Production Of Aminopeptidase Research Articles

The response of litter decomposition to phosphorus addition in typical temperate grassland in Inner Mongolia

The productivity of terrestrial ecosystems is limited by soil fertility, such assoil phosphorus (P). The decomposition of litter is the main process that affects nutrient cycling. To understand the characteristics of litter decomposition and nutrient release under P addition, we conducted a 2-year litter bag experiment at P addition rates (0–12.5 g P m−2 yr−1). All litter decomposed faster during the second growing season due to highly concentrated precipitation, and Stipa grandis litter decomposed faster (1.02–1.63 times) than Leymus chinensis under P treatments. The decomposition of L. chinensis and mixed litter was more thoroughly at 1 g P m−2 yr−1, while S. grandis decomposed more under 5 g P m−2 yr−1. P addition promoted nitrogen (N) and P immobilization and all litter reached its maximum at 12.5 g P m−2 yr−1. Phosphorus addition increased β-glucosidase production at 5 g P m−2 yr−1, but leucine aminopeptidase production was promoted at high P. The ratio of leucine aminopeptidase to phosphatase increased over time, indicating the N deficiency and P saturation during late stages of decomposition. Our results show that Inner Mongolia's grasslands was limited by P availability and that precipitation was important in the region's decomposition processes.

Production of aminopeptidase from soybean meal with nutrients supplementation by Bacillus licheniformis SWJS33: Feasibility and metabolic process

Enhancing aminopeptidase fermentation using cheap substrate is very necessary for industrial production and application of aminopeptidase. The suitability of using raw soybean meal (SBM) as substrate for aminopeptidase production by Bacillus licheniformis SWJS33 in submerged fermentation was first evaluated. SBM alone provided both nitrogen and carbon sources for SWJS33 to produce aminopeptidase. Supplementation of 3 g/L glucose and 15 g/L yeast extract further improved the aminopeptidase yield by 0.63 and 1.3 folds, respectively. Addition of yeast extract altered the metabolic process of SWJS33, including shortening the lag phase, increasing the maximum specific cell growth rate and maximum specific aminopeptidase formation rate, and changing the utilization process of nutrients during the fermentation. What's more, SWJS33 secreted aminopeptidase prior to endoproteases when quick-release nitrogen (e.g. yeast extract) was supplied. High dissolved oxygen was demanded during aminopeptidase fermentation. The aminopeptidase production was successfully up scaled to 20 L fermenter.

Induction of Extracellular Aminopeptidase Production by Peptides in Some Marine Bacterial Species.

Bacterial extracellular aminopeptidases are key enzymes in protein processing in oligotrophic seawater. To the best of our knowledge, the regulation of aminopeptidase production in microbes inhabiting seawater has not yet been reported. The present study attempted to experimentally clarify which organic materials affect bacterial extracellular aminopeptidase production by nutrient-rich and starved cells growing in artificial seawater using Photobacterium, Alteromonas, Ruegeria, and Sulfitobacter. In all four species, we found that peptides induced bacterial extracellular aminopeptidase production. Amino acids led to cell growth with markedly lower aminopeptidase production by Photobacterium and Sulfitobacter, but not by Alteromonas and Ruegeria. These results suggest that the extracellular aminopeptidases of marine bacteria are primarily produced on demand in response to the presence of relevant substrates (peptides) in seawater. Peptidyl substances may be regulatory nutrients for marine bacterial growth in aquatic environments.

Construction of a highly active secretory expression system via an engineered dual promoter and a highly efficient signal peptide in Bacillus subtilis

A strong promoter and highly efficient signal peptides are essential for the secretory overproduction of recombinant proteins in Bacillus subtilis. To enhance the limited overexpression capability of natural promoters, various strategies for promoter engineering have been developed and used to construct gene expression systems in B. subtilis and other hosts. By applying a semi-rational approach for promoter engineering, a series of expression plasmids containing single and dual promoters were constructed using aminopeptidase (AP) with an intrinsic signal peptide as the reporter protein. Of the single and dual promoters investigated, the dual promoter PgsiB-PHpaII gave the best performance. To optimize secretion efficiency, the signal peptide YncM was selected after screening a library containing 19 different Sec-type signal peptides. The AP activity detected in the supernatants of a recombinant strain containing the plasmid pBSG24-YncM was as high as 88.86U/mL. The capacity of the expression plasmid pBSG24-YncM was also evaluated with batch fermentation in a 5-L fermentor. Increased production of AP (205U/mL, equal to 1.7g/L) was achieved after 45h of fermentation. These results suggest that this expression system can be used for high-level protein expression in B. subtilis.

Heterologous expression of Gaeumannomyces graminis lipoxygenase in Aspergillus nidulans.

Aspergillus sp. contain ppo genes coding for Ppo enzymes that produce oxylipins from polyunsaturated fatty acids. These oxylipins function as signal molecules in sporulation and influence the asexual to sexual ratio of Aspergillus sp. Fungi like Aspergillus nidulans and Aspergillus niger contain just ppo genes where the human pathogenic Aspergillus flavus and Aspergillus fumigatus contain ppo genes as well as lipoxygenases. Lipoxygenases catalyze the synthesis of oxylipins and are hypothesized to be involved in quorum-sensing abilities and invading plant tissue. In this study we used A. nidulans WG505 as an expression host to heterologously express Gaeumannomyces graminis lipoxygenase. The presence of the recombinant LOX induced phenotypic changes in A. nidulans transformants. Also, a proteomic analysis of an A. nidulans LOX producing strain indicated that the heterologous protein was degraded before its glycosylation in the secretory pathway. We observed that the presence of LOX induced the specific production of aminopeptidase Y that possibly degrades the G. graminis lipoxygenase intercellularly. Also the presence of the protein thioredoxin reductase suggests that the G. graminis lipoxygenase is actively repressed in A. nidulans.

Arginine specific aminopeptidase from Lactobacillus brevis

The proteolytic system of lactic acid bacteria contribute to the development of flavor during the ripening of cheese through the generation of short peptides and free amino acids, which directly or indirectly act as flavor precursors. Newly isolated lactic acid bacteria (LAB) as well as those procured from culture collection centers were screened for the production of various substrate specific aminopeptidases. Among all the strains screened, L. brevis (NRRL B-1836) was found to produce quantifiable amount of intracellular arginine specific aminopeptidase (EC 3.4.11.6). The productivity of arginine aminopeptidase in 5 L fermentor was 36 IU/L/h. The Luedeking and Piret model was tested for intracellular production of aminopeptidase and the data seemed to fit well, as the correlation coefficient was 0.9964 for MRS. The αAP and βAP was 0.4865 and 0.0046, respectively in MRS medium indicating that the yield was predominantly depended on growth. The culture produced lactic acid and also tolerated pH 2.0-3.0 and 0.3-0.5% bile salts, the most important probiotic features.

Statistical optimization of l-leucine amino peptidase production from Streptomyces gedanensis IFO 13427 under submerged fermentation using response surface methodology

Response surface methodology (RSM) employing the central composite design (CCD) was used to optimize the fermentation medium for the production of l-leucine amino peptidase (LAP) from Streptomyces gedanensis IFO13427 under submerged fermentation. The design was employed by selecting substrate concentration, NaCl concentration and initial pH as model factors by ‘one variable at a time’ experiment. A second-order quadratic model and response surface method showed that the optimum conditions (soy bean 0.3%, NaCl, 0.03 M, and initial pH 7) resulted in the improvement of LAP production (25.69 IU/ml) as compared to the initial level (12.17 ± 0.23 IU/ml) after 72 h of fermentation, whereas its value predicted by the quadratic model was 24.56 IU/ml. Analysis of variance (ANOVA) showed a high coefficient of determination (R2) value of 0.9799, ensuring a satisfactory adjustment of the quadratic model with the experimental data. This is first report on LAP production by S. gedanensis using statistical experimental design and response surface methodology in submerged fermentation.

Production of aminopeptidase by marine heterotrophic nanoflagellates

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 34:129-137 (2004) - doi:10.3354/ame034129 Production of aminopeptidase by marine heterotrophic nanoflagellates B. R. Mohapatra1,2,*, K. Fukami 1Laboratory of Aquatic Environmental Sciences, Faculty of Agriculture, Kochi University, Otsu 200, Monobe, Nankoku, Kochi 783-8502, Japan 2Present address: CANMET Materials Technology Laboratory, Natural Resources Canada, 568 Booth Street, Ottawa, Ontario K1A 0G1, Canada *Email: bmohapat@nrcan.gc.ca ABSTRACT: In order to examine the hypothesis that the enzyme levels of heterotrophic nanoflagellates (HNF) are influenced by qualitatively different food bacteria, the production of aminopeptidase by an isolated marine HNF Jakoba libera-5(2) fed on natural communities of bacteria and 6 bacterial strains of 5 different taxonomic groups (Aeromonas, Bacillus, Coryneforms, Flavobacterium and Pseudomonas) were compared. The aminopeptidase activity (total and free) and abundance of J. libera-5(2) significantly differed with the types of food bacteria. The total and free aminopeptidase activities and abundance of J. libera-5(2) were at a maximum with Pseudomonas spp., followed by Flavobacterium sp. and natural communities of bacteria as prey. The values of total aminopeptidase activity of J. libera-5(2) with Pseudomonas spp., Flavobacterium sp. and natural communities of bacteria were 140 ± 6.78 to 285 ± 12.36, 123 ± 11.17 and 38 ± 0.56 μmol h-1 l-1, respectively, and those of free aminopeptidase activity were 83 ± 6.15 to 137 ± 5.83, 82 ± 12.18 and 8 ± 0.14 μmol h-1 l-1, respectively. J. libera-5(2) did not produce any detectable amounts of total and free aminopeptidase while grazing on Aeromonas, Bacillus and Coryneforms. The chemical characterization of partially purified aminopeptidase of J. libera-5(2) produced during grazing on a strain of Pseudomonas sp. indicated the enzyme to be metal-chelater-sensitive alkaline serine aminopeptidase with optimal activity at pH 8.0 and 30°C; it was not affected by the major cations of seawater, such as Na+, Ca2+ and Mg2+. The results suggest that at least some marine HNF may significantly contribute to the enzyme pool in marine environments while selectively grazing on bacteria. KEY WORDS: Aminopeptidase · Grazing · Heterotrophic nanoflagellates · Marine bacteria Full article in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 34, No. 2. Online publication date: February 04, 2004 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2004 Inter-Research.

Protein expression and secretion in the yeast Yarrowia lipolytica.

Strains and vectors for protein expression and secretion have been developed in the yeast Yarrowia lipolytica. Host strains were constructed with non-reverting auxotrophic markers, deletions of protease-encoding genes, and carrying a docking platform. To drive transcription, either the synthetic hp4d or the inducible POX2 promoter were used. Protein secretion is either directed by the targeting sequence of the alkaline extracellular protease or the extracellular lipase (LIP2p) signal sequence. We describe a set of vectors based on these promoters, targeting sequences and two URA3 alleles as selection markers. The wild-type URA3 allele, ura3d1, was used for single-copy integration and a mutant URA3 allele, ura3d4, was used to select for multi-copy integration into the genome. These vectors were used to express the Y. lipolytica extracellular lipase LIP2p and the Aspergillus oryzae leucine amino peptidase II. Lipase production under the control of the hp4d promoter by a strain containing a single copy reached 1000 U ml(-1) in shake flasks, while a strain containing multiple integrations reached 2000 U ml(-1) in shake flasks, 11500 U ml(-1) in batch and 90500 U ml(-1) in fed batch. Leucine amino peptidase production under the control of the hp4d promoter reached 320 mU ml(-1) in batch with a mono-copy lapA integrant and 28000 mU ml(-1) in fed batch with a multi-copy transformant.

Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin.

The kinetics, control, and efficiency of nisin-induced expression directed by the nisA promoter region were studied in Lactococcus lactis with transcriptional and translational fusions to the gusA reporter genes. In the nisin-producing L. lactis strain NZ9700, the specific beta-glucuronidase activity increased very rapidly after mid-exponential growth until the maximum level at the start of the stationary phase was reached. Expression of the gusA gene was also studied in L. lactis NZ9800, an NZ9700 derivative carrying a deletion in the structural nisA gene that abolishes nisin production, and in L. lactis NZ3900, an MG1363 derivative containing the regulatory nisRK genes integrated in the chromosome. In both strains, beta-glucuronidase activity was linearly dependent on the amount of nisin added to the medium. Without nisin, no beta-glucuronidase production was observed. To optimize translation initiation, an expression vector was constructed by fusing the gusA gene translationally to the start codon of the nisA gene. Use of the translational fusion vector yielded up to six times more beta-glucuronidase activity than the transcriptional fusion vector in these strains after induction by nisin. In this way, gene expression can be achieved in a dynamic range of more than 1,000-fold. The beta-glucuronidase activity was found to be up to 25-fold higher in extracts of strain NZ3900 than in extracts of strain NZ9800. This translational fusion vector was used for high-level production of aminopeptidase N, up to 47% of the total intracellular protein. These results clearly illustrate the potential of the nisin-inducible expression system for overproduction of desired proteins.

Regulation of the yeast vacuolar aminopeptidase Y gene (APY1) expression.

The steady-state levels of the aminopeptidase Y (APY1) transcript and the level of assayable aminopeptidase Y activity were measured under a variety of nutritional conditions. Specific APY1 mRNA is less abundant in cells grown in minimal medium than in cells grown in rich medium, while active enzyme levels follow just the opposite pattern. Aminopeptidase Y activity decreases when cells are deprived of glucose without a concomitant fall in mRNA levels. Production of aminopeptidase Y is not markedly affected by nitrogen regulation. APY1 gene expression is not disturbed in heat-shocked cells. Our data support the idea that the main control event in vacuolar aminopeptidase Y expression is a post-transcriptional step.

Production of Aminopeptidase from Skim Milk Whey Permeate Medium by Lactobacillus casei ssp. casei

Extracellular and intracellular aminopeptidases were produced from Lactobacillus casei ssp. casei grown in skim milk whey permeate, but those grown in MRS medium produced intracellular enzyme only. Extracellular enzyme was produced by intact cells rather than by cell lysis. The enzyme was highly unstable during storage at −20°C or −80°C or upon cell breakage. Aminopeptidase production from cells, regardless of their growth medium, was predominantly associated with growth. Cells released aminopeptidase on extraction with Triton X-100, phosphate buffer, or lysozyme in decreasing order of activity.

Regulation of the yeast vacuolar aminopeptidase Y gene ( APY1) expression

The steady-state levels of the aminopeptidase Y ( APY1) transcript and the levels of assayable aminopeptidase Y activity were measured under a variety of nutritional conditions. Specific APY1 mRNA is less abundant in cells grown in minimal medium than in cells grown in rich medium, while active enzyme levels follow just the opposite pattern. Aminopeptidase Y activity decreases when cells are deprived of glucose without a concomitant fall in mRNA levels. Production of aminopeptidase Y is not markedly affected by nitrogen regulation. APY1 gene expression is not disturbed in heat-shocked cells. Our data support the idea that the main control event in vacuolar aminopeptidase Y expression is a post-transcriptional step.

Culture conditions of Brevibacterium linens for production of proteolytic enzymes.

Proteinases and aminopeptidases of B. linens, which are involved in the protein breakdown of surface ripening cheeses, have potential application for accelerated ripening of hard cheese. Both enzyme activities were found to be higher in extracellular than intracellular preparations. Sodium chloride inhibited aminopeptidase production but not proteinase production. Aminopeptidase was produced at the stationary phase of the growth while proteinase was produced in the beginning of logarithmic phase. Optimum medium composition and characterization of crude enzyme are described.

A continuous culture study of the regulation of extracellular protease production in Vibrio SA1.

During growth of Vibrio SA1 in a lactate-limited chemostate in the presence of 2mM phenylalanine as an inducer, the rate of production of two proteolytic enzymes, namely an endopeptidase and an aminopeptidase, was dependent upon the dilution rate. An optimum in the rate of synthesis of both proteases was observed at a dilution rate of 0.23 h-1 and enzyme production only occurred between dilution rates of 0.06 and 0.45 h-1. Without inducer a low rate of aminopeptidase production was found with an optimum at 0.19 h-1, but only trace amounts of endopeptidase were detectable in the culture. In the presence of inducer the rate of enzyme production increased with increasing dilution rates over the range 0.06 to 0.23 h-1 which was explained by an increase in saturation of inducer sites. The progressive decrease in the rate of protease production at higher dilution rates was ascribed to an increasing effect of catabolite repression by the increasing concentration of the growth substrate. It was shown that 5 mM cyclic AMP could not relieve catabolite repression caused by glucose or lactate. Repression of protease production also occurred in the presence of higher concentrations (5 mM) phenylalanine and other amino acids and by ammonium ions. It is suggested that the energy-status of the cell may play an important role in the regulation of protease synthesis in Vibrio SA1.

Production of Aminopeptidase and Carboxypeptidase by Streptomyces peptidofaciens

Streptomyces peptidofaciens KY 2389, a new species isolated from a soil sample, exhibited the highest potencies in production of both aminopeptidase and carboxypeptidase among about 1,300 strains tested. Optimum pH values of both aminopeptidase and carboxypeptidase for Leu-β-naphthyl-amide and Cbz-Gly-Leu were 8.0. Aminopeptidase was thermostable and the activity was not lost by treatment at 70°C for 1 hr, in the presence of Ca2+. Carboxypeptidase was heat-labile and over 50% of the activity was lost by treatment at 60°C for 1 hr. Of the synthetic peptides tested, Leu-Gly-Gly and Cbz-Gly-Leu were the most suitable substrates for amino-peptidase and carboxypeptidase, respectively.

Regulation of proteolytic enzyme production by Aeromonas proteolytica. II. Extracellular aminopeptidase

In media containing simple nitrogen sources, the highest yields of aminopeptidase were obtained from Aeromonas proteolytica Merkel et al. when the organism was grown in asparagine or sodium nitrate supplemented with glycerol; growth in a medium containing enzymatically hydrolyzed casein, however, produced 10 times more enzyme than that observed with the best simple nitrogen sources. The presence of glycerol (0.4%) in a 0.2% asparagine medium resulted in 2.5 times more enzyme although growth was not similarly stimulated. When acid-hydrolyzed casein was the nitrogen source, aminopeptidase synthesis was about three times greater than that obtained with asparagine and glycerol but only one-third of that observed when enzymatically hydrolyzed casein was used. The production of aminopeptidase was greatly enhanced by the addition of small quantities of peptide mixtures to media containing either acid-hydrolyzed casein or asparagine as the primary nutrient source. The data suggest that the synthesis of aminopeptidase is under multiple controls which involve the action of short chain compounds and peptides.