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.

α-L-rhamnosidase: production, properties, and applications.

α-L-rhamnosidase [EC 3.2.1.40] belongs to glycoside hydrolase (GH) families (GH13, GH78, and GH106 families) in the carbohydrate-active enzymes (CAZy) database, which specifically hydrolyzes the non-reducing end of α-L-rhamnose. Αccording to the sites of catalytic hydrolysis, α-L-rhamnosidase can be divided into α-1, 2-rhamnosidase, α-1, 3-rhamnosidase, α-1, 4-rhamnosidase and α-1, 6-rhamnosidase. α-L-rhamnosidase is an important enzyme for various biotechnological applications, especially in food, beverage, and pharmaceutical industries. α-L-rhamnosidase has a wide range of sources and is commonly found in animals, plants, and microorganisms, and its microbial source includes a variety of bacteria, molds and yeasts (such as Lactobacillus sp., Aspergillus sp., Pichia angusta and Saccharomyces cerevisiae). In recent years, a series of advances have been achieved in various aspects of α-validates the above-described-rhamnosidase research. A number of α-L-rhamnosidases have been successfully recombinant expressed in prokaryotic systems as well as eukaryotic systems which involve Pichia pastoris, Saccharomyces cerevisiae and Aspergillus niger, and the catalytic properties of the recombinant enzymes have been improved by enzyme modification techniques. In this review, the sources and production methods, general and catalytic properties and biotechnological applications of α-L-rhamnosidase in different fields are summarized and discussed, concluding with the directions for further in-depth research on α-L-rhamnosidase.

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.

Increased Expression of Recombinant Chitosanase by Co-expression of Hac1p in the Yeast Pichia pastoris

BACKGROUND: Pichia pastoris is one of the most popular eukaryotic hosts for producing heterologous proteins, while increasing the secretion of target proteins is still a top priority for their application in industrial fields. Recently, the research effort to enhance protein production has focused on up-regulating the unfolded protein response (UPR). OBJECTIVE: We evaluated the effects of activated UPR via Hac1p co-expression with the promoter AOX1 (PAOX1) or GAP (PGAP) on the expression of recombinant chitosanase (rCBS) in P. pastoris. METHOD: The DNA sequence encoding the chitosanase was chemically synthesized and cloned into pPICZαA, and the resulting pPICZαA/rCBS was transformed into P. pastoris for expressing rCBS. The P. pastorisHAC1i cDNA was chemically synthesized and cloned into pPIC3.5K to give pPIC3.5K/Hac1p. The HAC1i cDNA was cloned into PGAPZB and then inserted with the HIS4 gene from pAO815 to construct the vector PGAPZB/Hac1p/HIS4. For co-expression of Hac1p, the two plasmids pPIC3.5K/Hac1p and PGAPZB/Hac1p/HIS4 were transformed into P. pastoris harboring the CBS gene. The rCBS was assessed based on chitosanase activity and analyzed by SDSPAGE. The enhanced Kar2p was detected with western blotting to evaluate UPR. RESULTS: Hac1p co-expression with PAOX1 enhanced rCBS secretion by 41% at 28°C. Although the level of UPR resulting from Hac1p co-expression with PAOX1 was equivalent to that with PGAP in terms of the quantity of Kar2p (a hallmark of the UPR), substitution of PGAP for PAOX1 further increased rCBS production by 21%. The methanol-utilizing phenotype of P. pastoris did not affect rCBS secretion with or without co-expression of Hac1p. Finally, Hac1p co-expression withPAOX1 or PGAP promoted rCBS secretion from 22 to 30°C and raised the optimum induction temperature. CONCLUSION: The study indicated that Hac1p co-expression with PAOX1 or PGAP is an effective strategy to trigger UPR of P. pastoris and a feasible means for improving the production of rCBS therein.

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.

Integrated Strategies for Enhancing the Expression of the AqCoA Chitosanase in Pichia pastoris by Combined Optimization of Molecular Chaperones Combinations and Copy Numbers via a Novel Plasmid pMC-GAP.

In our previous study, the chitosanase AqCoA and the chitooligosaccharides it produced were found to exhibit significant protective effects against fungal diseases. In this study, we enhanced the expression of AqCoA using the novel pMC-GAP that enables stable transformation of Escherichia coli, and built an integrated model based on the gene copy number, molecular chaperones, and protein production of AqCoA. In terms of gene dosage, the highest hydrolase activity was 0.32 U/ml in the strain with four copies, which was 1.78-fold higher than that of the strain with only one copy (0.18 U/ml). In addition, we found the chaperones such as PDI, ERO1, HAC1, YDJ1, SSE1, SSA4, and SSO2 improved protein expression. Furthermore, the PDI/ERO1, SSA4/SSE1, and YDJ1/SSO2 pairs synergistically increased the expression levels by 61%, 31%, and 42%, respectively. Finally, we investigated the combined effects of gene copy numbers and molecular chaperones on protein expression. The highest activity reached 2.32 U/ml in the strain with six integrated molecular chaperone expression cassettes and sixteen copies of the target gene, which was 13-fold higher than that of the control strain with only one copy (GAP-1AqCoA). Combined optimization of gene dosage and molecular chaperone combinations significantly increased the expression level of AqCoA, providing a powerful strategy to improve the expression of other heterologous proteins in P. pastoris.

High-level expression of a novel multifunctional GH3 family ß-xylosidase/α-arabinosidase/ß-glucosidase from Dictyoglomus turgidum in Escherichia coli.

A novel ß-xylosidase Dt-2286 from Dictyoglomus turgidum was cloned and overexpressed in Escherichia coli BL21 (DE3). Dt-2286 belonging to glycoside hydrolase (GH) family 3 encodes a polypeptide with 762 amino acid residues with a molecular weight of 85.1 kDa. By optimization of the growth and induction conditions, the activity of ß-xylosidase reached 273 U/mL, which is the highest yield reported to date from E. coli in a shake-flask. The optimal activities of the purified Dt-2286 were found at pH 5.0 and 98 °C. It also shows excellent thermostable/haloduric/organic solvent-tolerance. Dt-2286 was revealed to be a multifunctional enzyme with ß-xylosidase, α-arabinofuranoside, α-arabinopyranoside and ß-glucosidase activities, and Kcat/Km was 5245.316 mM-1 s-1, 2077.353 mM-1 s-1, 1626.454 mM-1 s-1, and 470.432 mM-1 s-1 respectively. Dt-2286 showed significant synergistic effects on the degradation of xylans, releasing more reduced sugars (up to 15.08 fold) by simultaneous addition with endoxylanase. Moreover, this enzyme has good activity in the hydrolysis of epimedium B, demonstrating its versatility in practical applications.

Optimisation of Recombinant Myrosinase Production in Pichia pastoris.

Myrosinase is a plant defence enzyme catalysing the hydrolysis of glucosinolates, a group of plant secondary metabolites, to a range of volatile compounds. One of the products, isothiocyanates, proved to have neuroprotective and chemo-preventive properties, making myrosinase a pharmaceutically interesting enzyme. In this work, extracellular expression of TGG1 myrosinase from Arabidopsis thaliana in the Pichia pastoris KM71H (MutS) strain was upscaled to a 3 L laboratory fermenter for the first time. Fermentation conditions (temperature and pH) were optimised, which resulted in a threefold increase in myrosinase productivity compared to unoptimised fermentation conditions. Dry cell weight increased 1.5-fold, reaching 100.5 g/L without additional glycerol feeding. Overall, a specific productivity of 4.1 U/Lmedium/h was achieved, which was 102.5-fold higher compared to flask cultivations.

Effects of the Transcription Factor Ace2 from Trichoderma reesei on Cellulase and Hemicellulase Expression in Trichoderma orientalis EU7-22.

Trichoderma orientalis (T. orientalis) EU7-22 has a complete cellulase system and shows a remarkable enzyme activity with high potential in the industry. Ace2 is an important transcriptional factor for cellulase and hemicellulase expression in Trichoderma reesei (T. reesei). However, the ace2 gene cannot be found in the genome of T. orientalis. Researches show that the mechanism of cellulase transcriptional regulation in T. orientalis keeps high similarity with T. reesei up till now. So, in this study, the ace2 of Trichoderma reesei QM9414 was heterologous expressed in T. orientalis EU7-22. As a result, xylanase activity and ß-glucosidase activity of ace2 heterogeneous expression strains are improved and total cellulase activity is decreased. The result of qPCR is in accordance with enzyme activities. This study provides a reference for an in-depth study on transcriptional regulation mechanisms of T. orientalis.

Improve Production of Pullulanase of Bacillus subtilis in Batch and Fed-Batch Cultures.

Pullulanase is a debranching enzyme that cleaves explicitly α-1,6 glycosidic bonds, which is widely used in starch saccharification, production of glucose, maltose, and bioethanol. The thermal-resistant pullulanase is isolated from a variety of microorganisms; however, the lack of industrial production of pullulanase has hindered the transformation of the laboratory to industry. In this study, the expensive maltose syrup and soybean meal powder were replaced with cheap corn starch and corn steep liquor, exhibiting 440 U/mL of pullulanase in shake flasks by changing the C/N value and the total energy of the medium. Subsequently, the cultivation conditions were explored in a 50-L and 50-m3 bioreactor. In batch culture, the pullulanase activity reached 896 U/mL, while it increased to 1743 U/mL in fed-batch culture by controlling the dissolved oxygen, pH, reducing sugar content, and temperature. Remarkably, the cultivation volume was enlarged to 50 m3 based on the technical parameters of fed-batch culture. The industrial production of pullulanase was successful, and the activity achieved 1546 U/mL. When the product was stored at room temperature (25 °C) for 6 months, the pullulanase activity was over 90%. The half-lives at 60 and 80 °C were 119.45 h and 51.18 h, respectively, which satisfied the industrial application requirements of pullulanase.

Secretory production in Escherichia coli of a GH46 chitosanase from Chromobacterium violaceum, suitable to generate antifungal chitooligosaccharides.

A chitosanase (CvCsn46) from Chromobacterium violaceum ATCC 12472 was produced in Escherichia coli, purified, and partially characterized. When subjected to denaturing polyacrylamide gel electrophoresis, the enzyme migrated as two protein bands (38 and 36 kDa apparent molecular masses), which were both identified as CvCsn46 by mass spectrometry. The enzyme hydrolyzed colloidal chitosan, with optimum catalytic activity at 50 °C, and two optimum pH values (at pH 6.0 and pH 11.0). The chitosanolytic activity of CvCsn46 was enhanced by some ions (Ca2+, Co2+, Cu2+, Sr2+, Mn2+) and DTT, whereas Fe2+, SDS and ß-mercaptoethanol completely inhibited its activity. CvCsn46 showed a non-Michaelis-Menten kinetics, characterized by a sigmoidal velocity curve (R2 = 0.9927) and a Hill coefficient of 3.95. ESI-MS analysis revealed that the hydrolytic action of CvCsn46 on colloidal chitosan generated a mixture of low molecular mass chitooligosaccharides, containing from 2 to 7 hexose residues, as well as D-glucosamine. The chitosan oligomers generated by CvCsn46 inhibited in vitro the mycelial growth of Lasiodiplodia theobromae, significantly reducing mycelium extension and inducing hyphal morphological alterations, as observed by scanning electron microscopy. CvCsn46 was characterized as a versatile biocatalyst that produces well-defined chitooligosaccharides, which have potential to control fungi that cause important crop diseases.

Glycoside hydrolase (PelAh) immobilization prevents Pseudomonas aeruginosa biofilm formation on cellulose-based wound dressing.

Bacterial cellulose (BC) is recognized as a wound dressing material well-suited for chronic wounds; however, it has no intrinsic antimicrobial activity. Further, the formation of biofilms can limit the effectiveness of the pre-saturation of BC with antimicrobial agents. Here, to hinder biofilm formation by P. aeruginosa, we immobilized the hydrolytic domain of PelA (a glycohydrolase involved in the synthesis of biofilm polysaccharide Pel) on the surface of BC. The immobilization of 32.35 ±â€¯1.05 mg PelAh per g BC membrane resulted in an eight-fold higher P. aeruginosa cell detachment from BC membrane, indicating reduced biofilm matrix stability. Further, 1D and 2D infrared spectroscopy analysis indicated systematic reduction of polysaccharide biofilm elements, confirming the specificity of immobilized PelAh. Importantly, BC-PelAh was not cytotoxic towards L929 fibroblast cells. Thus, we conclude that PelAh can be used in BC wound dressings for safe and specific protection against biofilm formation by P. aeruginosa.

A Review of the Role of Probiotic Supplementation in Dental Caries.

Dental diseases are among the common health issues experienced around the world. Dental caries is one of the most predominant oral diseases worldwide. Major factors associated with caries development include poor oral hygiene, the content of specific carbohydrates in the diet, dental biofilm formation, the cariogenic microbial load, reduction in salivary flow, insufficient fluoride exposure, gingival recession, genetic factors, and lack of personal attention to one's dental health. Several preventive measures have been implemented to reduce the risk of the development of caries. Probiotics are live microbes that when administered in suitable amounts confer health benefits on the host; they are recognized as potential adjunct therapeutic agents for several diseases. The present manuscript summarizes recent findings on the role of probiotics in dental caries prevention and the possible mechanisms of probiotic effects. Review of the literature indicates the regular consumption of probiotic products significantly reduced the risk of caries by inhibiting cariogenic bacteria and enriching commensal microbes in the oral cavity. Buffering the salivary pH, production of bacteriocin and enzymes (dextranase, mutanase, and urease), the capacity of competing for the adhesion and colonization on tooth surfaces are the possible mechanisms behind the beneficial effect of probiotics. Further studies are necessary to address the efficacy of long-term probiotic supplementation on the control of dental diseases and the influence of childhood probiotic supplementation on the risk of caries development.

High level production of a Bacillus amlyoliquefaciens chitosanase in Pichia pastoris suitable for chitooligosaccharides preparation.

Chitooligosaccharides (COS) are hydrolytic products of chitosan that are essential in functional food, medicine, and other fields due to their biological activities. Commercial COS are often prepared by the hydrolysis of chitosan by chitosanase. In this study, a glycoside hydrolase family 46 cluster B chitosanase from Bacillus amyloliquefaciens (BaCsn46B) was efficiently expressed in Pichia pastoris. The recombinant enzyme was secreted into the culture medium that reached a total extracellular protein concentration of 4.5 g/L with an activity of 8907.2 U/mL in a high cell density fermenter (5 L). The molecular mass of deglycosylated BaCsn46B was 29.0 kDa. Purified BaCsn46B exhibited excellent enzymatic properties, which had high specific activity (2380.5 U/mg) under optimal reaction conditions (55 °C and pH 6.5). BaCsn46B hydrolyzed chitosan yielded a series of COS with different degrees of polymerization by endo-type cleavage. The end hydrolytic products of BaCsn46B were chitobiose and chitotriose, while no monosaccharide yield was evident in the hydrolytic reaction. The excellent secreted expression level and hydrolytic performance make the enzyme a desirable biocatalyst for the industrial preparation of COS.

Medium optimization and kinetic modeling for the production of Aspergillus niger inulinase.

The goals of this study were to optimize the medium formulation for enhanced production of Aspergillus niger inulinase using Plackett-Burman Design (PBD) and to model the fermentation in optimal medium formulation. Results indicated that (NH4)2SO4 (negative effect), yeast extract and peptone (positive effect) were determined as significant factors affecting the inulinase production. Different media including Medium A (non-enriched), Medium B (contains both negative and positive factors) and Medium C (contains only positive factors) were formed and inulinase fermentations were performed. Findings showed that the best nutritional formulation was Medium C, which yielded to be 1011.02 U/mL, 834.28 U/mL, 1.22, 4383.44 U/mg, 4186 U/mg, 158.49 U/mL/day, 128.60 U/mL/day and 94.54% of PInulinase, SInulinase, I/S ratio, SInulinase, SSucrase, QInulinase, QSucrase and SUY, respectively. Additionally, fungal growth, enzyme or protein production and substrate consumption were modeled using the logistic model, Luedeking-Piret model, and modified Luedeking-Piret model, respectively, and found that enzyme or protein production was non-growth associated. Besides, maintenance value (Z) was lower than γ value, indicating that A. niger mainly utilizes the sugars for enzyme production and fungal growth. Consequently, optimum medium composition was successfully determined by PBD and also the kinetic models fitted the experimental data very well with high regression coefficient.

Inulinase production and mathematical modeling from carob extract by using Aspergillus niger.

The main objectives of the study were to produce inulinase from carob extract by Aspergillus niger A42 (ATCC 204447) and to model the inulinase fermentation in the optimum carob extract-based medium. In the study, carob extract was used as a novel and renewable carbon source in the production of A. niger inulinase. For medium optimization, eight different variables including initial sugar concentration (°Bx), (NH4 )2 HPO4 , MgSO4 .7H2 O, KH2 PO4 , NH4 NO3 , yeast extract, peptone, and ZnSO4 .7H2 O were employed. After fermentations, optimum medium composition contained 1% yeast extract in 5°Bx carob extract. As a result of the fermentation, the maximum inulinase activity, maximum invertase-type activity, I/S ratio, maximum inulinase- and invertase-type activity rates, maximum sugar consumption rate, and sugar utilization yield were 1507.03 U/ml, 1552.86 U/ml, 0.97, 175.82 and 323.76 U/ml/day, 13.26 g/L/day, and 98.52%, respectively. Regarding mathematical modeling, the actual inulinase production and sugar consumption data were successfully predicted by Baranyi and Cone models based on the model evaluation and validation results and the predicted kinetic values, respectively. Consequently, this was the first report in which carob extract was used in the production of inulinase as a carbon source. Additionally, the best-selected models can serve as universal equations in modeling the inulinase production and sugar consumption in shake flask fermentation with carob extract medium.

Production of thermostable endo-1,5-α-L-arabinanase in Pichia pastoris for enzymatically releasing functional oligosaccharides from sugar beet pulp.

Sugar beet pulp is an agricultural processing residue that is a rich source of the cell wall polysaccharide arabinan. Functional oligosaccharides, specifically feruloylated arabino-oligosaccharides (FAOs), can be isolated from sugar beet pulp through selective action by endo-arabinanase (glycoside hydrolase family 43). This study aimed to develop yeast (Pichia pastoris) as an efficient, eukaryotic platform to produce a thermophilic endo-1,5-α-L-arabinanase (TS-ABN) for extracting FAOs from sugar beet pulp. Recombinant TS-ABN was secreted into yeast culture medium at a yield of ~ 80 mg/L, and the protein exhibited specific enzyme activity, pH and temperature optimum, and thermostability comparable to those of the native enzyme. Treatment of sugar beet pulp with Pichia-secreted TS-ABN released FAOs recovered by hydrophobic chromatography at 1.52% (w/w). The isolated FAOs averaged seven arabinose residues per ferulic acid, and treatment of T84 human colon epithelial cells significantly increased expression of two key tight junction-related proteins-zonula occludens-1 and occludin-in a dose-dependent manner. This research establishes a biochemical platform for utilizing sugar beet pulp to produce value-added bioproducts with potential nutraceutical applications.

Engineering dual-glycan responsive expression systems for tunable production of heterologous proteins in Bacteroides thetaiotaomicron.

Genetically engineering intestinal bacteria, such as Bacteroides thetaiotaomicron (B. theta), holds potential for creating new classes of biological devices, such as diagnostics or therapeutic delivery systems. Here, we have developed a series of B. theta strains that produce functional transgenic enzymes in response to dextran and arabinogalactan, two chemically distinct glycans. Expression systems for single glycan induction, and a novel "dual-glycan" expression system, requiring the presence of both dextran and arabinogalactan, have been developed. In addition, we have created two different chromosomal integration systems and one episomal vector system, compatible with engineered recipient strains, to improve the throughput and flexibility of gene cloning, integration, and expression in B. theta. To monitor activity, we have demonstrated the functionality of two different transgenic enzymes: NanoLuc, a luciferase, and BuGH16C, an agarase from the human intestinal bacterium, Bacteroides uniforms NP1. Together this expression platform provides a new collection of glycan-responsive tools to improve the strength and fidelity of transgene expression in B. theta and provides proof-of-concept for engineering more complex multi-glycan expression systems.

Substrates' and products' inhibition in fructanase production by a new Kluyveromyces marxianus CF15 from Agave tequilana fructan in a batch reactor.

This study focuses on fructanase production in a batch reactor by a new strain isolated from agave juice (K. marxianus var. drosophilarum) employing different Agave tequilana fructan (ATF) concentrations as substrate. The experimental data suggest that the fructanase production may be inhibited or repressed by high substrate (50 g/L) and ethanol (20.7 g/L) concentrations present in culture medium. To further analyze these phenomena an unstructured kinetic mathematical model taking into account substrate and products inhibition was proposed and fitted. The mathematical model considers six reaction kinetics and the ethanol evaporation, and predicts satisfactorily the biomass, fructan, glucose, fructose, ethanol, and fructanase behavior for different raw material initial concentrations. The proposed model is the first to satisfactorily describe the production of fructanase from branched ATF with a new strain of K. marxianus.

Evaluation of carbon sources for the production of inulinase by Aspergillus niger A42 and its characterization.

Inulinases are used for the production of high-fructose syrup and fructooligosaccharides, and are widely utilized in food and pharmaceutical industries. In this study, different carbon sources were screened for inulinase production by Aspergillus niger in shake flask fermentation. Optimum working conditions of the enzyme were determined. Additionally, some properties of produced enzyme were determined [activation (Ea)/inactivation (Eia) energies, Q10 value, inactivation rate constant (kd), half-life (t1/2), D value, Z value, enthalpy (ΔH), free energy (ΔG), and entropy (ΔS)]. Results showed that sugar beet molasses (SBM) was the best in the production of inulinase, which gave 383.73 U/mL activity at 30 °C, 200 rpm and initial pH 5.0 for 10 days with 2% (v/v) of the prepared spore solution. Optimum working conditions were 4.8 pH, 60 °C, and 10 min, which yielded 604.23 U/mL, 1.09 inulinase/sucrase ratio, and 2924.39 U/mg. Additionally, Ea and Eia of inulinase reaction were 37.30 and 112.86 kJ/mol, respectively. Beyond 60 °C, Q10 values of inulinase dropped below one. At 70 and 80 °C, t1/2 of inulinase was 33.6 and 7.2 min; therefore, inulinase is unstable at high temperatures, respectively. Additionally, t1/2, D, ΔH, ΔG values of inulinase decreased with the increase in temperature. Z values of inulinase were 7.21 °C. Negative values of ΔS showed that enzymes underwent a significant process of aggregation during denaturation. Consequently, SBM is a promising carbon source for inulinase production by A. niger. Also, this is the first report on the determination of some properties of A. niger A42 (ATCC 204,447) inulinase.