Effect of the single mutation N9Y on the catalytical properties of xylanase Xyn11A from Cellulomonas uda: a biochemical and molecular dynamic simulation analysis.

Cellulomonas uda produces Xyn11A, moderately thermostable xylanase, with optimal activity at 50 °C and pH 6.5. An improvement in the biochemical properties of Xyn11A was achieved by site-directed mutagenesis approach. Wild-type xylanase, Xyn11A-WT, and its mutant Xyn11A-N9Y were expressed in Escherichia coli, and then both enzymes were purified and characterized. Xyn11A-N9Y displayed optimal activity at 60 °C and pH 7.5, an upward shift of 10 °C in the optimum temperature and an upward shift of 1 unit in optimum pH; also, it manifested an 11-fold increase in thermal stability at 60 °C, compared to that displayed by Xyn11A-WT. Molecular dynamics simulations of Xyn11A-WT and Xyn11A-N9Y suggest that the substitution N9Y leads to an array of secondary structure changes at the N-terminal end and an increase in the number of hydrogen bonds in Xyn11A-N9Y. Based on the significant improvements, Xyn11A-N9Y may be considered as a candidate for several biotechnological applications.

Charge Interactions in a Highly Charge-Depleted Protein.

Electrostatic forces are important for protein folding and are favored targets of protein engineering. However, interactions between charged residues are difficult to study because of the complex network of interactions found in most proteins. We have designed a purposely simple system to investigate this problem by systematically introducing individual and pairs of charged and titratable residues in a protein otherwise free of such residues. We used constant pH molecular dynamics simulations, NMR spectroscopy, and thermodynamic double mutant cycles to probe the structure and energetics of the interaction between the charged residues. We found that the partial burial of surface charges contributes to a shift in pKa value, causing an aspartate to titrate in the neutral pH range. Additionally, the interaction between pairs of residues was found to be highly context dependent, with some pairs having no apparent preferential interaction, while other pairs would engage in coupled titration forming a highly stabilized salt bridge. We find good agreement between experiments and simulations and use the simulations to rationalize our observations and to provide a detailed mechanistic understanding of the electrostatic interactions.

Domain architecture divergence leads to functional divergence in binding and catalytic domains of bacterial and fungal cellobiohydrolases.

Cellobiohydrolases directly convert crystalline cellulose into cellobiose and are of biotechnological interest to achieve efficient biomass utilization. As a result, much research in the field has focused on identifying cellobiohydrolases that are very fast. Cellobiohydrolase A from the bacterium Cellulomonas fimi (CfCel6B) and cellobiohydrolase II from the fungus Trichoderma reesei (TrCel6A) have similar catalytic domains (CDs) and show similar hydrolytic activity. However, TrCel6A and CfCel6B have different cellulose-binding domains (CBDs) and linkers: TrCel6A has a glycosylated peptide linker, whereas CfCel6B's linker consists of three fibronectin type 3 domains. We previously found that TrCel6A's linker plays an important role in increasing the binding rate constant to crystalline cellulose. However, it was not clear whether CfCel6B's linker has similar function. Here we analyze kinetic parameters of CfCel6B using single-molecule fluorescence imaging to compare CfCel6B and TrCel6A. We find that CBD is important for initial binding of CfCel6B, but the contribution of the linker to the binding rate constant or to the dissociation rate constant is minor. The crystal structure of the CfCel6B CD showed longer loops at the entrance and exit of the substrate-binding tunnel compared with TrCel6A CD, which results in higher processivity. Furthermore, CfCel6B CD showed not only fast surface diffusion but also slow processive movement, which is not observed in TrCel6A CD. Combined with the results of a phylogenetic tree analysis, we propose that bacterial cellobiohydrolases are designed to degrade crystalline cellulose using high-affinity CBD and high-processivity CD.

Convergent evolution of processivity in bacterial and fungal cellulases.

Cellulose is the most abundant biomass on Earth, and many microorganisms depend on it as a source of energy. It consists mainly of crystalline and amorphous regions, and natural degradation of the crystalline part is highly dependent on the degree of processivity of the degrading enzymes (i.e., the extent of continuous hydrolysis without detachment from the substrate cellulose). Here, we report high-speed atomic force microscopic (HS-AFM) observations of the movement of four types of cellulases derived from the cellulolytic bacteria Cellulomonas fimi on various insoluble cellulose substrates. The HS-AFM images clearly demonstrated that two of them (CfCel6B and CfCel48A) slide on crystalline cellulose. The direction of processive movement of CfCel6B is from the nonreducing to the reducing end of the substrate, which is opposite that of processive cellulase Cel7A of the fungus Trichoderma reesei (TrCel7A), whose movement was first observed by this technique, while CfCel48A moves in the same direction as TrCel7A. When CfCel6B and TrCel7A were mixed on the same substrate, "traffic accidents" were observed, in which the two cellulases blocked each other's progress. The processivity of CfCel6B was similar to those of fungal family 7 cellulases but considerably higher than those of fungal family 6 cellulases. The results indicate that bacteria utilize family 6 cellulases as high-processivity enzymes for efficient degradation of crystalline cellulose, whereas family 7 enzymes have the same function in fungi. This is consistent with the idea of convergent evolution of processive cellulases in fungi and bacteria to achieve similar functionality using different protein foldings.

Three-Enzyme Phosphorylase Cascade for Integrated Production of Short-Chain Cellodextrins.

Cellodextrins are linear ß-1,4-gluco-oligosaccharides that are soluble in water up to a degree of polymerization (DP) of ≈6. Soluble cellodextrins have promising applications as nutritional ingredients. A DP-controlled, bottom-up synthesis from expedient substrates is desired for their bulk production. Here, a three-enzyme glycoside phosphorylase cascade is developed for the conversion of sucrose and glucose into short-chain (soluble) cellodextrins (DP range 3-6). The cascade reaction involves iterative ß-1,4-glucosylation of glucose from α-glucose 1-phosphate (αGlc1-P) donor that is formed in situ from sucrose and phosphate. With final concentration and yield of the soluble cellodextrins set as targets for biocatalytic synthesis, three major factors of reaction efficiency are identified and partly optimized: the ratio of enzyme activity, the ratio of sucrose and glucose, and the phosphate concentration used. The efficient use of the phosphate/αGlc1-P shuttle for cellodextrin production is demonstrated and the soluble product at 40 g L-1 is obtained under near-complete utilization of the donor substrate offered (88 mol% from 200 mm sucrose). The productivity is 16 g (L h)-1 . Through a simple two-step route, the soluble cellodextrins are recovered from the reaction mixture in ≥95% purity and ≈92% yield. Overall, this study provides the basis for their integrated production.

Cellulomonas telluris sp. nov., an endoglucanase-producing actinobacterium isolated from Badain Jaran desert sand.

A Gram-stain-positive, aerobic bacterium, designated CPCC 204705T, was isolated from a desert soil sample, collected from the Badain Jaran desert. Growth of strain CPCC 204705T was observed at pH 6.0-8.0 and 15-37 °C, with optimal growth at 28 °C and pH 7.0. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain CPCC 204705T belonged to the genus Cellulomonas, showing the highest similarity (98.54 %) of 16S rRNA gene sequence to Cellulomonas oligotrophica JCM 17534T. The peptidoglycan type was A4ß, containing d-ornithine and d-glutamic acids as diagnostic amino acids. Rhamnose and galactose were detected in the whole-cell hydrolysate as diagnostic sugars. The major cellular fatty acids were anteiso-C15 : 0, anteiso-C15 : 1A, C14 : 0 and C16 : 0. The major menaquinone was MK-9 (H4) and the polar lipid system contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol mannoside, one unidentified lipid, one unidentified aminolipid and two unidentified aminophospholipids. The DNA-DNA hybridization value between strain CPCC 204705T and C. oligotrophica JCM 17534T was 7.1±0.4 %, and the value of average nucleotide identity between these two strains was 79.8 %. The DNA G+C content of strain CPCC 204705T was 75.4 mol%. Based on the results of physiological experiments, chemotaxonomic data, phylogenetic analysis and DNA-DNA hybridization value, strain CPCC 204705T should be classified as a novel Cellulomonas species. The name Cellulomonas telluris sp. nov. is proposed, with strain CPCC 204705T (=DSM 105430T=KCTC 39974T) as the type strain.

Quantifying cellulose accessibility during enzyme-mediated deconstruction using 2 fluorescence-tagged carbohydrate-binding modules.

Two fluorescence-tagged carbohydrate-binding modules (CBMs), which specifically bind to crystalline (CBM2a-RRedX) and paracrystalline (CBM17-FITC) cellulose, were used to differentiate the supramolecular cellulose structures in bleached softwood Kraft fibers during enzyme-mediated hydrolysis. Differences in CBM adsorption were elucidated using confocal laser scanning microscopy (CLSM), and the structural changes occurring during enzyme-mediated deconstruction were quantified via the relative fluorescence intensities of the respective probes. It was apparent that a high degree of order (i.e., crystalline cellulose) occurred at the cellulose fiber surface, which was interspersed by zones of lower structural organization and increased cellulose accessibility. Quantitative image analysis, supported by 13C NMR, scanning electron microscopy (SEM) imaging, and fiber length distribution analysis, showed that enzymatic degradation predominates at these zones during the initial phase of the reaction, resulting in rapid fiber fragmentation and an increase in cellulose surface crystallinity. By applying this method to elucidate the differences in the enzyme-mediated deconstruction mechanisms, this work further demonstrated that drying decreased the accessibility of enzymes to these disorganized zones, resulting in a delayed onset of degradation and fragmentation. The use of fluorescence-tagged CBMs with specific recognition sites provided a quantitative way to elucidate supramolecular substructures of cellulose and their impact on enzyme accessibility. By designing a quantitative method to analyze the cellulose ultrastructure and accessibility, this study gives insights into the degradation mechanism of cellulosic substrates.

A new enzymatic method assessing the impact of wastewater treatment plant effluents on the assimilative capacity of small rivers.

Microorganisms play an important role in maintaining a good water quality in rivers by degrading organic material, including toxic substances. In the present study, we analyzed the potential impact of municipal wastewater treatment plant (WWTP) effluents as a major stress factor on the assimilative capacity of small rivers. It was the aim to develop a new bioassay for assessing such impacts in the receiving rivers by measuring the activity of extracellular enzymes (exoenzymes) in bacteria. Therefore, we established a specific in-vitro assay to detect inhibitory effects of solid phase-enriched water samples on ß-glucosidase (BGL) activity of the actinobacterium Cellulomonas uda as a proxy for the microbial decomposition of organic substances and thus for the assimilative capacity of surface waters. We found significant reductions of BGL activity in the WWTP effluents and in the receiving waters directly downstream as well as a relative quick recovery over the further course of the water bodies. The new bioassay offers a promising tool for the assessment of the assimilative capacity in surface waters and a potential impact of WWTP effluents on this key ecosystem function. Abbreviations WWTP wastewater treatment plant BGL ß-glucosidase EU-WFD European Water Framework Directive FAU Formazin Attenuation Units PE population equivalents REF relative enrichment factor; SPE solid phase extraction MTBE methyl-tert-buthyl-ether DMSO dimethyl-sulfoxide NPG 4-nitrophenol-ß-d-glucopyranoside DOC dissolved organic carbon.

Product solubility control in cellooligosaccharide production by coupled cellobiose and cellodextrin phosphorylase.

Soluble cellodextrins (linear ß-1,4-d-gluco-oligosaccharides) have interesting applications as ingredients for human and animal nutrition. Their bottom-up synthesis from glucose is promising for bulk production, but to ensure a completely water-soluble product via degree of polymerization (DP) control (DP ≤ 6) is challenging. Here, we show biocatalytic production of cellodextrins with DP centered at 3 to 6 (~96 wt.% of total product) using coupled cellobiose and cellodextrin phosphorylase. The cascade reaction, wherein glucose was elongated sequentially from α-d-glucose 1-phosphate (αGlc1-P), required optimization and control at two main points. First, kinetic and thermodynamic restrictions upon αGlc1-P utilization (200 mM; 45°C, pH 7.0) were effectively overcome (53% → ≥90% conversion after 10 hrs of reaction) by in situ removal of the phosphate released via precipitation with Mg2+ . Second, the product DP was controlled by the molar ratio of glucose/αGlc1-P (∼0.25; 50 mM glucose) used in the reaction. In optimized conversion, soluble cellodextrins in a total product concentration of 36 g/L were obtained through efficient utilization of the substrates used (glucose: 98%; αGlc1-P: ∼80%) after 1 hr of reaction. We also showed that, by keeping the glucose concentration low (i.e., 1-10 mM; 200 mM αGlc1-P), the reaction was shifted completely towards insoluble product formation (DP ∼9-10). In summary, this study provides the basis for an efficient and product DP-controlled biocatalytic synthesis of cellodextrins from expedient substrates.

Characterization of a New DyP-Peroxidase from the Alkaliphilic Cellulomonad, Cellulomonas bogoriensis.

DyP-type peroxidases are heme-containing enzymes that have received increasing attention over recent years with regards to their potential as biocatalysts. A novel DyP-type peroxidase (CboDyP) was discovered from the alkaliphilic cellulomonad, Cellulomonas bogoriensis, which could be overexpressed in Escherichia coli. The biochemical characterization of the recombinant enzyme showed that it is a heme-containing enzyme capable to act as a peroxidase on several dyes. With the tested substrates, the enzyme is most active at acidic pH values and is quite tolerant towards solvents. The crystal structure of CboDyP was solved which revealed atomic details of the dimeric heme-containing enzyme. A peculiar feature of CboDyP is the presence of a glutamate in the active site which in most other DyPs is an aspartate, being part of the DyP-typifying sequence motif GXXDG. The E201D CboDyP mutant was prepared and analyzed which revealed that the mutant enzyme shows a significantly higher activity on several dyes when compared with the wild-type enzyme.

Secretome profile of Cellulomonas sp. B6 growing on lignocellulosic substrates.

AIMS: Lignocellulosic biomass deconstruction is a bottleneck for obtaining biofuels and value-added products. Our main goal was to characterize the secretome of a novel isolate, Cellulomonas sp. B6, when grown on residual biomass for the formulation of cost-efficient enzymatic cocktails. METHODS AND RESULTS: We identified 205 potential CAZymes in the genome of Cellulomonas sp. B6, 91 of which were glycoside hydrolases (GH). By secretome analysis of supernatants from cultures in either extruded wheat straw (EWS), grinded sugar cane straw (SCR) or carboxymethylcellulose (CMC), we identified which proteins played a role in lignocellulose deconstruction. Growth on CMC resulted in the secretion of two exoglucanases (GH6 and GH48) and two GH10 xylanases, while growth on SCR or EWS resulted in the identification of a diversity of CAZymes. From the 32 GHs predicted to be secreted, 22 were identified in supernatants from EWS and/or SCR cultures, including endo- and exoglucanases, xylanases, a xyloglucanase, an arabinofuranosidase/ß-xylosidase, a ß-glucosidase and an AA10. Surprisingly, among the xylanases, seven were GH10. CONCLUSIONS: Growth of Cellulomonas sp. B6 on lignocellulosic biomass induced the secretion of a diverse repertoire of CAZymes. SIGNIFICANCE AND IMPACT OF THE STUDY: Cellulomonas sp. B6 could serve as a source of lignocellulose-degrading enzymes applicable to bioprocessing and biotechnological industries.

Can a Charged Surfactant Unfold an Uncharged Protein?

Does sodium dodecyl sulfate (SDS) denature proteins through electrostatic SDS-protein interactions? We show that a protein completely lacking charged side chains is unfolded by SDS in a manner similar to charged proteins, revealing that formal protein charges are not required for SDS-induced protein unfolding or binding.

A single step electrochemical integration of gold nanoparticles, cholesterol oxidase, cholesterol esterase and mediator with polypyrrole films for fabrication of free and total cholesterol nanobiosensors.

The development of free and total cholesterol nanobiosensors based on a single step electrochemical integration of gold nanoparticles (AuNPs), cholesterol oxidase (COx), cholesterol esterase (CE) and a mediator with polypyrrole (PPy) films is described. The incorporation of the various components in the PPy films was confirmed by chronopotentiometry, cyclic voltammetry (CV), scanning electron microscopy, energy dispersive X-ray analysis (SEM-EDX), and Fourier transformed infrared (FTIR) spectroscopy. The free cholesterol, PPy-NO3--Fe(CN)64--AuNPs-COx, nanobiosensor achieved a minimum detectable concentration of 5 µM, a linear concentration range of 5-25 µM and a sensitivity of 1.6 µA cm-2 µM-1 in 0.05 M phosphate buffer (pH 7.00). For the total cholesterol, PPy-NO3--Fe(CN)64--AuNPs-COx-CE, nanobiosensor which also involved the co-incorporation of cholesterol esterase (CE) with the other components, the achieved performances include a minimum detectable total cholesterol concentration of 25 µM, a broader linear concentration range of 25-170 µM and a lower sensitivity of 0.1 µA µM-1 cm-2. Owing to its high selectivity, the presence of common interferants did not affect the total cholesterol measurement with the PPy-NO3--Fe(CN)64--AuNPs-COx-CE nanobiosensor. Both nanobiosensors were successfully used for direct and indirect determination of total cholesterol in human blood serum samples.

Characterisation of novel biomass degradation enzymes from the genome of Cellulomonas fimi.

Recent analyses of genome sequences belonging to cellulolytic bacteria have revealed many genes potentially coding for cellulosic biomass degradation enzymes. Annotation of these genes however, is based on few biochemically characterised examples. Here we present a simple strategy based on BioBricks for the rapid screening of candidate genes expressed in Escherichia coli. As proof of principle we identified over 70 putative biomass degrading genes from bacterium Cellulomonas fimi, expressing a subset of these in BioBrick format. Six novel genes showed activity in E. coli. Four interesting enzymes were characterised further. α-l-arabinofuranosidase AfsB, ß-xylosidases BxyF and BxyH and multi-functional ß-cellobiosidase/xylosidase XynF were partially purified to determine their optimum pH, temperature and kinetic parameters. One of these enzymes, BxyH, was unexpectedly found to be highly active at strong alkaline pH and at temperatures as high as 100 °C. This report demonstrates a simple method of quickly screening and characterising putative genes as BioBricks.

Cellulomonas fimi secretomes: In vivo and in silico approaches for the lignocellulose bioconversion.

Lignocellulose degradation is a challenging step for value added products and biofuels production. Cellulomonas fimi secretes complex mixtures of carbohydrate active enzymes (CAZymes) which synergistically degrade cellulose and hemicelluloses. Their characterization may provide new insights for enzymatic cocktails implementation. Bioinformatic analysis highlighted 1127 secreted proteins, constituting the in silico secretome, graphically represented in a 2DE map. According to Blast2GO functional annotation, many of these are involved in carbohydrates metabolism. In vivo secretomes were obtained, growing C. fimi on glucose, CMC or wheat straw for 24 h. Zymography revealed degradative activity on carbohydrates and proteomic analysis identified some CAZymes, only in secretomes obtained with CMC and wheat straw. An interaction between cellobiohydrolases is proposed as a strategy adopted by soluble multimodular cellulases. Such approach can be crucial for a better characterization and industrial exploitation of the synergism among C. fimi enzymes.

Characterization of a thermostable endoglucanase from Cellulomonas fimi ATCC484.

Bacteria in the genus Cellulomonas are well known as secretors of a variety of mesophilic carbohydrate degrading enzymes (e.g., cellulases and hemicellulases), active against plant cell wall polysaccharides. Recent proteomic analysis of the mesophilic bacterium Cellulomonas fimi ATCC484 revealed uncharacterized enzymes for the hydrolysis of plant cell wall biomass. Celf_1230 (CfCel6C), a secreted protein of Cellulomonas fimi ATCC484, is a novel member of the GH6 family of cellulases that could be successfully expressed in Escherichia coli. This enzyme displayed very little enzymatic/hydrolytic activity at 30 °C, but showed an optimal activity around 65 °C, and exhibited a thermal denaturation temperature of 74 °C. In addition, it also strongly bound to filter paper despite having no recognizable carbohydrate binding module. Our experiments show that CfCel6C is a thermostable endoglucanase with activity on a variety of ß-glucans produced by an organism that struggles to grow above 30 °C.

Cloning and characterization of two novel ß-glucosidase genes encoding isoenzymes of the cellobiase complex from Cellulomonas biazotea.

Enzymatic degradation of cellulosic waste to generate renewable biofuels has offered an attractive solution to the energy problem. Synergistic hydrolysis of cellulose residues requires the participation of three different types of cellulases - endoglucanases, exoglucanases, and ß-glucosidases (Bgl). Our group has been interested in using Bgl of Cellulomonas biazotea in studies designed to investigate cooperative action among different cellulases. We previously have cloned bgl genes encoding Cba and Cba3, which are C. biazotea Bgl isozymes representing two different Bgl families, respectively; specifically, Glycoside Hydrolase Family 3 (GH3) and Glycoside Hydrolase Family 1 (GH1). To gain an understanding of the complexity of Bgl in C. biazotea, we analyzed E. coli clones containing plasmids into which C. biazotea DNA had been inserted; these clones could hydrolyze 4-methylumbelliferyl ß-d-glucopyranoside (MUG) supplemented in solid agar media, suggesting they might contain bgl genes. Through restriction analysis and DNA sequencing, two novel bgl genes, designated cba4 and cba5 and encoding Cba4 (484 amino acids) and Cba5 (758 amino acids) were identified. Cba4 and Cba5 appear to be members of GH1 and GH3, respectively. Both Cba4 and Cba5 were concluded to be genuine cellobiases as each was found to enable their E. coli hosts to survive on media in which cellobiose was the sole carbon source. Despite lacking a typical secretory signal sequence, Cba4 and Cba5 are secretory proteins. Although they are isoenzymes, Cba, Cba3, Cba4, and Cba5 were shown to possess distinct substrate specificities. These four Bgl members may play important roles in hydrolyzing a wide variety of ß-glucosides including cellobiose and non-cellulosic substrates.

Expression of a codon-optimized ß-glucosidase from Cellulomonas flavigena PR-22 in Saccharomyces cerevisiae for bioethanol production from cellobiose.

Bioethanol is one of the main biofuels produced from the fermentation of saccharified agricultural waste; however, this technology needs to be optimized for profitability. Because the commonly used ethanologenic yeast strains are unable to assimilate cellobiose, several efforts have been made to express cellulose hydrolytic enzymes in these yeasts to produce ethanol from lignocellulose. The C. flavigenabglA gene encoding ß-glucosidase catalytic subunit was optimized for preferential codon usage in S. cerevisiae. The optimized gene, cloned into the episomal vector pRGP-1, was expressed, which led to the secretion of an active ß-glucosidase in transformants of the S. cerevisiae diploid strain 2-24D. The volumetric and specific extracellular enzymatic activities using pNPG as substrate were 155 IU L-1 and 222 IU g-1, respectively, as detected in the supernatant of the cultures of the S. cerevisiae RP2-BGL transformant strain growing in cellobiose (20 g L-1) as the sole carbon source for 48 h. Ethanol production was 5 g L-1 after 96 h of culture, which represented a yield of 0.41 g g-1 of substrate consumed (12 g L-1), equivalent to 76% of the theoretical yield. The S. cerevisiae RP2-BGL strain expressed the ß-glucosidase extracellularly and produced ethanol from cellobiose, which makes this microorganism suitable for application in ethanol production processes with saccharified lignocellulose.

Identification of an α-(1,4)-Glucan-Synthesizing Amylosucrase from Cellulomonas carboniz T26.

Amylosucrase, catalyzing the synthesis of α-(1,4)-glucan from sucrose, has been widely studied and used in carbohydrate biotransformation because of its versatile activities. In this study, a novel amylosucrase was characterized from Cellulomonas carboniz T26. The recombinant enzyme was overexpressed in Escherchia coli and purified by nickel affinity chromatography. It was determined to be a monomeric protein with a molecular mass of 72 kDa. The optimum pH and temperature for transglucosylation were measured to be pH 7.0 and 40 °C. The transglucosylation activity was significantly higher than the hydrolytic activity. The main product generated from sucrose was structurally determined to be α-(1,4)-glucan. A small amount of glucose was produced by hydrolysis, and sucrose isomers including turanose and trehalulose were generated as minor products. The ratio of hydrolytic, polymerization, and isomerization reactions was calculated to be 5.8:84.0:10.2. The enzyme favored production of long-chain insoluble α-glucan at lower temperature.

Amperometric triglyceride bionanosensor based on nanoparticles of lipase, glycerol kinase, glycerol-3-phosphate oxidase.

The nanoparticles (NPs) aggregates of lipase from porcine pancreas, glycerol kinase (GK) from Cellulomonas sp. and glycerol-3-phosphate oxidase (GPO) from Aerococcus viridanss were prepared by desolvation and glutaraldehyde crosslinking and functionalized by cysteamine. These enzyme nanoparticles (ENPs) were characterized by transmission electron microscopy (TEM) and Fourier transform infra red (FTIR) spectroscopy. The functionalzed ENPs aggregates were co-immobilized covalently onto polycrystalline Au electrode through thiolated bond. An improved amperometric triglyceride (TG) bionanosensor was constructed using this ENPs modified Au electrode as working electrode. Biosensor showed optimum current at 1.2 V within 5s, at pH 6.5 and 35 °C.A linear relationship was obtained between current (mA) and triolein concentration in lower concentration range,10-100 mg/dL and higher concentration range, 100-500 mg/dL. Limit of detection (LOD) of bionanosensor was 1.0 µg/ml. Percent analytical recovery of added trolein (50 and 100 mg/dL) in serum was 95.2 ± 0.5 and 96.0 ± 0.17. Within and between batch coefficients of variation (CV) were 2.33% and 2.15% respectively. A good correlation (R2 = 0.99) was obtained between TG values in sera measured by present biosensor and standard enzymic colorimetric method with the regression equation: y= (0.993x + 0.967). ENPs/Au electrode was used 180 times over a period of 3 months with 50% loss in its initial activity, when stored dry at 4 °C.