Cell disruption optimization and covalent immobilization of beta-D-galactosidase from kluyveromyces marxianus YW-1 for lactose hydrolysis in milk

β-D-galactosidase (EC 3.2.1.23) from Kluyveromyces marxianus YW-1, an isolate from whey, has been studied in terms of cell disruption to liberate the useful enzyme. The enzyme produced in a bioreactor on a wheat bran medium has been successfully immobilized with a view to developing a commercially usable technology for lactose hydrolysis in the food industry. Three chemical and three physical methods of cell disruption were tested and a method of grinding with river sand was found to give highest enzyme activity (720 U). The enzyme was covalently immobilized on gelatin. Immobilized enzyme had optimum pH and temperature of 7.0 and 40 °C, respectively and was found to give 49% hydrolysis of lactose in milk after 4 h of incubation. The immobilized enzyme was used for eight hydrolysis batches without appreciable loss in activity. The retention of high catalytic activity compared with the losses experienced with several previously reported immobilized versions of the enzyme is significant. The method of immobilization is simple, effective, and can be used for the immobilization of other enzymes.

Production of high fructose syrup from asparagus inulin using immobilized exoinulinase from Kluyveromyces marxianus YS-1

Extracellular exoinulinase from Kluyveromyces marxianus YS-1, which hydrolyzes inulin into fructose, was immobilized on Duolite A568 after partial puriWcation by ethanol precipitation and gel exclusion chromatography on Sephadex G-100. Optimum temperature of immobilized enzyme was 55 °C, which was 5 °C higher than the free enzyme and optimal pH was 5.5. Immobilized biocatalyst retained more than 90% of its original activity after incubation at 60 °C for 3 h, whereas in free form its activity was reduced to 10% under same conditions, showing a signiWcant improvement in the thermal stability of the biocatalyst after immobilization. Apparent Km values for inulin, raYnose and sucrose were found to be 3.75, 28.5 and 30.7 mM, respectively. Activation energy (Ea) of the immobilized biocatalyst was found to be 46.8 kJ/mol. Metal ions like Co2+ and Mn2+ enhanced the activity, whereas Hg2+ and Ag2+ were found to be potent inhibitors even at lower concentrations of 1 mM. Immobilized biocatalyst was eVectively used in batch preparation of high fructose syrup from Asparagus racemosus raw inulin and pure inulin, which
yielded 39.2 and 40.2 g/L of fructose in 4 h; it was 85.5 and 92.6% of total reducing sugars produced, respectively.

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

An extracellular exoinulinase( ^{2, 1}- ß- 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 50º c. Mn²⁺ and Ca²⁺ produced a 2A-fold and 1.2-fold enhancement in enzyme activity, whereas Hg²⁺ and Ag²⁺  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.

Optimization of medium and process parameters for the production of inulinase from a newly isolated Kluyveromyces marxianus YS-1

A newly isolated strain of Kluyveromyces marxianus YS-1 was used for the production of extra cellular inulinase in a medium containing inulin, meat extract, CaCl₂ and sodium dodecyl sulphate (SDS). Fermentation medium pH 6.5, cultivation temperature 30 °C and 5% (v/v) inoculum of 12 h-old culture were optimal for enzyme production (30.8 IU/ml) with a fermentation time of 72 h at shake flask level. Raw inulin (2%, w/v) extracted from dahlia tubers by processing at 15 kg/cm² for 10 min was optimum for bioreactor studies. Maximum enzyme production (55.4 IU/ml) was obtained at an agitation rate of 200 rpm and aeration of 0.75 vvm in a stirred tank reactor with a fermentation time of 60 h.

Production of inulinase from Kluyveromyces marxianus YS-1 using root extract of Asparagus racemosus

Roots of Asparagus racemosus were used as a source of inulin for the production of inulinase from Kluyveromyces marxianus YS-1. Root extract prepared at 10 kg/cm² for 10 min showed the maximum production of inulinase. Various parameters of fermentation have been examined in order to improve overall enzyme yield. Inulinase yield of 40.5 IU/mL in an optimized medium containing inulin (3.5%), beef extract (0.5%), MnSO₄ (0.5 mM), CoCl₂ (0.05 mM), SDS (0.4 mM) and pH 6.5 at 30 °C under agitation (150 rpm) for 60 h has been obtained at shake flask level. After optimization the enzyme production has been increased 6.1 times at flask level. In a bioreactor (working volume 1 L) fermentation medium containing 4% inulin has shown maximum inulinase production (47.3 IU/mL) under optimized parameters of 200 rpm, 0.75 vvm aeration at 30 °C after 60 h.

Production, purification and characterization of an extracellular inulinase from Kluyveromyces marxianus var. bulgaricus

The yeast Kluyveromyces marxianus var. bulgaricus produced large amounts of extracellular inulinase activity when grown on inulin, sucrose, fructose and glucose as carbon source, This protein has been purified to homogeneity by using successive DEAE-Trisacryl Plus and Superose 6 HR 10/30 columns. The purified enzyme showed a relative molecular weight of 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 77 kDa by gel filtration in Superose 6 HR 10/30, Analysis by SDS-PAGE showed a unique polypeptide band with Coomassie Blue stain and nondenaturing PAGE of the purified enzyme obtained from media with different carbon sources showed the band, too, when stained for glucose oxidase activity, the optimal hydrolysis temperature for sucrose, raffinose and inulin was 55 degrees C and the optimal pH for sucrose was 4.75, the apparent K-m values for sucrose, raffinose and inulin are 4.58, 7.41 and 86.9 mg/ml, respectively, Thin layer chromatography showed that inulinase from K. marxianus var. bulgaricus was capable of hydrolyzing different substrates (sucrose, raffinose and inulin), releasing monosaccharides and oligosaccharides, the results obtained suggest the hypothesis that enzyme production was constitutive.

Yacon (Polymnia sanchifolia) extract as a substrate to produce inulinase by Kluyveromyces marxianus var. bulgaricus

The inulinase production by yeast K marxianus var. bulgaricus growing in yacon extract was investigated. The microorganism showed good development in yacon, higher enzymatic activities were achieved at 30% and 40% (v/v) of extract. The cultivation temperature (20, 25, 30, 35, 40 degreesC) neither influenced the growth or the enzymatic activity. The optimum cultivation pH was 3.5. The highest activity was observed at 60 degreesC and pH 4.0. At temperature of 55 C and 60 C occurred sharp decrease in the enzyme activity. (C) 2004 Elsevier Ltd. All rights reserved.

Effects of culture conditions on the production of inulinase by Kluyveromyces marxianus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Sucrose hydrolysis by gelatin-immobilized inulinase from Kluyveromyces marxianus var. bulgaricus

The crude cell-free medium from a culture of Kluyveromyces marxianus var. bulgaricus was immobilized in a gelatin-water support, with an immobilization yield of 82.60% for inulinase activity. The optimum pH for both free and immobilized inulinase was the same (3.5) and the optimum temperatures were 55 degrees C for the free and 60 degrees C for the immobilized enzyme. The Arrhenius plots were linear and activation energies were 56.20 (free enzyme) and 20.27 kj/mol K (immobilized enzyme). The kinetic parameters were calculated by Lineweaver-Burk plots and the V-max and K-m were 37.60 IU/mg protein and 61.83 mM for the free inulinase and 31.45 IU/mg protein and 149.28 mM for the immobilized enzyme, respectively. The operational stability of the immobilized inulinase was studied in a continuous fixed-bed column reactor for 33 days, at the end of which the sucrose conversion was 58.12%. (c) 2008 Elsevier Ltd. All rights reserved.

Effect of Conditioning on the Production of Inulinase by Kluyveromyces marxianus var. bulgaricus through Fed-Batch Fermentation

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influence of nitrogen source and sucrose concentration on inulinase production by Kluyveromyces marxianus in fed-batch fermentation

This work studied the influence of nitrogen source and sucrose concentration in the feeding medium for biomass and inulinase production by Kluyveromyces marxianus var. bulgaricus. The results show that the best nitrogen source was a combination of 5 g/L of yeast extract and 10 g/L of peptone. Both cellular growth and enzymatic activity increased with sucrose concentration in the feeding medium (from 200 to 500 g/L). When the sucrose concentration reached 600 g/L, both cellular growth and enzymatic activity decreased.

Location of the β-galactosidase of the yeast Kluyveromyces marxianus var. marxianus ATCC 10022

During the growth of Kluyveromyces marxianus var. marxianus ATCC 10022 on lactose, peaks of glucose, but not β-galactosidase activity, were detected in culture medium. Harvested and washed whole cells produced glucose and galactose from lactose, or ortho-nitro-phenol from the chromogenic substrate ortho-nitro-phenyl-β-D-galactopyranoside (ONPG), indicating that β-galactosidase is physically associated with cells. ONPG hydrolysis by whole cells presented a monophasic kinetics (Km 36.6 mM) in lactose exponential growth phase cells, but a biphasic kinetics (Km 0.2 and 36.6 mM) in stationary growth phase cells. Permeabilization with digitonin or disruption of cells from both growth phases led to monosite ONPG hydrolysis (Km 2.2 to 2.5 mM), indicating that β-galactosidase is not located in the periplasm. In addition, the energy inhibitors fluoride or arsenate, as well as the uncouplercarbonyl cyanide m-chlorophenylhydrazone (CCCP) prevented ONPG hydrolysis by whole cells. These findings indicate that energy coupled transmembrane transport is the rate-limiting step for intracellular ONPG cleavage. The taxonomic and physiologic implications of the exclusive intracellular location of β-galactosidase of K. marxianus var. marxianus ATCC 10022 are discussed. © 1996 Kluwer Academic Publishers.

Estudo da bioconversão da lactose do soro de leite em bioetanol pela evedura Kluyveromyces marxianus 229

Pós-graduação em Engenharia e Ciência de Alimentos - IBILCE

Fermentative production of ribonucleotides from whey by Kluyveromyces marxianus: effect of temperature and pH

Ribonucleotides have shown many promising applications in food and pharmaceutical industries. The aim of the present study was to produce ribonucleotides (RNA) by Kluyveromyces marxianus ATCC 8,554 utilizing cheese whey, a dairy industry waste, as a main substrate under batch fermentation conditions. The effects of temperature, pH, aeration rate, agitation and initial cellular concentration were studied simultaneously through factorial design for RNA, biomass production and lactose consumption. The maximum RNA production (28.66 mg/g of dry biomass) was observed at temperature 30°C, pH 5.0 and 1 g/l of initial cellular concentration after 2 h of fermentation. Agitation and aeration rate did not influence on RNA concentration (p >0.05). Maximum lactose consumption (98.7%) and biomass production (6.0 g/l) was observed after 12 h of incubation. This study proves that cheese whey can be used as an adequate medium for RNA production by K. marxianus under the optimized conditions at industrial scale.

Produzione di bioetanolo da effluenti del settore lattiero-caseario con Kluyveromyces marxianus

Il siero di latte e la scotta sono effluenti provenienti rispettivamente dal processo di trasformazione del latte in formaggio e ricotta. Il siero di latte contiene minerali, lipidi, lattosio e proteine; la scotta contiene principalmente lattosio. Il siero può essere riutilizzato in diversi modi, come l'estrazione di proteine o per l’alimentazione animale, mentre la scotta è considerata solamente un rifiuto. Inoltre, a causa degli ingenti volumi di siero prodotti nel mondo, vengono a crearsi seri problemi ambientali e di smaltimento. Destinazioni alternative di questi effluenti, come le trasformazioni biotecnologiche, possono essere un modo per raggiungere il duplice obiettivo di migliorare il valore aggiunto dei processi agroindustriali e di ridurre il loro impatto ambientale. In questo lavoro sono state studiate le condizioni migliori per produrre bioetanolo dal lattosio del siero e della scotta. Kluyveromyces marxianus è stato scelto come lievito lattosio-fermentante. Sono state effettuate fermentazioni su scala di laboratorio aerobiche e anaerobiche in batch, fermentazioni semicontinue in fase dispersa e con cellule immobilizzate in alginato di calcio,. Diverse temperature sono state testate per migliorare la produzione di etanolo. Le migliori prestazioni, per entrambe le matrici, sono state raggiunte a basse temperature (28°C). Anche le alte temperature sono compatibili con buone rese di etanolo nelle fermentazioni con siero. Ottimi risultati si sono ottenuti anche con la scotta a 37°C e a 28°C. Le fermentazioni semicontinue in fase dispersa danno le migliori produzioni di etanolo, in particolare con la scotta. Invece, l'uso di cellule di lievito intrappolate in alginato di calcio non ha migliorato i risultati di processo. In conclusione, entrambi gli effluenti possono essere considerati adatti per la produzione di etanolo. Le buone rese ottenute dalla scotta permettono di trasformare questo rifiuto in una risorsa.