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1.
Kangkang Chen Song Liu Dongxu Zhang Miao Wang Zhongping Shi Guocheng Du Jian Chen 《Process Biochemistry》2013,48(5-6):782-787
Streptomyces transglutaminase (TGase) is naturally synthesized as a zymogen (pro-TGase), which is then processed to produce the active enzyme through removal of its pro-peptide. In this study, we investigated the effect of the pro-peptide on the secretion of Streptomyces hygroscopicus TGase in Escherichia coli by modifying its pro-peptide. Four N-terminal amino acid residues (Tyr12, Asn27, Asn30, and Arg32) in the pro-peptide predicted to interact with TGase region through hydrogen bonds. When the four amino acid residues were mutated into Ala, the secretion of TGase was partially or completely inhibited. Furthermore, deletion of the C-terminal α-helix37–42 in the pro-peptide concomitantly decreased the secretion of TGase. However, deletion of the C-terminal loop43–52 of the pro-peptide, resulted in increased secretion of TGase by approximately 70% as compared with the control native pro-peptide. These findings indicate that modification of the pro-peptide has a significant impact on the secretory efficiency of TGase in E. coli. 相似文献
2.
【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。 相似文献
3.
Kangkang Chen Song Liu Guangsheng Wang Dongxu Zhang Guocheng Du Jian Chen Zhongping Shi 《Journal of industrial microbiology & biotechnology》2013,40(3-4):317-325
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-helix37G?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. 相似文献
4.
BACKGROUND: Members of the subtilisin family of serine proteases usually have a conserved asparagine residue that stabilizes the oxyanion transition state of peptide-bond hydrolysis. Yeast Kex2 protease is a member of the subtilisin family that differs from the degradative subtilisin proteases in its high substrate specificity, it processes pro-alpha-factor, the precursor of the alpha-factor mating pheromone of yeast, and also removes the pro-peptide from its own precursor by an intramolecular cleavage reaction. Curiously, the mammalian protease PC2, a Kex2 homolog that is likely to be required for pro-insulin processing, has an aspartate in place of asparagine at the 'oxyanion hole'. RESULTS: We have tested the effect of making substitutions of the conserved oxyanion-hole asparagine (Asn 314) of the Kex2 protease. To do this, we have developed a rapid method of site-directed mutagenesis, involving homologous recombination of a polymerase chain reaction product in yeast. Using this method, we have substituted alanine or aspartate for Asn 314 in a form of Kex2 engineered for secretion. Transformants expressing the two mutant enzymes could be identified by failure either to produce mature alpha-factor or to mate. The Ala 314 enzyme was unstable but the Asp 314 enzyme accumulated to a high level, so that it could be purified and its activity towards various substrates tested in vitro. We found that, with three peptides that are good substrates of wild-type Kex2, the k(cal) of the Asp 314 enzyme was reduced approximately 4500-fold and its K(M) approximately 4-fold, relative to the wild-type enzyme. For the peptide substrate corresponding to the cleavage site of pro-alpha-factor, however, k(cat) of the Asp 314 enzyme was reduced only 125-fold, while the K(m) was increased 3-fold. Despite its reduced catalytic activity, however, processing of the mutant enzyme in vivo - by the intramolecular cleavage that removes its amino-terminal pro-domain - occurs at an unchanged rate. CONCLUSIONS: The effects of the Asn 314-Asp substitution reveal contributions to the reaction specificity of the Kex2 protease of substrate residues amino-terminal to the pair of basic residues at the cleavage site. Aspartate at the oxyanion hole appears to confer k(caf) discrimination between substrates by raising the energy barrier for productive substrate binding: this may have implications for pro-insulin processing by the PC2 protease, which has an aspartate at the equivalent position. The rate of intramolecular cleavage of pro-Kex2 may be limited by a step other than catalysis, presumably protein folding. 相似文献
5.
The pheromone-processing Kex2p endoprotease of Saccharomyces cerevisiae has been difficult to characterize due to its low level of expression in yeast cells. To overcome this problem, we have overexpressed Kex2p using the baculovirus/insect cell expression system. Spodoptera frugiperda Sf9 insect cells infected with a recombinant baculovirus, containing the complete KEX2 gene which encodes the Kex2p protease (814 amino acids), accumulate an 120-kDa functional form of the enzyme. The inhibition profile of the insect-cell-derived endoprotease is similar to that of the yeast enzyme. The recombinant infected insect cells also secrete into the medium about half of the total Kex2p activity produced. Deleting the carboxyl-terminal tail and the transmembrane domain of Kex2p (Kex2 delta p, 666 amino acids) does not measurably interfere with the enzyme characteristics and results in the secretion of up to 90% of the total enzyme activity. The truncated form, Kex2 delta p, of the endoprotease accumulates in the cell supernatant to 6.7 x 10(5) U/l. The molecular mass of the secreted forms for both the wild-type Kex2p and Kex2 delta p is the same (70 kDa) and is 50-kDa lower than the intracellular form. This result implicates a processing event which gives rise to shorter extracellular forms of both the wild-type Kex2p and Kex2 delta p and which trims their carboxy termini upsteam of amino acid 666. This processing event requires the integrity of the Ser385 of the Kex2p active site. 相似文献
6.
Liu XQ Yang XQ Xie FH Song LY Zhang GQ Qian SJ 《Protein expression and purification》2007,51(2):179-186
Since transglutaminase (TGase) have been widely used in industry, mass production of the enzyme is especially necessary. The mature TGase gene from Streptomyces fradiae was cloned into pET21a and overexpressed in Escherichia coli BL21(DE3). The recombinant TGase was formed as inclusion bodies, and its content was as high as 55% of the total protein content. The insoluble fractions were separated from cellular debris by centrifugation and solubilized with 8 M urea. With an on-column refolding procedure based on cation SP Fast Flow chromatography with dual-gradient, the active TGase protein was recovered efficiently from inclusion bodies. The final purified product was 95% pure detected by SDS-PAGE. Under appropriate experimental conditions, the protein yield and specific activity of the TGase were up to 53% and 21 U/mg, respectively. Furthermore, the refolded recombinant protein demonstrated nearly identical ability to polymerized BSA compared with that of native TGase. One hundred and five milligrams of refolded TGase protein was obtained from 3.2g wet weight cells in the 400 ml cell culture. 相似文献
7.
Yu YJ Wu SC Chan HH Chen YC Chen ZY Yang MT 《Applied microbiology and biotechnology》2008,81(3):523-532
A novel microbial transglutaminase (TGase) from the cultural filtrate of Streptomyces netropsis BCRC 12429 (Sn) was purified. The specific activity of the purified TGase was 18.2 U/mg protein with an estimated molecular
mass of 38 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The TGase gene of S. netropsis was cloned and an open reading frame of 1,242 bp encoding a protein of 413 amino acids was identified. The Sn TGase was synthesized
as a precursor protein with a preproregion of 82 amino acid residues. The deduced amino acid sequence of the mature S. netropsis TGase shares 78.9–89.6% identities with TGases from Streptomyces spp. A high level of soluble Sn TGase with its N-terminal propeptide fused with thioredoxin was expressed in E. coli. A simple and efficient process was applied to convert the purified recombinant protein into an active enzyme and showed
activity equivalent to the authentic mature TGase.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
8.
The N-terminal pro-peptide of 77 amino acid residues is essential for the folding of subtilisin, an alkaline serine protease from Bacillus subtilis. The synthetic pro-peptide has been shown to be capable of guiding the proper folding of denatured subtilisin to enzymatically active enzyme. Thus the pro-peptide serves as an intramolecular chaperone, which is removed by an autoprocessing reaction after the completion of the folding. With use of localized polymerase chain reaction random mutagenesis a total of 25 amino acid substitution mutations that affected subtilisin activities were isolated. These mutations occurred in a high frequency at the hydrophobic regions of the pro-peptide. For one of the mutations, M(-60)T, a second-site suppressor mutation, S(188)L, was isolated within the mature region. These results suggest that the pro-peptide consists of a few functional regions which interact with specific regions of the mature region of subtilisin during the folding process. 相似文献
9.
Y. Ohta H. Hojo S. Aimoto T. Kobayashi X. Zhu F. Jordan M. Inouye 《Molecular microbiology》1991,5(6):1507-1510
The amino-terminal pro-sequence consisting of 77 amino acid residues is required to guide the folding of secreted subtilisin E, a serine protease, into active, mature enzyme (ikemura et al., 1987). Furthermore, denatured subtilisin E can be folded to active enzyme in an intermolecular process with the aid of an exogenously added pro-subtilisin E, the active site of which was mutated (Zhu et al., 1989). In this report, we have synthesized the pro-peptide of 77 residues (corresponding to -1 to -77 in the sequence, where residue +1 is the N-terminal amino acid residue of the mature protein), and have found that it could intermolecularly complement the folding of denatured subtilisin E to active enzyme. Furthermore, we have found that the synthetic pro-peptide exhibits specific strong binding to the active mature enzyme by inhibiting it competitively at its active centre with an upper limit to a Ki of 5.4 x 10(-7). In contrast, synthetic pro-peptides corresponding to -44 to -77, -1 to -64 and -1 to -43 inhibited the enzyme with Ki values weaker by two orders of magnitude. The results indicate that the sequence extending from -1 to -77 is essential for specificity of interaction, perhaps generating a conformation that accounts for both roles found hitherto, i.e. specific binding to the active centre, and guiding of the refolding to active enzyme. Thus these results suggest that the pro-peptide functions as an intramolecular chaperone [corrected]. 相似文献
10.
Liu S Zhang D Wang M Cui W Chen K Liu Y Du G Chen J Zhou Z 《FEMS microbiology letters》2011,324(2):98-105
Streptomyces transglutaminase (TGase) is secreted as a zymogen (pro-TGase) in liquid cultures and is then processed by the removal of its N-terminal region, resulting in active TGase. To date, there is no report describing TGase (or pro-TGase) secretion in Escherichia coli. In this study, the pro-TGase from Streptomyces hygroscopicus was efficiently secreted by E.?coli BL21(DE3) using the TGase signal peptide or the pelB signal peptide. The secreted pro-TGase was efficiently transformed into active TGase by adding dispase to the culture supernatant of the recombinant strains. Mutational analysis showed that deletion of the first six amino acids of the N-terminal of the pro-region reduced the secretion of pro-TGase, and removal of the next 10 amino acids resulted in the formation of insoluble pro-TGase. These results suggest that the pro-region of TGase is essential for its efficient secretion and solubility in E.?coli. 相似文献
11.
Biophysical comparison of BMP-2, ProBMP-2, and the free pro-peptide reveals stabilization of the pro-peptide by the mature growth factor 总被引:5,自引:0,他引:5
Pro-forms of growth factors have received intensive scientific attention recently because in some cases different biological activities have been ascribed compared with the mature growth factors. Examples are the pro-apoptotic role of the nerve growth factor (NGF) proform (proNGF) or the latency of the transforming growth factor (TGF)-beta pro-form (proTGF-beta). To investigate a possible biological function of the pro-form of bone morphogenetic protein (BMP)-2, a member of the TGF-beta family, mature BMP-2, proBMP-2, and the isolated pro-peptide were recombinantly produced in Escherichia coli cells, and a biophysical comparison was performed. Protocols were developed that allowed efficient refolding and subsequent purification of the proteins. ProBMP-2 could be processed to an N-terminally truncated form of BMP-2, digit removed BMP-2 (drBMP-2), that possessed biological activity, i.e. it induced ectopic bone formation. Bone inducing activity was also displayed by proBMP-2. The three proteins were characterized both by fluorescence and CD spectroscopy. From these analyses, predominant beta-sheet secondary structural elements in the pro-peptide were deduced. The thermodynamic stability of the pro-peptide was determined by chemical unfolding assays. As in the case of NGF/proNGF, the mature part of BMP-2 stabilized the structure of the pro-peptide moiety. However, in contrast to NGF/proNGF, the pro-peptide did not stimulate oxidative folding of the mature part in vitro. 相似文献
12.
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。 相似文献
13.
Penicillin amidase from Alcaligenes faecalis is a recently identified N-terminal nucleophile hydrolase, which possesses the highest specificity constant (kcat/Km) for the hydrolysis of benzylpenicillin compared with penicillin amidases from other sources. Similar to the Escherichia coli penicillin amidase, the A. faecalis penicillin amidase is maturated in vivo from an inactive precursor into the catalytically active enzyme, containing one tightly bound Ca2+ ion, via a complex post-translational autocatalytic processing with a multi-step excision of a small internal pro-peptide. The function of the pro-region is so far unknown. In vitro addition of chemically synthesized fragments of the pro-peptide to purified mature A. faecalis penicillin amidase increased its specific activity up to 2.3-fold. Mutations were used to block various steps in the proteolytic processing of the pro-peptide to obtain stable mutants with covalently attached fragments of the pro-region to their A-chains. These extensions of the A-chain raised the activity up to 2.3-fold and increased the specificity constants for benzylpenicillin hydrolysis mainly by an increase of the turnover number (kcat). 相似文献
14.
Zhang L Zhang L Han X Du M Zhang Y Feng Z Yi H Zhang Y 《Applied microbiology and biotechnology》2012,93(6):2335-2343
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 MgCl2, 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 MgCl2 medium. Furthermore, total protease, metalloprotease, and serine protease were also synthesized at a faster rate in the medium
containing MgCl2. The results demonstrate that MgCl2 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 MgCl2 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 MgCl2 stress. 相似文献
15.
I. A. van Gemeren A. Beijersbergen W. Musters R. J. Gouka C. A. M. J. J. van den Hondel C. T. Verrips I. A. van Gemeren 《Applied microbiology and biotechnology》1996,45(6):755-763
A synthetic derivative of the cutinase cDNA of Fusarium solani pisi was expressed in Aspergillus awamori using the A. awamori endoxylanase II (exlA) promoter and terminator. The influence of the origin of the pre-sequence and the presence of a pro-sequence on the efficiency
of extracellular cutinase production was analysed in single-copy transformants containing an expression cassette integrated
at the pyrG locus. Transformants containing a construct encoding a direct, in-frame fusion of the xylanase pre-peptide to the mature
cutinase showed a 2-fold higher cutinase production level compared to strains containing constructs with an additional cutinase
pro-peptide. The effect of multicopy integration of the expression cassette on cutinase production was analysed in strains
with different numbers of a cutinase construct containing its own pre-prosequence. The multicopy strains showed a 6- to 12-fold
increased production of extracellular cutinase relative to the single-copy strains. No linear dose response relation to the
number of expression cassettes present in the strains was observed. The amount of active enzyme produced by the strains correlated
with the amount of cutinase-specific mRNA, suggesting that cutinase overproduction is not limited at the level of translation
or secretion.
Received: 3 August 1995/Received revision: 20 December 1995/Accepted: 8 January 1996 相似文献
16.
J A Dias 《Biology of reproduction》1985,33(4):835-843
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. 相似文献
17.
Subtilisin, an alkaline serine protease, is produced in the bacterium as pre-pro-subtilisin; the pre-peptide of 29 amino acid residues is the signal peptide essential for the secretion of prosubtilisin from the cytoplasm into the culture medium. On the other hand, the pro-peptide of 77 residues covalently linked to the amino terminal end of the subtilisin intramolecularly guides the folding of subtilisin into the active enzyme. Importantly, the pro-peptide is not required for the enzymatic activity and is removed intramolecularly by autoprocessing upon the completion of the protein folding. In this review, I will first summarize all the data concerning the functions of the subtilisin pro-peptide. On the basis of these results, I shall discuss a new general concept, an intramolecular chaperone to explain the essential role of the pro-peptide in protein folding. 相似文献
18.
alpha-Lytic protease is a 19.8-kDa protein secreted from the Gram-negative bacterium Lysobacter enzymogenes. We have cloned and sequenced the gene for this serine protease. The nucleotide sequence contains an open reading frame which codes for the 198-residue mature enzyme and a potential prepro-peptide, also of 198 residues. The COOH-terminal 49 residues of the pro-peptide are significantly homologous to the propeptides of Streptomyces griseus proteases A and B. We suggest that this pro-peptide region facilitates formation of the active enzyme. A region bridging the NH2-terminal pre- and pro-peptides is homologous to a maize inhibitor of serine proteases. We speculate that this region inhibits enzymatic activity of the prepro-enzyme. 相似文献
19.
J774, thioglycollate-elicited mouse peritoneal and BCG-induced rabbit alveolar macrophages all contain high levels of a triacylglycerol hydrolase (EC 3.1.1.3) (TGase) with optimal activity at pH 6.5. The J774 macrophages, a cell line deficient in the calcium-independent mannose 6-phosphate receptor, were found to secrete large quantities of the TGase into the culture medium. In contrast, mouse peritoneal and rabbit alveolar macrophages, which are both mannose 6-phosphate receptor-competent cell types, secreted much lower amounts of neutral TGase. The enzyme was localized in the lysosomes of rabbit alveolar macrophages. Addition of 25 mM NH4Cl induced a 6-fold increase in TGase secretion by alveolar macrophages, while 50 mM NH4Cl induced a 12-fold increase in TGase secretion. NH4Cl had no effect on TGase secretion by J774 macrophages. The TGase secreted by J774 macrophages was internalized by I-cell disease fibroblasts, increasing the cellular content of TGase 10-fold after 8 h. Internalization was inhibited 70% by the addition of 2 mM mannose 6-phosphate to the culture medium, but was not affected by 2 mM mannose or glucose 6-phosphate. After internalization, the neutral TGase was converted to a TGase with a pH optimum of 5.1. These data are consistent with the spontaneous release of a lysosomal enzyme precursor from a calcium-independent mannose 6-phosphate receptor-deficient cell line, indicating that the neutral TGase previously reported in several types of macrophages may be the precursor of the lysosomal acid TGase. 相似文献
20.
B Chaudhuri S E Latham S B Helliwell P Seeboth 《Biochemical and biophysical research communications》1992,183(1):212-219
The prepro sequence of the yeast prepro-alpha-factor, usually referred to as the alpha-factor leader, has often been used for the efficient secretion of heterologous proteins from the yeast Saccharomyces cerevisiae. The alpha-factor leader consists of a 19-amino acid N-terminal pre or signal sequence followed by a 66-amino acid proregion. After removal of the signal sequence during membrane translocation, the proregion is cleaved from the precursor protein by the Kex2 endoprotease only in a late Golgi compartment. Here we report that a modified Kex2 enzyme, containing at the C-terminus the HDEL tetrapeptide, cleaves the proregion from the alpha-factor leader--human insulin like growth factor-1 fusion protein in the endoplasmic reticulum. The processing of pro-proteins earlier in the secretion pathway could be helpful in defining the cellular function of the proregions present naturally in various eucaryotic precursor proteins. 相似文献