High-level expression and characterization of the thermostable leucine aminopeptidase Thelap from the thermophilic fungus Thermomyces lanuginosus in Aspergillus niger and its application in soy protein hydrolysis.

Leucine aminopeptidase (LAP), an exopeptidase that releases amino acid residues, especially leucine, from the N-terminus of polypeptides, is often applied to debitter protein hydrolysate in the food industry. However, there are no thermostable and high activity enzymes that can be used in the food industry. In this study, we obtained the highly active and thermostable leucine aminopeptidases screened from the thermophilic fungi Thermomyces lanuginosus, Talaromyces thermophilus, and Malbranchea cinnamomea. The activity of the recombinant leucine aminopeptidase Thelap was significantly increased to 2771.5 U/mL, as mediated by the CRISPR/Cas9 tool. The recombinant Thelap was easily purified from fermentation broth by Ni-affinity chromatography, and the specific activity of the purified Thelap was increased to 7449.6 U/mg. The recombinant Thelap showed optimal activity at pH 8.5 and 75 °C and remained above 70% of the maximum activity over a wide temperature range (30-80 °C). With regard to temperature stability, Thelap retained more than 90% activity when it was incubated at 65-75 °C for 2 h. K+ and Co2+ increased the enzyme activity of the recombinant Thelap, while Ba2+, Mn2+, Ni2+, Ca2+, Mg2+ and SDS inhibited its enzyme activity, and the inhibition capacity of Mg2+ was the weakest. Upon application in soy protein hydrolysis, Thelap could significantly increase the degree of hydrolysis and remove more hydrophobic amino acids from the N-terminal region of the polypeptide to decrease the bitterness.

Molecular identification and characterization of leucine aminopeptidase 2, an excretory-secretory product of Clonorchis sinensis.

Aminopeptidases serve vital roles in metabolism of hormones, neurotransmission, turnover of proteins and immunological regulations. Leucine aminopeptidases catalyze the hydrolysis of amino-acid residues from the N-terminus of proteins and peptides. In the present study, leucine aminopeptidase 2 (LAP2) gene of Clonorchis sinensis (C. sinensis) was isolated and identified from an adult cDNA library of C. sinensis. Recombinant CsLAP2 was expressed and purified in Escherichia coli BL21. The open reading frame of LAP2 contains 1,560 bp equivalent to 519 amino acids, a similarity analysis showed a relatively low homology with Homo sapiens (19.0 %), Trypanosoma cruzi (18.0 %), Mus musculus (19.3 %), and relatively high homology with Schistosoma mansoni (65.6 %). The optimum condition of rCsLAP2 enzyme activity was investigated using a fluorescent substrate of Leu-MCA at 37 °C and pH 7.5. The K (m) and V (max) values of rCsLAP2 were 18.2 µM and 10.7 µM/min, respectively. CsLAP2 gene expression can be detected at the stages of the adult worm, metacercaria, excysted metacercaria and egg of C. sinensis using real-time PCR, no difference was observed at the stages of the adult worm, metacercaria and egg. However, CsLAP2 showed a higher expression level at the stage of excysted metacercaria than the adult worm (3.90-fold), metacercaria (4.60-fold) and egg (4.59-fold). Histochemistry analysis showed that CsLAP2 was located at the tegument and excretory vesicle of metacercaria, and the tegument and intestine of adult worm. The immune response specific to rCsLAP2 was characterized by a mixed response patterns of Th1 and Th2, indicating a compounded humoral and cellular immune response. The combined results from the present study indicate that CsLAP2 was an important antigen exposed to host immune system, and probably implicated as potential role in interaction with host cells in clonorchiasis.

Genetic variation in a gradient of environmental variability: marine bivalvia (Mollusca).

Six bivalve mollusk species were sampled for genetic variability at two enzyme synthesizing loci. The effective number of alleles and absolute number of alleles decreased with depth of burial within the sediment, intertidally, and with depth of water, subtidally. It is proposed that environmental variability regulates genetic variability at these two loci.

Leucine aminopeptidase in the ixodid tick Haemaphysalis longicornis: endogenous expression profiles in midgut.

We previously identified a cDNA from the ixodid tick Haemaphysalis longicornis that encodes leucine aminopeptidase, HlLAP. Functionally, recombinant HlLAP effectively hydrolyzed synthetic amino acid derivatives. Here, we investigated the temporal expression profiles of midgut HlLAP in adult H. longicornis parthenogenetic ticks from the starting of blood feeding until just before the onset of oviposition. Midgut HlLAP transcript expression level was higher during post-engorgement period than that during feeding period. Endogenous HlLAP in the midgut was also observed with higher expression level during post-engorgement period. Histological localization of HlLAP was in the cytosol of midgut epithelial cells, notably the newly differentiated basophilic cells at post-engorgement. Our data suggested that HlLAP was dominantly localized in basophilic cells, where it may play regulatory roles in protein biosynthesis and degradation.

A new method to evaluate the enzyme-suppressing activity of a leucine aminopeptidase 3 inhibitor.

The expression of leucine aminopeptidase 3 (LAP3) is associated with the prognosis for and malignant transformation of many types of tumors. Therefore, a LAP3 inhibitor may represent a new strategy for cancer therapy. Evaluating the suppression of enzyme activity by an LAP3 inhibitor is essential. Right now, leucine aminopeptidases (LAPs) purified from the porcine kidneys are the only enzymes that can be used to evaluate the suppression of enzyme activity by an LAP3 inhibitor. This approach cannot accurately reflect the suppression of human LAP3 by an inhibitor. The current study developed a new method with which to evaluate the suppression of enzyme activity by an LAP3 inhibitor. Total protein from K562 cells seldom catalyzed the LAP3 substrate. A lentivirus was used to induce K562 cells to overexpress LAP3 (K562-LAP3). After puromycin screening, flow cytometry data indicated that 98.8% of cells expressed green fluorescent protein. The expression of LAP3 in K562-LAP3 cells was also assessed using Western blotting. K562-LAP3 cells were lysed with ultrasonication. Total protein was used as an enzyme source and L-leucine p-nitroaniline hydrochloride was used as a substrate to measure enzyme activity. Total protein from K562-LAP3 cells catalyzed the substrate more than that from K562 cells did. The LAP3 inhibitor ubenimex was used as a positive control to evaluate the suppression of LAP3 enzyme activity. Results indicated that ubenimex significantly inhibited the enzyme activity of LAP3. This approach provides a convenient and accurate way to evaluate the suppression of enzyme activity by an LAP3 inhibitor.

High-Level Expression in Escherichia coli, Purification and Kinetic Characterization of LAPTc, a Trypanosoma cruzi M17-Aminopeptidase.

The M17 leucyl-aminopeptidase of Trypanosoma cruzi (LAPTc) is a novel drug target for Chagas disease. The objective of this work was to obtain recombinant LAPTc (rLAPTc) in Escherichia coli. A LAPTc gene was designed, optimized for its expression in E. coli, synthesized and cloned into the vector pET-19b. Production of rLAPTc in E. coli BL21(DE3)pLysS, induced for 20 h at 25 °C with 1 mM IPTG, yielded soluble rLAPTC that was catalytically active. The rLAPTc enzyme was purified in a single step by IMAC. The recombinant protein was obtained with a purity of 90% and a volumetric yield of 90 mg per liter of culture. The enzymatic activity has an optimal pH of 9.0, and preference for Leu-p-nitroanilide (appKM = 74 µM, appkcat = 4.4 s-1). The optimal temperature is 50 °C, and the cations Mg2+, Cd2+, Ba2+, Ca2+ and Zn2+ at 4 mM inhibited the activity by 60% or more, while Mn2+ inhibited by only 15% and addition of Co2+ activated by 40%. The recombinant enzyme is insensitive toward the protease inhibitors PMSF, TLCK, E-64 and pepstatin A, but is inhibited by EDTA and bestatin. Bestatin is a non-competitive inhibitor of the enzyme with a Ki value of 881 nM. The enzyme is a good target for inhibitor identification.

Production of L-leucine aminopeptidase by selected Streptomyces isolates.

AIMS: To screen various Streptomyces cultures producing L-leucine aminopeptidase (LAP). METHODS AND RESULTS: Twenty-one Streptomyces strains were screened for LAP production. The best three producers were found to be Streptomyces mobaraensis NRRL B-3729, Streptomyces gedanensis IFO 13427, and Streptomyces platensis NRRL 2364. pH optima of the three enzymes were in the range of 8.0-8.5 and the temperature optima varied between 50 and 65 degrees C. LAP of S. mobaraensis was stable at 60 degrees C and pH 8.5 for 60 min. Metal ion salts, CoCl(2).6H(2)O and ZnSO(4).7H(2)O in 0.7 mmol l(-1) concentration enhanced the relative enzyme activity in all three enzymes. Molecular mass of LAP of S. mobaraensis was found to be approx. 37 kDa. CONCLUSIONS: Streptomyces mobaraensis NRRL B-3729, S. gedanensis IFO 13427, and S. platensis NRRL 2364 were found to be good producers of extracellular LAP. The approx. 37 kDa enzyme of S. mobaraensis is considerably thermostable. SIGNIFICANCE AND IMPACT OF THE STUDY: A good number of Streptomyces were screened and the ability of the aminopeptidases to release a particular N-terminal amino acid along with its good thermal stability makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate.

Expression, purification and characterization of two leucine aminopeptidases of the blood fluke, Schistosoma mansoni.

Schistosomiasis is a major neglected tropical disease (NTD) and considered the most important of the human helminthiases in terms of morbidity and mortality. Whereas treatment with praziquantel has been effective since the 1980s, the potential for the emergence of drug resistance has propelled the search for new interventions. Studies have revealed key roles of proteases in parasitic helminths during establishment of infection, tissue invasion, immune evasion, parasite feeding and development throughout the different developmental stages, pinpointing them as possible candidates. The leucine aminopeptidases (LAPs), members of the M17 family of Zn-metalloproteases, preferentially cleave leucine (Leu) residues at the N-terminal end of proteins and short peptides. These enzymes display broad proteolytic activities beyond Leu hydrolysis and are involved in processing, maturation, activation and/or degradation of substrates. As a vaccine immunogen, LAP induces protection against infection with the liver fluke Fasciola hepatica. Herein, two LAPs, SmLAP1 (Smp_030000) and SmLAP2 (Smp_083870) of the human blood fluke Schistosoma mansoni were cloned, expressed, purified and biochemically characterized. The enzymes differed in activity against diagnostic substrates, including leucine, methionine and arginine, with an optimal pH of 8.0. The activity increased in the presence of Mg+2 and Mn+2, and was inhibited by bestatin, a specific inhibitor of aminopeptidase. In addition, 1,10-phenanthroline and EDTA inhibited the enzymatic activity of SmLAP2. Finally, immunolocalization using antibodies specific for SmLAP1 and SmLAP2 identified the expression of these proteases in the egg and adult developmental stages of S. mansoni, and in intestinal epithelia, vitelline cells and sub-tegumental regions of the parasite. Characterization of schistosome proteases not only enhances understanding of the biology of schistosomes and schistosomiasis, but may also provide novel intervention approaches.

Characterization of Aspergillus sojae Isolated from Meju, Korean Traditional Fermented Soybean Brick.

Initially, we screened 18 Aspergillus sojae-like strains from Aspergillus spp. isolated from meju (Korean traditional fermented soybean brick) according to their morphological characteristics. Because members of Aspergillus section Flavi are often incorrectly identified because of their phylogenetic similarity, we re-identified these strains at the morphological and molecular genetic levels. Fourteen strains were finally identified as A. sojae. The isolates produced protease and α-amylase with ranges of 2.66-10.64 and 21.53-106.73 unit/g-initial dry substrate (U/g-IDS), respectively, which were equivalent to those of the koji (starter mold) strains employed to produce Japanese soy sauce. Among the isolates and Japanese koji strains, strains SMF 127 and SMF 131 had the highest leucine aminopeptidase (LAP) activities at 6.00 and 6.06 U/g-IDS, respectively. LAP plays an important role in flavor development because of the production of low-molecular-weight peptides that affect the taste and decrease bitterness. SMF 127 and SMF 131 appeared to be non-aflatoxigenic because of a termination point mutation in aflR and the lack of the polyketide synthase gene found in other A. sojae strains. In addition, SMF 127 and SMF 131 were not cyclopiazonic acid (CPA) producers because of the deletion of maoA, dmaT, and pks/nrps, which are involved in CPA biosynthesis. Therefore, A. sojae strains such as SMF 127 and SMF 131, which have high protease and LAP activities and are free of safety issues, can be considered good starters for soybean fermentations, such as in the production of the Korean fermented soybean products meju, doenjang, and ganjang.

Genetic and environmental effects on the expression of peptidases and larval viability in Drosophila melanogaster.

The peptidase system in Drosophila melanogaster, consisting of dipeptidase-A, dipeptidase-B, dipeptidase-C and the leucine aminopeptidases, was used as a model to study the adaptive significance of enzyme activity variation. The involvement of the peptidases in osmoregulation has been suggested from the ubiquitous distribution of peptidase activities in nearly all tissues and the high concentration of amino acids and oligopeptides in the hemolymph. Under this hypothesis, larvae counteract increases in environmental osmotic stress by hydrolyzing peptides into amino acids both intra- and extracellularly to increase physiological osmotic concentration. The expression of the peptidases was studied by assaying for peptidase activities in third instar larvae of isogenic lines, which were reared under increasing levels of environmental osmotic stress using either D-mannitol or NaCl. Second and third chromosome substitution isogenic lines were used to assess the relative contribution of regulatory and structural genes in enzyme activity variation. Results indicate that: (1) genetic variation exists for peptidase activities, (2) the effect of osmotic stress is highly variable among peptidases, (3) changes in peptidase activities in response to osmotic stress depend on both genetic background and osmotic effector and (4) peptidase activities are correlated with each other, but these phenotypic correlations depend on genetic background, osmotic effector, and level of osmotic stress. Osmotic concentration in the larval hemolymph is correlated with leucine aminopeptidase activity, but changes in hemolymph osmotic concentration in response to environmental osmotic stress depend on the osmotic effector in the environment. Although these findings suggest that genetic and environmental factors contribute significantly toward the expression of enzymes with similar functions, a relative larval viability study of genotypes that differed significantly in dipeptidase-B (DIP-B) activity revealed that low DIP-B activity did not confer any measurable reduction in larval viability under increasing levels of environmental osmotic stress. These negative results suggest that, either DIP-B does not play a major role in osmoregulation or differential osmoregulation is not related to egg to adult viability in these tests.

Protein, leucine aminopeptidase, esterase, acid phosphatase and photosynthetic responses of oleander (Nerium oleander L.) during cold acclimation and freezing treatments.

Six-month-old oleander (Nerium oleander L.) pot plants, derived from vegetative propagation by cuttings, were tested for their ability to cold hardening. Damage of the non-acclimated (NA) plants was visible when treated by low freezing temperatures (below -2 degrees C). The responses of total proteins, leucine aminopeptidase (LAP), esterase (EST) and acid phosphatase (ACP) isoforms of NA and cold-acclimated (CA; 4 degrees C for 14 days) plants were compared using polyacrylamide gel electrophoresis. These molecular markers were also compared in NA and CA plants which received for 2h temperatures of 0, -2, -4, -6 and -8 degrees C. A new 38-kDa polypeptide appeared from day 7 to 14 during the acclimation treatment in the bark extracts and on day 14 in the leaf extracts. The above-mentioned polypeptide band (38 kDa) strongly appeared in all freezing treatments (0, -2, -4, -6 and -8 degrees C) in both bark and leaf extracts of the CA plants. Alterations in the number and the intensity of LAP and EST isoforms as well as in the intensity of ACP isoforms were observed in both bark and leaf of the CA oleander plants. A newly expressed EST isoform is proposed as biochemical marker for the cold acclimation treatment. CO2 assimilation rates (A) as well as transpiration rates (E) in NA plants were positive in 0 degrees C and negative in all temperatures below zero in the freezing treatments. In contrast, CO2 assimilation rates (A) and transpiration rates (E) were positive in CA plants in all temperatures of freezing treatment. A significant decrease (P<0.05) in chlorophyll (Chl) a, Chl a+b concentration and Chl a/b ratio were noticed in oleander plants during the acclimation treatment (from day 0 to 14), while Chl b concentration was unchanged at the respective time. On the other hand, no significant (P<0.05) differences were observed in the freezing treatments.

Possible involvement of placental peptidases that degrade gonadotropin-releasing hormone (GnRH) in the dynamic pattern of placental hCG secretion via GnRH degradation.

The presence of an extrahypothalamic gonadotropin releasing hormone (GnRH) in human placenta is well known and this decapeptide is presumed to play an important role in the regulation of the function and growth of human placenta. Immunohistochemistry showed that neutral endopeptidase 24.11 (NEP), a candidate of the responsible enzyme of GnRH degradation, is highly expressed on the cell surface of trophoblasts. Hydrolysis of GnRH by human villi was studied by measuring liberated amino acids using high performance liquid chromatography. The GnRH degrading activity was 1.53 times higher after incubation with the membrane fraction of first trimester villi than that after incubation with the membrane fraction of term villi. Phosphoramidon, a potent inhibitor of NEP, reduced the liberated amino acids to about a half, suggesting that NEP is a responsible enzyme for GnRH degradation. Ubenimex, which can inhibit several aminopeptidases, also reduced the liberated amino acids to about 50 per cent. O-phenanthroline, EDTA, and thiorphan could inhibit GnRH degradation but inhibitors of post proline endopeptidase could not. Furthermore, GnRH degrading activity of the membrane fraction was reduced remarkably after the membrane fraction was immunotitrated by anti NEP and anti placental leucine aminopeptidase (P-LAP) IgG. In conclusion, NEP and P-LAP are responsible enzymes for GnRH degradation in human villi.

Model building and quantitative analysis of a tandem immuno-capturing assay as a screening tool for breast cancer.

The onset of breast cancer appears to occur, on average, a decade earlier in Mexican women in comparison to American or European women. Early detection and prevention of breast cancer are of crucial importance to increase survival and improve quality of life. Based on the molecular elucidation of critical events leading to breast carcinogenesis, a tandem immuno-capturing blood test was developed as a quantitative population screening assay in view of providing a cost-effective and non-invasive alternative to population screening. Clinical analysis of 63 Mexican women within an age group of 35-70, revealed that Interstron activity increases from 800+/-65 IUJPA (Interstron Units) in the asymptomatic normal women to 994+/-100 IUJPA in the symptomatic/benign group, reaching 1289+/-81 IUJPA in the cancerous group. Accordingly, activity thresholds were established at 800 and 1200 IUJPA respectively, encompassing three risk groups: (i) Healthy Otherwise Normal (<800 IUJPA); (ii) Grey Risk Area (>800 and <1200 IUJPA), and (iii) At Risk group (>1200 IUJPA). Taking into account both baseline and clinical case reports, the Healthy Otherwise Normal group and the At Risk group were mostly homogeneous in nature, comprising a population of normal and cancer patients respectively. The Grey Risk group is heterogeneous, likely reflecting a transitional nature towards a potential early stage of breast disease development. Based on these results, a screening algorithm was developed as the underlining principle for population surveillance encompassing over 30,000 Mexican women. The current screening results have enabled us to objectively prioritize medical attention to approximately 1 in 8 women out of the general population mapped within the At Risk group. Overall, our findings suggest that monitoring Interstron activity units provides a valuable quantitative screening analysis as to selectively streamline the population of women in need of early medical counseling and/or mammography, thereby enhancing both the quality and cost-effectiveness of preventative population surveillance programs targeting breast cancer.

Cyclic changes in salivary lactate dehydrogenase, peroxidase and leucine aminopeptidase during menstrual cycle.

Salivary lactate dehydrogenase (LDH), peroxidase (Px) and leucine aminopeptidase (LAP) activities were scanned in both normally ovulating and anovulating women during entire menstrual cycle. In ovulating women, all the three enzymes exhibited significant increase in the activity on or before the onset of ovulation which was monitored by the shift of the basal body temperature (BBT) as well as the ferning pattern of the cervical mucus. The peak maximum at the midcycle was several times higher than the previous day value in all the six normal women. In anovulatory women, no such remarkable change in the enzyme activities was found throughout the cycle. Salivary LDH and LAP showed peak at the midcycle and at the same time required short time for assay, so the present results are strongly suggestive that the determination of salivary enzyme content may be a convenient method for detecting the day of ovulation.

[Biosynthesis of leucine aminopeptidase by Xanthomonas rubrilineans 67 in culture with various concentrations of nitrogen containing compounds]. / Biosintez leitsinaminopeptidazy kul'turoi Xanthomonas rubrilineans 67 pri var'irovanii azotosoderzhashchikh komponentov sredy.

The dynamics of consumption of amine and ammonium nitrogen and glucose in the process of Xanthomonas rubrilineans 67 growth and biosynthesis of leucine aminopeptidase was studied. It was shown that the rate of leucine, alanine and glycine consumption as a source of amine nitrogen out of 16 amino acids was the highest during the fermentation. Addition of these three amino acids or their mixtures to the medium at definite stages of the fermentation process increased the leucine aminopeptidase biosynthesis by 50 to 100 per cent. Ammonium nitrogen was not used by X.rubrilineans 67. The consumption of glucose during the fermentation was even: by the 24th hour of the process the medium contained about 10 per cent of the glucose initial concentration. The optimal temperature for the culture growth and leucine aminopeptidase biosynthesis was determined. It was shown to be 28 degrees C. Higher aeration increased the culture productivity.

Histochemical study on the activity of some oxidative and hydrolytic enzymes in the normal human endometrium blood vessels and at various intervals after applying the intra uterine contraceptive device.

The paper intends to study the cytoenzymological modifications of the human endometrium blood vessels after applying the intrauterine contraceptive device for a long period of time. The results were different according to the: type of the enzyme-ATP-ase, Lactatdehydrogenase, Leucineaminopeptidase, NADH2-citochrome-c-reductase, the components of the blood vessel wall (intima, media, adventitia), the blood vessel diameter, the hormonal cyclic stage.

Simultaneous investigation of the rat serum and liver response in an acute toxic substances exposure.

Some biochemical parameters were simultaneously investigated in serum and hepatic homogenate in acute experiment with three pesticides in rat. The one-dose exposure response was relatively well correlated with the chemical toxicity and showed the statistically significant values, preferentially placed 48 hours after dosing. The liver morphological alteration might be associated with the serum and tissue comportment of the bioenzymological factors. The various genotoxicity induced by the three pesticides, appreciated by the frequency of micronucleus induction in the bone marrow cells, completes the data about the one-dose exposure effects of the chemical in rat.

Expression of leucine aminopeptidase 3 (LAP3) correlates with prognosis and malignant development of human hepatocellular carcinoma (HCC).

Leucine aminopeptidases (LAPs) were associated with tumor cell proliferation, invasion and/or angiogenesis. LAP3 is one important member of this family. However, its clinical significance and biological function in hepatocellular carcinoma (HCC) remains unknown. In the present study, we demonstrated that LAP3 expression was significantly up-regulated in HCC tissues as well as cells and was closely correlated with lower differentiation, positive lymph node metastasis and high Ki-67 expression, indicating a poor prognosis. Then cell viability assays, flow cytometry assays, wound-healing assays and matrigel invasion assays were performed to demonstrate that LAP3 promoted HCC cells proliferation by regulating G1/S checkpoint in cell cycle and advanced HCC cells migration. Furthermore, we discovered that knockdown LAP3 will enhance the sensitivity of HCC cells to cisplatin, thus promoting the cell death of HCC cells. Collectively, our results indicated that up-regulated expression of LAP3 might contribute to the proliferation and metastasis of HCC. Our data gains greater insight into the cancer-promoting role of LAP3 and its functions in HCC cells, possibly providing potential therapeutic strategies for clinical trials.

Production of leucine amino peptidase in lab scale bioreactors using Streptomyces gedanensis.

Studies were conducted on the production of leucine amino peptidase (LAP) by Streptomyces gedanensis to ascertain the performance of the process in shake flask, parallel fermenter and 5-L fermenter utilizing soy bean meal as the carbon source. Experiments were conducted to analyze the effects of aeration and agitation rate on cell growth and LAP production. The results unveiled that an agitation rate of 300 rpm, 50% dissolved oxygen (DO) upholding and 0.15 vvm strategies were the optimal for the enzyme production, yielding 22.72 ± 0.11 IU/mL LAP in parallel fermenter which was comparable to flask level (24.65 ± 0.12 IU/mL LAP) fermentation. Further scale-up, in 5-L fermenter showed 50% DO and 1 vvm aeration rate was the best, producing optimum and the production was 20.09 ± 0.06 IU/mL LAP. The information obtained could be useful to design a strategy to improve a large-scale bioreactor cultivation of cells and production of LAP.