NaCl对轮枝链霉菌产谷氨酰胺转胺酶的促进作用研究

为了提高轮枝链霉菌发酵生产谷氨酰胺转胺酶的产量,研究了3种无机盐(NaCl、MgCl₂、KCl)对发酵产酶的影响。研究表明0.5%MgCl₂、0.5%NaCl均可促进菌体产酶,其中添加NaCl后谷氨酰胺转胺酶酶活提高最为显著。SDS-PAGE图谱分析显示,在各取样点实验组谷氨酰胺转胺酶酶原和成熟酶的总量高于对照,而且实验组成熟酶的增加也比对照快,从而显示NaCl是通过促进酶原的分泌和谷氨酰胺转胺酶的成熟,从而提高酶活、提早产酶。对NaCl的添加量进行优化,表明NaCl的最适添加量为0.5%,在此条件下,与对照相比,酶活水平提高了12%以上,发酵终点提前了约15h。     

Leishmania species: evidence for transglutaminase activity and its role in parasite proliferation

Albeit transglutaminase (TGase) activity has been reported to play crucial physiological roles in several organisms including parasites; however, there was no previous report(s) whether Leishmania parasites exhibit this activity. We demonstrate herein that TGase is functionally active in Leishmania parasites by using labeled polyamine that becomes conjugated into protein substrates. The parasite enzyme was about 2- to 4-fold more abundant in Old World species than in New World ones. In L. amazonensis, comparable TGase activity was found in both promastigotes and amastigotes. TGase activity in either parasite stage was optimal at the basic pH, but the enzyme in amastigote lysates was more stable at higher temperatures (37-55 degrees C) than that in promastigote lysates. Leishmania TGase differs from mouse macrophage (M Phi) TGase in two ways: (1) the parasite enzyme is Ca(2+)-independent, whereas the mammalian TGase depends on the cation for activity, and (2) major protein substrates for L. amazonensis TGase were found within the 50-75 kDa region, while those for the M Phi TGase were located within 37-50 kDa. The potential contribution of TGase-catalyzed reactions in promastigote proliferation was supported by findings that standard inhibitors of TGase [e.g., monodansylcadaverine (MDC), cystamine (CS), and iodoacetamide (IodoA)], but not didansylcadaverine (DDC), a close analogue of MDC, had a profound dose-dependent inhibition on parasite growth. Myo-inositol-1-phosphate synthase and leishmanolysin (gp63) were identified as possible endogenous substrates for L. amazonensis TGase, implying a role for TGase in parasite growth, development, and survival.     

The pro-peptide of Streptomyces mobaraensis transglutaminase functions in cis and in trans to mediate efficient secretion of active enzyme from methylotrophic yeasts

Transglutaminase (TGase) from the actinomycete Streptomyces mobaraensis is a useful enzyme in the food industry, and development of an efficient production system for it would be desirable. Herein we report secretion of TGase in an enzymatically active form by methylotrophic yeasts as expression hosts. Secretory production of active TGase required a pro-peptide from TGase. When an artificial Kex2-endopeptidase recognition site was placed between the pro-peptide and mature TGase, secretion and in vitro maturation of TGase depended on Kex2-dependent cleavage. Unexpectedly, coexpression of unlinked pro-peptide with mature TGase yielded efficient secretion of the active enzyme. These results indicate that the pro-peptide from TGase functions not only in an intramolecular but also in an intermolecular manner. Site-directed mutagenesis of putative N-glycosylation sites increased the productivity of the active TGase further. A recombinant Candida boidinii strain was found to secrete active TGase up to 1.83 U/ml (about 90 mg/l) after 119 h of cultivation.     

Cell type-specific activation of intracellular transglutaminase 2 by oxidative stress or ultraviolet irradiation: implications of transglutaminase 2 in age-related cataractogenesis

Transglutaminase (TGase) 2 is a ubiquitously expressed enzyme that modifies proteins by cross-linking or polyamination. An aberrant activity of TGase 2 has implicated its possible roles in a variety of diseases including age-related cataracts. However, the molecular mechanism by which TGase 2 is activated has not been elucidated. In this report, we showed that oxidative stress or UV irradiation elevates in situ TGase 2 activity. Neither the expression level nor the in vitro activity of TGase 2 appeared to correlate with the observed elevation of in situ TGase 2 activity. Screening a number of cell lines revealed that the level of TGase 2 activation depends on the cell type and also the environmental stress, suggesting that unrecognized cellular factor(s) may specifically regulate in situ TGase 2 activity. Concomitantly, we observed that human lens epithelial cells (HLE-B3) exhibited about 3-fold increase in in situ TGase 2 activity in response to the stresses. The activated TGase 2 catalyzed the formation of water-insoluble dimers or polymers of alphaB-crystallin, betaB(2)-crystallin, and vimentin in HLE-B3 cells, providing evidence that TGase 2 may play a role in cataractogenesis. Thus, our findings indicate that in situ TGase 2 activity must be evaluated instead of in vitro activity to study the regulation mechanism and function of TGase 2 in biological and pathological processes.     

Deletion combined with saturation mutagenesis of N-terminal residues in transglutaminase from Streptomyces hygroscopicus results in enhanced activity and thermostability

Transglutaminase (TGase) is an important industrial enzyme that catalyzes the cross-linking of proteins. In this study, the N-terminal residues were deleted and substituted to improve the activity and thermostability of Streptomyces hygroscopicus TGase. Seven N-terminal residues of TGase were chosen to be deleted individually. The mutated TGase missing the first four residues showed an increase in specific activity of 32.92%. The fifth residue (E5) in the N-terminus was then selected for substitution with the 19 other amino acids. The mutant replacing the fifth residue with an aspartic acid exhibited a 1.85-fold higher specific activity and a 2.7-fold longer half-life at 50 °C when compared with the wild-type enzyme. The melting temperature of the mutated TGase increased from 68.9 to 79.1 °C by circular dichroism spectroscopy analysis. This study showed that substitution combined with deletion of the N-terminal amino acids could enhance the activity and thermostability of TGase.     

Transglutaminase activity and embryonal carcinoma cell differentiation

Murine embryonal carcinoma (EC) cells induced to differentiate by retinoic acid (RA) modulate transglutaminase (TGase) activity shortly after exposure to the inducer. Compounds that inhibit TGase enzyme activity in vitro can successfully block RA induced EC cell differentiation in culture. These observations suggest that TGase may play a role in mediating RA induced EC cell differentiation.     

Transglutaminase activity in testicular homogenates and serum-free Sertoli cell cultures

Transglutaminase (EC 2.3.2.13) (TGase) activity has been localized in homogenates of rat Leydig cells and seminiferous tubules and is present in cytosol and membrane fractions. The enzyme has a requirement for Ca2+ and when the acceptor substrate casein was deleted from the assay mixture, incorporation of [14C]putrescine into cytosolic and membrane fractions occurred. Transglutaminase was also detected in Sertoli cells cultured in serum-free medium. Sertoli cells reside within the seminiferous tubule and are involved in normal spermatogenesis. Sertoli cell TGase has a strict requirement for Ca2+ and is not activated by Mg2+. Activation of the enzyme occurs with as little as 0.3 microM Ca2+; however, consistent with intracellular calcium levels, maximum stimulation occurred at 1.9 mM Ca2+. Sertoli cell TGase activity is markedly stimulated if the cells are cultured in 10% fetal bovine serum rather than in serum-free medium. Inhibition of Sertoli cell TGase by monodansylcadaverine concomitantly decreased the response of the cells to follicle-stimulating hormone (FSH)-induced secretion of cAMP but did not change basal cAMP levels. These data suggest that TGase may play a facilitative rather than an absolute role in activation of Sertoli cells by FSH and the resultant secretion of cellular products. This may occur through modulation of activities of membrane and cytosolic components by TGase.     

Involucrin cross-linking by transglutaminase 1. Binding to membranes directs residue specificity

The transglutaminase 1 (TGase 1) enzyme is essential for the assembly of the cell envelope barrier in stratified squamous epithelia. It is usually bound to membranes, but to date most studies with it have involved solution assays. Here we describe an in vitro model system for characterizing the function of TGase 1 on the surface of synthetic lipid vesicles (SLV) of composition similar to eukaryote plasma membranes. Recombinant baculovirus-expressed human TGase 1 readily binds to SLV and becomes active in cross-linking above 10 microM Ca2+, in comparison to above 100 microM in solution assays, suggesting that the membrane surface is important for enzyme function. Involucrin also binds to SLV containing 12-18% phosphatidylserine and at Ca2+ concentrations above 1 microM. In reactions of involucrin with TGase 1 enzyme in solution, 80 of its 150 glutamines serve as donor residues. However, on SLV carrying both involucrin and TGase 1, only five glutamines serve as donors, of which glutamine 496 was the most favored. As controls, there was no change in specificity toward the glutamines of other substrates used by free or SLV-bound TGase 1 enzyme. We propose a model in which involucrin and TGase 1 bind to membranes shortly after expression in differentiating keratinocytes, but cross-linking begins only later as intracellular Ca2+ levels increase. Furthermore, the data suggest that the membrane surface regulates the steric interaction of TGase 1 with substrates such as involucrin to permit specific cross-linking for initiation of cell envelope barrier formation.     

GTP is required to stabilize and display transamidation activity of transglutaminase 2

Transglutaminase 2 (TGase 2) is a bifunctional enzyme that catalyzes calcium-dependent transamidation and GTP binding/hydrolysis. The transamidation activity is proposed to be associated with several neurodegenerative disorders such as Alzheimer's and Hungtinton's disease. However, the regulation mechanism by which TGase 2 causes neurodegeneration is unknown. In this study, we show that two activities of TGase 2 have a differential stability; transamidation activity is less stable than GTP hydrolytic activity, and that GTP was required to stabilize and to display transamidation activity. Moreover, GTP binding-defective mutant of TGase 2 did not show any transamidation activity in transfection experiments. These results indicate that GTP binding is crucial for transamidation activity of TGase 2, suggesting that protein cross-linking by TGase 2 might be associated with G-protein coupled receptor signaling system. Thus, our data could contribute to understand the regulation of TGase 2 activity and TGase 2-associated pathogenesis.     

Transglutaminase activity changes during the hypersensitive reaction, a typical defense response of tobacco NN plants to TMV

The occurrence of glutamyl polyamines (PAs) and changes in activity and levels of transglutaminase (TGase, EC 2.3.2.13), the enzyme responsible for their synthesis, are reported during the progression of the hypersensitive reaction (HR) of resistant NN tobacco plants ( Nicotiana tabacum L. cv. Samsun) to tobacco mosaic virus (TMV). Mature leaves of tobacco were collected over 0–72 h after inoculation with TMV or phosphate buffer (mock). In vivo synthesis of polyamine glutamyl derivatives (glutamyl PAs), catalyzed by TGase activity, was evaluated after supplying labeled putrescine (Pu, a physiological substrate of TGase) to leaves. Results show that, starting from 24 h, mono-( γ -glutamyl)-Pu and bis-( γ -glutamyl)-Sd were recovered in TMV-inoculated samples but not in mock-inoculated ones; 2 days later, in the former, the amount of glutamyl derivatives further increased. An in vitro radiometric assay showed that, in TMV-inoculated leaves, TGase activity increased from 24 h onwards relative to mock controls. An immunoblot analysis with AtPng1p polyclonal antibody detected a 72-kDa protein whose amount increased at 72 h in TMV-inoculated leaves and in the lesion-enriched areas. A biotin-labeled cadaverine incorporation assay showed that TGase activity occurred in S1 (containing soluble proteins), S2 (proteins released by both cell walls and membranes) and S3 (membrane intrinsic proteins) fractions. In S3 fraction, where changes were the most relevant, TGase activity was enhanced in both mock-inoculated and TMV-inoculated samples, but the stimulation persisted only in the latter case. These data are discussed in the light of a possible role of TGase activity and glutamyl PAs in the defense against a viral plant pathogen.     

Cloning of Transglutaminase Gene from Streptomyces fradiae and its Enhanced Expression in the Original Strain

The transglutaminase (TGase) gene of Streptomyces fradiae was cloned. It had an ORF of 1242 bp, encoding a presumed prepro-region of 82 amino acids and a mature TGase of 331 amino acids. Enhanced expression of the TGase was achieved by introducing another copy of TGase gene into the original host genome which was driven by the strong constitutive promoter, “ermE up”, and shown to be expressed at the mRNA and protein levels. TGase activity in the recombinant strain (3.2 U/ml) was improved 1.3-fold when compared to that normally expressed in the original strain (2.4 U/ml). The specific enzyme activity in the recombinant strain (3.8 U/mg) was double that of the original strain (1.9 U/mg).     

Enhancement of Streptomyces transglutaminase activity and pro-peptide cleavage efficiency by introducing linker peptide in the C-terminus of the pro-peptide

Streptomyces transglutaminase (TGase) has been widely used in food, pharmaceutical and textile industries. Streptomyces TGase is naturally synthesized as zymogen (pro-TGase), which is then processed to produce active enzyme by removing its N-terminal pro-peptide. Although the pro-peptide is essential for TGase folding and secretion, few studies have been reported on improving the properties of TGase by pro-peptide engineering. In this study, we developed a new approach to improve the properties of TGase based on pro-peptide engineering. When the α-helix^37G?42S in pro-peptide was substituted with three glycines and three alanines respectively, the mutants exhibited higher specific activity and the efficiency of pro-peptide cleavage was enhanced. To further improve the properties of TGase, relevant mutations were constructed by introducing linker peptides in the C-terminus of the pro-peptide. Mutants with GS (GGGGS) and PT (PTPPTTPT) linker peptide exhibited 1.28 fold and 1.5 fold higher specific activity than the wild-type enzyme, respectively. This new method could be used to improve the properties of TGase by pro-peptide modification, which is a promising technology for creating unique TGase with various beneficial properties.     

拉达克轮丝菌TGase在大肠杆菌中的分子改造

【背景】谷氨酰胺转氨酶是一种能够催化酰基转移反应的酶,催化各种蛋白质分子之间或之内发生交联反应,在食品、化妆品、医药等领域具有重要的潜在价值。【目的】克隆来自拉达克轮丝菌(Streptoverticillium ladakanum) B1的谷氨酰胺转氨酶(TGase)基因并对其进行分子改造,使其在大肠杆菌中获得高效异源表达。【方法】分别克隆来自拉达克轮丝菌谷氨酰胺转氨酶的自身前导肽(pro)和除前导肽以外的成熟谷氨酰胺转氨酶(TGase)基因,以pET-22b为表达载体构建pro、TGase共表达和融合表达两种表达模式,在这两种表达模式的基础上进一步用定点突变的方法对成熟TGaseN端前4个氨基酸进行改造,检测不同表达模式以及突变对酶活的影响。【结果】当采用前导肽与TGase共表达时,可以直接得到活性形式的TGase,比酶活达到37.71 U/mg。在融合表达的基础上,将TGaseN端前3个氨基酸DSD突变为AAA,比酶活达到14.04U/mg,相较于原始表达模式提高了14.05%。【结论】前导肽与TGase共表达可以直接产生活性TGase,对于融合表达模式,合适位点的突变有利于提高TGase酶活。     

The acropetal wave of developmental cell death of tobacco corolla is preceded by activation of transglutaminase in different cell compartments

The activity of transglutaminase (TGase), an enzyme responsible for polyamine conjugation to proteins, was analyzed in relationship to developmental cell death (DCD) during the flower life span stages of the tobacco (Nicotiana tabacum) corolla. As the DCD exhibits an acropetal gradient, TGase was studied in corolla proximal, medial, and distal parts. TGase was immunorecognized by three TGase antibodies; the main 58-kD band decreased during corolla life, whereas a 38-kD band localized progressively from basal to distal parts. The former was present in the soluble, microsomal, plastidial (together with the 38-kD band), and cell wall fractions. The endogenous TGase activity increased during DCD reaching a maximum soon after the corolla opening. The activity maximum shifted from proximal to distal part, preceding the DCD acropetal pattern. A similar activity increase was observed by the exogenous TGase substrate (histidine(6)-Xpr-green fluorescent protein). Subcellular activities were detected in (1) the microsomes, where TGase activity is in general higher in the proximal part, peaking at the corolla opening; (2) the soluble fraction, where it is present only in the proximal part at senescence; (3) the plastids, where it shows an increasing trend; and (4) cell walls, prevailing in the distal part and progressively increasing. These data suggest a relationship between DCD and TGase; the latter, possibly released in the cell wall through the Golgi vesicles, could cooperate to cell wall strengthening, especially at the abscission zone and possibly during corolla shape change. The plastid TGase, stabilizing the photosystems, could sustain the energy requirements for the senescence progression.     

Effect of Pepsin Inhibitor (S-PI) on the Growth of Rhodotorula glutinis No. K-24

A tissue-type transglutaminase (TGase) was purified from liver tissue of the red sea bream, Pagrus major, by ion-exchange chromatography and heparin-Sepharose affinity chromatography. Its activity was assessed using a fluorometric assay to measure the incorporation of monodansylcadaverine into N,N′-dimethyl casein. The molecular mass of purified TGase was estimated to be 78kDa by SDS–polyacrylamide gel electrophoresis. The enzyme required Ca²⁺ to express its activity, although 10 mM Sr²⁺ also activated the enzyme fully. TGase activity was maximal at pH 9.0–9.5, and the enzyme was strongly inhibited by sulfhydryl reagents. The purified enzyme catalyzed the cross-linking of myosin heavy chain obtained from Alaska pollack, resulting in gelation of an actomyosin solution. The partial amino acid sequence of this fish TGase showed divisionally significant similarity to TGase from guinea pig liver.     

Transglutaminase in sporulating cells of Bacillus subtilis

We screened various Bacillus species producing transglutaminase (TGase), measured as labeled putrescine incorporated into N,N-dimethylcasein. As a result, we detected TGase activity in sporulating cells of B. subtilis, B. cereus, B. alvei and B. aneurinolyticus, and found TGase activity related to sporulation. TGase activity of Bacillus subtilis was detected in lysozyme-treated sporulating cells during late sporulation, but not in cells without lysozyme treatment or the supernatant of the culture broth. TGase was found to be localized on spores. TGase was preliminarily purified by gel filtration chromatography for characterization. Its activity was eluted in the fractions indicating a molecular weight of approximately 23 kDa. TGase could cross-link and polymerize a certain protein. The enzyme was strongly suggested to form epsilon-(gamma-glutamyl)lysine bonds, which were detected in the spore coat proteins of B. subtilis. The activity was Ca(2+)-independent like the TGases derived from Streptoverticillium or some plants. It is suggested that TGase is expressed during sporulation and plays a role in the assembly of the spore coat proteins of the genus Bacillus.     

Intracellular distribution of active and inactive transglutaminase in stimulated cultured C6 glioma cells

The cellular distribution of active and inactive transglutaminase (TGase) was studied in C6 glioma cells before and during stimulation by a serum-containing medium. The activity of the enzyme was determined in the soluble and insoluble fractions obtained by freezing and thawing the cells, followed by centrifugation at 12,000g for 5 min. In the soluble fractions, the activity of TGase decreased 2.5 h post-stimulation and increased after 5 and 8 h. In the corresponding insoluble fractions, no significant changes in the activity of the enzyme were noted up to 8 h after stimulating the cells with fresh medium. An immunological approach was next used to determine the quantity of TGase antigen during the stimulation of the cultured glioma cells. In the soluble fraction, the quantity of the antigen decreases significantly at 2.5, 5, and 8 h. In contrast, in the insoluble fraction, a significant increase in TGase antigen was detected 8 h after the addition of fresh medium. Cycloheximide completely inhibited the increase in the quantity of TGase antigen in the insoluble fraction, 8 h post-stimulation, while actinomycin D caused a partial inhibition. Trypsin, neuraminidase, or Sendai viruses increased the activity of TGase significantly, when added to nonstimulated cells. Trypsin had no effect on TGase activity when added to the cells 2 h after stimulation with a serum-containing medium. These findings suggest that an inactive form of the enzyme is present in the insoluble cellular fraction. A model has been proposed to explain the variations in TGase activity, its distribution and translocation during cellular stimulation.     

Transglutaminase-catalyzed modification of cytoskeletal proteins by polyamines during the germination of Malus domestica pollen

 We investigated polyamine linkage to different structural proteins in pollen of Malus domestica Borkh. cv Red Chief at different phases of germination. This linkage has the characteristics of covalent linkages, indeed, it could be catalyzed by transglutaminase (TGase; EC 2.3.2.13). This assumption is supported by: (1) formation of a labelled TCA pellet and selective labelling of endogenous proteins by covalent binding of radioactive polyamines and (2) cross-reactivity of two different polyclonal antibodies against mammalian TGases; western blot analysis allowed us to detect a protein of about 80 kDa in both rehydrated ungerminated and germinated pollen. TGase activity was high at 90 min after germination and was influenced by Ca²⁺ supply only in the rehydrated ungerminated pollen. Extraction by Triton X-100 suggests that pollen TGase was at least partially membrane-bound. The enzyme catalyzed the incorporation of polyamines mainly into proteins having a molecular mass of 43 kDa and 52–58 kDa in both ungerminated and germinated pollen. These bands matched immunolabelled spots identified by mouse monoclonal anti-actin and anti-α-tubulin antibodies. Supplying exogenous actin and tubulin in a cell-free extract of rehydrated ungerminated and germinated pollen enhanced the activity. Autoradiography of the SDS-PAGE of these samples clearly showed that both actin and tubulin were substrates of TGase. Thus, the pollen TGase may be involved in the rapid cytoskeletal rearrangement which takes place during rehydration of ungerminated pollen and organization and growth of pollen tubes. Received: 9 August 1996 / Revision accepted: 26 October 1996