TGase Activity Research Articles

Characterization of amine donor and acceptor sites for tissue type transglutaminase using a sequence from the C-terminus of human fibrillin-1 and the N-terminus of osteonectin

Transglutaminase (TGase)-modified proteins are commonly observed in a wide range of biological systems. Therefore, the identification of TGase substrates and respective consensus sites may contribute to a better understanding of the physiological role of TGase. In this study, we identified enzyme-specific properties of two peptide sequences, EDGFFKI, derived from human fibrillin-1, and the previously characterized APQQEA, derived from human osteonectin. EDGFFKI was identified in a previous publication as an amine donor substrate for tissue TGase; APQ 3Q 4EA is an amine acceptor for this enzyme. A widely-used lysine donor mimic, monodansylcadaverine (MDC), was used as a control. EDGFFKI crosslinked specifically only to Q 3 of the acceptor probe. The EDGFFKI sequence also showed enzyme specificity for tissue TGase while no reaction was observed with plasma TGase (Factor XIIIa), consistent with its natural occurrence in vivo. Using this substrate in biotinylated form we demonstrate its value as a tracer probe to detect endogenous TGase activity in human tissues as well as to target potential amine acceptor substrates via an enzyme-directed site-specific labeling. The results of this study show natively derived EDGFFKI and APQQEA are better and more specific indicators of endogenous tissue TGase activity as compared to a small molecule probe; this may be important in diagnostic applications. The specificity with which matrix sequences APQQEA and EDGFFKI interact with tissue TGase but not plasma TGase may also be crucial for understanding and controlling the function of these TGases in vivo and in tissue engineering.

Synthesis and Evaluation of a New Fluorescent Transglycosylase Substrate: Lipid II-Based Molecule Possessing a Dansyl-C20 Polyprenyl Moiety

The preparation of a novel fluorescent lipid II-based substrate for transglycosylases (TGases) is described. This substrate has characteristic structural features including a shorter lipid chain, a fluorophore tag at the end of the lipid chain rather than on the peptide chain, and no labeling with a radioactive atom. This fluorescent substrate is readily utilized in TGase activity assays to characterize TGases and also to evaluate the activities of TGase inhibitors.

Transglutaminase participates in the blockade of neurotransmitter release by tetanus toxin: evidence for a novel biological function

Inhibition of neuroexocytosis by tetanus neurotoxin (TeNT) involves VAMP-2/synaptobrevin-2 cleavage. However, deletion of the TeNT activity does not completely abolish its inhibitory action. TeNT is a potent activator of the cross-linking enzyme transglutaminase 2 (TGase 2) in vitro. The role of the latter mechanism in TeNT poisoning was investigated in isolated nerve terminals and intact neurons. TeNT-induced inhibition of glutamate release from rat cortical synaptosomes was associated with a simultaneous activation of neuronal transglutaminase (TGase) activity. The TeNT-induced blockade of neuroexocytosis was strongly attenuated by pretreatment of either live Aplysia neurons or isolated nerve terminals with specific TGase inhibitors or neutralizing antibodies. The same treatments completely abolished the residual blockade of neuroexocytosis of a non-proteolytic mutant of TeNT light chain. Electrophysiological studies indicated that TGase activation occurs at an early step of TeNT poisoning and contributes to the inhibition of transmitter release. Bioinformatics and biochemical analyses identified synapsin I and SNAP-25 as potential presynaptic TGase substrates in isolated nerve terminals, which are potentially involved in the inhibitory action of TeNT. The results suggest that neuronal TGase activity plays an important role in the regulation of neuroexocytosis and is one of the intracellular targets of TeNT in neurons.

Compatible and self-incompatible pollination in Pyrus communis displays different polyamine levels and transglutaminase activity

The polyamine (PA) content and the transglutaminase (TGase) activity have been investigated in Pyrus communis pollination with compatible and self-incompatible (SI) pollen in order to deepen their possible involvement in the progamic phase of plant reproduction. The PA distribution as free, perchloric acid (PCA)-soluble and PCA-insoluble fractions in ungerminated (UGP), germinating pollen (GP), styles and pollinated styles with compatible and SI pollens is discussed in the light of a possible role during pollination. Generally, the conjugated PAs both in PCA-soluble and PCA-insoluble fractions were higher than the free form. Within the conjugated PAs, the PCA-insoluble ones were the highest with the exception of the not pollinated styles. As TGase mediates some of the effects of PAs by covalently binding them to proteins, the activity of this enzyme, never checked before in styles and pollinated styles, was examined. In the SI styles, the TGase activity is higher in comparison to style-pollinated with compatible pollen, and high molecular mass cross-linked products were formed, suggesting an involvement of TGase in SI response. This is the first evidence on the presence of this enzyme activity in not pollinated and pollinated styles.

Transglutaminase II interacts with rac1, regulates production of reactive oxygen species, expression of snail, secretion of Th2 cytokines and mediates in vitro and in vivo allergic inflammation

Transglutaminase II (TGase II) is a protein cross-linking enzyme with diverse biological functions. Here we report the role of TGase II in allergic inflammation. Antigen stimulation induced expression and activity of TGase II by activation of NF-κB in rat basophilic leukemia (RBL2H3) cells. This induction of TGase II was dependent on FcɛRI and EGFR. Interaction between TGase II and rac1 was induced following antigen stimulation. TGase II was responsible for the increased production of reactive oxygen species, expression of prostaglandin E2 synthase (PGE2 synthase) and was responsible for increased secretion of prostaglandin E2. ChIP assay showed that TGase II, through interaction with NF-κB, was responsible for the induction of histone deacetylase-3 (HDAC3) and snail by direct binding to promoter sequences. HDAC3 and snail induced by TGase II, exerted transcriptional repression on E-cadherin. Snail exerted negative effect on expression of MMP-2, and secretion of Th2 cytokines. Inhibition of matrix metalloproteinase-2 (MMP-2) inhibited secretion of Th2 cytokines. In vivo induction of TGase II was observed in Balb/c mouse model of IgE antibody-induced passive cutaneous anaphylaxis. Chemical inhibition of TGase II exerted negative effect on IgE-dependent passive cutaneous anaphylaxis. Chemical inhibition of TGase II by cystamine exerted negative effect on Balb/c mouse model of phorbol myristate acetate (PMA)-induced atopic dermatitis. These results suggest novel role of TGase II in allergic inflammation and TGase II can be developed as target for the development of allergy therapeutics.

Substrate specificity of microbial transglutaminase as revealed by three-dimensional docking simulation and mutagenesis

Transglutaminases (TGases) are used in fields such as food and pharmaceuticals. Unlike other TGases, microbial transglutaminase (MTG) activity is Ca2+-independent, broadening its application. Here, a three-dimensional docking model of MTG binding to a peptide substrate, CBZ-Gln-Gly, was simulated. The data reveal CBZ-Gln-Gly to be stretched along the MTG active site cleft with hydrophobic and/or aromatic residues interacting directly with the substrate. Moreover, an oxyanion binding site for TGase activity may be constructed from the amide groups of Cys64 and/or Val65. Alanine mutagenesis verified the simulated binding region and indicated that large molecules can be widely recognized on the MTG cleft.

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.

Striatal Expression of a Calmodulin Fragment Improved Motor Function, Weight Loss, and Neuropathology in the R6/2 Mouse Model of Huntington's Disease

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder, caused by a polyglutamine expansion in the huntingtin protein (htt). Increasing evidence suggests that transglutaminase (TGase) plays a critical role in the pathophysiology of HD possibly by stabilizing monomeric, polymeric and aggregated htt. We previously reported that in HEK293 and SH-SY5Y cells expression of a calmodulin (CaM)-fragment, consisting of amino acids 76-121 of CaM, decreased binding of CaM to mutant htt, TGase-modified htt and cytotoxicity associated with mutant htt and normalized intracellular calcium release. In this study, an adeno-associated virus (AAV) that expresses the CaM-fragment was injected into the striatum of HD transgenic R6/2 mice. The CaM-fragment significantly reduced body weight loss and improved motor function as indicated by improved rotarod performance, longer stride length, lower stride frequency, fewer low mobility bouts and longer travel distance than HD controls. A small but insignificant increase in survival was observed in R6/2 mice with CaM-fragment expression. Immunoprecipitation studies show that expression of the CaM-fragment reduced TGase-modified htt in the striatum of R6/2 mice. The percentage of htt-positive nuclei and the size of intranuclear htt aggregates were reduced by the CaM-fragment without striatal volume changes. The effects of CaM-fragment appear to be selective, as activity of another CaM-dependent enzyme, CaM-dependent kinase II, was not altered. Moreover, inhibition of TGase-modified htt was substrate-specific since overall TGase activity in the striatum was not altered by treatment with the CaM-fragment. Together, these results suggest that disrupting CaM-htt interaction may provide a new therapeutic strategy for HD.

Transglutaminase-1 protects renal epithelial cells from hydrogen peroxide-induced apoptosis through activation of STAT3 and AKT signaling pathways

Our recent studies showed that transglutaminase-1 (TGase-1) is uniquely expressed in mouse renal proximal tubular cells (RPTC) and mediates cell proliferation. In this study, we investigated the role of TGase-1 in cell survival and the survival signaling pathways regulated by TGase-1 in RPTC following oxidant injury. Exposure of RPTC to hydrogen peroxide (H2O2) resulted in apoptosis and an increase in TGase activity. Inhibition of TGase activity with monodansylcadervine (MDC), a TGase inhibitor, or knockdown of TGase-1 with small interference (si)RNA enhanced apoptosis and decreased cell survival in H2O2-treated RPTC. Conversely, overexpression of TGase-1 rendered RPTC more resistant to H2O2 toxicity and MDC treatment blocked this response. Concurrent with RPTC apoptosis, phosphorylation of AKT, signal transducer and activator of transcription-3 (STAT3), and glucogen synthase kinase-3beta (GSK-3beta) were observed. Pretreatment of cells with MDC or TGase-1 siRNA inhibited phosphorylation of all these molecules. Inhibition of either the AKT or STAT3 pathway potentiated H2O2-induced cell death and increased GSK-3beta activity by dephosphorylation at serine 9. Furthermore, treatment with GSK-3beta inhibitors reduced H2O2-induced apoptosis and abolished the death-promoting effect of AKT and STAT3 inhibition. Therefore, we have identified TGase-1 as a novel survival factor in renal epithelial cells and it contributes to cell survival through activation of the AKT and STAT3 signaling pathways following oxidant injury.

The mechanism of transglutaminase 2 inhibition with glucosamine: implications of a possible anti-inflammatory effect through transglutaminase inhibition

Although many efforts on revealing mechanism of the constitutive activation of NF-κB in cancer cells contributed to understanding canonical pathways, largely it remains to be determined for therapeutic approaches. Recently, we found that increased expression of transglutaminase 2 (TGase 2) appears to be responsible for constitutive activation of NF-κB in certain types of cancer cells. In previous studies, we demonstrated that TGase 2 inhibition markedly increases anti-cancer drug sensitivity in drug resistance cancer cells. Therefore, we develop safe and effective TGase 2 inhibitors for therapeutic approach. We screened a chemical library of natural compounds using in vitro TGase 2 activity assay. The salient discovery was that glucosamine (GlcN), a known anti-inflammatory substance, inhibited the cross-linking activity of TGase 2. We tested, through a biochemical analysis including kinetics, whether the GlcN and GlcN analogs specifically inhibit TGase 2. We also determined the inhibitory mechanism using conformational change of TGase 2. We found that the primary amine of GlcN plays a key role in TGase 2 inhibition. We also demonstrated that GlcN reversed TGase 2-mediated I-κBα polymerization in vitro. Interestingly, the metabolite of GlcN, glucosamine-6-phosphate (GlcN6P), inhibited TGase 2 activity via binding to the GTP-binding site with better efficiency than GlcN. In the native gel electrophoresis, it was clearly observed that GlcN6P binds to TGase 2 directly as an allosteric inhibitor. We concluded that GlcN inhibits TGase 2 activity by direct contact. GlcN and its metabolite GlcN6P can down-regulate constitutive activation of NF-κB in vivo via inhibition of TGase 2.

New mutations in the transglutaminase 1 gene in three families with lamellar ichthyosis

Autosomal recessive lamellar ichthyosis (LI) is a severe skin disorder characterized by generalized hyperkeratosis. Gene mutation in transglutaminase 1 (TGM1), which mediates cross-links in the formation of the cell envelope during terminal differentiation of epidermis, has been identified as a cause of LI. To determine mutations of TGM1 gene in three Chinese families with LI. The TGM1 gene was sequenced to identify disease-causing mutations in the three families with LI. One of the results was confirmed by using reverse transcriptase PCR and in situ hybridization. An in situ transglutaminase (TGase) 1 assay was performed to estimate TGase 1 activity in the patients' skin. Four novel mutations of keratinocyte TGase1 (Q203X, D254N, R687H and IVS4 + 1G-->T) were found in the three families. No TGase 1 mRNA was detected in patient skin using RT-PCR and in situ hybridization, and the in situ TGase assay showed that there was no or decreased TGase 1 activity in patient skin. Our findings suggest that four novel mutations in TGM1 gene result in decrease or absence of TGase activity in the skin and, as a consequence, cause the phenotype of LI.

Kinetin affects the level of chloroplast polyamines and transglutaminase activity during senescence of barley leaves.

We analysed the level of polyamines (PAs) bound to thylakoids and the level and activity of thylakoid transglutaminases throughout barley leaf senescence, retarded by kinetin. The level of PAs bound to thylakoids changed in senescing barley leaves: bound putrescine (PU) and spermidine (SD) increased throughout senescence, whereas bound spermine (SM) decreased. Kinetin diminished the increase in thylakoid-bound PU and SD and almost completely abolished the decrease of the bound SM. These data suggest different roles of PU/SD and SM in thylakoid degradation. Immunodetection of transglutaminases (TGase) in thylakoid fraction revealed three bands of 33, 58 and 78 kDa. During senescence the intensity of all bands increased and it was correlated with an increase in TGase activity. Kinetin down-regulated the accumulation of the 58- and 78-kDa TGases and the TGase activity. We postulate that formation of covalent bonds between PAs and proteins by TGase is involved in chloroplast senescence. The kinetin-mediated preservation of low TGase levels and activity throughout leaf senescence may represent an important component of the mechanism of kinetin action in the retardation of leaf senescence.

Polyamines and transglutaminase actions: Polyamine und Transglutaminase-Wirkungen

Polyamines (putrescine, spermidine and spermine), secreted by the prostate gland, occur in high concentrations in the seminal fluid of many species, including man and rat. The physiological significance of seminal polyamines is still obscure, however. It has been postulated that polyamines may serve as amine donor substrates for transglutaminases (TGases), enzymes catalyzing protein crosslinking by the formation of gamma-glutamyl-lysine or bis(gamma-glutamyl)polyamine cross-bridges. We have analyzed TGase-activities and polyamine content of the various rat prostate lobes and rat seminal vesicles. Highest TGase activities were observed in the coagulating gland (anterior prostate) and the dorsolateral prostate, whereas very little TGase activity was present in the ventral prostate gland. In contrast, polyamine concentrations were highest in the ventral prostate but low in coagulating glands. Seminal vesicles, and in particular seminal vesicle secretions, contained low polyamine levels and intermediate TGase activity. Levels of protein-bound polyamines, not extractable by perchlorid acid, did not correlate with TGase-activities in ventral prostate and coagulating glands, suggesting an extracellular rather than intracellular function of prostatic TGase and polyamines. The observation that Km-values of rat prostate TGase for all three polyamines (57-120 microM, using N,N-dimethylated casein as protein substrate) were well below seminal polyamine concentrations is compatible with a regulatory role of polyamines in the process of seminal clot formation.

An in situ and in vitro investigation for the transglutaminase potential in tissue engineering

Transglutaminases (TGases) constitute a family of enzymes that stabilize protein assemblies by gamma-glutamyl-epsilon-lysine crosslinks. The role of tissue transglutaminase (TGase 2) in several pathophysiologies, wound healing applications, biomaterials functionalization, and drug delivery systems provides grounds for its use in tissue engineering. Herein, we initially studied the endogenous TGase activity and expression under normal (skin, duodenum, colon, and small bowel) and pathophysiological (keloid scar) conditions on cadaveric human tissues. Successful inhibition was achieved using low concentrations of BOC-DON-QIV-OMe (0.1 mM and 1 mM for normal skin and keloid scar, respectively), iodoacetamide (0.1 mM and 1 mM for normal skin and keloid scar, respectively), and cystamine dihydrochloride (1 mM and 10 mM for normal skin and keloid scar, respectively), whilst di-BOC-cystamine was found ineffective even at 100 mM concentration. Secondly, the addition of exogenous guinea pig liver transglutaminase (gpTGase) onto the inhibited tissues and collagen scaffolds was studied, and results presented advocate its use as potential tissue adhesive and drug delivery tool. However, the investigation of its crosslinking extent using second harmonic generation microscopy and differentially scanning calorimetry revealed rather poor stabilization function. Overall, our study indicates that TGase 2 has a role as a biological glue to consolidate various micro-structural components of tissues and biomaterials.

Development of an isoenzyme-specific colorimetric assay for tissue transglutaminase 2 cross-linking activity

Tissue transglutaminase (TGase 2) belongs to the multigene transglutaminase family of Ca 2+-dependent protein cross-linking enzymes. Based on the transamidation activity of TGase 2, a novel colorimetric assay has been developed using covalently coupled spermine to carboxy-substituted polystyrene plates and biotinylated pepT26, an excellent acyl-donor substrate, highly specific for TGase 2. The assay is based on the incorporation of the gamma-carboxamide of glutamine of pepT26 into the immobilized spermine. The amount of biotinylated pepT26 bound to the plate, as measured by the activity of streptavidin-peroxidase, is directly proportional to the TGase activity. The colorimetric procedure showed a good correlation ( r = 0.995) with the commonly used radiometric filter paper method for TGase2, and provides linear dose–response curves over a wide range of hrTGase2 concentrations (2.5–40 μU/ml). In addition, the assay shows higher sensitivity when compared with our previous TG-colorimetric test (more than 50-fold increase) and other existing assays. PepT26 displays strong reactivity with TGase 2, and no reactivity with TGases 1, 3, and FXIII. The procedure constitutes a rapid, TG2-specific, sensitive, and nonisotopic method for the measurement of TGase 2 activity in as low as 4 ng of hrTGase 2 and purified guinea pig liver transglutaminase, and 1.25 μg of guinea pig liver extracts.

Advanced Glycation End Products Increase Transglutaminase Activity in Primary Porcine Tenocytes

ObjectivesTendon abnormalities, such as increased stiffness, thickness, and excess calcification, occur commonly in patients with diabetes mellitus and cause considerable disability. These changes are frequently attributed to increased cross-linking of...

Transglutaminase-3 Enzyme: A Putative Actor in Human Hair Shaft Scaffolding?

The family of transglutaminases (TGase) is known to be involved in terminal differentiation processes in the epidermis. These enzymes contribute also to the physical resistance and the preservation of the hair follicle structure. Our particular interest in hair fiber keratinization led us to focus on the TGase 3, exclusively expressed in the hair shaft. To date its function is still to be elucidated, thus we have developed a multidisciplinary approach in order to define the localization, activity, and substrates of TGase 3. The hair fiber is characterized by the expression of specific proteins essentially consisting of keratin intermediate filaments and keratin-associated proteins (KAPs), which are essential for the formation of a rigid hair shaft through their extensive disulfide cross-links. Gel electrophoresis combined with mass spectrometry experiments revealed an unexpected protein migration pattern, suggesting the existence of covalent interactions other than disulfide bonds. Western blot and amino-acid analysis revealed the presence of gamma-glutamyl-epsilon-lysine isopeptide linkages that could constitute this second covalent network. Our hypothesis is that TGase 3-driven specific isopeptide bonds between intermediate filaments and KAPs participate to the progressive scaffolding of the hair shaft.

Transglutaminase activity in the hematopoietic tissue of a crustacean, Pacifastacus leniusculus, importance in hemocyte homeostasis

BackgroundTransglutaminases (TGases) form a group of enzymes that have many different substrates and among the most well known are fibrin for Factor XIIIa and the clotting protein in crustaceans. We also found that TGase is an abundant protein in the hematopoietic tissue (Hpt) cells of crayfish and hence we have studied the possible function of this enzyme in hematopoiesis.ResultsTGase is one of the most abundant proteins in the Hpt and its mRNA expression as well as enzyme activity is very high in the Hpt cells, lesser in the semi-granular hemocytes and very low in the granular cells. In cultured hematopoietic tissues, high activity was present in cells in the centre of the tissue, whereas cells migrating out of the tissue had very low TGase activity. RNAi experiments using dsRNA for TGase completely knocked down the transcript and as a result the cell morphology was changed and the cells started to spread intensely. If astakine, a cytokine directly involved in hematopoiesis, was added the cells started to spread and adopt a morphology similar to that observed after RNAi of TGase. Astakine had no effect on TGase expression, but after a prolonged incubation for one week with this invertebrate cytokine, TGase activity inside and outside the cells was completely lost. Thus it seems as if astakine addition to the Hpt cells and RNAi of TGase in the cell culture will lead to the same results, i.e. loss of TGase activity in the cells and they start to differentiate and spread.ConclusionThe results of this study suggest that TGase is important for keeping the Hpt cells in an undifferentiated stage inside the hematopoietic tissue and if expression of TGase mRNA is blocked the cells start to differentiate and spread.This shows a new function for transglutaminase in preventing hematopoietic stem cells from starting to differentiate and migrate into the hemolymph, whereas their proliferation is unaffected. Astakine is also important for the hematopoiesis, since it induces hemocyte synthesis in the Hpt but now we also show that it in some unknown way participates in the differentiation of the Hpt cells.

Determination of Transglutaminase Activity Using Fluorescence Spectrophotometer

An improved fluorometric assay for determining the activity of the microbial transglutaminase (TGase) in the culture medium samples has been developed. The assay procedure measures the fluorescence enhancement due to the incorporation of monodansyl cadaverine (Substrate A) into pentafluorophenylester of CBZ-Gln-Gly (Substrate Q) at λexc. 260 nm and λem 538 nm. The effect of the competitive inhibitors in the culture medium samples on TGase activity was determined. The assay was combined with HPLC method for determining enzyme activity as an international unit (IU). Enzymatic reaction was monitored by HPLC and the rate of product formation was measured via amine substrate consumption rate. A conversion factor was obtained using HPLC and fluorescence spectrophotometer data together. This was formulated for quantification of TGase activity as IU using fluorometric assay reported in this study. The detection limit of the assay was determined as 0.0014 IU (0.5 mg). TGase activity remained linear upto the enzyme concentraion of 20 mg. This technique dramatically decreases the incubation time of enzyme to a few minutes of activity measurement.

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.