Total Extracellular Protein Research Articles

Injury-induced enzymatic methylation of aging collagen in the extracellular matrix of blood vessels.

As a result of blood vessel injury, protein D-aspartyl/L-isoaspartyl carboxyl methyltransferase (PIMT), a normally intracellular enzyme, becomes trapped within the meshwork of the vascular extracellular matrix where it can methylate substrate proteins. In this investigation we examined the distribution of such altered aspartyl-containing substrate proteins in the vascular wall. Nearly 90% of all the altered aspartyl residues were inaccessible to intracellular PIMT. Proteins of the extracellular matrix were found to be the major repository of altered aspartyl-containing polypeptides in the blood vessel wall, accounting for approximately 70% of the total amount. Proteolytic cleavage of extracellular matrix proteins with cyanogen bromide (CNBr) revealed that collagens account for most of the altered aspartyl-containing proteins of the ECM. As a consequence of blood vessel injury, both type I and type III collagen along with other proteins were found to become methylated by injury-released PIMT. It is estimated that 1 cm of vein contains on the order of 5 x 10(14) altered aspartyl residues involving between 1% and 5% of the total extracellular protein.

A perfusion air-lift bioreactor for high density plant cell cultivation and secreted protein production

A new bioreactor design that allows continuous perfusion cultivation of plant cell suspensions is described in this paper. This design incorporates an internal cell settling zone with an external-loop air-lift bioreactor. The settling zone is created by inserting a baffle plate into the upper portion of the downcomer. Using this bioreactor, Anchusa officinalis suspension culture was cultivated to a cell density of 27.2 g l −1 DW in 14 days at a perfusion rate of 0.123 per day. The maximum total extracellular protein concentration attained 1.11 g l −1. Complete cell retention was achieved throughout the culture during which the maximum packed cell volume (PCV) exceeded 80%. In comparison, the maximum cell density and extracellular protein concentration in the batch culture were 12.6 g l −1 DW and 0.47 g l −1, respectively. SDS-PAGE of the extracellular protein samples revealed two major bands at 58 and 47 kDa, each accounted for approximately 45% of the total secreted proteins.

Efficient flotation of yeast cells grown in batch culture

A fast flotation assay was used to select new floating yeast strains. The flotation ability did not seem to be directly correlated to total extracellular protein concentration of the culture. However, the hydrophobicity of the cell was definitely correlated to the flotation capacity. The Saccharomyces strains (FLT strains) were highly hydrophobic and showed an excellent flotation performance in batch cultures without additives (flotation agents) and with no need for a special flotation chamber or flotation column. A stable and well-organized structure was evident in the dried foam as shown by scanning electron microscopy which revealed its unique structure showing mummified cells (dehydrated) attached to each other. The attachment among the cells and the high protein concentration of the foams indicated that proteins might be involved in the foam formation. The floating strains (strains FLT) which were not flocculent and showed no tendency to aggregate, were capable of growing and producing ethanol in a synthetic medium containing high glucose concentration as a carbon source. The phenomenon responsible for flotation seems to be quite different from the flocculation phenomenon.

Overproduction of Serratia marcescens metalloprotease (SMP) from the recombinant Serratia marcescens strains

To overproduce Serratia marcescens metalloprotease(SMP), various recombinant plasmids encoding SMP gene were constructed and the SMP productivities from the recombinant S. marcescens strains were examined. The recombinant S. marcescens strains indispensably required proteinaceous substrates such as casein for the extracellular production of SMP. We obtained maximum 9,100U/ml of SMP from the culture supernatant of S. marcescens ATCC27117 containing a regulatory plasmid pTSP2 encoding SMP gene fused with a strong trc99a promoter and its repressor gene lacIq, which is about 23 times higher than that of wild type strain. SMP produced from the recombinant S. marcescens(pTSP2) was 88.3% of total extracellular proteins.

High-level production of recombinant glutamic acid-specific protease from Bacilllus licheniformis in Bacillus subtilis expression system

To obtain large quantities of glutamic acid-specific protease isolated originally from Bacillus licheniformis (BLase), an expression plasmid was constructed by inserting the BLase gene into a plasmid vector (pUB110) for Bacillus subtilis. B. subtilis strain ISW1214 harboring the resultant recombinant plasmid containing the coding and 5′-promoter and 3′-terminator regions of BLase gene secreted approximately 0.25 g/ l of BLase in a culture medium contained in a 90- l jar fermentor, corresponding to nearly 10 times the natural production level and resulting in a stable large-scale production. The amount of BLase in the culture medium accounted for roughly 60% of the total extracellular proteins secreted from the recombinant strain, simplifying enzyme purification.

Regulation of β-xylosidase formation by xylose in Trichoderma reesei

The soft-rot fungus Trichoderma reesei forms β-xylosidase (EC 3.2.1.37) activity during cultivation on xylan and xylose, but not on glucose. When mycelia precultivated on glycerol were washed and transferred to fresh medium without a carbon and nitrogen source, β-xylosidase formation was induced by xylan, xylobiose and xylose. A supply of 4 mm xylose and a pH of 2.5 provided optimal conditions for induction. β-Xylosidase accounted for the major portion of total extracellular protein under these conditions, and could be purified to physical homogeneity by a single anion exchange chromatography step. A recombinant strain of T. reesei that carries multiple copies of the homologous xylanase II-encoding gene has a six-fold increased xylanase activity, but forms comparable β-xylosidase activities. This shows that the rate of xylan hydrolysis has no effect on the induction of β-xylosidase. Methyl-β-d-xyloside inhibited β-xylosidase competitively and was a weak β-xylosidase inducer. The induction by xylobiose and xylan was strongly enhanced by the simultaneous addition of methyl-β-d-xylosidese and xylan or xylobiose. The results suggest that a slow supply of xylose is a trigger for β-xylosidase induction.

The relationship between phenol oxidase activity, soluble protein and ergosterol with growth of Lentinus species in oak sawdust logs

The growth of four strains of the shiitake mushroom Lentinus edodes in solid substrate fermentation in synthetic oak sawdust logs was studied over a 14-week period. Total extracellular phenol oxidase activity and soluble protein were monitored and biomass estimated as the ergosterol content of the fermented sawdust. It was observed that two of the strains had a similar pattern of phenol oxidase activity with two cycles with maxima at 2 and 8 weeks of mycelial growth prior to fruiting. With the other two strains there was a maximum at week 4. For each strain, phenol oxidase activity increased with the cold shock used to induce fruiting. Phenol oxidase activity was not found to be correlated with either soluble protein or total fungal biomass in the fermented sawdust, which were correlated for each strain. Quantification of biomass from submerged liquid culture on the basis of dry weight and ergosterol contents showed that the strains fell into the same two groups with respect to the ergosterol to biomass ratio, which was markedly lower than that for a strain of L. lepideus.

Purification and characterization of a chitosanase from Streptomyces N174

A highly efficient chitosanase producer, the actinomycete N174, identified by chemotaxonomic methods as belonging to the genus Streptomyces was isolated from soil. Chitosanase production by N174 was inducible by chitosan or d-glucosamine. In culture filtrates the chitosanase accounted for 50–60% of total extracellular proteins. The chitosanase was purified by polyacrylic acid precipitation, CM-Sepharose and gel permeation chromatography. The maximum velocity of chitosan degradation was obtained at 65° C when the pH was maintained at 5.5. The enzyme degraded chitosans with a range of acetylation degrees from 1 to 60% but not chitin or CM-cellulose. The enzyme showed an endo-splitting type of activity and the end-product of chitosan degradation contained a mixture of dimers and trimers of d-glucosamine.

Functional half life of the α-amylase mRNA ofBacillus amyloliquefaciens

The functional half life of α-amylase mRNA was investigated inBacillus amyloliquefaciens CCM 3502. The amount of mRNA was followed indirectly: the amount of protein translated by preformed mRNA was measured after transcription was terminated by rifampicin. The radioactivity of14C-leucine-labeled extracellular protein produced by drug-treated cells was a measure of the quantity of α-amylase, because this amount formed, in standard experiments, at least 90% of the total extracellular proteins. The effect of rifampicin and chloramphenicol was studied at the level of mRNA transcription and translation. Negligible radio-activity of the extracellular protein produced by cells after the addition of chloramphenicol (200 μg·ml−1) proved that α-amylase is formed de novo, and no form of preformed enzyme is secreted. From the radioactivity of extracellular proteins synthesized after the inhibition of mRNA by rifampicin, the half life of α-amylase mRNA was calculated to be 26.9 min at 30°C. The half life of mRNA for cellular proteins was calculated to be 2.9 min.

Characteristics of batch culture of a recombinantbacillus brevis excreting a foreign esterase

The productivity of extracellular enzyme was evaluated in batch culture using a protein hyperexcreting host, Bacillus brevis HPD31(5) harboring pHSC131, which carried a gene (est) encoding esterase activity from Bacillus stearother mophilus. Optimum temperature and pH for the bacterial growth and the production of extracellular esterase were found to be 35 degrees C and pH 6.5, by using the standard medium (GPY) containing neomycin as a selective pressure, Under the cultivation condition employed, cell growth reached 5 g dry cell weight/L, while the extracellular esterase activity amounted to 4.5 U/mL. Most (79%-92%) of the esterase produced was excreted into the medium. pHSC131 was stably retained in the host cell during cultivation in the presence of neomycin. However, in the absence of neomycin, the plasmid was completely lost from the host after 12-h cultivation accompanied by decreases in both esterase activity and production of total extracellular protein. The copy number of the plasmid was estimated to be approximately 7 throughout the cultivation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the excreted proteins showed the presence of a protein having an apparent molecular weight of 32,000, which equals to the value predicted from the DNA sequence of the est gene.

Effect of phosphate on antibiotic and extracellular protein production by Myxococcus coralloides D.

The effect of inorganic phosphate concentrations on antibiotic and extracellular protein production by Myxococcus coralloides D have been examined. Antibiotic production by growing cells of this myxobacterium was maximal at phosphate concentrations of 10-20 mM, but was inhibited by concentrations higher than 20 mM. The total extracellular protein and the extracellular protein per cell ratio were independent of phosphate levels in the culture broth.

Substrate specificity, de novo synthesis and partial purification of polyphenol oxidase derived from the wood-decay fungus,Coriolus versicolor

Coriolus versicolor, a white-rot Basidiomycete, secretes cellulolytic and ligninolytic enzymes as well as polyphenol oxidase (PPO). Whereas the former degrade wood polymers, the latter can convert diphenols to diquinones and oligomerize syringic acid, a lignin derivative. Certain phenolic compounds can serve as disease-resistance factors controlling the proliferation of wood-decay fungi within host tissues. BecauseC. vesicolor can be ‘batch-cultured’, overproduction and enhanced secretion of enzymes of biological and commercial interests are feasible. Reported here are the results of attempts to define the timed appearances of intracellular and extracellular PPO, to assess substrate specificity as well as distinguish synthesis versus activation of intracellular PPO and to partially purify extracellular PPO. These efforts were to provide data enabling cell-free synthesis of PPO, cloning of the gene(s) for the oxidase and the establishment of its subcellular route of secretion. Whereas two protein peaks (6 and 12 days in a 16 day time-course) were observed for dialyzed mycelial homogenates, the homogenates' PPO specific activity rose between 4 and 12 days and then declined. Total extracellular protein content climbed from 6 to 15 days for dialyzed growth medium and the medium's PPO specific activity rose at 4 days post-inoculation and except at 9 days increased linearly to 15 days. When aliquots of dialyzed 12 and 15 day media were added to PPO assay mixtures containing catechol and either syringic or gallic acids, statistically significant differences in PPO specific activity between phenolic substrates were noted. Supplementation of cultures with 1.91 μg cycloheximide ml growth medium−1 (control, growth medium only) together with 0.5 μCi [14C]-leucine revealed that cycloheximide inhibited PPO activity and suppressed [14C]-leucine incorporation into TCA-insoluble cytoplasmic protein. As for PPO partial purification, growth medium dialysis followed by 0–30% (NH4)2SO4 fractionation and subsequent 12 000×g dialyzate centrifugation yielded a 3.27-fold enhancement in PPO specific activity within the 12 000×g supernatant. Chromatography of the latter upon DEAE-Sephadex indicated that PPO exchanged with the DEAE counterion as it could be eluted with high ionic strength salt. These results suggest that: the occurrences of intracellular and extracellular PPO are time-dependent, intracellular PPO is de novo synthesized, the preferred substrate for extracellular PPO appears to be catechol and extracellular PPO can be partially purified by a combination of dialysis and ammonium sulfate fractionation as well as possibly DEAE chromatography and/or Sephadex G-150 gel filtration.

Effects of tetracyclines on the production of extracellular proteins by members of the propionibacteriaceae.

The effect of tetracyclines on the synthesis of proteins in Propionibacterium avidum and P. acnes was examined. Synthesis of an extracellular lipase by P. avidum was slightly more sensitive to inhibition by tetracycline than total (cellular and extracellular) protein synthesis. The effect of tetracycline and other analogues on the synthesis of secreted proteins was also examined in P. avidum and P. acnes by protein radiolabelling experiments. In all cases the synthesis of secreted proteins was only about 2-fold more sensitive to inhibition by tetracyclines than total protein synthesis. These results contrast with previously published findings in Escherichia coli which show that synthesis of secreted proteins is highly susceptible to inhibition by tetracycline. The implications of these findings in relation to inhibition of membrane bound ribosomes by tetracyclines are discussed.

Effects of tetracyclines on the production of extracellular proteins by members of the propionibacteriaceae

The effect of tetracyclines on the synthesis of proteins in Propionibacterium avidum and P. acnes was examined. Synthesis of an extracellular lipase by P. avidum was slightly more sensitive to inhibition by tetracycline than total (cellular and extracellular) protein synthesis. The effect of tetracycline and other analogues on the synthesis of secreted proteins was also examined in P. avidum and P. acnes by protein radiolabelling experiments. In all cases the synthesis of secreted proteins was only about 2-fold more sensitive to inhibition by tetracyclines than total protein synthesis. These results contrast with previously published findings in Escherichia coli which show that synthesis of secreted proteins is highly susceptible to inhibition by tetracycline. The implications of these findings in relation to inhibition of membrane bound ribosomes by tetracyclines are discussed.

Regulation of extracellular proteins and α-amylase secretion by temperature inBacillus subtilis

alpha-Amylase was found to be the main protein secreted by Bacillus subtilis, corresponding to 90, 87 and 60% of total extracellular proteins at 30, 40 and 45 degrees C, respectively. A change in temperature can affect the pattern of proteins secreted as detected by gel electrophoresis. 14C-Leucine incorporation into extracellular proteins and their proportion at the end of the growth phase was higher at 30 degrees C than that at 40 or 45 degrees C. The effect of temperature on alpha-amylase synthesis as determined by its enzymic activity and on the extracellular protein synthesis followed a similar pattern.

Optically monitoring Baker's yeast (Saccharomyces cerevisiae) growing in an air-fluidized/expanded potato starch matrix

In order to study cell behavior in solid fermentation processes, model systems using gelatin and starch have been developed to track Baker's yeast growth. The difficulty in estimating the cell concentration within solid materials arises because both the solid material and the cellular material contribute to the measurement (such as optical resistance). In general, however, the two materials cannot be easily separated, hence the need to measure the cells along with the solid supporting material. A simple spectrophotometric method has previously been shown to work well in both aerated submerged batch cultures and aerated static solid cultures. The optical approach is applied here to monitor a more complex solidified system: cell growth in a novel air-fluidized/expanded bed of yeast growing on a starch matrix. Conventional assays for reducing sugar, total extracellular protein, and extracellular lysine were also applied to monitor yeast behavior in this new system.

Lack of pleiotropic compensation in extracellular protein production by hypoproducing variants of Staphylococcus simulans biovar staphylolyticus.

The changes in bacterial density, total extracellular protein and activities of three extracellular enzymes were monitored during growth of wild-type Staphylococcus simulans biovar staphylolyticus, a representative pleiotropic variant that produced decreased levels of the three extracellular enzymes, and a variant that produced only 5 of the 14 extracellular proteins secreted by the wild-type organism. Both variants produced less total extracellular protein than did the parental organism. SDS-PAGE of the proteins secreted by these hypoproducing variants showed that all of the extracellular proteins were produced in decreased amounts. No pleiotropic compensation in extracellular protein production was observed for these hypoproducing variants of S. simulans biovar staphylolyticus.

Color Variants of Aureobasidium pullulans Overproduce Xylanase with Extremely High Specific Activity

Xylanase activity from naturally occurring color variants of Aureobasidium pullulans was associated with extracellular monomeric proteins of 20 to 21 kilodaltons. Xylanase represented nearly half the total extracellular protein, with a yield of up to 0.3 g of xylanase per liter. The specific activity of partially purified xylanase exceeded 2,000 IU/mg. Xylanase from typically pigmented strains appeared similar to that from color variants with respect to molecular weight, pH and temperature optima, and specific activity of purified (but not crude) enzyme. However, xylanase from typical strains made up only about 1.0% of total extracellular protein. Xylanase from strains of Cryptococcus albidus was associated with abundant proteins of about 43 kilodaltons and showed much lower specific activity.

Protein complexity of central nervous system cell lines

The total extracellular proteins and the most abundant 960 intracellular proteins of clonal CNS nerve and glial cell lines were examined by quantitative 2-dimensional acrylamide gel electrophoresis. While less than 0.2% of the intracellular proteins differ among the 5 nerve and 4 glial cell lines studied, over 65% of the extracellular proteins vary in distribution between the 2 major classes of CNS cells. These data indicate that the phenotypic complexity of nerve and glia populations is similar and that most of the protein complexity is in extracellular molecules.

Pretranslational regulation of type I collagen, fibronectin, and a 50-kilodalton noncollagenous extracellular protein by dexamethasone in rat fibroblasts.

The effect of dexamethasone on the synthesis of total cellular and extracellular proteins and specifically on the synthesis of type I procollagen chains, fibronectin, and a 50-kDa extracellular noncollagenous polypeptide was examined in cultured rat dermal fibroblasts. A slight but consistent inhibition of total protein synthesis by dexamethasone was dose and time dependent. Treatment of cells with 1 microM dexamethasone for 24 h while abolishing procollagen synthesis nearly completely (less than 95%) had the opposite effect (5-7-fold increase) on the synthesis of an extracellular noncollagenous 50-kDa polypeptide. Dexamethasone did not significantly affect the rates of synthesis of fibronectin. Cell-free translation of mRNA from dexamethasone-treated cells revealed corresponding changes in the steady-state levels of functional mRNAs coding for procollagens, the 50-kDa polypeptide, and fibronectin. Northern blot hybridization using nick-translated cDNA plasmids coding for pro-alpha 1(I), fibronectin, and cytoplasmic beta-actin mRNA corroborated the data obtained from cell-free translation experiments. Run-off transcription assays using nuclei from cells treated with 1 microM dexamethasone for 24 h revealed that glucocorticoid treatment did not significantly affect the rate of transcription of type I collagen genes; similarly, the rate of transcription of fibronectin and cytoplasmic beta-actin genes also remained unchanged under these conditions. An analysis of the kinetics of decay of radiolabeled mRNA coding for pro-alpha 1(I), pro-alpha 2(I), and fibronectin in dexamethasone-treated cells revealed that procollagen mRNAs were turned over at an accelerated rate in glucocorticoid-treated cells. These data suggest that dexamethasone regulates type I collagen gene expression by preferentially decreasing the stability of pro-alpha 1(I) and pro-alpha2(I) mRNAs. Although dexamethasone increased the levels of translatable mRNAs coding for a 50-kDa polypeptide, the molecular mechanism(s) of how hormone exerts this effect remains unknown.