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Critical Reviews in Food Science and Nutrition, 46:197–205 (2006) Copyright C Taylor and Francis Group, LLC ISSN: 1040-8398 DOI: 10.1080/10408690590957296

Sources, Properties and Suitability of New Thermostable Enzymes in Food Processing
´ JOZEF SYNOWIECKI, BEATA GRZYBOWSKA and ANNA ZDZIEBLO
Department of Food Chemistry, Technology and Biotechnology, Chemical Faculty, Gdansk University ofTechnology, ul. Gabriela Narutowicza 11/12, 80-952 Gdansk, Poland

Investigations concerning recombinant α-amylases from Pyrococcus woesei and thermostable α-glucosidase from Thermus thermophilus indicate their suitability for starch processing. Furthermore, the study of recombinant β-galactosidase from Pyrococcus woesei suitable for purpose of low lactose milk and whey production are alsopresented. The activity of this enzyme in a wide pH range of 4.3–6.6 and high thermostability suggests that it can be used for processing of dairy products at temperatures which restrict microbial growth during a long operating time of continuous-flow reactor with an immobilized enzyme system. Preparation of recombinant α-amylase and β-galactosidase was facilitated by cloning and expression of genes fromPyrococcus woesei in Escherichia coli host. Satisfactory level of recombinant enzymes purification was achieved by thermal precipitation of native proteins originated from Escherichia coli. The obtained α-amylase has maximal activity at pH 5.6 and 93◦ C. The half-life of this preparation (pH 5.6) at 90◦ C and 110◦ C was 11 h and 3.5 h, respectively, and retained 24% of residual activity followingincubation for 2 h at 120◦ C. An advantageous attribute of recombinant α-amylase is independence of its activity and stability on calcium salt. α-Glucosidase from Thermus thermophilus also not require metal ions for stability and retained about 80% of maximal activity at pH range 5.8–6.9. Thus, this enzyme can be used together with recombinant α-amylase. Keywords α-amylase, Pyrococcus woesei,β-galactosidase, Thermus thermophilus, α-glucosidase

INTRODUCTION Enzymes from extreme thermophilic and hyperthermophilic microorganisms often retain the activity even above 100◦ C and are more thermostable as well as more resistant than their mesophilic counterparts to organic solvents, detergents, low and high pH, and other denaturing agents.1,10,65,69 Incorporation of water miscible organicsolvent allows the yield of reverse reactions to increase, e.g., during plastein formation.8,10,56 Application of thermozymes limits probability of microbial contamination during the long operating time of the reactors and causes inactivation of undesired enzymes originating from food material. Moreover, small activities of thermostable enzymes at low temperatures allow to terminate the reaction just bycooling. Additional benefits of the high-temperature catalysis include: enhanced solubility of the reagents and decreased viscosity of the reaction environment. It improves the mixing and pumping

when concentrated substrate solutions are used. These advantages are important in many industrial applications.7,24,34,44,60 Unconventional growth conditions of many extremophiles, low cell yields, andsometimes generation of toxic or corrosive metabolites cause technological difficulties during large-scale enzyme production. Hyperthermophiles grow optimally at 90◦ C or above and a majority of these microorganisms are strictly anaerobic. Usually acetate, H2 , CO2 , and sometimes H2 S or sulphuric acid are formed as fermentation products.1,27 Generally, most of thermophilic bacteria and archeaproduce the desired enzymes in an amount not sufficient to large-scale enzyme production. Expression of the genes responsible for synthesis of the thermostable enzymes in mesophilic host could help to solve this problem. ISOLATION AND PURIFICATION OF THE THERMOSTABLE RECOMBINANT ENZYMES The great differences in thermostability cause that after heating of the cell-free extract the native proteins...
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