Maximum Enzyme Activity Research Articles

Enhancement of alkaline protease production in recombinant Bacillus licheniformis by response surface methodology

Alkaline protease is widely used in the food, detergent, and pharmaceutical industries because of its comparatively great hydrolysis ability and alkali tolerance. To improve the ability of the recombinant Bacillus licheniformis to produce alkaline protease, single-factor experiments and response surface methodology (RSM) were utilized to determine and develop optimal culture conditions. The results showed that three factors (corn starch content, soybean meal content, and initial medium pH) had significant effects on alkaline protease production (P < 0.05), as determined through the Plackett‒Burman design. The maximum enzyme activity was observed with an optimal medium composition by central composite design (CCD): corn starch, 92.3 g/L; soybean meal, 35.8 g/L; and initial medium pH, 9.58. Under these optimum conditions, the alkaline protease activity of strain BL10::aprE was 15,435.1 U/mL, 82% higher than that in the initial fermentation medium. To further investigate the application of the optimum fermentation medium, the overexpressed strain BL10::aprE/pHYaprE was cultured using the optimized medium to achieve an enzyme activity of 39,233.6 U/mL. The present study achieved the highest enzyme activity of alkaline protease by B. licheniformis at the shake-flask fermentation level, which has important application value for large-scale production.Graphical

Identification of a Strain Degrading Ammonia Nitrogen, Optimization of Ammonia Nitrogen Degradation Conditions, and Gene Expression of Key Degrading Enzyme Nitrite Reductase

In recent years, nitrogen pollutants have become one of the main causes of water pollution and eutrophication; thus, it is very important to increase the research on nitrogen removal in wastewater. In this study, a bacterium with outstanding ammonia nitrogen degradation capability was isolated from piggery wastewater and identified as Bacillus tequilensis (designated as A2). The ammonia nitrogen degradation rate of A2 reached the highest level (95%) when the incubation temperature was 42 °C, the initial pH was 7, the seed volume was 5%, the rotation speed was 160 r·min−1, the C/N was 10:1, and the carbon source was sodium citrate. A new nitrite reductase gene was successfully expressed in E. coli BL21 (DE3), and the result showed that the enzyme gene contained 2418 bp and 805 encoding amino acids, the recombinant enzyme was purified through an Ni2+ affinity chromatography column, it had a molecular weight of about 94 kDa, it displayed the maximum enzyme activity at 40 °C and pH 6.0, it exhibited good stability in the range of 25 °C to 35 °C, and it showed a pH of 6.0 to 7.0. A 1 mM concentration of Fe3+ promoted the enzyme activity, followed by a 1 mM concentration of Fe2+ and Mg2+. The kinetic parameters of Km, Kcat, and the Vmax of NiR-A2 were calculated to be 1.37 μmol·mL−1, 4.9 × 102 s−1, and 23.75 μmol·mg−1·min−1, respectively. This strain shows good prospects for wastewater treatment, especially in the treatment of high concentration ammonia nitrogen and nitrite degradation, because of its tolerance to and high degradation rate of high concentrations of ammonia nitrogen and high nitrite.

Comparative assessment on lignocellulose degrading enzymes and bioethanol production from spent mushroom substrate of Calocybe indica and Volvariella volvacea.

In the current study, we compared the production of extracellular lignocellulose degrading enzymes and bioethanol from the spent mushroom substrate (SMS) of Calocybe indica and Volvariella volvacea. From SMS at different stages of the mushroom development cycle, ligninolytic and hydrolytic enzymes were analysed. The activities of lignin-degrading enzymes, including lignin peroxidase (LiP), laccase, and manganese peroxidase (MnP) were maximal in the spawn run and primordial stages, while hydrolytic enzymes including xylanase, cellobiohydrolase (CBH), and carboxymethyl cellulase (CMCase) showed higher activity during fruiting bodies development and at the end of the mushroom growth cycle. SMS of V. volvacea showed relatively lower ligninase activity than the SMS of C. indica, but had the maximum activity of hydrolytic enzymes. The enzyme was precipitated with acetone and further purified with the DEAE cellulose column. The maximum yield of reducing sugars was obtained after hydrolysis of NaOH (0.5M) pretreated SMS with a cocktail of partially purified enzymes (50% v/v). After enzymatic hydrolysis, the total reducing sugars were 18.68 ± 0.34g/l (SMS of C. indica) and 20.02 ± 0.87g/l (SMS of V. volvacea). We observed the highest fermentation efficiency and ethanol productivity (54.25%, 0.12g/l h) obtained from SMS hydrolysate of V. volvacea after 48h at 30 ± 2°C, using co-culture of Saccharomyces cerevisiae MTCC 11,815 and Pachysolen tannophilus MTCC 1077.

Extraction, purification and characterization of papain enzyme from papaya

Papain is one of the widely used enzymes in the industry. This study aimed to extract papain enzyme from various parts (i.e. latex, leaves, peels and seeds) of papaya using distilled water and citric phosphate buffer and subsequently purified with a two-step salt precipitation method. The optimum protein concentration of purified papain was obtained with citric phosphate buffer (0.159-0.319 mg/mL) and distilled water (0.119-0.347 mg/ mL). The protein concentration was almost similar for crude extracted with both the solvent. The purification procedure enhanced up to 1.56 fold when extracted with distilled water and 0.83 fold when extracted with citric phosphate buffer. Latex, leaves, peels and seeds showed maximum enzyme activity at 60oC and pH 8.0 as well as better thermal stability at 60oC for 30 mins. Findings highlighted that extracted papain enzyme from waste parts of papaya could be utilized as a potential source of industrial application in terms of waste to value-added products.

Production of ethanol from xylan by indigenous xylanolytic and ethanologenic bacteria isolated from fruit wastes

Xylan-containing fruit wastes can be valorized efficiently by xylanolytic microbes for the production of biofuels. The current study aimed to isolate and screen ethanol-producing bacteria from rotten fruit wastes that ferment xylose. Sixteen bacteria were isolated by spread-plate method from apple, banana, guava, and grape wastes on a solid medium supplemented with 5% xylan. The potential of ten bacterial isolates to degrade xylose was checked by biochemical tests. Bacterial strains XA2, XG1, and XG3 showed maximum CO2 production in Durham tubes. XA2, XG2, and XG3 showed maximum zones (1–2.6 cm) by Congo-red staining. XG2, XG3, and XA2 showed maroon and pink color with triphenyltetrazolium demonstrating degradation of xylan. The selected bacterial isolates were subjected to ferment 5% xylan-supplemented minimal medium for ten days to investigate ethanologenesis. Bacterial isolate XG2 produced maximum ethanol of 3.069 ± 0.738 g/L on day 8. The sugar contents in the fermentation medium continued to be decreased as the experiments proceeded up to 10 days. Maximum enzyme activity was observed by both XA2 and XG2 as 0.817 ± 0.036 and 0.917 ± 0.059 μmol/min/L. Strains XA2 and XG2 were characterized as Enterococcus faeceum XA2 and Bacillus cereus XG2 (Accession Nos. OM971654 and OM970803) by comparing their 16 S rRNA gene sequences with the available sequences in GenBank each having resemblance of 98 %. The present study highlights the dual capacity of bacteria to degrade xylan as well as promote ethanol production by employing xylan as a carbon source.

Physico-Chemical Characterization of an Exocellular Sugars Tolerant Β-Glucosidase from Grape Metschnikowia pulcherrima Isolates.

A broad variety of microorganisms with useful characteristics in the field of biotechnology live on the surface of grapes; one of these microorganisms is Metschnikowia pulcherrima. This yeast secretes a β-glucosidase that can be used in fermentative processes to liberate aromatic compounds. In this work, the synthesis of an exocellular β-glucosidase has been demonstrated and the optimal conditions to maximize the enzyme's effectiveness were determined. There was a maximum enzymatic activity at 28 °C and pH 4.5. Furthermore, the enzyme presents a great glucose and fructose tolerance, and to a lesser extent, ethanol tolerance. In addition, its activity was stimulated by calcium ions and low concentrations of ethanol and methanol. The impact of terpene content in wine was also determined. Because of these characteristics, β-glucosidase is a good candidate for use in enology.

Concentrating omega-3 fatty acids in Nannochloropsis oceanica oil by using enzyme immobilized nano-silica systems

Burning agricultural waste causes local air pollution and global warming. Agricultural waste, such as rice husks, contains silica reserves that can be processed into nano silica and used as a substrate for the immobilization of enzymes. The present study attempts to develop efficient silica-based nano-biocatalysts for concentrating omega-3 fatty acids in algal oil by hydrolyzing specific short-chain fatty acids. In this regard, tetraethyl orthosilicate (TEOS) and rice husk ash (RHA) were used as precursors to obtain nano-silica supports. Candida rugosa lipase (CRL) was then physically adsorbed and chemically cross-linked onto the created silica nano supports. The immobilized nano-systems created using the physical adsorption approach showed the maximum binding efficiency and enzymatic activity of CRL. The protein content of the CRL physically adsorbed in TEOS was 925 mg/g, while the protein loading in the RHA systems was 938 mg/g. However, the enzymatic activity of the selected systems for hydrolyzing para nitro-phenol palmitate (100 mM PBS buffer at pH-7.2, for 10 min at 37 °C) was 56 units/mg for the RHA system and 707 units/mg for the TEOS system. These two nano-enzyme systems were utilized further to hydrolyze the Nannochloropsis oceanica oil, and a physically adsorbed CRL TEOS system was found to have a 2.5-fold enrichment in eicosapentaenoic acid (EPA) which is used in food and pharmaceutical applications.

Characterization of a salt-resistant isocitrate lyase gene from mangrove wetland using shotgun metagenomic sequencing

Isocitrate lyase (ICL), as the key enzyme in the glyoxylate metabolic pathway, plays an important role in metabolic adaptation to environmental changes. In this study, metagenomic DNA from the soil and water microorganism collected from the Dongzhai Harbor Mangroves (DHM) reserve, in Haikou City, China, was high-throughput sequenced using an Illumina HiSeq 4000 platform. The icl121 gene, encoding an ICL with the highly conserved catalytic pattern IENQVSDEKQCGHQD was identified. Then, this gene was subcloned into the pET-30a vector and overexpressed in Escherichia coli BL21 (DE3) cells. The maximum enzymatic activity of the recombinant ICL121 protein is 9.47×102U/mg occurring at pH 7.5 and 37°C. Furthermore, as a metalo-enzyme, ICL121 can utilize the appropriate concentrations of Mg2+, Mn2+, and Na+ ion as cofactors to exhibit high enzymatic activity. In particular, the novel metagenomic-derived icl121 gene displayed distinct salt tolerance (NaCl) and might be useful for generating salt-tolerant crops in the future.

Biochemical Characterization of γ-Glutamyl Transpeptidase from Bacillus altitudinis IHB B1644 and Its Application in the Synthesis of l-Theanine

An extracellular γ-glutamyl transpeptidase (GGT) produced from Bacillus altitudinis IHB B1644 was purified to homogeneity employing ion-exchange chromatography. GGT comprised two subunits of 40 and 22 kDa determined by SDS-PAGE. The maximum enzyme activity was optimal at pH 9 and 37 °C. The purified enzyme was stable from pH 5-10 and <50 °C. Steady-state kinetic studies revealed a Km value of 0.538 mM against γ-GpNA. For substrate specificity, GGT showed highest affinity for l-methionine. The inhibitors' effect demonstrated that serine or threonine and tryptophan residues are essential for enzyme activity. l-Theanine production was optimized by employing a one-variable-at-a-time approach with 60-65% conversion rate. The final reaction consisted of 20 mM l-glutamine, 200 mM ethylamine hydrochloride, and 10 U mL-1 enzyme concentration at 37 °C in Tris-Cl (50 mM, pH 9) for 5 h. l-Theanine was purified using a Dowex 50W X 8 hydrogen form resin and confirmed by HPLC and 1H NMR spectroscopies.

Biofilm-Based Biocatalysis for Galactooligosaccharides Production by the Surface Display of β-Galactosidase in Pichia pastoris

Galactooligosaccharides (GOS) are one of the most important functional oligosaccharide prebiotics. The surface display of enzymes was considered one of the most excellent strategies to obtain these products. However, a rough industrial environment would affect the biocatalytic process. The catalytic process could be efficiently improved using biofilm-based fermentation with high resistance and activity. Therefore, the combination of the surface display of β-galactosidase and biofilm formation in Pichia pastoris was constructed. The results showed that the catalytic conversion rate of GOS was up to 50.3% with the maximum enzyme activity of 5125 U/g by screening the anchorin, and the number of the continuous catalysis batches was up to 23 times. Thus, surface display based on biofilm-immobilized fermentation integrated catalysis and growth was a co-culture system, such that a dynamic equilibrium in the consolidated integrative process was achieved. This study provides the basis for developing biofilm-based surface display methods in P. pastoris during biochemical production processes.

Isolation, identification, and optimization of the fibrinolytic protease-producing strains

Cardiovascular complications due to thrombosis have become one of the main causes of death worldwide. The high cost and undesirable side effects of existing thrombolytic agents have led researchers to isolate potential strains that produce fibrinolytic enzymes for therapeutic applications. Fibrinolytic enzymes, especially of microbial origin, are recognized as potential therapeutic candidates for thrombosis. In this study, isolation, identification, and optimization of fibrinolytic protease enzyme-producing strains were performed using fermentative protein sources. Fibrinolytic protease-producing strains were selected by analyzing the isolated strains on skim milk agar medium. The selected strains were examined on blood agar and fibrin plate medium, and the ones showing high enzymatic activity were determined. The strain determined to have the highest activity was identified as Acinetobacter johnsonii TR01 by 16S rRNA analysis. The maximum fibrinolytic protease production of the strain occurred at 60°C and pH 7.0. Under different medium conditions used for enzyme production, fructose was found to be the best carbon source, while yeast extract and peptone were the best nitrogen sources. It was observed that CaCl2, KH2PO4, andMgSO4 components had a negative effect, while MnCl2and ZnC4H6O4 components had a positive effect on enzyme production. The medium composition for maximum enzyme activity (8.30IU/ml) determined by Response Surface Methodology was 14.22g/L fructose, 11.190g/L yeast extract, 14.22g/L peptone, 0.5g/L MnCl2, and 0.5g/L ZnC4H6O4.

Production of Cellulase from the Municipal Waste Residue by a Novel Cellulolytic Fungi

Cellulases catalyze the hydrolysis of 1,4-β-D-glycosidic linkages in cellulose and play a significant role in nature by recycling polysaccharide debris. This enzyme has enormous potential in industries such as textile wet processing, bio-stoning of denim fabric, biopolishing of textile fibres, softening of garments and removal of excess dye from the fabrics. Therefore, the research focused on obtaining new cellulose-producing microorganisms with higher specific activities and greater efficiency. By identifying a good strain, improving the production medium and using an alternative carbon source such as waste residue, this study aimed to lower the manufacturing cost of cellulase. This study was designed to assess the cellulase production by fungi isolated from water, soil, straw, dung, leaf and goat manure. In the present research, cellulase-producing fungal isolates obtained from waste samples were identified by morphological and microscopic features. On Congo red test, the largest zone of hydrolysis was found as 1.2 cm. From the morphological and microscopic test, the fungal strain was expected as Aspergillus sp. The assay of the enzyme cellulase was performed by measuring the release amount of reducing sugar. Optimization of process parameters was carried out for the isolate to maximize enzyme yield. On optimization, isolated fungal species showed maximum enzyme activity at a temperature of 30 °C and pH 6. Under optimized conditions of temperature and pH, agitation at 200 rpm with a 1 L/m air flow rate showed better cellulase activity. Cellulase production from Aspergillus sp. can be an advantage as the enzyme production rate is normally higher as compared to other fungi.

Isolation and identification of high-yielding alkaline phosphatase strain: a novel mutagenesis technique and optimization of fermentation conditions

Isolating and screening enzyme-producing strains from microorganisms and the commercial production of ALPs from microorganisms are of increasing interest. In this work, isolation and identification of high-yielding alkaline phosphatase strain were carried out using atmospheric and room temperature plasma mutagenesis (ARTP) for optimization of fermentation conditions. A strain of alkaline phosphatase-producing bacteria was screened from soil and identified by 16S rRNA gene sequencing as Bacillus amyloliquefaciens and named S-1. This strain had an alkaline phosphatase activity of 2594.73 U/L. Later, mutagenesis breeding of the alkaline phosphatase-producing S-1 strain was conducted using (ARTP), from which a higher alkaline phosphatase-producing positive mutant strain S-52 was screened. A central combination of five factors, including corn starch, yeast extract, metal ions, fermentation temperature and inoculum ratio, was then used to influence the activity of alkaline phosphatase. Results from the response surface methodology showed that the maximum enzyme activity of alkaline phosphatase was 12,110.6 U/L at corn starch, yeast extract and magnesium ions concentrations of 17.48 g/L, 18.052 g/L and 0.744 g/L, respectively; fermentation temperature of 37.192 °C; and inoculation ratio of 5.59%. This study is important for further exploring ARTP mutagenesis in B. amyloliquefaciens and the commercialization of ALPs.

Antarctic yeasts as a source of L-asparaginase: Characterization of a glutaminase-activity free L-asparaginase from psychrotolerant yeast Leucosporidium scottii L115

Microorganisms from extreme environments, such as the Antarctic ecosystems, have a great potential to produce enzymes with novel characteristics. Within this context, L-asparaginase (ASNase) obtained from yeast species has been poorly studied. In this study, yeasts isolated from samples collected at Admiralty Bay (King George Island, Antarctica) were tested to produce ASNase. From an initial screening of 40 strains, belonging to 13 different species, Leucosporidium scottii L115 produced an ASNase activity (LsASNase activity: 6.24 U g-1 of dry cell weight) with the lowest glutaminase activity. The LsASNase was purified 227-fold, with a specific activity of 137.01 U mg-1 at 37 °C, without glutaminase activity. Moreover, the maximum enzyme activity was observed at pH 7.5 and at a temperature of 55 °C. The enzyme is a multimer of 462 kDa, presenting a single band of 53 kDa molecular mass in reduced conditions; after PGNase F treatment, a single band of 45 kDa was observed. The enzymatic kinetic evaluation revealed an allosteric regulation of the enzyme and the kinetic parameters were determined at 37 °C, pH 7.0 as substrate affinity constant, K0.5 = 233 μM, kcat = 54.7 s−1 and Hill coefficient, nH = 1.52, demonstrating positive cooperativity by the enzyme and the substrate. This is the first study to report L. scottii as a source of glutaminase-activity free L-asparaginase, an acute lymphoblastic leukemia drug feature suitable for the treatment of asparagine synthetase negative cancer cells.

Deglycosylation Differentially Regulates Weaned Porcine Gut Alkaline Phosphatase Isoform Functionality along the Longitudinal Axis

Gut alkaline phosphatases (AP) dephosphorylate the lipid moiety of endotoxin and other pathogen-associated-molecular patterns members, thus maintaining gut eubiosis and preventing metabolic endotoxemia. Early weaned pigs experience gut dysbiosis, enteric diseases and growth retardation in association with decreased intestinal AP functionality. However, the role of glycosylation in modulation of the weaned porcine gut AP functionality is unclear. Herein three different research approaches were taken to investigate how deglycosylation affected weaned porcine gut AP activity kinetics. In the first approach, weaned porcine jejunal AP isoform (IAP) was fractionated by the fast protein-liquid chromatography and purified IAP fractions were kinetically characterized to be the higher-affinity and lower-capacity glycosylated mature IAP (p < 0.05) in comparison with the lower-affinity and higher-capacity non-glycosylated pre-mature IAP. The second approach enzyme activity kinetic analyses showed that N-deglycosylation of AP by the peptide N-glycosidase-F enzyme reduced (p < 0.05) the IAP maximal activity in the jejunum and ileum and decreased AP affinity (p < 0.05) in the large intestine. In the third approach, the porcine IAP isoform-X1 (IAPX1) gene was overexpressed in the prokaryotic ClearColiBL21 (DE3) cell and the recombinant porcine IAPX1 was associated with reduced (p < 0.05) enzyme affinity and maximal enzyme activity. Therefore, levels of glycosylation can modulate plasticity of weaned porcine gut AP functionality towards maintaining gut microbiome and the whole-body physiological status.

Characterization of an α-L-Arabinofuranosidase GH51 from the Brown-rot Fungus Gloeophyllum trabeum.

Woody biomass is anticipated to be a resource for a decarbonized society, but the difficulty of isolating woody components is a significant challenge. Brown-rot fungi, a type of wood rotting fungi, decompose hemicellulose particularly efficiently. However, there are few reports on the hemicellulases from brown-rot fungi. An α-L-arabinofuranosidase belonging to glycoside hydrolase family 51 (GH51) from the brown-rot fungus Gloeophyllum trabeum (GtAbf51A) was cloned and characterized in the present study. Analyses of the phylogeny of GH51 enzymes in wood rotting fungi revealed the existence of two groups, intercellular and extracellular enzymes. After deglycosylation, the recombinant GtAbf51A produced by Pichia pastoris appeared on SDS-PAGE as approximately 71,777 daltons, which is the expected molecular weight based on the amino acid sequence of GtAbf51A. Maximum enzyme activity occurred between pH 2.2 and 4.0 and at 50 °C, while it was stable between pH 2.2 and 10.0 and up to 40 °C. Due to the presence of a signal peptide, GtAbf51A was thought to hydrolyze polysaccharide containing arabinose. However, the hydrolysis rate of arabinosyl linkages in polysaccharides was only 3-5 % for arabinoxylan and 18 % for arabinan. GtAbf51A, in contrast, efficiently hydrolyzed arabinoxylooligosaccharides, particularly O-α-L-arabinofuranosyl-(1→3)-O-β-D-xylopyranosyl-(1→4)-β-D-xylopyranose, which is the principal product of GH10 β-xylanase. These data suggest that GtAbf51A cooperates with other xylan-degrading enzymes, such as β-xylanase, to degrade xylan in nature.

A green approach to copper biodetoxification and sustainable agriculture application by vermicomposting in pig manure

A green approach of copper (Cu) contaminated pig manure composting by earthworm Eisenia fetida was optimized. This work aims to assess the relationship between the bio-fertility properties and bioaccumulation of Cu during vermicomposting with five different doses of Cu. The optimal concentration of copper largely promoted the enrichment of nitrogen, phosphorus, and potassium, but the biological activities of earthworms could be inhibited once the Cu concentration exceed the threshold. When the Cu doses at 300 mg kg−1, the nutrient recovery rate (Irecovery) of available nitrogen, phosphorus and potassium reached their highest value, concomitant with largest C/N ratio reduced at 46.01 %. Moreover, nutrients recovery mechanism of total phosphorus increased up to 0.11 % h−1 and higher bioaccumulations in faces and intestine were detected by 1.79 and 0.99, respectively, during vermicomposting. The maximal enzyme activity rates (kmax) indicate that the enzyme activities, such as ROS and SOD, are sensitive bioindicators, which can be used to estimate the stress response of earthworms and Cu biotoxicity. The maximum specific growth rate (μmax) of the actinomycetes (TAct) increased gradually from 0.02 to 0.04 with the increase of Cu doses, but total fungi (TF) showed different response to μmax, which decreased firstly and then increased. It was demonstrated that Cu influenced the gut microbial community to vary the bio-fertility properties and bioaccumulation of Cu in the pig manure. All the findings refer that the vermicomposting could be the sustainable agricultural practices.

Production, purification, characterization and application of two novel endoglucanases from buffalo rumen metagenome

BackgroundLignocellulose biomass is the most abundant and renewable material in nature. The objectives of this study were to characterize two endoglucanases TrepCel3 and TrepCel4, and determine the effect of the combination of them (1.2 mg TrepCel3, 0.8 mg TrepCel4) on in vitro rumen fermentation characteristics. In this study, three nature lignocellulosic substrates (rice straw, RS; wheat straw, WS; leymus chinensis, LC) were evaluated for their in vitro digestibility, gas, NH3-N and volatile fatty acid (VFA) production, and microbial protein (MCP) synthesis by adding enzymatic combination.MethodsTwo endoglucanases’ genes were successfully expressed in Escherichia coli (E. coli) BL21 (DE3), and enzymatic characteristics were further characterized. The combination of TrepCel3 and TrepCel4 was incubated with lignocellulosic substrates to evaluate its hydrolysis ability.ResultsThe maximum enzymatic activity of TrepCel3 was determined at pH 5.0 and 40 °C, while TrepCel4 was at pH 6.0 and 50 °C. They were stable over the temperature range of 30 to 60 °C, and active within the pH range of 4.0 to 9.0. The TrepCel3 and TrepCel4 had the highest activity in lichenan 436.9 ± 8.30 and 377.6 ± 6.80 U/mg, respectively. The combination of TrepCel3 and TrepCel4 exhibited the highest efficiency at the ratio of 60:40. Compared to maximum hydrolysis of TrepCel3 or TrepCel4 separately, this combination was shown to have a superior ability to maximize the saccharification yield from lignocellulosic substrates up to 188.4% for RS, 236.7% for wheat straw WS, 222.4% for LC and 131.1% for sugar beet pulp (SBP). Supplemental this combination enhanced the dry matter digestion (DMD), gas, NH3-N and VFA production, and MCP synthesis during in vitro rumen fermentation.ConclusionsThe TrepCel3 and TrepCel4 exhibited the synergistic relationship (60:40) and significantly increased the saccharification yield of lignocellulosic substrates. The combination of them stimulated in vitro rumen fermentation of lignocellulosic substrates. This combination has the potential to be a feed additive to improve agricultural residues utilization in ruminants. If possible, in the future, experiments in vivo should be carried out to fully evaluate its effect.

Nanoparticles Mimicking Oxidase Activity and their Application in Synthesis of Neurodegenerative Therapeutic Drug L‐DOPA

AbstractNanozymes are nanomaterials with intrinsic enzyme‐like activity and show high operational stability as compared to native enzymes. They have gathered the attention of researchers due to their unique physico‐chemical properties and diverse applications. In the present study, screening of different nanoparticles (zinc oxide, iron oxide, selenium and titanium oxide) was performed to evaluate their polyphenol oxidase (PPO) activity. PPO is a biotechnologically important enzyme and its application includes wastewater treatment, food processing and synthesis of l‐3,4‐dihydroxyphenylalanine (L‐dopa, a precursor of dopamine neurotransmitter). Out of all nanoparticles used in this study, zinc oxide nanoparticles (ZnO NPs) showed maximum enzyme activity of 75 %. ZnO NPs demonstrated high thermal stability and can be used upto 80°C. The colloidal solution of nanoparticles showed storage stability upto 6 months at room temperature and showed high activity in the pH range of 6–9 as compared to free enzyme. The nanoparticles were reused till 6th cycle retaining more than 80 % oxidase activity. To study its potential as a catalyst, it was used for the synthesis of L‐dopa from L‐tyrosine and a good productivity of 100 mg/L (0.1 g/L) was obtained. Thus, ZnO nanozymes can be used as an alternate to oxidase enzyme for synthesis of this therapeutic drug.

Exo-polygalacturonase production enhancement by Piriformospora indica from sugar beet pulp under submerged fermentation using the response surface methodology.

This study proposed a novel and cost-effective approach to enhance and optimize the exo-polygalacturonase from P. indica, a root endophytic fungus. In the current investigation, the impact of ammonium sulfate, sugar beet pulp (SBP), and glucose as variables on induction of exo-polygalacturonase from P. indica was optimized using the central composite design (CCD) of response surface methodology (RSM) under submerged fermentation (SmF). Additionally, determination of the exo-polygalacturonase molecular weight and in situ analysis was performed. The optimal reaction conditions, which resulted in the highest enzyme activity, were observed in the following conditions: ammonium sulfate (4g/L), SBP (20g/L), and glucose (60g/L). Under the optimized condition, the maximum enzyme activity reached 19.4U/ml (127U/mg), which increased by 5.84 times compared to non-optimized conditions. The exo-polygalacturonase molecular weight was estimated at 60 KDa. In line with the bioinformatic analysis, the exo-polygalacturonase sequence of P. indica showed similarity with Rhizoctonia solani's and Thanateporus cucumeris. These results indicated that SBP acts as a cheap and suitable inducer of exo-polygalacturonase production by P. indica in submerged cultivation. The outcome of this study will be useful for industries to decrease environmental pollution with cost-effective approaches.