Expression and biochemical characterization of a novel thermostable alkaline ß-1,3-1,4-glucanase (lichenase) from an alkaliphilic Bacillus lehensis G1.

New thermostable ß-1,3-1,4-glucanase (lichenase) designated as Blg29 was expressed and purified from a locally isolated alkaliphilic bacteria Bacillus lehensis G1. The genome sequence of B. lehensis predicted an open reading frame of Blg29 with a deduced of 249 amino acids and a molecular weight of 28.99 kDa. The gene encoding for Blg29 was successfully amplified via PCR and subsequently expressed as a recombinant protein using the E. coli expression system. Recombinant Blg29 was produced as a soluble form and further purified via immobilized metal ion affinity chromatography (IMAC). Based on biochemical characterization, recombinant Blg29 showed optimal activity at pH9 and temperature 60 °C respectively. This enzyme was stable for more than 2 h, incubated at 50 °C, and could withstand ∼50 % of its activity at 70 °C for an hour and a half. No significant effect on Blg29 was observed when incubated with metal ions except for a small increase with ion Ca2+. Blg29 showed high substrate activity towards lichenan where Vm, Km, Kcat, and kcat/Km values were 2040.82 µmolmin‾1mg‾1, 4.69 mg/mL, and 986.39 s‾1 and 210.32 mLs‾1mg‾1 respectively. The high thermostability and activity make this enzyme useable for a broad prospect in industry applications.

Microbially-derived cocktail of carbohydrases as an anti-biofouling agents: a 'green approach'.

Enzymes, also known as biocatalysts, display vital properties like high substrate specificity, an eco-friendly nature, low energy inputs, and cost-effectiveness. Among their numerous known applications, enzymes that can target biofilms or their components are increasingly being investigated for their anti-biofouling action, particularly in healthcare, food manufacturing units and environmental applications. Enzymes can target biofilms at different levels like during the attachment of microorganisms, formation of exopolymeric substances (EPS), and their disruption thereafter. In this regard, a consortium of carbohydrases that can target heterogeneous polysaccharides present in the EPS matrix may provide an effective alternative to conventional chemical anti-biofouling methods. Further, for complete annihilation of biofilms, enzymes can be used alone or in conjunction with other antimicrobial agents. Enzymes hold the promise to replace the conventional methods with greener, more economical, and more efficient alternatives. The present article explores the potential and future perspectives of using carbohydrases as effective anti-biofilm agents.

Solid-state fermented brewer's spent grain enzymatic extract increases in vitro and in vivo feed digestibility in European seabass.

Brewer's spent grain (BSG) is the largest by-product originated from the brewery industry with a high potential for producing carbohydrases by solid-state fermentation. This work aimed to test the efficacy of a carbohydrases-rich extract produced from solid-state fermentation of BSG, to enhance the digestibility of a plant-based diet for European seabass (Dicentrarchus labrax). First, BSG was fermented with A. ibericus to obtain an aqueous lyophilized extract (SSF-BSG extract) and incorporated in a plant-based diet at increasing levels (0-control; 0.1%, 0.2%, and 0.4%). Another diet incorporating a commercial carbohydrases-complex (0.04%; Natugrain; BASF) was formulated. Then, all diets were tested in in vitro and in vivo digestibility assays. In vitro assays, simulating stomach and intestine digestion in European seabass, assessed dietary phosphorus, phytate phosphorus, carbohydrates, and protein hydrolysis, as well as interactive effects between fish enzymes and dietary SSF-BSG extract. After, an in vivo assay was carried out with European seabass juveniles fed selected diets (0-control; 0.1%, and 0.4%). In vitro digestibility assays showed that pentoses release increased 45% with 0.4% SSF-BSG extract and 25% with Natugrain supplemented diets, while amino acids release was not affected. A negative interaction between endogenous fish enzymes and SSF-BSG extract was observed in both diets. The in vivo digestibility assay corroborated in vitro data. Accordingly, the dietary supplementation with 0.4% SSF-BSG increased the digestibility of dry matter, starch, cellulose, glucans, and energy and did not affect protein digestibility. The present work showed the high potential of BSG to produce an added-value functional supplement with high carbohydrases activity and its potential contribution to the circular economy by improving the nutritional value of low-cost and sustainable ingredients that can be included in aquafeeds.

Production of α-rhamnosidases from Lactobacillus plantarum WCFS1 and their role in deglycosylation of dietary flavonoids naringin and rutin.

This work addresses the amino acid sequence, structural analysis, biochemical characterization and glycosidase activity of two recombinant α-rhamnosidases, Ram1 and Ram2, from Lactobacillus plantarum WCFS1. The substrate specificity of both enzymes towards the disaccharide rutinose and natural dietary flavonoids naringin and rutin was also determined and compared to that of a commercial multienzyme complex (Pectinex Ultra Passover, PPO). Ram1 is a less acidic- and heat-active enzyme than Ram2 and exhibited a high activity towards pNP-α-L-rhamnopyranoside, but it was unable to hydrolyze neither rutinose, naringin or rutin. In contrast, Ram2 enzyme showed a substrate specificity towards α-(1➔6) glycosidic flavonoids, such as rutin, and the disaccharide rutinose. The mechanism of action of Ram2 towards rutin was elucidated and revealed the potential cost-effective and selective production of the monoglycosylated flavonoid isoquercetin (quercetin-3-O-glucoside). PPO efficiently converted both naringin and rutin into their corresponding aglycones. These findings revealed the potential usefulness of PPO for the improvement of sensory properties of beverages through debittering of citrus juices, as well as the potential use of Ram2 to selectively produce isoquercetin, a highly valued and bioactive flavonoid whose production is not currently affordable.

Characterization and Modeling of Thermostable GH50 Agarases from Microbulbifer elongatus PORT2.

Viewing the considerable potential of marine agar as a source for the sustainable production of energy as well as nature-derived pharmaceutics, this work investigated the catalytic activity of three novel GH50 agarases from the mesophilic marine bacterium Microbulbifer elongatus PORT2 isolated from Indonesian coastal seawaters. The GH50 agarases AgaA50, AgaB50, and AgaC50 were identified through genome analysis; the corresponding genes were cloned and expressed in Escherichia coli BL21 (DE3). All recombinant agarases hydrolyzed ß-p-nitrophenyl galactopyranoside, indicating ß-glycosidase characteristics. AgaA50 and AgaB50 were able to cleave diverse natural agar species derived from Indonesian agarophytes, indicating a promising tolerance of these enzymes for substrate modifications. All three GH50 agarases degraded agarose, albeit with remarkable diversity in their catalytic activity and mode of action. AgaA50 and AgaC50 exerted exolytic activity releasing differently sized neoagarobioses, while AgaB50 showed additional endolytic activity in dependence on the substrate size. Surprisingly, AgaA50 and AgaB50 revealed considerable thermostability, retaining over 75% activity after 1-h incubation at 50 °C. Considering the thermal properties of agar, this makes these enzymes promising candidates for industrial processing.

Ultrasound-assisted enzymatic extraction of Corni Fructus alpha-glucosidase inhibitors improves insulin resistance in HepG2 cells.

Corni Fructus (CF) is a traditional medicine and beneficial food with multifaceted protective effects against diabetes and its complications. Since alpha-glucosidase inhibitors (GIs) are promising first-choice oral antihyperglycemic drugs for diabetes, we examined whether GIs from CF (GICF) are useful for diabetes treatment. Therefore, GICF was extracted by ultrasound-assisted enzymatic extraction (UAEE) that is optimized by a three-level, four-factor Box-Behnken design and determined by ultra-performance liquid chromatography. Compared to 36.31 mg g-1 without enzyme treatment, the GICF yield increased to 70.44 mg g-1via UAEE under optimum conditions (0.5% compound enzyme extracted in 23 min at 46 °C and pH 4.8). The activity (91.99%) of GICF was as predicted (93.28%). When GICF was used in an insulin-resistant HepG2 cell model, it significantly ameliorated the glucose metabolism in a dose-dependent manner. Our findings indicate that UAEE may be an innovative method for functional food extraction and a potential strategy for high-quality food ingredient (such as GI) production with high efficiency and productivity.

Production and immobilization of ß-glucanase from Aspergillus niger with its applications in bioethanol production and biocontrol of phytopathogenic fungi.

ß-Glucanase has received great attention in recent years regarding their potential biotechnological applications and antifungal activities. Herein, the specific objectives of the present study were to purify, characterize and immobilize ß-glucanase from Aspergillus niger using covalent binding and cross linking techniques. The evaluation of ß-glucanase in hydrolysis of different lignocellulosic wastes with subsequent bioethanol production and its capability in biocontrol of pathogenic fungi was investigated. Upon nutritional bioprocessing, ß-glucanase production from A. niger EG-RE (MW390925.1) preferred ammonium nitrate and CMC as the best nitrogen and carbon sources, respectively. The soluble enzyme was purified by (NH4)2SO4, DEAE-Cellulose and Sephadex G200 with 10.33-fold and specific activity of 379.1 U/mg protein. Tyrosyl, sulfhydryl, tryptophanyl and arginyl were essential residues for enzyme catalysis. The purified ß-glucanase was immobilized on carrageenan and chitosan with appreciable yield. However, the cross-linked enzyme exhibited superior activity along with remarkable improved thermostability and operational stability. Remarkably, the application of the above biocatalyst proved to be a promising candidate in liberating the associate lignocellulosic reducing sugars, which was utilized for ethanol production by Saccharomyces cerevisiae. The purified ß-glucanase revealed an inhibitory effect on the growth of two tested phytopathogens Fusarium oxysporum and Penicillium digitatum.

High level expression of a xyloglucanase from Rhizomucor miehei in Pichia pastoris for production of xyloglucan oligosaccharides and its application in yoghurt.

The xyloglucanase gene (RmXEG12A) from Rhizomucor miehei CAU432 was successfully expressed in Pichia pastoris. The highest xyloglucanase activity of 25,700 U mL-1 was secreted using high cell density fermentation. RmXEG12A was optimally active at pH 7.0 and 65 °C, respectively. The xyloglucanase exhibited the highest specific activity towards xyloglucan (7915.5 U mg-1). RmXEG12A was subjected to hydrolyze tamarind powder to produce xyloglucan oligosaccharides with the degree of polymerization (DP) 7-9. The hydrolysis ratio of xyloglucan in tamarind powder was 89.8%. Moreover, xyloglucan oligosaccharides (2.0%, w/w) improved the water holding capacity (WHC) of yoghurt by 1.1-fold and promoted the growth of Lactobacillus bulgaricus and Streptococcus thermophiles by 2.3 and 1.6-fold, respectively. Therefore, a suitable xyloglucanase for tamarind powder hydrolysis was expressed in P. pastoris at high level and xyloglucan oligosaccharides improved the quality of yoghurt.

Structural model and functional properties of an exo-polygalacturonase from Neosartorya glabra.

A purified exo-polygalacturonase of Neosartorya glabra (EplNg) was successfully characterized. EplNg native presented 68.2 kDa, with 32% carbohydrate content. The deglycosylated form showed 46.3 kDa and isoelectric point of 5.4. The identity of EplNg was confirmed as an exo-polygalacturonase class I (EC 3.2.1.67) using mass spectrometry and Western-Blotting. Capillary electrophoresis indicated that only galacturonic acid was released by the action of EplNg on sodium polypectate, confirming an exoenzyme character. The structural model confers that EplNg has a core formed by twisted parallel ß-sheets structure. Among twelve putative cysteines, ten were predicted to form disulfide bridges. The catalytic triad predicted is composed of Asp223, Asp245, and Asp246 aligned along with a distance in 4-5 Å, suggesting that EplNg probably does not perform the standard inverting catalytic mechanism described for the GH28 family. EplNg was active from 30 to 90 °C, with maximum activity at 65 °C, pH 5.0. The Km and Vmax determined using sodium polypectate were 6.9 mg·mL-1 and Vmax 690 µmol·min-1.mg-1, respectively. EplNg was active and stable over a wide range of pH values and temperatures, confirming the interesting properties EplNg and provide a basis for the development of the enzyme in different biotechnological processes.

A novel fungal metal-dependent α-L-arabinofuranosidase of family 54 glycoside hydrolase shows expanded substrate specificity.

Trichoderma genus fungi present great potential for the production of carbohydrate-active enzymes (CAZYmes), including glycoside hydrolase (GH) family members. From a renewability perspective, CAZYmes can be biotechnologically exploited to convert plant biomass into free sugars for the production of advanced biofuels and other high-value chemicals. GH54 is an attractive enzyme family for biotechnological applications because many GH54 enzymes are bifunctional. Thus, GH54 enzymes are interesting targets in the search for new enzymes for use in industrial processes such as plant biomass conversion. Herein, a novel metal-dependent GH54 arabinofuranosidase (ThABF) from the cellulolytic fungus Trichoderma harzianum was identified and biochemically characterized. Initial in silico searches were performed to identify the GH54 sequence. Next, the gene was cloned and heterologously overexpressed in Escherichia coli. The recombinant protein was purified, and the enzyme's biochemical and biophysical properties were assessed. GH54 members show wide functional diversity and specifically remove plant cell substitutions including arabinose and galactose in the presence of a metallic cofactor. Plant cell wall substitution has a major impact on lignocellulosic substrate conversion into high-value chemicals. These results expand the known functional diversity of the GH54 family, showing the potential of a novel arabinofuranosidase for plant biomass degradation.

Heterologous Expression of Thermogutta terrifontis Endo-Xanthanase in Penicillium verruculosum, Isolation and Primary Characterization of the Enzyme.

Heterologous endo-xanthanase (EX) from the thermophilic planktomycete Thermogutta terrifontis strain was obtained using Penicillium verruculosum 537 (ΔniaD) expression system with the cellobiohydrolase 1 gene promoter. Homogeneous EX with a molecular weight of 23.7 kDa (pI 6.5) was isolated using liquid chromatography methods. This xanthan degrading enzyme also possesses the enzymatic activity towards CM-cellulose, ß-glucan, curdlan, lichenan, laminarin, galactomannan, xyloglucan but not towards p-nitrophenyl derivatives of ß-D-glucose, mannose and cellobiose. The temperature and pH optima of EX were 55°C and 4.0, respectively; the enzyme exhibited 90% of its maximum activity in the temperature range 50-60°C and pH 3-5.

Exo-inulinase production from Aspergillus fumigatus NFCCI 2426: purification, characterization, and immobilization for continuous fructose production.

Aspergillus fumigatus was found to produce thermostable exo-inulinase (EC 3.8.1.80; 38 U/ml) on inulin-rich infusions. Exo-inulinase (14.6 U/mg) was immobilized on glutaraldehyde activated Ca-alginate beads for continuous generation of fructose by hydrolyzing sucrose, chicory, and dandelion substrates. Immobilization of enzyme was confirmed by microscopic and spectroscopic techniques. The exo-inulinase was purified using ion-exchange (1.30-folds) and size-exclusion chromatography (2.71-folds). The purified exo-inulinase showed 64 kDa band on gel and was optimally active at 60 °C and pH 6.0. Kinetic constants, Km and Vmax of purified exo-inulinase, were 5.88 mM and 1.66 µM/min, respectively, and its relative activity was found to be enhanced (125.8%) in the presence of calcium ion. Immobilized preparation was utilized for continuous generation of fructose from chicory juice (26 to 70%) and dandelion root extracts (16 to 24%) by recycling upto five cycles, respectively. In comparison to other sweeteners, such as sucrose, fructose is considered as a healthy alternative. The present study demonstrated the use of immobilized exo-inulinase in continuous generation of fructose from some underutilized plant sources that can be used in food industry. PRACTICAL APPLICATION: Thermostable exo-inulinase produced by A. fumigatus was immobilized on calcium alginate matrix and was employed for continuous hydrolysis of chicory juice and dandelion root extract for generation of fructose syrup.

An Antifungal Chitosanase from Bacillus subtilis SH21.

Bacillus subtilis SH21 was observed to produce an antifungal protein that inhibited the growth of F. solani. To purify this protein, ammonium sulfate precipitation, gel filtration chromatography, and ion-exchange chromatography were used. The purity of the purified product was 91.33% according to high-performance liquid chromatography results. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that the molecular weight of the protein is 30.72 kDa. The results of the LC-MS/MS analysis and a subsequent sequence-database search indicated that this protein was a chitosanase, and thus, we named it chitosanase SH21. Scanning and transmission electron microscopy revealed that chitosanase SH21 appeared to inhibit the growth of F. solani by causing hyphal ablation, distortion, or abnormalities, and cell-wall depression. The minimum inhibitory concentration of chitosanase SH21 against F. solani was 68 µg/mL. Subsequently, the corresponding gene was cloned and sequenced, and sequence analysis indicated an open reading frame of 831 bp. The predicted secondary structure indicated that chitosanase SH21 has a typical a-helix from the glycoside hydrolase (GH) 46 family. The tertiary structure shared 40% similarity with that of Streptomyces sp. N174. This study provides a theoretical basis for a topical cream against fungal infections in agriculture and a selection marker on fungi.

Purification and characterization of a cold-adapted κ-carrageenase from Pseudoalteromonas sp. ZDY3.

κ-Carrageenase (EC3.2.1.83) is a class of glycoside hydrolase, which can be used for hydrolysis of κ-carrageenan to κ-carrageenan oligosaccharides. In this study, a bacterium, identified as Pseudoalteromonas sp. ZDY3 isolated from rotten algae, was capable to degrade κ-carrageenan. The κ-carrageenase produced by Pseudoalteromonas sp. ZDY3 was purified to homogeneity and named as CgkZDY3. The accurate molecular mass of CgkZDY3 was determined through LC-HRMS, and a posttranslational removal of C-terminal end of the protein was discovered. CgkZDY3 had strict hydrolysis specificity to κ-carrageenan, the values of Km and kcat/Km of CgkZDY3 were 3.67 mg mL-1 and 53.0 mL mg-1 s-1, respectively. CgkZDY3 was a cold-adapted κ-carrageenase with excellent storage stability of both the temperature below 35 °C and a wide pH range, and was an endo-type κ-carrageenase with high hydrolysis rate, oligosaccharides with different degrees of polymerization can be obtained by controlling the hydrolysis time, and the final products were κ-neocarrabiose and κ-neocarratetraose. These properties are of great significance for production of κ-carrageenan oligosaccharides with different polymerization degrees under process control.

Therapeutic Activity of Type 3 Streptococcus pneumoniae Capsule Degrading Enzyme Pn3Pase.

PURPOSE: Streptococcus pneumoniae (Spn) serotype 3 (Spn3) is considered one of the most virulent serotypes with resistance to conventional vaccine and treatment regimens. Pn3Pase is a glycoside hydrolase that we have previously shown to be highly effective in degrading the capsular polysaccharide of type 3 Spn, sensitizing it to host immune clearance. To begin assessing the value and safety of this enzyme for future clinical studies, we investigated the effects of high doses of Pn3Pase on host cells and immune system. METHODS: We assessed the enzyme's catalytic activity following administration in mice, and performed septic infection models to determine if prior administration of the enzyme inhibited repeat treatments of Spn3-challenged mice. We assessed immune populations in mouse tissues following administration of the enzyme, and tested Pn3Pase toxicity on other mammalian cell types in vitro. RESULTS: Repeated administration of the enzyme in vivo does not prevent efficacy of the enzyme in promoting bacterial clearance following bacterial challenge, with insignificant antibody response generated against the enzyme. Immune homeostasis is maintained following high-dose treatment with Pn3Pase, and no cytotoxic effects were observed against mammalian cells. CONCLUSIONS: These data indicate that Pn3Pase has potential as a therapy against Spn3. Further development as a drug product could overcome a great hurdle of pneumococcal infections.

Functional Characterization of a New GH107 Endo-α-(1,4)-Fucoidanase from the Marine Bacterium Formosa haliotis.

Fucoidans from brown macroalgae are sulfated fucose-rich polysaccharides, that have several beneficial biological activities, including anti-inflammatory and anti-tumor effects. Controlled enzymatic depolymerization of the fucoidan backbone can help produce homogeneous, defined fucoidan products for structure-function research and pharmaceutical uses. However, only a few endo-fucoidanases have been described. This article reports the genome-based discovery, recombinant expression in Escherichia coli, stabilization, and functional characterization of a new bacterial endo-α-(1,4)-fucoidanase, Fhf1, from Formosa haliotis. Fhf1 catalyzes the cleavage of α-(1,4)-glycosidic linkages in fucoidans built of alternating α-(1,3)-/α-(1,4)-linked l-fucopyranosyl sulfated at C2. The native Fhf1 is 1120 amino acids long and belongs to glycoside hydrolase (GH) family 107. Deletion of the signal peptide and a 470 amino acid long C-terminal stretch led to the recombinant expression of a robust, minimized enzyme, Fhf1Δ470 (71 kDa). Fhf1Δ470 has optimal activity at pH 8, 37-40 °C, can tolerate up to 500 mM NaCl, and requires the presence of divalent cations, either Ca2+, Mn2+, Zn2+ or Ni2+, for maximal activity. This new enzyme has the potential to serve the need for controlled enzymatic fucoidan depolymerization to produce bioactive sulfated fucoidan oligomers.

Metagenomic insights into the diversity of carbohydrate-degrading enzymes in the yak fecal microbial community.

BACKGROUND: Yaks are able to utilize the gastrointestinal microbiota to digest plant materials. Although the cellulolytic bacteria in the yak rumen have been reported, there is still limited information on the diversity of the major microorganisms and putative carbohydrate-metabolizing enzymes for the degradation of complex lignocellulosic biomass in its gut ecosystem. RESULTS: Here, this study aimed to decode biomass-degrading genes and genomes in the yak fecal microbiota using deep metagenome sequencing. A comprehensive catalog comprising 4.5 million microbial genes from the yak feces were established based on metagenomic assemblies from 92 Gb sequencing data. We identified a full spectrum of genes encoding carbohydrate-active enzymes, three-quarters of which were assigned to highly diversified enzyme families involved in the breakdown of complex dietary carbohydrates, including 120 families of glycoside hydrolases, 25 families of polysaccharide lyases, and 15 families of carbohydrate esterases. Inference of taxonomic assignments to the carbohydrate-degrading genes revealed the major microbial contributors were Bacteroidaceae, Ruminococcaceae, Rikenellaceae, Clostridiaceae, and Prevotellaceae. Furthermore, 68 prokaryotic genomes were reconstructed and the genes encoding glycoside hydrolases involved in plant-derived polysaccharide degradation were identified in these uncultured genomes, many of which were novel species with lignocellulolytic capability. CONCLUSIONS: Our findings shed light on a great diversity of carbohydrate-degrading enzymes in the yak gut microbial community and uncultured species, which provides a useful genetic resource for future studies on the discovery of novel enzymes for industrial applications.

Thermostable and alkalistable exopolygalacturonase of Bacillus pumilus dcsr1: Characteristics and applicability.

The bioactive form of thermostable and alkali stable pectinase of Bacillus pumilus dcsr1 is a homodimer of the molecular mass of 60 kDa with a pI of 4.6. The enzyme is optimally active at 50 °C and pH 10.5, and its Michaelis constant (Km), maximum rate of reaction (Vmax), activation energy (Ea), and temperature quotient (Q10) values (for citrus pectin) are 0.29 mg mL-1, 116 µmole mg-1 min-1, 74.73 KJmol-1 and 1.57, respectively. The enzyme has a shelf life of one and a half years at room temperature as well as 4 °C. The activity of the enzyme is stimulated by Mn2+ and Ca2+ and inhibited by Hg+, Cd2+, Co2+, Zn2+, Fe2+, Pb2+, EDTA and urea to a varied extent. The conformational studies of the enzyme revealed a high ß-sheet content in the bioactive dimer, and high α-helix in the inactive monomer. The Circular Dichroism (CD) spectra of the dimer in the presence of inhibitors suggested a marked decrease in ß-sheet, and a significant increase in α-helix, suggesting a key role of ß-sheets in the enzyme catalysis. Based on the end product analysis, the enzyme is an exopolygalacturonase with a unique ability of transglycosylation. When ramie fibers were treated with the enzyme, removal of gummy material (pectin) was visible, confirming its applicability in the degumming process.

Partial purification and characterization of Aspergillus niger inulinase produced from sugar-beet molasses in the shaking incubator and stirred-tank bioreactors.

The objectives of this study were to purify Aspergillus niger inulinase produced from sugar-beet molasses in the shaking incubator (100 mL) and stirred-tank bioreactors (5-L and 30-L) by using some downstream processes and to determine enzyme kinetics and characterization. The results showed that the best centrifuge-time combination was 16,873 ×g-5 min with the purification coefficient of 1.4. Besides, with the ultrafiltration process, the inulinase activities yielded using the shaking incubator, pH-controlled/uncontrolled small-scale bioreactors, and large-scale bioreactor were increased from 1101.3, 2079.2, 1561.3, and 787.5 U/mL to 12,065.2, 21,789.0, 11,296.9, and 2948.1 U/mL with purification coefficients of 5.33, 1.38, 1.46, and 1.67, respectively. Additionally, for the inulinase from shaking incubator and pH-uncontrolled bioreactor, the values of Km for inulin and sucrose were 17.8 and 49.4 mg/mL and 28.8 and 25.9 mg/mL, respectively. As the enzyme amount added to the substrate increased, the activity also increased. Mn2+ is the activator of the enzyme, and Cu2+ and Ag+ are inhibitors of the enzyme. The molecular weight of inulinase has been determined to be between 60 and 70 kDa. Consequently, this study ensures valuable and significant information about the purification and characterization of inulinase for industrial implementations.

Purification, thermodynamics and kinetic characterization of fungal endoinulinase for the production of fructooligosaccharides from inulin.

Three-step purification technique (isopropanol precipitation, ion-exchange and size-exclusion chromatography) was used for the purification of an endoinulinase from the culture broth of Aspergillus tritici BGPUP6. The molecular mass of purified endoinulinase was found to be 53.45 kDa and 53.70 kDa by denatured protein gel (SDS-PAGE) and size-exclusion (Sephadex G-100) chromatographic analysis, respectively. Higher Km (0.90 mM), Vmax (19.60 mM/min·mg), Kcat (1.3 × 10-3/min) and Vmax/Km ratio (21.77/min·mg) of purified endoinulinase for inulin than stachyose depicts its higher affinity towards inulin. Purified enzyme was found stable in a pH range 4.0-7.0 with an optimal pH 5.5. The optimal temperature of purified biocatalyst was 55 °C with thermostability in the range of 50-70 °C. D-value and Z-value for endoinulinase at 55 °C was found to be 100.08 h and 11.62 °C, respectively. Thermodynamics inactivation parameters (ΔG, ΔH and ΔS) of endoinulinase shows its wide range thermal stability. Endoinulinase activity was enhanced by CaCl2 and MnSO4, while CuSO4, CoCl2, AgNO3, CdCl2, NiCl2, ZnSO4, BaCl2, HgCl2 and EDTA inhibited the activity of enzyme. Purified endoinulinase was successfully used for the production of fructooligosaccharides from inulin.