indicated similar size ( approximately 37 kDa) for all three enzymes. TGase was the dominating protein band on SDS PAGE for two Streptomyces strains in SSF extracts. Other enzymes were present in smaller quantities. Streptomyces mobaraensis NRRL B-3729, S. paucisporogenes ATCC 12596 and S. platensis NRRL 2364 strains were successfully propagated under SSF conditions on crushed/milled liver kidney bean and green mung bean to obtain good level of TGase. Owing to much reduced production cost and direct applicability, SSF TGase without downstream processing (cheap in situ enzyme, crude enzyme) may be an excellent candidate for some nonfood applications.

Optimization of microbial transglutaminase production using experimental designs

In prokaryotes, transglutaminase (TGase) has been found only in actinomycetes from the genus Streptoverticillium. The role of this TGase, as well as the mechanism regulating the enzyme expression, are still unknown. In order to improve TGase production by Streptoverticillium cinnamoneum CBS 683.68 and simultaneously elucidate the relationship between growth and TGase activity, we decided to study these two responses using different designs of statistical analysis. Among the five factors tested, casein, glycerol, peptones, yeast extract and oligoelements, only oligoelements were found to have no effect either on growth or on TGase production in a complete factorial design. The two factors casein and glycerol were found to have a highly significant effect on both dry weights and TGase activity in a Box-Behnken design used to improve the model. Finally, the TGase activity was increased three times to reach 0.331±0.038 U/ml with optimum concentrations of casein (38.4 g/l) and glycerol (31.2 g/l) calculated with the help of a composite design. In the course of these experiments, the two responses varied in the same way, demonstrating that growth and TGase production were tightly correlated under the conditions described. However, TGase was produced during the stationary phase of growth in optimized medium, indicating that the enzyme production could be induced.