Enzyme Units Research Articles

Structures of a dimodular nonribosomal peptide synthetase reveal conformational flexibility.

Nonribosomal peptide synthetases (NRPSs) are biosynthetic enzymes that synthesize natural product therapeutics using a modular synthetic logic, whereby each module adds one aminoacyl substrate to the nascent peptide. We have determined five x-ray crystal structures of large constructs of the NRPS linear gramicidin synthetase, including a structure of a full core dimodule in conformations organized for the condensation reaction and intermodular peptidyl substrate delivery. The structures reveal differences in the relative positions of adjacent modules, which are not strictly coupled to the catalytic cycle and are consistent with small-angle x-ray scattering data. The structures and covariation analysis of homologs allowed us to create mutants that improve the yield of a peptide from a module-swapped dimodular NRPS.

Moving modules drive biosynthesis

Biosynthetic Enzymes Modular biosynthesis of small molecules—where enzyme units can be swapped in and out of assembly line complexes to produce desired products—is a distant goal in the lab despite a huge diversity of modular systems in nature. Part of the challenge is in understanding how modules interact and hand off intermediates. Reimer et al. determined crystal structures of portions of a nonribosomal peptide synthetase, including a full dimodule. Module positioning differed between these structures even when the same intermediate was attached to the enzyme. The authors used small-angle x-ray scattering to confirm that large conformational changes are possible during biosynthesis and handoff between modules. Science , this issue p. [eaaw4388][1] [1]: /lookup/doi/10.1126/science.aaw4388

Recovery of starch from cassava bagasse for cyclodextrin production by sequential treatment with α-amylase and cyclodextrin glycosyltransferase

Abstract This work aimed at the enzymatic recovery of starch from cassava bagasse using α-amylase and the production of cyclodextrins (CDs) by cyclodextrin glycosyltransferase (CGTase). Cassava bagasse containing 50% (w/w) starch (dry basis) was hydrolyzed by α-amylase until a maximum starch recovery as a maltooligosaccharide solution. Afterwards, this solution was used for the production of CDs using CGTase as biocatalyst. Optimal conditions for starch extraction using α-amylase were 60 °C and 0.15 mL enzyme/kg dry bagasse, with 93% starch recovery. Several operational parameters were evaluated to optimize the CD yields, such as starch dextrose equivalent (DE), temperature and CGTase loading. The starch DE did not significantly influence the production of CDs, resulting in a similar yield for all experiments (43%). The highest productivity of CDs (93.6 mg of CDs per enzyme Unit) was obtained using an enzyme loading of 2.5 U/g starch for 1 h at 60 °C and pH 6.0. After 6 h of reaction at 60 °C and pH 6.0, the CD yield reached a value of 50%. These findings did indicate that cassava bagasse is a promising feedstock for the production of CDs using this bi-enzymatic system.

Role of putrescine in ovary and embryo development in fruit bat Cynopterus sphinx during embryonic diapause.

The aim of this study was to evaluate the effect of putrescine on ovarian activity and the rate of embryonic development in Cynopterus sphinx during delayed development. The result showed the presence of a rate-limiting enzyme, ornithine decarboxylase-1, in both ovary and utero-embryonic unit of C. sphinx suggests a synthesis of putrescine in these sites. The corpus luteum showed increased, whereas utero-embryonic unit showed decreased production of putrescine during delayed development as compared with the normal development. The bat treated in vivo with putrescine during delayed development showed increase in progesterone and estradiol synthesis, correlated with increased expression of luteinizing hormone receptor, steroidogenic acute receptor protein, and 3β-hydroxysteroid dehydrogenase through extracellular signal-regulated kinase (ERK1/2)-mediated pathway in the ovary; but showed increase in the weight and expression of progesterone receptor (PR), B-cell lymphoma 2, proliferating cell nucleus antigen, and vascular endothelial growth factor proteins in utero-embryonic unit. The in vitro treatment of putrescine showed stimulatory whereas treatment with an inhibitor of putrescine, 2-difluoromethylornithine caused an inhibitory effect on ovarian progesterone synthesis and cell proliferation, and cell survival in the utero-embryonic unit. In conclusion, the putrescine showed two separate roles during embryonic diapause, high concentration of putrescine in the ovary may support corpus luteum and basal synthesis of progesterone, whereas a low level of putrescine causes retarded embryonic development by inhibiting cell proliferation in the utero-embryonic unit. The bat treated with putrescine either directly promotes cell proliferation, cell survival, and angiogenic activities or acts indirectly increasing PR on utero-embryonic unit thereby activating development in delayed embryo in C. sphinx.

Antiphosphatidylserine/prothrombin complex antibodies as a determinant of prothrombotic plasma fibrin clot properties in patients with antiphospholipid syndrome

BackgroundAntiphosphatidylserine/prothrombin complex (aPS/PT) antibodies are recognized as a marker for antiphospholipid syndrome (APS). Dense and poorly lysable fibrin clots occur in thrombotic APS. Compact clots predict thromboembolism, but determinants of the unfavorable clot phenotype remain unknown in APS. We hypothesized that elevated aPS/PT antibodies determine unfavorable clot features. MethodsIn a cohort study involving 124 consecutive patients with thrombotic APS, we measured at baseline plasma fibrin clot permeability (Ks), efficiency of fibrinolysis (clot lysis time, CLT), and turbidity (off anticoagulation) along with immunoglobulin (Ig)G/IgM aPS/PT. During follow‐up, symptomatic thromboembolic events were recorded. ResultsElevated IgG and IgM aPS/PT antibodies >30 international enzyme units (UI) were detected in 54.8% and 42.7% of APS patients, including 76.2% and 54% of lupus anticoagulant‐ (LA, n = 63) positive patients, respectively. Elevated IgG and IgM aPS/PT antibodies predicted low Ks (lower quartile, <6 × 10−9 cm2; odds ratio [OR] = 5.93, 95% confidence interval [CI] 2.09‐16.82 and OR = 11.79, 95% CI 4.10‐33.92) and prolonged CLT (top quartile, ≥116 min; OR = 4.85, 95% CI 2.42‐25.07 and OR = 6.04, 95% CI 2.42‐15.07). No such associations were observed for anticardiolipin or β2‐glycoprotein I antibodies or LA presence. During follow‐up (median 72.5, range 66‐83 months), thromboembolic events observed in 32 (26.7%, 4.6%/year) patients were independently predicted by IgG aPS/PT antibodies >30 UI (hazard ratio [HR] = 3.04, 95% CI 1.20‐8.88) and low Ks (HR = 3.00, 95% CI 1.41‐6.50). ConclusionsWe identified aPS/PT antibodies as a determinant of denser and poorly lysable plasma fibrin clot formation in APS patients. The association of elevated aPS/PT antibodies with thromboembolism in APS could be at least in part mediated by prothrombotic clot properties.

Directed evolution of a 6-phosphogluconate dehydrogenase for operating an enzymatic fuel cell at lowered anodic pHs

Most enzymatic fuel cells (EFCs) are designed for operating at physiological conditions and cannot stand for low pHs due to the enzyme denaturation. Improving the acid tolerance of EFCs represents the increased robustness and diversity of their practical applications. In this study, we successfully constructed a three-enzyme pathway-catalyzed EFC consisting of two dehydrogenases and a diaphorase that can be operated at the anodic pH of 5.4. The acid-intolerance of one enzyme, 6-phosphogluconate dehydrogenase, was overcome by enzyme engineering. After the directed evolution including three rounds of error-prone PCR and a petri-dish-based double-layer screening method, the best 6-phosphogluconate dehydrogenase mutant (named as 3-3) exhibited a 42-fold increase in catalytic efficiency at pH 5.4 compared to the original enzyme. The electrochemical measurements revealed that the EFC equipped with this mutant achieved a maximum power density of 0.13 mW cm−2 at pH 5.4, more than 10-fold higher than that with the same enzyme unit loading but at pH 7.3. Under a high loading amount of enzymes, the EFC yielded a maximum power density of ∼0.5 mW cm−2, and that with the mutant also exhibited an improved stability. This study demonstrates that lowering anodic pH may be a useful strategy for constructing high-performance and robust EFCs and the concerns for enzyme deactivation can be addressed by enzyme engineering.

Enzymatic Production of Fish Protein Hydrolysates in A Pilot Plant Scale

Protease enzyme produced from Bacillus sp was employed to hydrolyze fish protein hydrolysates (FPH) under controlled conditions at a batch-pilot plant scale-process. Thirty kilograms of fish meat was mincedand mixed with 60 liters of water in 100 liters stainless steel vessel and 20,000 units of protease enzyme was added per kg of fish. Hydrolysis of fish was carried out at 55 oC for 6 hours. Multi stage of filtration were done to separate the FPH from unhydrolized fish residue. Mass balance were carried out to determine the rate of hydrolysis and yields. W ithout pH adjustment, 80% of substrate hydrolyzed could be achieved in 6 hour at 55 °C. Three kinds of products were recovered from the process, i.e solid residue, liquid FPH as filtration product, and spray dried FPH. Hydrolysis of 30 kg of fish meat substrate producing 1.7-2.0 kg of unhydrolyzed residue and 70 L of liquid FPH. Afterspray drying process of liquid FPH, 13 kg of FPH powder was recovered. The proximate and amino acid analysis of spray dried FPH showed that the FPH containing 20% of protein, rich in amino acids especially lysine and leucineand the residue still had 85,36% of protein (dry basis) that could be utilized for other purpose.

The method to detect sIgE to thyroid peroxidase in cases of the patients with autoimmune and allergic diseases

Aim of this research has been to create a method to detect sIgE to thyroid peroxidase (TPO) in serum of the patients with autoimmune and allergic diseases.Materials and methods. In this research native affine-purified TPO (more the 95% clean, enzyme activity 60±15 of enzyme unit per mg) with immune dominant elements only (confirmed by inhibitory ELISA) has been used. The detection of sIgE in serum, combined with TPO, was carried out with monoclonal antibodies to IgE (8E/4F4) conjugated with horseradish peroxidase.Results. In serum of patients with bronchial asthma, allergic rhinitis and chronic spontaneous urticarial sIgE to TPO has been detected in 18—21% of cases. The healthy people and the patients with autoimmune thyroiditis had no sign of sIgE to TPO in their serum.Conclusion. The detection of sIgE to TPO in cases of patients with chronic spontaneous urticarial and allergic diseases will make possible to perform the early diagnosis of thyroid autoimmune disorders and provide timely and adequate therapy.

Design, synthesis and biological evaluation of 4-aminoquinoline-guanylthiourea derivatives as antimalarial agents

Guanylthiourea (GTU) has been identified as an important antifolate antimalarial pharmacophore unit, whereas, 4-amino quinolones are already known for antimalarial activity. In the present work molecules carrying 4-aminoquinoline and GTU moiety have been designed using molecular docking analysis with PfDHFR enzyme and heme unit. The docking results indicated that the necessary interactions (Asp54 and Ile14) and docking score (−9.63 to −7.36 kcal/mmol) were comparable to WR99210 (−9.89 kcal/mol). From these results nine molecules were selected for synthesis. In vitro analysis of these synthesized compounds reveal that out of the nine molecules, eight show antimalarial activity in the range of 0.61–7.55 μM for PfD6 strain and 0.43–8.04 μM for PfW2 strain. Further, molecular dynamics simulations were performed on the most active molecule to establish comparative binding interactions of these compounds and reference ligand with Plasmodium falciparum dihydrofolate reductase (PfDHFR).

Bioelectrochemical TiN|Tsfdh Catalyst for CO2 Reduction to Hcooh

The ever-increasing worldwide energy demand and anthropogenic emissions produced by the combustion of fossil fuels, along with the consequent increment in the global average temperature, have pushed the scientific community towards the development of energy vectors with minimal carbon footprint. Electrochemical -reduction is an approach that holds potential for exploiting the cyclic reduction and oxidation of carbon-based fuels. As of today, high efficiency in these systems can only be achieved by employing precious metals, which suffer from both low natural availability and high costs as well as unsatisfactory energy efficiencies and lack of conversion product specificity. Recently, the development of a novel hybrid technology based on the combined use of biological organisms or molecules and nanomaterials has created great opportunities to produce renewable fuels and chemicals from the reduction of with a minimal overpotential. According to this, the enzymatic electrosynthesis (EES) exploits pure enzymes to catalyze reactions with higher transformation efficiency, higher activity under controlled experimental conditions and higher selectivity towards both specific substrates and products. The first step of CO2 reduction enzymatic process consists of the production of formic acid using the catalytic properties of the enzyme formate dehydrogenase (FDH) (EC 1.2.1.2.). Unlike most metallic catalysts, this enzyme reversibly catalyzes the transformation of CO2 to formate as the only product of the reaction. In this contest, we used the NAD-dependent FDH from the aerobic bacterium Thiobacillus sp. KNK65MA (TsFDH) for its superior -reducing activity (Kcat= 0.318 s-1)[1]. Surface chemistry and surface morphology play a key role in the interaction between the enzyme and the electrode, ultimately affecting the successful development of a bioelectrochemical system (BES). In fact, the atomic composition of the material and its structure affect enzyme immobilization, which in turn influences the electrochemical performance of the EES. In this context, titanium nitride (TiN) can be efficiently used as a scaffold for enzyme immobilization by exploiting its surface-exposed Ti4+ atoms for the binding. As such, TiN has been described as a promising general catalyst support material. Indeed, TiN features high electrical conductivity, good biocompatibility, as well as outstanding oxidation and acid corrosion resistance, together with a hybrid metallic/ceramic behaviour[2]. The aim of this work is to realize a nanostructured TiN scaffold featuring an increased available surface area to enhance the interface between the enzyme and the electrode. To this end, we developed a novel hybrid device where the FDH enzyme from Thiobacillus sp. KNK65MA (EC 1.2.1.2) is deposited on a nanostructured mesoporous support of TiN fabricated by Pulsed Laser Deposition (Fig.1a). This deposition method allows the production of a biocompatible nanostructured support with high surface area and a tree-like morphology. Its high porosity and high specific area maximize the contact with the enzyme, thus improving the efficiency of the EES for reduction. In order to identify the most suitable film morphology, the immobilization process on TiN support with different porosity was assessed by enzymatic assay after drop-casting (active area of 1 cm2). The results show that there is an increase in the amount of TsFDH that is bound to the TiN nanostructure when the porosity, and therefore the surface area, of the nanostructure increases. For the more porous morphology the percentage of adsorbed TsFDH is 48%, corresponding to 1 µg of specifically-adsorbed TsFDH. After, a calibration curve was performed to determine the maximum binding capacity of the TiN support. The result shows that, by increasing the concentration of the drop-casted TsFDH, the enzyme unit calculated on the surface increases until a TsFDH loading quantity of 1,14 mg is used, corresponding to 59 µg of immobilized protein. To assess the CO2 reduction electrochemical performance of the TiN|TsFDH electrode, chronoamperometric measurements were done at a constant potential of -0.45VRHE. The TiN support alone shows a stable reduction current (-130 µA ), due to hydrogen evolution (HER) occurring on the surface of the electrode (blue line, Figure 1b). Then, we tested the CO2 reduction activity of the enzyme-conjugated electrode (green trace, Figure 1b). A reductive current density with higher magnitude of -190 µA was registered, showing the actual operation of the BES system. After test, the solution was examined by 1H NMR spectroscopy. The peak signal at 8.455 ppm in NMR spectra demonstrates that the formic acid is the only product of the catalytic reaction performed by TsFDH. This result was used to quantify the amount of formic acid synthetized in the BES. During a 3 h reaction period, the electrosynthesis of formic acid was about 5.3±0.4 μmol under potential applied of -0.45VRHE. [1]H.Choe;PLOS ONE(2014). [2]P.H.;Electrochimica Acta(2010) Figure 1

Determination of antioxidant enzyme activity and phenolic contents of some species of the Asteraceae family from medicanal plants

Medicnal and aromatic plants are used in different fields depending on the amount of antioxidant and phenolic substances they have. This study was conducted to determine the antioxidant enzyme activity and phenolic contents of some species of the Asteraceae family (Achillea biebersteinii Afan., Achillea millefolium L., Achillea nobilis L., Artemisia absinthium L., Artemisia alba Turra., Artemisia dracunculus L., Artemisia santanicum L., Artemisia vulgaris L., Carthamus tinctorius L., Centaurea cyanus L., Echinacea pallida Nutt., Echinacea purpurea L. Moench., Grindelia robusta Nutt., Helianthus annuus L., Helichrysum plicatum L., İnula helenium L. and Santolina chamaecyparissus L.) for medicines, additive food supplement and cosmetic sector. In, this study, the antioxidant amount of some medicinal and aromatic plants with different properties belonging to Asteraceae family and hormones, phenolic substance and vitamin C and vitamin E were determined. E. purpurea, A. nobilis, G. robusta and S. chamaecyparissus species have more antioxidant, enzymes, hormones and some phenolic substances such as vitamin C and vitamin E, than other species in Asteracae. According to study result, highest amount of gibberellic acid, salicylic acid, indole acetic acid (IAA), abscisic acid (ABA), catalase (CAT) enzyme activity, peroxidase (POD) enzyme activity, superoksid dismutase (SOD) enzyme activity, ascorbate peroxidase (AxPOD) enzyme activity, malondialdehyte (MDA), hydrogen peroxide, total antioxidant, total phenolic, total carotenoid amount, vitamin E contents, vitamin C contents, chlorogenic acid contents, caffeic acids, chloric acid and proantocyanidin were determined in the A. millefolium (819.46 ng μl−1), A. dracunculus (91.29 ng μl−1), S. cyparissus L. (1985.08 enzyme unit (EU) g leaf−1), S. chamaecyparissus (415.22 EU g leaf−1) E. purpurea (83.78 EU g leaf−1), A. nobilis (41.91 EU g leaf−1), S. chamaecyparissus (140.85 nmol g−1), E. purpurea (10.49 μmol g−1) A. nobilis (1464 μmol Trolox Equivalents (TE) per g−1), E. purpurea (13.34 mg gallic acid (GA) per g−1 fresh weight (FW)), E. purpurea (29.41 g vitamine A 100−1), G. robusta (33.63 g-Alfa tocopherol eg 100 g−1), A. nobilis (20.70 g 100g−1), E. purpurea (13.51 mg g−1 fw), A. nobilis and S. chamaecyparissus (1.83 mg g−1 fw), S. chamaecyparissus (143.84 mg g−1 fw) and A. nobilis (83.63%) species, respectively. According to the results obtained, it is determined that there are significant changes in the contents of the medicinal and aromatic plants depending on the region and climatic conditions.

Capability of immobilised glucoamylase on mesostructured cellular foam silica to hydrolyse tapioca starch

Mesoporous size siliceous-based materials have been associated with immobilisation of free glucoamylase and soluble starches hydrolysis. As ethanol and other chemicals can be produced from insoluble feed such as tapioca, understanding of glucoamylase immobilised on mesostructured cellular foam (MCF) silica to hydrolyse the starch is required. To get the knowledge on this process for development of a large-scale operation, free glucoamylase SQzyme AGP was immobilised on MCF silica (9.2T-3D) by direct adsorption and used the product to hydrolyse tapioca. The free enzyme was able to be immobilised onto the carrier with the efficiencies of 39.20-84.84% where the operational factors of temperature, agitation speed and pH affected the immobilisation process highly. During the enzymatic hydrolysis, effects of temperature and agitation speed were high. The dextrose equivalent (DE) values of 1.21-81.70% were obtained. The optimum temperature and agitation speed were 70°C and 140 rpm. Other factors such as buffer pH, starch concentration and enzyme unit activity showed low effects on the hydrolysis process. Excellent DE were obtained at the conditions of pH, tapioca concentration and enzyme unit activity of 4.6, 3 mg mL−1 and 2351 U, respectively.

Eco-friendly anti-felting treatment of wool top based on biosurfactant and enzymes

Anti-felting processes with low environmental impacts are important for improving wool fiber attributes, especially if fiber is intended for the organic market. The aim of this study was to assess the potential of proteolytic enzymes, alone or in combination with Bacillus subtilis O9 biosurfactant (surfactin), to reduce felting of Merino wool top. Different concentrations of extracellular proteolytic extracts from Bacillus sp. G51 and Bacillus patagoniensis PATO5T were used to treat wool top. As indicated by the felt-ball test results, a significant decrease in wool felting tendency was achieved using 50 and 150 Enzyme Units (EU)/g wool of PATO5T and G51 proteases, respectively. Neither of these treatments caused a significant reduction in wool tensile strength. The addition of a pre-treatment step with biosurfactant surfactin, at a concentration above the Critical Micelle Concentration (CMC), further reduced felting tendency of wool. The combination of biosurfactant pre-treatment followed by B. patagoniensis PATO5T protease treatment rendered the significantly smallest felt-ball density (0.049 ± 0.004 g/cm3), without a significant loss in tensile strength. In PATO5T proteolytic extract, enzyme aggregates ≥100 kDa account for about 50% of the proteolytic activity. This could reduce the velocity of protease diffusion into wool fiber, promoting its action on wool surface. According to our knowledge, this is the first report that demonstrates the usefulness of a biosufactant in an environmental-friendly process aimed at reducing wool top felting.

Pilot-scale production of a highly thermostable α-amylase enzyme fromThermotoga petrophilacloned intoE. coliand its application as a desizer in textile industry

In this study, the industrial applications of a highly thermostable α-amylase as a desizer in the textile industry was evaluated. The cloned gene was expressed in different media (ZBM, LB, ZYBM9, and ZB) with IPTG (isopropyl β-d-1-thiogalactopyranoside) used as an inducer. Lactose was also used as an alternate inducer for the T7 promoter system in E. coli. For the large-scale production of the enzyme, different parameters were optimized. The maximum enzyme production was achieved when the volume of medium was 70% of the total volume of fermenter with a 2.0 vvm air supply and 20% dissolved oxygen at a 200 rpm agitation rate. Under all the optimized conditions, the maximum enzyme production was 22.08 U ml−1 min−1 with lactose (200 mM) as an inducer in ZBM medium. The desizing potential of the purified α-amylase enzyme was calculated with different enzyme concentrations (50–300 U ml−1) at different temperatures (50–100 °C), and pHs (4–9) with varying time intervals (30–120 min). The highest desizing activity was found when 150 U ml−1 enzyme units were utilized at 85 °C and at 6.5 pH for 1 h.

Catalytic Performance of Food Additives Alum, Flocculating Agent, Al(SO4)3, AlCl3, and Other Lewis Acids in Microwave Solvolysis of Hardwoods and Recalcitrant Softwood for Biorefinery

The development of a novel pretreatment system using catalysts with high safety and low cost is pivotal to establish lignocellulosic biorefinery. We evaluated 16 Lewis acid catalysts for microwave solvolysis pretreatment to enhance enzymatic saccharification of woody biomass and found that very cheap and safe food additives, alum, are effective for the pretreatment, giving high sugar yield comparable to that of AlCl3, a strong Lewis acid catalyst effective both for softwood and hardwood. In microwave solvolysis of Japanese cedar, Paraserianthes falcataria and Eucalyptus globulus using alum in ethylene glycol/water (9/1, w/w), the maximum sugar yields after enzymatic saccharification reached 47.8, 51.0, and 59.7% based on the weight of each wood. The same reactions in glycol/water (9/1, w/w) gave the sugar yield, 34.5, 54.1, and 58.5%, indicating differential reactivity of the Lewis acid/solvent system depending on wood species. We found that efficiency and selectivity of pretreatment with a flocculating and astringent agent, Al2(SO4)3, was promoted by microwave. Reaction of Japanese beech wood with Al2(SO4)3 in 50% aqueous 1-propanol by microwave and conventional heating revealed that microwave irradiation suppressed excessive degradation of carbohydrates into furfural derivatives. Saccharification of the wood pretreated by microwave with 8 and 1 filter paper units of cellulolytic enzymes gave sugar yields per wood of 50.3 and 43.9%, whereas the same reaction in an autoclave resulted in 48.9 and 34.7% yields, demonstrating that microwave irradiation accelerated the saccharification and the effect was remarkable with a lower enzyme dosage.

Optimization of parameters for the production of biodiesel from rubber seed oil using onsite lipase by response surface methodology.

In the present study, we demonstrate the properties of rubber seed oil (RSO) and its potential application for producing biodiesel by lipase-catalyzed transesterification. Rubber seed contains 39.45% of oil on a dry weight basis. Lipase was secreted from novel bacterial isolate of Pseudomonas aeruginosa strain BUP2. Response Surface Methodology (RSM) cum Box-Behnken Design (BBD) was employed to optimize the combined effect of different independent parameters namely oil-methanol ratio, enzyme unit, reaction temperature and reaction time. Biodiesel yield of 99.52% was obtained in the validation experiments at the optimal level of lipase (750 U), methanol ratio (1:10), temperature (45°C) and time (4h). The fuel properties of biodiesel obtained under the validation condition met the specifications as mentioned in ASTM D6751 and EN 14214 standards. Biodiesel aliquots were characterized using thin-layer chromatography (TLC), gas chromatography (GC), fourier transform infra-red spectroscopy (FTIR) and elemental analysis. The present study demonstrates an important application of a potential substitute for fossil fuel from raw feedstocks with high economic value.

Gelatin production using fish wastes by extracted alkaline protease from Bacillus licheniformis.

An effort was made to produce gelatin from Common carp wastes using extracted alkaline protease from Bacillus licheniformis PTCC 1595. Fermentation was performed by submerged media for 48h and 72h. The hydrolyzing enzyme was added in 5, 10, 15, 20, and 25 units per gram of wastes powder for hydrolysis. The produced gelatin was compared with commercial bovine gelatin with regard to some rheological and physicochemical properties. The yield of gelatin production was also determined as a result of hydroxyproline extraction from fish wastes. SDS-PAGE was performed for enzyme and gelatins. For enzyme, two bands were achieved with 39 and 10.5kDa molecular weight which were separated passing through a 15kDa UF filter. Both gelatins showed β, α1, and α2 chains as basic components, but the fish waste gelatin showed narrow bands. In conclusion, foam expansion and water holding capacity were approximately equal in both gelatin types used for food industry application. The results indicated that using 20 units of enzyme per gram of waste powder was the optimum amount of enzyme application. Further, fish wastes were concluded to be a practical source for gelatin production.

Improving palm kernel cake nutrition using enzymatic hydrolysis optimized by Taguchi method.

Enzymatic hydrolysis of palm kernel cake to improve the quality of substrates with multi-response criteria based on the Taguchi orthogonal array. Nine experimental runs were performed based on an L9 orthogonal array. Percent substrate, incubation time, and enzyme units were optimized considering multiple performance characteristics. Analysis of variance was also applied to identify the most significant factors. Results determined percent substrate as the most important factor for enzymatic hydrolysis followed by incubation time and enzyme units. Enzymatic hydrolysis conditions were optimized as percent substrate, incubation time and enzyme units at 14%, 6h and 750 units, respectively. Tests were conducted to compare experimental and model results. The experimental result (protein release) at optimal condition were three times higher than the predicted mode.

A comparative study for the isolation and characterization of mannoproteins from Saccharomyces cerevisiae yeast cell wall

Cell wall yeast, recovered upon the production of yeast extract, was investigated as an abundant source of mannoproteins. Selected isolation methods were evaluated for the recovery of mannoproteins, in terms of yield, mannan and protein recovery yield, mannoproteins content and mannan to protein ratio in extracted mannoproteins. The results showed that heat treatment and sodium dodecyl sulfate (SDS) extraction led to a lower yield compared to the enzymatic treatment; and no glycosylated proteins could be obtained upon the SDS extraction. As compared to other methods, the enzymatic approach, based on the use of Zymolyase®, exhibiting a high β-1,3-glucanase activity, resulted in the highest yield, mannoproteins content and mannan to protein ratio. A 5-level, 2-variable central composite rotatable design contributed to the better understanding of the effects of independent variables, reaction time and enzyme units, on the efficiency of the enzymatic treatment and to the better modulation of their actions. The effects of enzyme units and reaction time on the mannoproteins content and the mannan to protein ratio exhibited the same patterns. Comparison of predicted and experimental values validated the established predicted models, which can be used to identify the conditions for the isolation of mannoproteins with well-defined molecular properties.

N-Acetyl-d-glucosamine Production by a Chitinase of Marine Fungal Origin: a Case Study of Potential Industrial Significance for Valorization of Waste Chitins

Chitin is a linear homo-polymer of N-acetyl-D-glucosamine (GlcNAc) and the second most abundant biopolymer after cellulose. Several industries rely on the bioprocesses for waste chitin recycle and hydrolysis by chitinase (EC 3.2.1.14) for potential healthcare applications through the production of its monomeric subunit, GlcNAc. In the present study, a chitinase-producing fungus (named as MFSRK-S42) was isolated from the marine water sample of North Bay of the Andaman and Nicobar Islands. It was identified as Aspergillus terreus by morphological and molecular characterization methods leveraging the internal transcribed spacer between 18S rRNA and 5.8S rRNA. Chitinase that was isolated from the fermentation broth of marine Aspergillus terreus was used to carry out biotransformation of chitineaceous wastes. Prior to the enzymatic hydrolysis step, chitins from different sources were characterized for the presence of characteristic functional groups, grain size distribution, and surface morphology. Enzymatic hydrolysis of 50mg/ml substrate with six units of enzyme incubated for 5days revealed 15, 36.5, 40, and 46mg/ml GlcNAc production from ground prawn shell, chitin flakes, colloidal prawn shell, and swollen chitin respectively under standardized conditions, as determined by HPLC. In this study, 30, 73, 80, and 92% GlcNAc yields were observed from ground prawn shell, chitin flakes, colloidal prawn shell, and swollen chitin conversion respectively. The HPLC-eluted product was confirmed as GlcNAc by the presence of characteristic functional groups in FTIR and 244Da molecular weight peak in HRMS analyses.