Immobilization on chitosan of a thermophilic βglycosidase expressed inSaccharomyces cerevisiae

D’Auria, Sabato; Pellino, F; Cara, Francesco La; Barone, Roberto; Rossi, Mos È; Nucci, Roberto

doi:10.1007/bf02785698

Cited by 22 publications

(17 citation statements)

References 6 publications

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“…process for the conversion of lactose or other saccharides has not been widely studied so far (D'Auria et al, 1996;Febbraio et al, 1998;Fischer et al, 1996;Saito et al, 1994). We report here the covalent attachment of SsbGly and CelB in high yields onto Eupergit C, CPG, and chitosan.…”

Section: Immobilized Thermostable B-glycosidasesmentioning

confidence: 91%

“…The commercial chitosan was pretreated (D'Auria et al, 1996), then lyophilized and stored at room temperature before use. The immobilization used the protocol of D' Auria et al (1996) with slight modi®cations: approximately 40 U of each enzyme were added to 50 mg of chitosan in a total volume of 1.5 mL, and the resulting suspensions were shaken at 100 rpm for 2 h. After a further 4 h in the presence of 2.5% (wt/vol) of glutardialdehyde, the mixture was centrifuged at 1,200 rpm for 2 min.…”

Section: Chitosanmentioning

confidence: 99%

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Development of an ultrahigh‐temperature process for the enzymatic hydrolysis of lactose. IV. Immobilization of two thermostable β‐glycosidases and optimization of a packed‐bed reactor for lactose conversion

Petzelbauer

Kühn

Splechtna

et al. 2002

Biotech & Bioengineering

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Recombinant hyperthermostable beta-glycosidases from the archaea Sulfolobus solfataricus (Ss beta Gly) and Pyrococcus furiosus (CelB) were covalently attached onto the insoluble carriers chitosan, controlled pore glass (CPG), and Eupergit C. For each enzyme/carrier pair, the protein-binding capacity, the immobilization yield, the pH profiles for activity and stability, the activity/temperature profile, and the kinetic constants for lactose hydrolysis at 70 degrees C were determined. Eupergit C was best among the carriers in regard to retention of native-like activity and stability of Ss beta Gly and CelB over the pH range 3.0-7.5. Its protein binding capacity of approximately 0.003 (on a mass basis) was one-third times that of CPG, while immobilization yields were typically 80% in each case. Activation energies for lactose conversion by the immobilized enzymes at pH 5.5 were in the range 50-60 kJ/mol. This is compared to values of approximately 75 kJ/mol for the free enzymes. Immobilization expands the useful pH range for CelB and Ss beta Gly by approximately 1.5 pH units toward pH 3.5 and pH 4.5, respectively. A packed-bed enzyme reactor was developed for the continuous conversion of lactose in different media, including whey and milk, and operated over extended reaction times of up to 14 days. The productivities of the Eupergit C-immobilized enzyme reactor were determined at dilution rates between 1 and 12 h(-1), and using 45 and 170 g/L initial lactose. Results of kinetic modeling for the same reactor, assuming plug flow and steady state, suggest the presence of mass-transfer limitation of the reaction rate under the conditions used. Formation of galacto-oligosaccharides in the continuous packed-bed reactor and in the batch reactor using free enzyme was closely similar in regard to yield and individual saccharide components produced.

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Section: Immobilized Thermostable B-glycosidasesmentioning

confidence: 91%

Section: Chitosanmentioning

confidence: 99%

Development of an ultrahigh‐temperature process for the enzymatic hydrolysis of lactose. IV. Immobilization of two thermostable β‐glycosidases and optimization of a packed‐bed reactor for lactose conversion

Petzelbauer

Kühn

Splechtna

et al. 2002

Biotech & Bioengineering

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“…An ultra high temperature process for the enzymatic hydrolysis of lactose for the production of novel oligosaccharides was developed by Petzelbauer et al (2000) using β-glycosidases from S. solfataricus and P. furiosus. D'Auria et al (1996) developed a continuous cellobiose hydrolysis bioreactor system for glucose production by immobilizing a β-glycosidase isolated from S. solfataricus and expressed in Saccharomyces cerevisiae. The system can be run at a high flow rate and has a high degree of conversion, productivity and operational stability (D'Auria et al 1996).…”

Section: Archaeal Enzymes Of Biotechnological Interestmentioning

confidence: 99%

“…D'Auria et al (1996) developed a continuous cellobiose hydrolysis bioreactor system for glucose production by immobilizing a β-glycosidase isolated from S. solfataricus and expressed in Saccharomyces cerevisiae. The system can be run at a high flow rate and has a high degree of conversion, productivity and operational stability (D'Auria et al 1996).…”

Section: Archaeal Enzymes Of Biotechnological Interestmentioning

confidence: 99%

Perspectives on biotechnological applications of archaea

Schiraldi

Giuliano

Rosa

2002

Archaea

100

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Summary Many archaea colonize extreme environments. They include hyperthermophiles, sulfur-metabolizing thermophiles, extreme halophiles and methanogens. Because extremophilic microorganisms have unusual properties, they are a potentially valuable resource in the development of novel biotechnological processes. Despite extensive research, however, there are few existing industrial applications of either archaeal biomass or archaeal enzymes. This review summarizes current knowledge about the biotechnological uses of archaea and archaeal enzymes with special attention to potential applications that are the subject of current experimental evaluation. Topics covered include cultivation methods, recent achievements in genomics, which are of key importance for the development of new biotechnological tools, and the application of wild-type biomasses, engineered microorganisms, enzymes and specific metabolites in particular bioprocesses of industrial interest.

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“…Polyacrylamide-entrapped cells (PEC), eggwhite-entrapped cells (EWEC) and chitosan-entrapped cells (CEC) were prepared following previously described methods [2,5]. Ca-alginate gel beads [0.3±0.1 cm diameter, 2% (w/v) final gel concentration] were prepared by the method of Smidsrod and Skjak-Braek [17].…”

Section: Methodsmentioning

confidence: 99%

Evaluation of a high temperature immobilised enzyme reactor for production of non-reducing oligosaccharides

Schiraldi

Lernia

Giuliano

et al. 2003

J IND MICROBIOL BIOTECHNOL

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There is interest in the production of non-reducing carbohydrates due to their potential application in various industrial fields, particularly the food industry. In this paper, we describe the development of an immobilised cell bioprocess for the synthesis of non-reducing maltodextrins at high temperatures. The trehalosyl-dextrins-forming enzyme (TDFE) isolated from the thermoacidophilic archaeon Sulfolobus solfataricus (strain MT4), was recently expressed at high yields in Escherichia coli (strain Rb-791). Here, we evaluate different matrices, such as polyacrylamide gel, crude egg white, chitosan and calcium alginate for their effectiveness in immobilising whole recombinant E. coli cells subjected to prior thermal permeabilisation. Calcium-alginate based gels formed a solid biocatalyst with a good activity yield and the best enzymatic stability at the operating temperature (75 degrees C). Therefore, these beads were used to pack a glass column reactor to perform the bioconversion of interest. Optimal operating parameters were defined in relation to the substrate stream flow-rate and the substrate-to-biocatalyst ratio. The production of trehalosylmaltotetraose from maltohexaose reached equilibrium with a constant of about 2.6 at 75 degrees C. The bioreactor was exploited for production of trehalosylmaltodextrins from a commercial mixture of maltodextrins, achieving a productivity of 106.5 mg ml(-1) h(-1) (g biocatalyst)(-1) with ~40% conversion when using a 30% (w/v) solution.

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Immobilization on chitosan of a thermophilic βglycosidase expressed inSaccharomyces cerevisiae

Cited by 22 publications

References 6 publications

Development of an ultrahigh‐temperature process for the enzymatic hydrolysis of lactose. IV. Immobilization of two thermostable β‐glycosidases and optimization of a packed‐bed reactor for lactose conversion

Development of an ultrahigh‐temperature process for the enzymatic hydrolysis of lactose. IV. Immobilization of two thermostable β‐glycosidases and optimization of a packed‐bed reactor for lactose conversion

Perspectives on biotechnological applications of archaea

Evaluation of a high temperature immobilised enzyme reactor for production of non-reducing oligosaccharides

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