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1.
Aspergillus strains are being considered as potential hosts for recombinant heterologous protein production because of their excellent
extracellular enzyme production characteristics. However, Aspergillus proteases are problematic in that they modify and degrade the heterologous proteins in the extracellular medium. In previous
studies we observed that media adjustments and maintenance of a filamentous morphology greatly reduced protease activity and
that a low concentration of the aspartic protease inhibitor pepstatin inhibited the latter protease activity to the extent
of approximately 90%. In this paper we report that when the serine protease inhibitor chymostatin is used in combination with
pepstatin 99–100% of total protease activity in Aspergillus cultures is inhibited. In protease assays a concentration of 30 μM chymostatin combined with 0.075 μM pepstatin was required
for maximum inhibition. Inhibitor concentrations of chymostatin and pepstatin of 120 and 0.3 μM, respectively, when added
to Aspergillus cultures, has no significant effect on biomass production, glucose utilization or culture pH pattern. The potential of using
these protease inhibitors in cultures of recombinant Aspergillus strains producing heterologous proteins will now be investigated to determine if the previously observed recombinant protein
denaturing effects of Aspergillus proteases can be negated. 相似文献
2.
Extracellular proteases produced by the wood-degrading fungus Phanerochaete chrysosporium under ligninolytic and non-ligninolytic conditions 总被引:2,自引:0,他引:2
S. Balachandra Dass Carlos G. Dosoretz C. Adinarayana Reddy Hans E. Grethlein 《Archives of microbiology》1995,163(4):254-258
When subjected to nitrogen limitation, the wood-degrading fungus Phanerochaete chrysosporium produces two groups of secondary metabolic, extracellular isoenzymes that depolymerize lignin in wood: lignin peroxidases and manganese peroxidases. We have shown earlier the turnover in activity of the lignin peroxidases to be due in part to extracellular proteolytic activity. This paper reports the electrophoretic characterization of two sets of acidic extracellular proteases produced by submerged cultures of P. chrysosporium. The protease activity seen on day 2 of incubation, during primary growth when nitrogen levels are not known to be limiting, consisted of at least six proteolytic bands ranging in size from 82 to 22 kDa. The activity of this primary protease was strongly reduced in the presence of SDS. Following the day 2, when nitrogen levels are known to become limiting and cultures become ligninolytic, the main protease activity (secondary protease) consisted of a major proteolytic band of 76 kDa and a minor band of 25 kDa. The major and minor secondary protease activities were inhibited by phenylmethylsulfonyl fluoride and pepstatin A, respectively. When cultures were grown in the presence of excess nitrogen (non-ligninolytic condition), the primary protease remained the principal protease throughout the culture period. These results identify and characterize a specific proteolytic activity associated with conditions that promote lignin degradation. 相似文献
3.
Summary A simple and efficient medium for callus tissue culture from garlic to obtain maximal proteolytic activity is described. Murashige
and Skoog basal medium supplemented with 4.44 μM naphthaleneacetic acid (NAA) and 0.54 μM benzyladenine (BA) resulted in the best biomass production and protease expression. The protease activity belongs to the
class of cysteine proteases since they are inhibited by E64 and Leupeptin and also they are activated by 2-mercaptoethanol and cysteine. They showed good thermal stability. Three active
protease bands were found in zymograms of Allium sativum. The in vitro system revealed a significantly higher protease level than storage and embryo tissues of in vivo bulbs. 相似文献
4.
Although host proteases are often considered to have a negative impact upon heterologous protein production by filamentous
fungi, relatively little is known about the pattern of their appearance in recombinant fungal bioprocesses. In the present
study, we investigated extracellular proteases from a filamentous fungus, Aspergillus niger B1-D, genetically modified to secrete hen egg white lysozyme (HEWL). Our findings indicate that extracellular protease activity
is only detected after the carbon source is completely utilised in batch cultures. The proteases are predominantly acid proteases
and have optimal temperature for activity at around 45°C. Their activity could be partially inhibited by protease inhibitors,
indicating the existence of at least four kinds of proteases in these culture fluids, aspartic-, serine-, cysteine-, and metallo-proteases.
Oxygen enrichment does not have any noticeable effects on extracellular protease activity except that the onset of protease
activity appears earlier in oxygen enrichment runs. Oxygen enrichment stimulates HEWL production substantially, and we propose
that it is related to fungal morphology. Thermal stress imposed by raising process temperature (from 25 to 30 and 35°C) in
early exponential phase, led to appearance of protease activity in the medium following the heat shock. Continued cultivation
at high temperatures significantly reduced HEWL production, which was associated with increased activity of the extracellular
proteases in these cultures. 相似文献
5.
Jacques Labarère 《Archives of microbiology》1980,124(2-3):269-274
In Podospora anserina five proteolytic enzymes were characterized by chromatographic procedures. Three of these (proteases A, B and C) were found in the cell extracts of growing cultures and the other two (proteases III and IV) were revealed by studies on protoplasmic incompatibility. During growth, only protease C, an acidic enzyme, was active in crude extracts. From the stationary and the poststationary stages this activity decreased and finally disappeared, whereas a neutral serine protease (activity B) became active in crude extracts. A close relationship was observed between the proteolytic activity of the culture filtrates and the intracellular protease(s) concomitantly active in the crude extracts. None of the proteases associated with protoplasmic incompatibility was detected, both in the extra- and intracellular spaces. Qualitative variations in the proteolytic activities during stationary and post-stationary stages depended on the presence of specific genes and mutations: the mod C mutation suppressing protoplasmic incompatibility, inhibits the progressive decrease of protease C and, furthermore, the presence of non allelic incompatibility genes have for consequence the substitution of serine protease B by serine protease A during the poststationary stage. 相似文献
6.
Micromonospora echinospora differentiates in both submerged and surface cultures producing abundant dark spores after a period of vegetative mycelial
growth. In submerged batch cultures, under either carbon or nitrogen limiting conditions, protease activity was found to coincide
with sporulation indicating a relationship between proteolytic activity and differentiation in this organism. Further evidence
for this link was provided from surface grown cultures wherein sporulation was inhibited by the serine protease inhibitors
TLCK and TPCK. The association between proteolysis and differentiation apparent in this organism correlates with evidence
of a similar phenomenon observed in the streptomycetes, suggesting that this may be a common response associated with differentiation
in filamentous actinomycetes. 相似文献
7.
Juan Giarrizzo José Bubis Antonieta Taddei 《World journal of microbiology & biotechnology》2007,23(4):553-558
Streptomyces violaceoruber produces two different classes of mycelium, the substrate and the aerial mycelium. Since proteases have been associated with
morphological turnover processes in other Streptomyces species, the presence of excretory/secretory proteolytic activities was investigated here in S. violaceoruber culture supernatants. Various polypeptide bands, with apparent molecular masses ranging from 40 to 180 kDa, were detected
in soy trypticase broth (STB) culture media supernatants following 72 h of growth, using Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE). Zymograms showed the presence of five proteolytic enzymes (Spvio1–5), which migrated as bands
of 167.7, 130.7, 110.7, 48.3 and 40.9 kDa, respectively. The characterization of these proteases by specific inhibitors showed
that Spvio1–4 belong to the serine protease group and Spvio5 corresponds to a cysteine protease. Additionally, Spvio2 and
5 were inhibited by a mixture of EDTA and EGTA, indicating that both require divalent cations. The protease pattern obtained
in STB enriched with glucose was identical to that obtained in STB. However, Spvio3 and 4 were absent when nitrogen was added
to the culture medium. Cell death was fluorescently detected following 72 h of S. violaceoruber growth in STB and in STB that was enriched with glucose. On the contrary, no cell death was detected in nitrogen-enriched
STB media. Additionally, the formation of the aerial mycelium was impaired in solid cultures of STB media enriched with nitrogen.
These results demonstrate that the composition of the media influences the morphological turnover of the colony and the pattern
of excreted/secreted proteases from S. violaceoruber, and suggest that Spvio3 and 4 are involved in the aerial mycelium formation. 相似文献
8.
A general activity probe was synthesized and applied to the supernatant of a filamentous fungus, Ophiostoma, culture to identify directly the secreted serine proteases by covalent enzyme labeling. The activity probe contained a chemically
reactive group that reacted with, and thus covalently labeled, the serine residues of only active proteases and not heat-inactivated
proteases. The activity probe also contained a fluorescent group that allowed for the subsequent visualization of the captured
proteases in 1-D gels and their identification by N-terminal sequencing. This use of a chemical probe led to the rapid discovery of subtilisin-like serine protease of Ophiostoma. Two hypothetical proteins were also captured, with one being a probable endopeptidase K type of protease. 相似文献
9.
Summary The production of extracellular alkaline proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on casein pH 9.5 at 37 °C. The highest alkaline proteolytic activity (38 U/ml) was verified for culture medium containing glucose and casein at 1% (w/v) as substrates, obtained from cultures developed at 25 °C for 6 days. Cultures developed in Vogel medium with glucose at 2% (w/v) and 0.2% (w/v) NH4NO3 showed higher proteolytic activity (27 U/ml) when compared to the cultures with 1% of the same sugar. Optimum temperature was 40 °C and the half-lives at 40, 45 and 50 °C were 90, 25 and 18 min, respectively. Optimum pH of enzymatic activity was 9.5 and the enzyme was stable from pH 6.0 to 12.0. 相似文献
10.
Tremacoldi C.R. Watanabe N.K. Carmona E.C. 《World journal of microbiology & biotechnology》2004,20(6):639-642
The production of extracellular acid proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three
different temperatures during 10 days. The proteolytic activity was determined on haemoglobin pH 5.0 at 37 °C. The highest
acid proteolytic activity (80 U/ml) was observed in culture medium containing glucose and gelatin at 1%(w/v) at 30 °C at the
third day of incubation. Cultures developed in Vogel medium with glucose at 2%(w/v) showed at about 45% of proteolytic activity
when compared to the cultures with 1% of the same sugar. The optimum pH of enzymatic activity was 2.0 and the enzyme was stable
at pH values ranging from 2.0 to 4.0. The optimum temperature was 40 °C and the half-lives at 40, 45 and 50 °C were 30, 10
and 5 min, respectively.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
11.
Morphological changes of a steroid transforming filamentous fungus Rhizopus nigricans were studied by altering submerged growth conditions at inoculum sizes previously determined to favor pelleted growth. Beside
the main classification between pelleted and clumpy growth forms, the size, concentration and structure of pellets were characterized
at different cultivating temperature, initial pH value, medium composition, additives, and aeration conditions. Initial pH
below 4 and above 7, the presence of Ca2+ and Tween-80 gave rise to the clumpy growth, otherwise pelleted growth prevailed. Among tested factors the pellet size was
mainly influenced by the inoculum size and the presence of baffles and Ca2+ in cultivation medium. The formation of smooth pellets, prerequisite for further application in the process of steroid biotransformation,
resulted in cultivations at lower temperature, high agitation rates in shaken cultures without baffles and at high nitrogen
concentration.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
12.
The insect cell line BTI-TN-5B1-4 (High Five) is frequently used to express recombinant proteins in large amounts using the baculovirus expression system. However, extensive proteolytic degradation of recombinant proteins is often encountered. Furthermore, we have observed that recombinant proteins migrate in SDS-PAGE in agreement with poly-ubiquitinated forms of the protein, suggesting a ubiquitin/proteasome degradation pathway. Here, we describe a systematic study unraveling the effect of adding proteasome inhibitors or specific protease inhibitors to the growth medium of High Five insect cells infected with recombinant baculovirus. Furthermore, protease inhibitors were added to the lysis buffer to establish the most efficient way to inhibit proteolytic activity after lysis of baculovirus-infected cells expressing recombinant proteins. We conclude that a combination of adding protease inhibitors to the growth medium and to the lysis buffer minimizes the proteolytic activity in High Five cells. The most efficient protease inhibitors were E-64 in the growth medium together with Leupeptin in the lysis buffer at concentrations higher than with available cocktails of inhibitors. The optimal treatment of High Five cells is different from the optimal treatment of Sf9 cells. For proteins susceptible to ubiquitinylation, a treatment of insect cell cultures with the proteasome inhibitor MG132 (LLL) leads to a considerable reduction of the yield of production of recombinant protein. 相似文献
13.
Jean-Christophe Vuillemard Sylvie Terré Stéphane Benoit Jean Amiot 《Applied microbiology and biotechnology》1988,27(5-6):423-431
Summary
Serratia marcescens and Myxococcus xanthus cells were immobilized in calcium alginate gel beads. Immobilization under various conditions had no effect on the extracellular proteolytic activity of S. marcescens cells. Protease production seemed rather to depend on the free cells in the medium. However, the stability over time of enzyme production was enhanced, as immobilization increased protease production half-life from 5 to 12 days. On the other hand, Myxococcus xanthus produced proteases inside the gel beads which could diffuse into the medium. The proteolytic activity increased as a function of the initial cell content of the beads and of the bead inoculum. Compared to free cells, immobilized cells of Myxococcus xanthus could produce 8 times more proteolytic activity, with a very low free-cell concentration in the medium. 相似文献
14.
M. Vidyasagar S. Prakash S. K. Jayalakshmi K. Sreeramulu 《World journal of microbiology & biotechnology》2007,23(5):655-662
An extremely halophilic Chromohalobacter sp. TVSP101 was isolated from solar salterns and screened for the production of extracellular halothermophilic protease.
Identification of the bacterium was done based upon biochemical tests and the 16S rRNA sequence. The partially purified enzyme
displayed maximum activity at pH 8 and required 4.5 M of NaCl for optimum proteolytic activity. In addition, this enzyme was
thermophilic and active in broad range of temperature 60–80°C with 80°C as optimum. The Chromohalobacter sp. required 4 M NaCl for its optimum growth and protease secretion and no growth was observed below 1 M of NaCl. The initial
pH of the medium for growth and enzyme production was in the range 7.0–8.0 with optimum at pH 7.2. Various cations at 1 mM
concentration in the growth medium had no significant effect in enhancing the growth and enzyme production but 0.5 M MgCl2 concentration enhanced enzyme production. Casein or skim milk powder 1% (w/v) along with 1% peptone proved to be the best
nitrogen sources for maximum biomass and enzyme production. The carbon sources glucose and glycerol repressed the protease
secretion. Immobilization of whole cells in absence of NaCl proved to be useful for continuous production of halophilic protease. 相似文献
15.
The culture liquids of three Xanthomonas campestris pv. campestris strains were found to possess proteolytic activity. The culture liquid of strain B611 with the highest proteolytic activity was fractionated by salting-out with ammonium sulfate, gel filtration, and ion-exchange chromatography. The electrophoretic analysis of active fractions showed the presence of two proteases in the culture liquid of strain B611, the major of which was serine protease. The treatment of cabbage seedlings with the proteases augmented the activity of peroxidase in the cabbage roots by 28%. 相似文献
16.
Summary Biotechnology has become an important tool to produce plant secondary metabolites and proteases are among them. Although pineapple
plants have been found to produce proteases, most of the biotechnological investigations on this crop have been focused on
propagation. The procedure involving the use of temporary immersion bioreactors is one of the most outstanding because of
its high multiplication rate. We previously recorded specific protease activity in the culture medium during the pre-elongation
step of this protocol. Therefore we decided to modify this phase, looking for an increase of protease excretion. Three independent
experiments were performed to evaluate the effects of culture duration, and levels of gibberellic acid (GA) and 6-benzyladenine
(BA). The following indicators were recorded: shoot fresh mass per bioreactor; and protein concentration, proteolytic activity,
and specific protease activity in culture media. As happens in investigations focused on protease production, the specific
protease activity was the most important indicator recorded here. It maximized at 21 d of culture. Moreover, GA (4.2 μM) increased specific activity in the culture medium while BA produced a negative effect. Results shown here demonstrate that
conditions adquate for propagation purposes (15-d pre-elongation phase; 2.8 μM GA; 2.2 μM BA) are not necessarily adequate for protease excretion. 相似文献
17.
Margaret E. Katz Pam K. Flynn Patricia A. vanKuyk Brian F. Cheetham 《Molecular & general genetics : MGG》1996,250(6):715-724
The extracellular proteases ofAspergillus nidulans are known to be regulated by carbon, nitrogen and sulphur metabolite repression. In this study, a mutant with reduced levels of extracellular protease was isolated by screening for loss of halo production on milk plates. Genetic analysis of the mutant showed that it contains a single, recessive mutation, in a gene which we have designatedxprE, located on chromosome VI. ThexprE1 mutation affected the production of extracellular proteases in response to carbon, nitrogen and, to a lesser extent, sulphur limitation. Three reversion mutations,xprF1, xprF2 andxprG1, which suppressxprE1, were characterised. BothxprF andxprG map to chromosome VII but the two genes are unlinked. ThexprF1, xprF2 andxprG1 mutants showed high levels of milk-clearing activity on medium containing milk as a carbon source but reduced growth on a number of nitrogen sources. Evidence is presented that thexprE1 andxprG1 mutations alter expression of more than one protease and affect levels of alkaline protease gene mRNA. 相似文献
18.
Fábio M. Maciel Cristiane M. C. Salles Claudio A. Retamal Valdirene M. Gomes Olga L. T. Machado 《Acta Physiologiae Plantarum》2011,33(5):1867-1875
Jasmonates are signaling molecules that play key roles in wound response and regulate the biosynthesis of many defensive proteins,
including proteases. In this study, we investigate the effects of wounding and methyl jasmonate (MJ) application on the protein
expression pattern of Ricinus communis L. leaves and on proteolytic activity. Gelatin zymography demonstrated that both MJ and mechanical wounding induce alterations
in the proteolytic pattern of castor bean leaves (R. communis L.). Expression of two cysteine proteases (38 and 29 kDa) was induced by the treatments employed; however, MJ induced a higher
protease level than mechanical wounding during the stress period (24, 48, and 72 h). The increase in protease activity mirrors
the decline in soluble protein content and rubisco degradation that may indicate initiation of senescence in castor plants.
The 29 kDa protease has an acidic optimal pH; whereas the 38 kDa protease has a neutral optimum activity. Both proteases were
almost completely inhibited by E-64 and cystatin. The significant induction of these proteins by MJ suggests a possible role
of cysteine proteases in leaf senescence as well as their involvement in regulating both the wound response and MJ in castor
bean plants. 相似文献
19.
Purification and characterization of proteases from developing normal maize endosperm and high lysine opaque-2 maize endosperm
have been carried out with a view to understand their role in storage protein modification. At day 15, normal maize endosperm
had two types of proteolytic enzymes, namely, protease I and protease II, while at day 25 protease n disappeared and in place
protease III appeared. However, in opaque-2 maize endosperm at both the stages only one type of enzyme (protease I) was present.
These proteases had many properties in common-optimum pH and temperature were respectively, 5.7and 40°C; their activity was
inhibited to the extent of 75 –93 % by p-chloromercuribenzoate; trypsin inhibitor inhibited the activity more at early stages
of endosperm development; all proteases cleaved synthetic substrates p-tosyl-L-arginine methylesler and N-benzoyl-L-tyrosine
ethyl ester and poly-L-glutamic acid. TheKm values of day 15 and 25 normal maize endosperm proteases ranged from 2.73–3.30, while for opaque-2 maize endosperm protease
I it was 3.33 mg azocasein per ml assay medium. These enzymes, however, differed with respect to proteolytic activity towards
poly-L-lysine. Only normal maize endosperm protease III at day 25 followed by protease II at day 15 showed high activity towards
this homopolypeptide suggesting thereby their role in determining the quality of normal maize endosperm protein.
Part of Ph.D. thesis submitted by the first author 相似文献
20.
Sarita W. Nazareth Judith D. Sampy 《International biodeterioration & biodegradation》2003,52(4):207-214
This study on the lignocellulases in broth cultures of the basidiomycete Panus tigrinus indicates that laccase and xylanase enzymes are constitutive and cellulase is inducible. In stationary culture at 28°C, the greatest laccase and xylanase activity was observed after growth for approximately nine days. Laccase production was dependent on the presence, and the particular brand, of malt extract in the growth medium. While production of laccase was enhanced by growth at 37°C and 42°C, xylanase was not. Raising the pH of the growth medium from pH 5.6 to pH 7.0 did not affect xylanase production, but laccase production was reduced at the higher pH. In shake culture, growth was pelleted and biomass lower than in stationary culture, and synthesis of both enzymes was strongly inhibited. Cultures of P. tigrinus decolourised Poly R-478 and the toxic triphenyl methane dye, crystal violet. It was also shown to degrade a natural lignocellulosic waste, sawdust. 相似文献