Partial purification and characterization of exoinulinase from kluyveromyces marxianus YS-1 for preparation of high-fructose syrup

An extracellular exoinulinase( 2, 1-&nbsp;&szlig;- D fructan fructanohydrolase, EC 3.2.1.7), which catalyzes the hydrolysis of inulin into fructose and glucose, was purified 23.5-fold by ethanol precipitation, followed by Sephadex G-100 gel permeation from a cell-free extract of Kluyveromyces marxianus YS-1. The partially purified enzyme exhibited considerable activity between pH 5 to 6, with an optimum pH of 5.5, while it remained stable(100%) for 3 h at the optimum temperature of 50&ordm; c. Mn2+ and Ca2+ produced a 2A-fold and 1.2-fold enhancement in enzyme activity, whereas Hg2+ and Ag2+&nbsp; completely inhibited the inulinase. A preparation of the partially purified enzyme effectively hydrolyzed inulin, sucrose, and raffinose, yet no activity was found with starch, lactose, and maltose. The enzyme preparation was then successfully used to hydrolyze pure inulin and raw inulin from Asparagus racemosus for the preparation of a high-fructose syrup. In a batch system, the exoinulinase hydrolyzed 84.8% of the pure inulin and 86.7% of the raw Asparagus racemosus inulin, where fructose represented 43.6mg/ml and 41.3mg/ml, respectively.<br /

Solid-State Fermentation of Carrot Pomace for the Production of Inulinase by Penicillium oxalicum BGPUP-4

Inulinaze su važna skupina industrijskih enzima što se koriste u proizvodnji kukuruznog sirupa i fruktooligosaharida. Polifruktan inulin uobičajeno se upotrebljava za proizvodnju inulinaza, vrlo skupih suspstrata. Raznovrsni se agroindustrijski otpad koristi za ekonomičnu proizvodnju inulinaza. U ovom je radu odabrana komina mrkve kao podloga za proizvodnju inulinaze fermentacijom na čvrstoj podlozi s pomoću plijesni Penicillium oxalicum BGPUP-4. Komina mrkve dobra je podloga za bioprocese jer je bogata topljivim i netopljivim ugljikohidratima. Optimalna proizvodnja inulinaze iz komine mrkve postignuta je pomoću centralno složenog rotacijskog dizajna i metodologije odzivnih površina. Provedeno je 15 eksperimenata za utvrđivanje optimalnog raspona triju nezavisnih varijabli za proizvodnju inulinaze, i to: udjela vlage od 70 do 90 %, vremena inkubacije od 4 do 6 dana, te pH-vrijednosti od 5,0 do 7,0. Za proizvodnju inulinaze pri temperaturi od 30 °C upotrijebljena je komina mrkve na čvrstoj podlozi koja sadržava 0,5 % inulina, 0,2 % NH4H2PO4, 0,2 % NaNO3, 0,2 % KH2PO4, 0,05 % MgSO4·7H2O i 0,001 % FeSO4·7H2O. Pri optimalnim uvjetima (udjel vlage od 90 %, vrijeme inkubacije od 4 dana i pH-vrijednost od 7,0) proizvedeno je 322,10 IU inulinaze po gramu suhe tvari. Omjer inulinaze i invertaze (I/S) također je bio velik (3,38), što upućuje na zaključak da enzim ima inulinaznu aktivnost. Koeficijent višestruke korelacije (R) za proizvodnju inulinaze bio je 0,9995; a za omjer I/S 0,9947. Vrijednost R blizu 1 potvrđuje odličnu korelaciju između eksperimentalnih i predviđenih rezultataInulinases are an important class of industrial enzymes which are used for the production of high-fructose syrup and fructooligosaccharides. Inulin, a polyfructan, is generally employed for the production of inulinase, which is a very expensive substrate. A number of agroindustrial residues have been used for cost-effective production of inulinases. In the present study, carrot pomace was selected as a substrate for the production of inulinase by Penicillium oxalicum BGPUP-4 in solid-state fermentation. Carrot pomace is one of the good substrates for bioprocesses, because it is rich in soluble and insoluble carbohydrates. A central composite rotatable design (CCRD) used in response surface methodology was employed for the optimal production of inulinase from carrot pomace. Using CCRD, 15 runs were practiced to optimize the range of three independent variables: moisture content (70-90 %), incubation time (4-6 days) and pH (5.0-7.0) for inulinase production. Carrot pomace supplemented with 0.5 % inulin as an inducer, 0.2 % NH4H2PO4, 0.2 % NaNO3, 0.2 % KH2PO4, 0.05 % MgSO4·7H2O and 0.001 % FeSO4·7H2O was used for the production of inulinase in solid-state fermentation at 30 °C. Inulinase production (322.10 IU per g of dry substrate) was obtained under the optimized conditions, i.e. moisture content of 90 %, incubation time 4 days and pH=7.0. The corresponding inulinase/invertase (I/S) ratio (3.38) was also high, which indicates the inulolytic nature of the enzyme. Multiple correlation coefficients R for inulinase production and I/S ratio were 0.9995 and 0.9947, respectively. The R value very close to one indicates an excellent correlation between experimental and predicted results

Enhanced exoinulinase production from Kluyveromyces marxianus YS-1 using response surface methodology

Optimization of critical medium components for exoinulinase production by Kluyveromyces marxianus YS-1 at shake-flask was investigated using response surface methodology (RSM) based on a central composite rotatable design (CCRD). A five-level with five factors CCRD was used to evaluate the influence of related factors including concentration of inulin, meat extract, calcium chloride, sodium dodecyl sulphate and medium pH. Optimum values obtained by RSM were 2% inulin, 2.17% meat extract, 0.65 mM calcium chloride, 0.10 mM sodium dodecyl sulphate and pH 5.5. Optimized medium projected a theoretical exoinulinase production of 63.61 IU/mL and biomass yield of 0.965 (OD600/10). Multiple correlation coefficient R was 0.9976 and 0.9605 for exoinulinase production and biomass yield, respectively, which being close to one, justified an excellent correlation between the predicted and experimental values. Maximum productivity of exoinulinase (64.05 IU/mL) obtained experimentally by RSM was more than double in comparison to earlier findings using classical one-variable-at-a-time technique

Purification and characterization of a mucin specific mycelial lectin from Aspergillus gorakhpurensis: application for mitogenic and antimicrobial activity.

BackgroundLectins are carbohydrate binding proteins or glycoproteins that bind reversibly to specific carbohydrates present on the apposing cells, which are responsible for their ability to agglutinate red blood cells, lymphocytes, fibroblasts, etc. Interest in lectins has been intensified due to their carbohydrate specificity as they can be valuable reagents for the investigation of cell surface sugars, purification and characterization of glycoproteins. The present study reports the purification, characterization and evaluation of mitogenic and antimicrobial potential of a mycelial lectin from Aspergillus gorakhpurensis.MethodsAffinity chromatography on mucin-sepharose column was carried out for purification of Aspergillus gorakhpurensis lectin. The lectin was characterized for physico-chemical parameters. Mitogenic potential of the lectin was evaluated against splenocytes of Swiss albino mice by MTT assay. Antimicrobial activity of the purified lectin has also been evaluated by disc diffusion assay.ResultsSingle-step affinity purification resulted in 18.6-fold purification of the mycelial lectin. The molecular mass of the lectin was found to be 70 kDa and it was composed of two subunits of 34.8 kDa as determined by gel filtration chromatography, SDS-PAGE and MALDI-TOF analysis. pH optima of the lectin was found to be 6.5-9.5, while optimum temperature for lectin activity was 20-30 °C. Lectin was stable within a pH range of 7.0-10.5 and showed fair thermostability. EDTA did not affect lectin activity whereas it was found susceptible to the denaturants tested. MTT assay revealed strong mitogenic potential of A. gorakhpurensis lectin at a concentration upto 150 µg/mL. Antimicrobial activity assay showed its potent antibacterial activity against Bacillus cereus, Staphylococcous aureus and Escherichia coli and marginal antifungal activity against Saccharomyces cerevisiae.ConclusionThis is the first report on the mitogenic and antimicrobial potential of Aspergillus gorakhpurensis lectin. The results will provide useful guidelines for further research in clinical applications of this lectin

Protozoa lectins and their role in host-pathogen interactions

Lectins are proteins/glycoproteins of non-immune origin that agglutinate red blood cells, lymphocytes, fibroblasts, etc., and bind reversibly to carbohydrates present on the apposing cells. They have at least two carbohydrate binding sites and their binding can be inhibited by one or more carbohydrates. Owing to carbohydrate binding specificity of lectins, they mediate cell-cell interactions and play role in protozoan adhesion and host cell cytotoxicity, thus are central to the pathogenic property of the parasite. Several parasitic protozoa possess lectins which mediate parasite adherence to host cells based on their carbohydrate specificities. These interactions could be exploited for development of novel therapeutics, targeting the adherence and thus helpful in eradicating wide spread of protozoan diseases. The current review highlights the present state knowledge with regard to protozoal lectins with an emphasis on their haemagglutination activity, carbohydrate specificity, characteristics and also their role in pathogenesis notably as adhesion molecules, thereby aiding the pathogen in disease establishment

Antimicrobial activity of purified <i>A. gorakhpurensis</i> lectin.

<p>Antimicrobial activity of purified <i>A. gorakhpurensis</i> lectin.</p

Molecular mass determination of <i>A. gorakhpurensis</i> lectin by MALDI-TOF.

<p>Molecular mass determination of <i>A. gorakhpurensis</i> lectin by MALDI-TOF.</p

Summary of purification of <i>A. gorakhpurensis</i> lectin.

<p>The lectin was purified by affinity chromatography on mucin-sepharose column and fractions (1.0 mL) were collected. Bound lectin, eluted by EDTA (0.02 M) was recovered in only 2 fractions. Combined fractions were dialysed and assayed for lectin activity and protein content.</p><p>Summary of purification of <i>A. gorakhpurensis</i> lectin.</p

Sodium Dodecyl Sulphate-Polyacrylamide Gel of <i>A. gorakhpurensis</i> lectin.

<p>Lane 1: Molecular weight markers from top: myosin (201.2 kDa); β-galactosidase (120.3 kDa); bovine serum albumin (100.2 kDa); ovalbumin (55.9 kDa); carbonic anhydrase (38.3 kDa); soyabean trypsin inhibitor (29.7 kDa) and lysozyme (20.7 kDa), Lane 2: Porcine stomach mucin-Sepharose 4B fraction, Lane 3: Crude.</p