Demonstrated Enzyme Activity Research Articles

Enhancing enzyme immobilization: Fabrication of biosilica-based organic-inorganic composite carriers for efficient covalent binding of D-allulose 3-epimerase

D-allulose, an ideal low-calorie sweetener, is primarily produced through the isomerization of d-fructose using D-allulose 3-epimerase (DAE; EC 5.1.3.30). Addressing the gap in available immobilized DAE enzymes for scalable commercial D-allulose production, three core-shell structured organic-inorganic composite silica-based carriers were designed for efficient covalent immobilization of DAE. Natural inorganic diatomite was used as the core, while 3-aminopropyltriethoxysilane (APTES), polyethyleneimine (PEI), and chitosan organic layers were coated as the shells, respectively. These tailored carriers successfully formed robust covalent bonds with DAE enzyme conjugates, cross-linked via glutaraldehyde, and demonstrated enzyme activities of 372 U/g, 1198 U/g, and 381 U/g, respectively. These immobilized enzymes exhibited an expanded pH tolerance and improved thermal stability compared to free DAE. Particularly, the modified diatomite with PEI exhibited a higher density of binding sites than the other carriers and the PEI-coated immobilized DAE enzyme retained 70.4 % of its relative enzyme activity after ten cycles of reuse. This study provides a promising method for DAE immobilization, underscoring the potential of using biosilica-based organic-inorganic composite carriers for the development of robust enzyme systems, thereby advancing the production of value-added food ingredients like D-allulose.

Desensitization using pegaspargase in the era of commercially available Erwinia: A single-institution report on efficacy, cost, and resource utilization.

Pegaspargase is a therapeutic enzyme that is utilized in treatment regimens targeting pediatric acute lymphoblastic leukemia. However, many patients experience hypersensitivity reactions, requiring discontinuation of the therapy. Historically, this necessitated switching to an alternative form of the drug, most commonly asparaginase Erwinia chrysanthemi; however, in recent years this was difficult due to drug shortages and eventually commercial discontinuation. We report here our experience performing pegaspargase desensitizations in patients with prior hypersensitivity reactions. Patients with a clinical hypersensitivity reaction to pegaspargase were identified. When due for their next dose, patients were admitted to the pediatric intensive care unit, bone marrow transplant unit, or oncology unit, and underwent desensitization utilizing a rigorous premedication and multistep dilution-based protocol. Serum asparaginase activity levels were drawn after desensitization to assess for therapeutic levels of enzyme activity. We identified 11 patients who underwent a total of 33 desensitizations to pegaspargase and calaspargase pegol-mknl. No patients experienced clinically significant hypersensitivity reactions necessitating stopping the infusion, nor administration of rescue medications. All serum asparaginase activity levels collected demonstrated enzyme activity levels above predefined therapeutic thresholds. Cost analysis revealed substantial savings when patients received asparaginase desensitization over the now commercially available asparaginase E. chrysanthemi (recombinant) rywn. Performing desensitization to pegaspargase in the pediatric acute lymphoblastic leukemia population is feasible, safe, and effective. It is financially advantageous over available alternative approaches, and requires fewer injections and presentations to care.

Substitution of acidic residues near the catalytic Glu131 leads to human HYAL1 activity at neutral pH via charge-charge interactions.

Human hyaluronidase 1 (HYAL1) and PH20 play vital roles in degrading hyaluronic acids through the substrate-assisted double displacement mechanism. While HYAL1, a lysosomal enzyme, functions optimally under acidic conditions, PH20, a sperm surface hyaluronidase, displays a broader pH range, from acidic to neutral. Our objective was to extend HYAL1's pH range towards neutral pH by introducing repulsive charge-charge interactions involving the catalytic Glu131, increasing its pKa as the proton donor. Substituting individual acidic residues in the β3-loop (S77D), β3'-β3″ hairpin (T86D and P87E), and at Ala132 (A132D and A132E) enabled HYAL1 to demonstrate enzyme activity at pH 7, with the mutants S77D, P87E, and A132E showing the highest activity in the substrate gel assay. However, double and triple substitutions, including S77D/T86D/A132E as found in the PH20 configuration, did not result in enhanced activity compared to single substitutions. Conversely, PH20 mutants with non-acidic substitutions, such as D94S in the β3-loop and D103T in the β3'-β3″ hairpin, significantly reduced activity within the pH range of 4 to 7. However, the PH20 mutant E149A, reciprocally substituted compared to A132E in HYAL1, exhibited activity similar to PH20 wild-type (WT) at pH 7. In a turbidimetric assay, HYAL1 mutants with single acidic substitutions exhibited activity similar to that of PH20 WT at pH 7. These results suggest that substituting acidic residues near Glu131 results in HYAL1 activity at neutral pH through electrostatic repulsion. This study highlights the significance of charge-charge interactions in both HYAL1 and PH20 in regulating the pH-dependent activity of hyaluronidases.

Salmonid polysialyltransferases to generate a variety of sialic acid polymers

The human polysialyltransferases ST8Sia II and ST8Sia IV catalyze the transfer of several Neu5Ac residues onto glycoproteins forming homopolymers with essential roles during different physiological processes. In salmonids, heterogeneous set of sialic acids polymers have been described in ovary and on eggs cell surface and three genes st8sia4, st8sia2-r1 and st8sia2-r2 were identified that could be implicated in these heteropolymers. The three polysialyltransferases from the salmonid Coregonus maraena were cloned, recombinantly expressed in HEK293 cells and the ST8Sia IV was biochemically characterized. The MicroPlate Sialyltransferase Assay and the non-natural donor substrate CMP-SiaNAl were used to demonstrate enzyme activity and optimize polysialylation reactions. Polysialylation was also carried out with natural donor substrates CMP-Neu5Ac, CMP-Neu5Gc and CMP-Kdn in cell-free and cell-based assays and structural analyses of polysialylated products using the anti-polySia monoclonal antibody 735 and endoneuraminidase N and HPLC approaches. Our data highlighted distinct specificities of human and salmonid polysialyltransferases with notable differences in donor substrates use and the capacity of fish enzymes to generate heteropolymers. This study further suggested an evolution of the biological functions of polySia. C. maraena ST8Sia IV of particular interest to modify glycoproteins with a variety of polySia chains.

Tween® Preserves Enzyme Activity and Stability in PLGA Nanoparticles.

Enzymes, as natural and potentially long-term treatment options, have become one of the most sought-after pharmaceutical molecules to be delivered with nanoparticles (NPs); however, their instability during formulation often leads to underwhelming results. Various molecules, including the Tween® polysorbate series, have demonstrated enzyme activity protection but are often used uncontrolled without optimization. Here, poly(lactic-co-glycolic) acid (PLGA) NPs loaded with β-glucosidase (β-Glu) solutions containing Tween® 20, 60, or 80 were compared. Mixing the enzyme with Tween® pre-formulation had no effect on particle size or physical characteristics, but increased the amount of enzyme loaded. More importantly, NPs made with Tween® 20:enzyme solutions maintained significantly higher enzyme activity. Therefore, Tween® 20:enzyme solutions ranging from 60:1 to 2419:1 mol:mol were further analyzed. Isothermal titration calorimetry analysis demonstrated low affinity and unquantifiable binding between Tween® 20 and β-Glu. Incorporating these solutions in NPs showed no effect on size, zeta potential, or morphology. The amount of enzyme and Tween® 20 in the NPs was constant for all samples, but a trend towards higher activity with higher molar rapports of Tween® 20:β-Glu was observed. Finally, a burst release from NPs in the first hour with Tween®:β-Glu solutions was the same as free enzyme, but the enzyme remained active longer in solution. These results highlight the importance of stabilizers during NP formulation and how optimizing their use to stabilize an enzyme can help researchers design more efficient and effective enzyme loaded NPs.

Optimization of Culture Conditions for Protease Production using Three Strains of Bacillus

The aim of this work was to determine the effect of a few external factors on bacterial growth and the production of enzymes with a proteolytic effect in three strains of Bacillus: CMS5 (Bacillus subtilis), CMS4 (Bacillus sp.) and SPo5 14′ (Bacillus velenzensis) isolated from squashes packed in traditionally prepared cassava leaves, but also to determine the best source of carbon and nitrogen. All three strains have the ability to actively degrade milk casein. The strains were grown in Luria Bertani medium and the suspension from the cell culture was used to measure optical density and demonstrate enzyme activity on a petri dish containing skim milk. Several parameters were verified including the influence of temperature, pH, and carbon and nitrogen source on growth and enzyme production. Growth was possible from 25 to 60°C with an optimal temperature of 30°C after 24 hours. Enzyme production was observed from 25 to 55°C with an optimum at 37°C. For pH, growth and enzyme production was possible from pH 5.7 and 9 with an optimum of 7 in all three strains. Among the sources of carbons used, galactose is the best source for growth after 24 h in all three strains, and starch for production. Among nitrogen sources, Bacto-peptone is best for growth as well as production.

Expression of matrix metalloproteinases in Naegleria fowleri and their role in invasion of the central nervous system.

Naegleria fowleri is a free-living amoeba found in freshwater lakes and ponds and is the causative agent of primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system (CNS). PAM occurs when amoebae attach to the nasal epithelium and invade the CNS, a process that involves binding to, and degradation of, extracellular matrix (ECM) components. This degradation is mediated by matrix metalloproteinases (MMPs), enzymes that have been described in other pathogenic protozoa, and that have been linked to their increased motility and invasive capability. These enzymes also are upregulated in tumorigenic cells and have been implicated in metastasis of certain tumours. In the present study, in vitro experiments linked MMPs functionally to the degradation of the ECM. Gelatin zymography demonstrated enzyme activity in N. fowleri whole cell lysates, conditioned media and media collected from invasion assays. Western immunoblotting indicated the presence of the metalloproteinases MMP-2 (gelatinase A), MMP-9 (gelatinase B) and MMP-14 [membrane type-1 matrix metalloproteinase (MT1-MMP)]. Highly virulent mouse-passaged amoebae expressed higher levels of MMPs than weakly virulent axenically grown amoebae. The functional relevance of MMPs in media was indicated through the use of the MMP inhibitor, 1,10-phenanthroline. The collective in vitro results suggest that MMPs play a critical role in vivo in invasion of the CNS and that these enzymes may be amenable targets for limiting PAM.

ADAMTS‐4_v1 Is a Splice Variant of ADAMTS‐4 That Is Expressed as a Protein in Human Synovium and Cleaves Aggrecan at the Interglobular Domain

ObjectiveWe previously described a messenger RNA variant of ADAMTS4 (ADAMTS4_v1) in human synovial cell cocultures obtained from patients with osteoarthritis (OA). This RNA message has been found only in OA synovium and, if translated, would result in a protein identical to ADAMTS-4, except that the C-terminal spacer domain would be different. The purpose of this study was to determine whether ADAMTS4_v1 is translated into a protein, is expressed in vivo, and acts as a functional aggrecanase.MethodsPolyclonal antibodies were raised against unique C-terminal sequences of ADAMTS-4_v1. An immunohistochemical study of human OA synovium was performed. A mammalian expression vector coding for FLAG-tagged human ADAMTS4 was mutated to contain the different sequences of ADAMTS4_v1, and the resultant plasmid was used to transfect HEK 293 cells. ADAMTS-4_v1 produced by these cells was purified via the FLAG epitope, and the ability of this recombinant protein to cleave aggrecan, biglycan, and decorin was investigated.ResultsAn antibody specific for ADAMTS-4_v1 was found to bind to the synovial membrane surface on cryosections, and the protein was detected in cell lysates from synovium obtained from OA patients. The recombinant ADAMTS-4_v1 demonstrated enzyme activity toward the target substrate in a commercial aggrecanase 1 enzyme-linked immunosorbent assay and was also found to cleave aggrecan at the pathologically important Glu373↓374Ala aggrecanase site.ConclusionADAMTS-4_v1 is expressed as a protein in vivo in human OA synovium, functions as an aggrecanase, and cleaves other proteoglycan substrates. This splice variant may be a major contributor to loss of aggrecan from the superficial zone of OA cartilage.

The effect of neonatal gene therapy with a gamma retroviral vector on cardiac valve disease in mucopolysaccharidosis VII dogs after a decade

Mucopolysaccharidosis VII (MPS VII) is due to deficient activity of the lysosomal enzyme β-glucuronidase (GUSB) and results in the accumulation of glycosaminoglycans (GAGs). This study determined the long-term effect of neonatal intravenous injection of a gamma retroviral vector (RV) on cardiac valve disease in MPS VII dogs. Transduced hepatocytes secreted GUSB into the blood for up to 11years at levels similar to or greater than those achieved with enzyme replacement therapy (ERT). Valve regurgitation and thickening were scored from 0 (normal) to +4 (severely abnormal). At 1year, untreated MPS VII dogs had mitral regurgitation, mitral valve thickening, aortic regurgitation, and aortic valve thickening scores of 2.3±0.7, 2.3±0.6, 1.8±0.5, and 1.6±0.7, respectively, which were higher than the values of 0.6±0.1, 0.1±0.4, 0.3±0.8, and 0.1±0.4, respectively, in treated MPS VII dogs. Treated MPS VII dogs maintained low aortic regurgitation and aortic valve thickening scores in their lifetime. Although mitral regurgitation and mitral valve thickening scores increased to 2.0 at ≥8years of age in the treated MPS VII dogs, older normal dogs from the colony had similar scores, making it difficult to assess mitral valve disease. Older treated dogs had calcification within the mitral and the aortic valve annulus, while GUSB staining demonstrated enzyme activity within the mitral valve. We conclude that neonatal RV-mediated gene therapy reduced cardiac valve disease in MPS VII dogs for up to 11years, and propose that neonatal initiation of ERT should have a similar effect.

Phylogenetic variation in glycosidases and glycanases acting on plant cell wall polysaccharides, and the detection of transglycosidase and trans‐β‐xylanase activities

Wall polysaccharide chemistry varies phylogenetically, suggesting a need for variation in wall enzymes. Although plants possess the genes for numerous putative enzymes acting on wall carbohydrates, the activities of the encoded proteins often remain conjectural. To explore phylogenetic differences in demonstrable enzyme activities, we extracted proteins from 57 rapidly growing plant organs with three extractants, and assayed their ability to act on six oligosaccharides 'modelling' selected cell-wall polysaccharides. Based on reaction products, we successfully distinguished exo- and endo-hydrolases and found high taxonomic variation in all hydrolases screened: β-D-xylosidase, endo-(1→4)-β-D-xylanase, β-D-mannosidase, endo-(1→4)-β-D-mannanase, α-D-xylosidase, β-D-galactosidase, α-L-arabinosidase and α-L-fucosidase. The results, as GHATAbase, a searchable compendium in Excel format, also provide a compilation for selecting rich sources of enzymes acting on wall carbohydrates. Four of the hydrolases were accompanied, sometimes exceeded, by transglycosylase activities, generating products larger than the substrate. For example, during β-xylosidase assays on (1→4)-β-D-xylohexaose (Xyl₆), Marchantia, Selaginella and Equisetum extracts gave negligible free xylose but approximately equimolar Xyl₅ and Xyl₇, indicating trans-β-xylosidase activity, also found in onion, cereals, legumes and rape. The yield of Xyl₉ often exceeded that of Xyl₇₋₈, indicating that β-xylanase was accompanied by an endotransglycosylase activity, here called trans-β-xylanase, catalysing the reaction 2Xyl₆ → Xyl₃ + Xyl₉. Similar evidence also revealed trans-α-xylosidase, trans-α-arabinosidase and trans-α-arabinanase activities acting on xyloglucan oligosaccharides and (1→5)-α-L-arabino-oligosaccharides. In conclusion, diverse plants differ dramatically in extractable enzymes acting on wall carbohydrate, reflecting differences in wall polysaccharide composition. Besides glycosidase and glycanase activities, five new transglycosylase activities were detected. We propose that such activities function in the assembly and re-structuring of the wall matrix.

Confutation of the existence of sequence-conserved cytochrome P450 enzymes in Plasmodium falciparum

The aim of the present study was to find evidence for a homologous protein of the mammalian cytochrome P450 family member CYP2B1/B2 in Plasmodium falciparum at the nucleic acid level. Prior research had demonstrated enzyme activity in the parasite comparable to mammalian CYP1A, 2A, 2B and 2E enzymes and presence of CYP enzymes by spectrophotometric and electrophoretic analyses. In recent years, the transcriptome/proteome data of P. falciparum and other Plasmodium spp. have been published and we performed an in silico analysis to identify putative cytochrome P450 family members in the parasite. This analysis failed to identify homologs to CYP1A, 2A, 2B and 2E enzymes in Plasmodium. A prior study had also claimed the presence of a conserved CYP2B1/B2 gene in the parasite by using Northern analysis with a rat CYP2B1/B2 probe. We have repeated this analysis by cloning a rat CYP2B1/B2 cDNA and using it as a hybridization probe against total RNA extracted from P. falciparum K1 and 3D7 clones but did not obtain positive results. This is consistent with the transcriptome/proteome sequence data and suggests that the genus Plasmodium contains either only highly diverged CYP proteins which are not easily identified by their primary sequence or that they have been functionally replaced by other enzymes. It is suggested that further studies are performed that allow isolation and identification of such proteins through their functional activities.

Carbapenemases in Enterobacteria, Hong Kong, China, 2009

Carbapenemases in Enterobacteria, Hong Kong, China, 2009

Crystal structure of native cinnamomin isoform III and its comparison with other ribosome inactivating proteins

Crystal structure of native cinnamomin isoform III and its comparison with other ribosome inactivating proteins Arezki Azzi,1,2y Tao Wang,1y Dao-Wei Zhu, Yong-Shui Zou, Wang-Yi Liu, and Sheng-Xiang Lin* 1 Structural Biology Platform, Institute of Biochemistry and Cell Biology (IBCB), Shanghai Institutes of Biological Sciences (SIBS), Chinese Academy of Sciences, China 2 Laboratory of Oncology and Molecular Endocrinology, CHUL Research Center (CHUQ) and Laval University, Quebec, Canada

Enzyme hydration, activity and flexibility: A neutron scattering approach

Recent measurements have demonstrated enzyme activity at hydrations as low as 3%. The question of whether the hydration-induced enzyme flexibility is important for activity is addressed by performing picosecond dynamic neutron scattering experiments on pig liver esterase powders at various temperatures as well as solutions. At all temperatures and hydrations investigated here, significant quasielastic scattering intensity is found in the protein, indicating the presence of anharmonic, diffusive motion. As the hydration increases a temperature-dependent dynamical transition appears and strengthens involving additional diffusive motion. At low temperature, increasing hydration resulted in lower flexibility of the enzyme. At higher temperatures, systems containing sufficient number of water molecules interacting with the protein exhibit increased flexibility. The implication of these results is that, although the additional hydration-induced diffusive motion and flexibility at high temperatures in the enzyme detected here may be related to increased activity, they are not required for the enzyme to function.

Enzyme Activity and Flexibility at Very Low Hydration

Recent measurements have demonstrated enzyme activity at hydrations as low as 3%. This raises the question of whether hydration-induced enzyme flexibility is important for activity. Here, to address this, picosecond dynamic neutron scattering experiments are performed on pig liver esterase powders at 0%, 3%, 12%, and 50% hydration by weight and at temperatures ranging from 120 to 300 K. At all temperatures and hydrations, significant quasielastic scattering intensity is found in the protein, indicating the presence of anharmonic, diffusive motion. As the hydration increases, a temperature-dependent dynamical transition appears and strengthens involving additional diffusive motion. The implication of these results is that, although the additional hydration-induced diffusive motion in the protein detected here may be related to increased activity, it is not required for the enzyme to function.

Effects of environmental anoxia and different periods of reoxygenation on oxidative balance in gills of the estuarine crab Chasmagnathus granulata

We investigated the effects of anoxia (8 h) and different periods of reoxygenation (20 and 40 min) on the oxidative balance in anterior and posterior gills of the crab Chasmagnathus granulata. Enzyme activity of catalase and GST was increased in the gills of the animals submitted to anoxia, and SOD activity was decreased. These enzymes returned approximately to control levels during the anoxia recovery time. These results demonstrated enzyme activities change with variations in environmental oxygen levels. The posterior gills showed a higher antioxidant enzyme activity than anterior gills. In the gills, there were no changes in the non-enzymatic antioxidant system (TRAP) during anoxia. On the other hand, during anoxia recovery, an increase of TRAP in both gills was observed. Anoxia does not change lipid peroxidation (TBARS) in the gills. During anoxia recuperation, an increase in levels of TBARS was observed. Thus the results demonstrate that C. granulata has a similar strategy of preparation for oxidative stress as observed in other intertidal species, enabling the crabs to survive in an environment with extreme variations in physical and chemical characteristics, such as salt marshes.

Functional characterization of the K257R and G319E-hGALE alleles found in patients with ostensibly peripheral epimerase deficiency galactosemia

Epimerase deficiency galactosemia is an autosomal recessive condition resulting from the impairment of UDP-galactose 4′-epimerase (hGALE). Although a small number of clinically severe patients have been reported who exhibit “generalized” GALE deficiency, the vast majority exhibit an apparently benign “peripheral” form of the disorder in which enzyme impairment is restricted to the circulating red and white blood cells. Previously, preliminary data were reported suggesting that GALE deficiency is 10-fold more common among African-Americans than among non-African-Americans, and that two missense mutations, K257R and G319E, are found in at least some of these patients. We report here functional studies of these alleles involving expression of the substituted human enzymes in a null-background strain of yeast. Although under normal assay conditions both substituted proteins demonstrate enzyme activities indistinguishable from the wild-type, one (G319E) demonstrates mild impairment under conditions of substrate limitation. No impairments are evident under conditions of cofactor (NAD) limitation. These results are consistent with the apparently benign status of peripheral epimerase deficiency galactosemia, but leave open the question of why patients with these substitutions demonstrate GALE deficiency in their red blood cells. While the possibility remains that K257R and G319E may cause tissue-specific impairments not recapitulated in vitro or in yeast, an equally if not more plausible explanation suggested by interspecies sequence alignments is that both substitutions may be polymorphisms that exist in linkage disequilibrium with other, as yet unidentified causal mutations.

Differential induction and regulation of matrix metalloproteinases in osteoarthritic tissue and fluid synovial fibroblasts

Objectives: To investigate the secretion profiles of matrix metalloproteinases (MMP) and their inhibitors (TIMP) in synovial fluid-derived fibroblasts and to compare them with those of tissue-derived fibroblasts. Methods: Fibroblast cultures established from synovial tissues (TSC) and fluids (FSC) of the same OA patients were stimulated with tumor necrosis factor(TNF)-α, interleukin(IL)-1α, IL-1β, IL-6 and a combination of TNFα and IL-1β. Cocultures of fibroblasts and cartilage were stimulated either with the cytokine combination or with osteoarthritic synovial fluid. Secretion of MMP-1, MMP-3, MMP-8, MMP-13, TIMP-1, and TIMP-2 was measured by enzyme-linked immunosorbent assay. Gelatin zymography and immunoblotting were performed to demonstrate enzyme activity. Results: TNFα, IL-1α, and IL-1β led to marked increases in MMP-1 and MMP-3 release (up to 4.2-fold and 547-fold, respectively) by synovial fibroblasts, whereas secretion of MMP-13 was induced by concomitant administration of TNFα and IL-1β. Expression of intracellular MMP-8 was stimulated by cytokines, but adhesion of synovial fibroblasts to cartilage was required for the release. Throughout the study, significantly higher levels of secreted MMPs were observed in stimulated FSC compared to TSC cultures. Furthermore, increases in MMP secretion were not accompanied by increases in secreted TIMP-1 and TIMP-2, resulting in marked imbalances between enzyme and inhibitor levels. Conclusions: The results provide strong evidence for a significant impact of synovial-derived MMPs on cartilage destruction in OA. In this context, fibroblasts present in the synovial fluid appeared to play an outstanding role.

Enzyme production by obligate intestinal anaerobic bacteria isolated from oscars ( Astronotus ocellatus), angelfish ( Pterophyllum scalare) and southern flounder ( Paralichthys lethostigma)

The important role of the normal anaerobic intestinal flora has been elucidated in man and some domesticated animals; however, the presence of intestinal anaerobes, let alone their importance, in lower vertebrates is largely unknown. The purpose of this research was to isolate and to identify the obligate anaerobic intestinal flora of the freshwater angelfish ( Pterophyllum scalare), oscars ( Astronotus ocellatus) and the marine southern flounder ( Paralichthys lethostigma). Anaerobic intestinal flora were successfully isolated and cultivated in a Bactron II anaerobe chamber; however, many of these isolates could not be identified using available biochemical databases and may represent new undescribed taxa. The types of obligate anaerobes isolated differed among the fish, for example, only the genus Clostridium was recovered from southern flounder, while both Clostridium and Gram negative genera including Fusobacterium, Bacteroides and Porphorymonas were recovered from oscars and angelfish. Isolates were tested for the presence of constitutive enzymes using the APIZYM system. Anaerobic bacteria isolated from the gastrointestinal tracts demonstrated enzyme activities of acid and alkaline phosphatases, C4 and C8 esterases, C14 lipases, arylamidases and glycosidases. Enzyme activity differed with isolates. Generally, Gram negative genera demonstrated enzymes for the breakdown of carbohydrates, while Clostridium demonstrated enzymes for the breakdown of proteins. All genera produced phosphatases possibly used for absorption of nutrients.

A glutamate mutase is involved in the biosynthesis of the lipopeptide antibiotic friulimicin in Actinoplanes friuliensis.

Actinoplanes friuliensis produces the lipopeptide antibiotic friulimicin. This antibiotic is active against gram-positive bacteria such as multiresistant Enterococcus and Staphylococcus strains. It consists of 10 amino acids that form a ring structure and 1 exocyclic amino acid to which an acyl residue is attached. By a reverse genetic approach, biosynthetic genes were identified that are required for the nonribosomal synthesis of the antibiotic. In close proximity two genes (glmA and glmB) were found which are involved in the production of methylaspartate, one of the amino acids of the peptide core. Methylaspartate is synthesized by a glutamate mutase mechanism, which was up to now only described for glutamate fermentation in Clostridium sp. or members of the family ENTEROBACTERIACEAE: The active enzyme consists of two subunits, and the corresponding genes overlap each other. To demonstrate enzyme activity in a heterologous host, it was necessary to genetically fuse glmA and glmB. The resulting gene was overexpressed in Streptomyces lividans, and the fusion protein was purified in an active form. For gene disruption mutagenesis, a host-vector system was established which enables genetic manipulation of Actinoplanes spp. for the first time. Thus, targeted inactivation of biosynthetic genes was possible, and their involvement in friulimicin biosynthesis was demonstrated.