Extracellular Enzyme Preparation Research Articles

Characterization of putative mannoprotein in Kluyveromyces lactis for lactase production

Lactase is a member of the β-galactosidase family of enzymes that can hydrolyze lactose into galactose and glucose. However, extracellular lactase production was still restricted to the process of cell lysis. In this study, lactase-producing Kluyveromyces lactis JNXR-2101 was obtained using a rapid and sensitive method based on the fluorescent substrate 4-methylumbelliferyl-β-d-galactopyranoside. The purified enzyme was identified as a neutral lactase with an optimum pH of 9. To facilitate extracellular production of lactase, a putative mannoprotein KLLA0_E01057g of K. lactis was knocked out. It could effectively promote cell wall degradation and lactase production after lyticase treatment, which showed potential on other extracellular enzyme preparation. After optimizing the fermentation conditions, the lactase yield from mannoprotein-deficient K. lactis JNXR-2101ΔE01057g reached 159.62 U/mL in a 5-L fed-batch bioreactor.

Novel sialidase from non-pathogenic bacterium Oerskovia paurometabola strain O129.

Bacterial sialidases are enzymes that are involved in a number of vital processes in microorganisms and in their interaction with the host or the environment. Their wide application for scientific and applied purposes requires the search for highly effective and non-pathogenic producers. Here, we report the first description of sialidase from Oerskovia paurometabola. The extracellular enzyme preparation was partially purified. The presence of sialidase was confirmed in native PAGE treated with the fluorogenic substrate 4MU-Neu5Ac. Maximum enzyme activity was registered at 37°C and in the pH range of 4.0-5.5. The influence of metal ions and EDTA was examined. It was demonstrated that EDTA, Mn2+ and Ba2+ ions inhibit the sialidase activity to different extent, while Cd2+, Fe2+ and Fe3+ have stimulating effect on it. These features are studied for the first time concerning sialidase of Oerskovia representative. Cell bound sialidase and sialate aldolase were also established.

Biodegradation of fibrillated oil palm trunk fiber by a novel thermophilic, anaerobic, xylanolytic bacterium Caldicoprobacter sp. CL-2 isolated from compost.

Oil palm trunk (OPT) is one of the most promising lignocellulosic bioresources. To develop effective biodegradation, thermophilic, anaerobic microorganisms were screened from bovine manure compost using fibrillated OPT (f-OPT) pretreated by wet disk milling as the substrate. One thermophilic, anaerobic bacterium, strain CL-2, whose 16S rDNA gene has 98.6% sequence identity with that of Caldicoprobacter faecale DSM 20678T, exhibited high degradation activity (32.7% reduction in total dry solids of f-OPT). Strain CL-2 did not use cellulose as a carbon source, but used hemicelluloses such as xylan, arabinoxylan, starch and pectin at 70 °C. Phylogenetic and morphologic analyses and the polysaccharide use suggest that CL-2 may be classified as a novel species of Caldicoprobacter, named Caldicoprobacter sp. CL-2. To characterize enzymatic activities of CL-2, extracellular enzymes were prepared from culture broth using beechwood xylan as the carbon source. The extracellular enzymes showed high xylanase activity, but low cellulase activity, suggesting that f-OPT degradation may depend on xylanase activity. To understand the xylanase system of CL-2, a major xylanase was cloned and characterized. The xylanase (CalXyn11A) had a modular structure consisting of a glycoside hydrolase (GH) family-11 domain and a family 36 carbohydrate-binding module. CalXyn11A did not show f-OPT degradation activity, but a strong synergistic effect was observed when CalXyn11A was added to the extracellular enzyme preparation. These results indicate that, rather than working alone, CalXyn11A has an important role in enhancing total lignocellulose degradation activity by cooperation with other GHs.

The culture or co-culture of Candida rugosa and Yarrowia lipolytica strain rM-4A, or incubation with their crude extracellular lipase and laccase preparations, for the biodegradation of palm oil mill wastewater

Wastewater generated by the palm oil industry contains a high concentration of oil, grease and phenolic compounds, which makes it difficult to treat. The aim of this research was to evaluate the ability of free Candida rugosa and Yarrowia lipolytica rM-4A yeast cell cultures, as a single yeast strain and as a co-culture, as well as their crude extracellular enzyme preparations, to reduce the total phenolic compounds, triglycerides and chemical oxygen demand (COD) level in palm oil mill effluent (POME) that contains high amounts total phenolic compounds (9783 mg L−1) and oil and grease (7762 mg L−1). Culture with Y. lipolytica rM-4A effectively reduced the total phenolic content in undiluted POME, resulting in removal of 36% of the total phenolic content after 96 h. The highest removal of triglyceride (98.5%) and COD (60.3%) were observed in undiluted POME treated by the co-culture of C. rugosa and Y. lipolytica for 120 h. Triglyceride was removed rapidly by the extracellular lipase produced by C. rugosa, reaching 93% degradation after 48 h. This research revealed the excellent potential of both crude extracellular enzyme preparations and the co-culture of the two yeasts in the pretreatment of high fat and oil containing POME.

Evaluation and Optimization of Extracellular Digestive Enzymes from Bacillus spp. Isolated from Curds

Objective: The aim of the present study was to evaluate the digestive enzyme activity of the four Bacillus spp. and to optimize the physical parameters. Methods: The enzymes were produced by submerged fermentation supplementing enzyme specific substrates. The fermentation broths were centrifuged and the supernatants were used as source of crude enzyme. Amylase activity was determined by 3, 5-dinitro salicylic acid method using starch as substrate while copper soap method was used to evaluate lipase activity. Further, protease activity was measured by Lowry’s method; whereas, phytase activity was assayed using sodium phytate as substrate. All the enzymes were optimized for pH, temperature and substrate concentration. The total protein content per one mL of the crude enzyme in the supernatant was quantified by Lowry’s method. Results: All the four tested isolates B. subtilis GS 1, B. cereus GS 3, B. cereus GS 199 and B. subtilis GS 547 showed high extracellular digestive enzyme activity at pH range 5 to 8 and temperature 20 to 50°C. Conclusion: The four tested B. subtilis GS 1, B. cereus GS 3, B. cereus GS 199 and B. subtilis GS 547 could be promising extracellular digestive enzyme producing isolates. Further, evaluation of in vivo efficacy and safety in animal models and clinical trials would be helpful in assisting digestive enzyme deficiencies using these extracellular enzyme preparations or whole cell bacteria.

THERAPEUTIC L-ASPARAGINASE ACTIVITY OF BACTERIA ISOLATED FROM MARINE SEDIMENTS

L-Asparaginase, a therapeutic enzyme used in lymphoblastic leukemia and lymphosarcoma chemotherapy which is derived mostly from the bacterial sources Escherichia coli and Erwinia sp. The long term administration of the drug leads to the development of resistant tumours and anaphylactic shock in certain individuals. Hence serologically different L-Asparaginase from novel microbial sources with enhanced therapeutic potential and immunological characteristics is an essential requirement. The marine bacteria having diverse range of potential enzymes might be a source for L-Asparaginase with novel properties, which are still unexplored. In this study, we have screened marine bacteria isolated from the coastal regions of Kerala which showed both intra and extra cellular L-Asparaginase activity. Bacillus sp. (Accession no KF142395) was found to have the highest extracellular enzyme activity (2.31 IU/ml) while Shewanella sp. (Accession no KF142390) showed maximum intracellular Asparaginase activity (2.16 IU/ml).The crude extracellular enzyme preparation from Bacillus sp. had cytotoxic effect on HL60 cell line with an IC50 value of 12.5µg/ml.

Binary Interactions of Antagonistic Bacteria with Candida albicans Under Aerobic and Anaerobic Conditions.

We used both aerobic and anaerobic liquid co-cultures, prepared with Luria Bertani broth, to study the effect of bacteria on the survival of Candida albicans in the external environment, away from an animal host. The bacteria were represented by Aeromonas hydrophila, Bacillus cereus, Bacillus subtilis, Clostridium, Enterobacter, Klebsiella pneumoniae, Kluyvera ascorbata and Serratia marcescens. Under aerobic conditions, the yeast's growth was inhibited in the presence of bacterial growth; however, under anaerobic conditions, yeast and bacterial growth in co-cultures was similar to that observed for pure cultures. Subsequent assays revealed that the majority of bacterial strains aerobically produced extracellular hydrolytic enzymes capable of yeast cell wall hydrolysis, including chitinases and mannan-degrading enzymes. In contrast, except for the A. hydrophila strain, these enzymes were not detected in anaerobic bacterial cultures, nor was the antimicrobial compound prodigiosin found in anaerobic cultures of S. marcescens. When we suspended C. albicans cells in crude extracellular enzyme preparations from K. pneumoniae and S. marcescens, we detected no negative effect on yeast viability. However, we found that these preparations enhance the toxicity of prodigiosin towards the yeast, especially in combination with mannan-degrading enzymes. Analyses of the chitin and mannan content of yeast cell walls revealed that less chitin was produced under anaerobic than aerobic conditions; however, the levels of mannan, known for its low permeability, remained the same. The latter phenomenon, as well as reduced production of the bacterial enzymes and prodigiosin, may contribute to anaerobic growth and survival of C. albicans in the presence of bacteria.

Antagonistic effect of chitinolytic Pseudomonas and Bacillus on growth of fungal hyphae and spores of aflatoxigenic Aspergillus flavus

Abstract Use of biological control methods has become a promising strategy to counteract deleterious fungal contaminations and substantial post harvest losses. One of the major biological control methods is found in the use of fungal cell wall-degrading enzymes such as chitinases, which hydrolyze the homopolymer of β-1,4-1inked N-acetylglucosamine residues exposed on the fungal cell surface. In this study, antagonistic effects of the chitinolytic enzyme producing isolates of Pseudomonas fluorescens PB27, Bacillus cereus B1 and Bacillus thuringiensis K1 against the aflatoxigenic fungus Aspergillus flavus were investigated. Germination of the spores of A. flavus was inhibited by up to nearly 20% after co-incubation with P. fluorescens PB27 for 72 h, whereas the fungal spores were less affected when incubated with either B. cereus B1 or B. thuringiensis K1. When P. fluorescens or its extracted extracellular enzyme preparation was sprayed on the surface of hazelnuts, the growth of A. flavus was reduced nearly 25%. Scanning electron microscopy observations of hyphae and spores revealed that these enzymes induce numerous ultrastructural morphological changes during spore germination and mycelium growth. Since chitinolytic enzymes have not been shown to be toxic to humans or the environment, based on the observations in this study, an understanding of the functional properties and antagonistic effect of this natural antifungal agent can be practical in the management of post-harvest losses caused by food pathogenic and aflatoxin producing fungi.

Two new β-glucosidases from ethanol-fermenting fungus Mucor circinelloides NBRC 4572: enzyme purification, functional characterization, and molecular cloning of the gene

Two β-glucosidases (BGLs 1 and 2) were purified to homogeneity from the extracellular enzyme preparations of the ethanol-fermenting Mucor circinelloides NBRC 4572 statically grown on rice straw. BGLs 1 and 2 are monomeric glycoproteins whose apparent molecular masses (Ms) are around 78 kDa, which decreased by approximately 10 kDa upon enzymatic deglycosylation. Both BGLs showed similar enzyme characteristics in optimal temperature and pH, stability, and inhibitors. They were active against a wide range of aryl-β-glucosides and β-linked glucose oligosaccharides. Their amino acid sequences shared 81% identity and exhibited less than 60% identity with the known family-3 BGLs. Considering properties such as reduced inhibition by ethanol, glucose, and cellobiose, low transglucosylation activity, wider substrate range, less binding affinity to lignocellulosic materials, and abundant expression, BGL1 is likely to be more suitable for bioethanol production than BGL2 via simultaneous saccharification and fermentation of rice straw with M. circinelloides.

Performance of wood‐rotting fungi‐based enzymes on enzymic saccharification of rice straw

For effective saccharification of rice straw we focused on enzyme preparations from wood-rotting fungi that have the ability to degrade cell wall polysaccharides and lignin. We tested extracellular enzyme preparations from 14 species of fungi for saccharification activity and examined the factor for saccharification by statistical analysis. An enzyme preparation from Schizophyllum commune had the highest saccharification activity of rice straw. This preparation contained highly active endo-β-xylanase, endo-β-glucanase (CMCase), β-d-glucosidase and acetylxylan esterase. Correlation analysis of the 14 enzyme preparations demonstrated that acetylxylan esterase was closely related to saccharification activity in rice straw. Multiple regression analysis also showed that acetylxylan esterase had an important role in saccharification. Ligninolytic enzymes, which are characteristic of white-rot fungi, did not contribute to saccharification activity of rice straw. Deacetylation is an essential factor for saccharification of rice straw and enzyme preparations for saccharification need to contain highly active acetylxylan esterase as well as highly active cellulolytic and xylanolytic enzymes, but not ligninolytic ones.

Polysaccharidase and glycosidase production of avicel grown rumen fungusOrpinomycessp. GMLF5

Extracellular and cell-associated enzyme preparations were obtained from ruminal anaerobic fungi Orpinomyces sp. GMLF5 grown in culture containing microcrystalline cellulose (avicel) as sole energy source and degradation capacities of the preparations towards several polysaccharides and glycosides were studied. Fungus showed substantial increases in xylanase, carboxymethyl cellulase (CMCase), lichenase, amylase, beta-xylosidase, beta-glucosidase and alpha-L-arabinofuranosidase activities between 72 and 168 hours. High amounts of cell associated beta-xylosidase were noted in 4 and 5 days old cultures. Optimum temperature and pH of the polysaccharidases were found at 50 degrees C and 6.0-6.5, respectively. Xylanase was found to be virtually stable at 50 degrees C, CMCase and lichenase were stable at 40 degrees C for 200 min, however amylase was found more sensitive to heat treatment. The fibrolytic enzymes of the isolate GMLF5 were observed to be capable of hydrolyze the avicel.

A modified spectrophotometric assay to estimate deglycosylation of steroidal saponin to sapogenin by mixed ruminal microbes

The lack of a method for measuring deglycosylation of saponins in ruminal fluid has limited the ability to investigate the impact of these compounds on rumen microorganisms. A simple spectrophotometric assay was adapted and a protocol developed to enable measurement of steroidal saponin and sapogenin in ruminal fluid. The procedure was used for in vitro determination of deglycosylation activity of rumen bacteria obtained from cattle fed or not fed Yucca schidigera saponin, and to determine the relative deglycosylase activities of extracellular and cell-associated enzymes from ruminal content. Modifications to the spectrophotometric assay (i.e. heating time shortened to 10 min and 0.5 mL dH(2)O added to the reaction mixture) improved the stability of the optical density (425 nm) of the chromophore for up to 24 h post-reaction. Centrifugation (12 000 x g, 20 min) enabled differential estimations of steroidal saponin and sapogenin in ruminal fluid. Steroidal saponin added to defaunated ruminal fluid (dRF) or clarified ruminal fluid (cRF) was recovered completely from the mixture as saponin + sapogenin (99.1% and 100.6%, respectively), whereas saponin recovery from the supernatant of dRF was greatly reduced (P < 0.001) compared to that from supernatant of cRF (58.5 vs. 98.7%). Saponin recoveries from the supernatants of dRF and cRF did not differ between donor cattle fed or not fed Yucca schidigera saponin (59.2 vs. 57.3% and 98.4 vs. 99.3%, respectively). The majority (89-90%) of saponin added to a ruminal extracellular enzyme preparation was recoverable in supernatant after 24 h, compared with only 26-32% remaining in supernatant from incubation with a cell-associated enzymes fraction. Mixed rumen bacteria deglycosylate steroidal saponin to sapogenin, at activity levels unaffected by prior exposure to saponin, but they were unable to degrade the sapogenin core structure. Deglycosylation activity occurred primarily in the cell-associated enzyme fraction of ruminal content.

Isolation and culture of a marine bacterium degrading the sulfated fucans from marine brown algae.

Fucoidans are matrix polysaccharides from marine brown algae, consisting of an α-l-fucose backbone substituted by sulfate-ester groups and masked with ramifications containing other monosaccharide residues. In spite of their interest as biologically active compounds in a number of homologous and heterologous systems, no convenient sources with fucanase activity are available yet for the degradation of the fucalean algae. We here report on the isolation, characterization, and culture conditions of a bacterial strain capable of degrading various brown algal fucoidans. This bacterium, a member of the family Flavobacteriaceae, was shown to secrete fucoidan endo-hydrolase activity. An extracellular enzyme preparation was used to degrade the fucoidan from the brown alga Pelvetia canaliculata. End products included a tetrasaccharide and a hexasaccharide made of the repetition of disaccharidic units consisting of α-1→3-l-fucopyranose-2-sulfate-α-1→4-l-fucopyranose-2,3-disulfate, with the 3-linked residues at the nonreducing end.

Milk-clotting protease production by Nocardiopsis sp. in an inexpensive medium

The milk-clotting and proteolytic activities of extracellular enzyme preparations from Nocardiopsis sp. were investigated under different culture conditions. A soybean flour medium was used, with concentrations of soybean flour and of glucose varying from 0.25 to 1% w/v and from 0 to 1% w/v, respectively. Growth was monitored with 2 ml samples withdrawn from the culture medium at 8-h intervals, for determination of total protein, proteolytic activity, milk-clotting activity and sugar reduction. The best milk-clotting protease production, with a specific activity of 24.49 U/mg at 40 h, was obtained in the glucose-free medium containing soybean flour 1% w/v.

New enzymes with potential for PET surface modification

This work describes newly isolated organisms and their potential to modify the surface of polyethylene terephthalate (PET). Out of the different screening processes, four bacterial and five fungal strains were isolated. A PET model substrate was synthesized (bis (benzoyloxyethyl) terephthalate) and used in the screening process, mimicking the polymer in its crucial properties and having the advantage of defined hydrolysis products. On this model substrate, extracellular enzyme preparations from the isolated microorganisms showed a maximum activity of 8.54 nkat/L. All enzyme preparations showed esterase activity on p-nitrophenyl-acetate while no activity was found on p-nitrophenyl decanoate or p-nitrophenyl palmitate. Increased hydrophilicity of PET fabrics after enzyme treatment was found based on rising height and water dissipation measurements.

Optimization of extracellular endoxylanase, endoglucanase and peroxidase production by Streptomyces sp. F2621 isolated in Turkey.

To determine the effect of environmental conditions on the production of extracellular lignocellulose-degrading enzymes by Streptomyces sp. F2621 and to assess the potential use of these enzymes in the hydrolysis of lignocellulose material. The production of extracellular lignocellulose-degrading enzymes, endoxylanase, endoglucanase and peroxidase during the growth of Streptomyces sp. F2621 in basal salts-yeast extract medium containing different carbon sources and the effect of a number of environmental parameters (e.g. carbon sources and concentrations, pH and temperature) were investigated. The highest endoxylanase (22.41 U ml(-1)) and peroxidase (0.58 U ml(-1)) activities were obtained after 2-4 days of incubation at 30 degrees C in a basal salts medium containing 0.4% (w/v) oat spelt xylan and 0.6% (w/v) yeast extract, corresponding to C : N ratio of 6 : 1. Cell-free extracellular enzyme preparations from the strain were capable of releasing both sugar and aromatic compounds during incubation with eucalyptus paper pulp, straw and xylan. Overall, 9.3% hydrolysis of xylan occurred after 24-h incubation. However the rates of hydrolysis of paper pulp and straw were approximately twofold less than xylan hydrolysis, although the total percentage hydrolysis of available substrate (24.5% and 16.3%, respectively) was greater than xylan hydrolysis. The high levels of enzyme production achieved under batch cultivation conditions, coupled with no significant production of endoglucanase during the growth phase of organism and the release of both sugar and aromatic compounds from paper pulp and straw signify the suitability for these enzymes for industrial applications such as pulp and paper production. The results highlight the environmental conditions for the production of extracellular lignocellulose-degrading enzymes by Streptomyces sp. F2621 and suggest the use of streptomycetes and/or their enzymes in industrial processes.

Biodeinking of mixed office waste paper by alkaline active cellulases from alkalotolerant Fusarium sp.

Two extracellular alkalistable 1,4-β- d-glucan-4-glucanohydrolase (EC 3.2.1.4) fractions, namely Endo A and Endo B were separated from the culture filtrate of an alkalotolerant Fusarium strain by gel chromatography on Bio Gel P-100 column. The viscometric characterization of enzyme indicated that the Endo B was 1.9 times more random in action as compared to Endo A. Both the endoglucanases showed comparable patterns of their ability to release short fibers in the reaction mixture when filter paper was used as the substrate. The average size of released short fibers was determined by scanning electron microscopy ranged between 20 and 100 μm. Adsorption characteristics of the endoglucanases indicated that the enzyme complex acts prominently at the surface of substrate rather than at their cracked edges. The X-ray diffractograms showed that the crystallinity of the substrates does not vary significantly in the initial stages of the enzymatic action. The extracellular enzyme preparation was found to be suitable for deinking of mixed office waste papers. The enzyme treatment resulted in the increase in brightness with the reduction in ink counts of the recycled paper. Based on the distinct properties of endoglucanases a probable mechanism of enzymatic deinking process is presented schematically.

Purification and characterization of Lip2 and Lip3 isoenzymes from a Candida rugosa pilot-plant scale fed-batch fermentation

Previous purification of a crude extracellular enzyme preparation from Candida rugosa ATCC 14830 pilot-plant fed-batch fermentations showed the presence of two lipase isoenzymes, Lip2 and Lip3, differing in their molecular masses (58 and 62 kDa, respectively). These enzymes were purified but the lipases were forming active aggregates with a molecular mass higher than 200 kDa. In this work we developed a purification method following three steps: ammonium sulfate precipitation, sodium cholate treatment and ethanol/ether precipitation, and anion exchange chromatography which allowed the sequential disaggregation of the isoenzymes. Pure and monomeric Lip2 and Lip3 were characterized according to p I, glycosylation and activity for p-nitrophenol esters and triacylglycerols of varying acyl chain. Lip3 was the best catalyst for the hydrolysis of the simple esters and triacylglycerols with short and medium acyl chains.

Rapid detection of malto-oligosaccharide-forming bacterial amylases by high performance anion-exchange chromatography.

High performance anion-exchange chromatography with pulsed amperometric detection was applied for the rapid analysis of malto-oligosaccharides formed by extracellular enzyme preparations from 49 starch-degrading bacterial strains isolated from soil and compost samples. Malto-oligosaccharide-forming amylases, indicated by a predominant formation of maltohexaose from starch, were produced by enzyme preparations from four of the isolates growing at pH 7.0 and 10.

Characterization of the lipase and esterase multiple forms in an enzyme preparation from a Candida rugosa pilot-plant scale fed-batch fermentation

Characterization of crude extracellular enzyme preparations from Candida rugosa ATCC 14830 pilot-plant fed-batch fermentations showed the presence of multiple forms of lipases and esterases when a synthetic medium with oleic acid as a carbon source was used. Four major components were identified. An esterase of about 43 kDa, which was purified from the enzyme preparation by hydrophobic interaction and anion exchange chromatography. The N-terminal sequence for this esterase was homologous to acyl-CoA thioesterases YTE-1 and YTE-2 from C. rugosa [Diczfalusy MA, Alexson SEH. Arch Biochem Biophys 1996;334:104–112]. A 50.4-kDa protein with unknown activity whose sequence did not show any correspondence to previously described sequences reported in protein data banks. Also, two lipase isoforms were separated by means of hydrophobic interaction and ion-exchange chromatography. N-terminal sequencing of these proteins revealed that they corresponded to Lip2 and Lip3 lipases but not to Lip1, the major isoform usually present in crude lipase commercial preparations from C. rugosa (e.g. Sigma). Besides, these lipases were found to be tightly associated to a high-molecular-weight polysaccharide co-secreted by C. rugosa during the fermentation. These four proteins also have been characterized in terms of glycosylation degree.