Abstract
BackgroundThe development of a new cold-active β-D-galactosidases and microorganisms that efficiently ferment lactose is of high biotechnological interest, particularly for lactose removal in milk and dairy products at low temperatures and for cheese whey bioremediation processes with simultaneous bio-ethanol production.ResultsIn this article, we present a new β-D-galactosidase as a candidate to be applied in the above mentioned biotechnological processes. The gene encoding this β-D-galactosidase has been isolated from the genomic DNA library of Antarctic bacterium Arthrobacter sp. 32c, sequenced, cloned, expressed in Escherichia coli and Pichia pastoris, purified and characterized. 27 mg of β-D-galactosidase was purified from 1 L of culture with the use of an intracellular E. coli expression system. The protein was also produced extracellularly by P. pastoris in high amounts giving approximately 137 mg and 97 mg of purified enzyme from 1 L of P. pastoris culture for the AOX1 and a constitutive system, respectively. The enzyme was purified to electrophoretic homogeneity by using either one step- or a fast two step- procedure including protein precipitation and affinity chromatography. The enzyme was found to be active as a homotrimeric protein consisting of 695 amino acid residues in each monomer. Although, the maximum activity of the enzyme was determined at pH 6.5 and 50°C, 60% of the maximum activity of the enzyme was determined at 25°C and 15% of the maximum activity was detected at 0°C.ConclusionThe properties of Arthrobacter sp. 32cβ-D-galactosidase suggest that this enzyme could be useful for low-cost, industrial conversion of lactose into galactose and glucose in milk products and could be an interesting alternative for the production of ethanol from lactose-based feedstock.
Highlights
The development of a new cold-active β-D-galactosidases and microorganisms that efficiently ferment lactose is of high biotechnological interest, for lactose removal in milk and dairy products at low temperatures and for cheese whey bioremediation processes with simultaneous bio-ethanol production
Commercially available β-galactosidase preparations (e.g. Lactozym – Novo Nordisk, Maxilact – DSM Food Specialties) applied for lactose hydrolysis contain Kluyveromyces lactis β-galactosidase naturally intracellularly biosynthesized by K. lactis strains
Its optimum activity was observed at about 50°C. It showed over 50% of activity at pH 5.5–7.5 at 30°C and was not considerably inactivated by Ca2+ ions what can be of interest in industrial ethanol production from cheese whey by means of brewing Saccharomyces cerevisiae strains or by recombinant strains that simultaneously utilize glucose and galactose
Summary
The development of a new cold-active β-D-galactosidases and microorganisms that efficiently ferment lactose is of high biotechnological interest, for lactose removal in milk and dairy products at low temperatures and for cheese whey bioremediation processes with simultaneous bio-ethanol production. Nowadays low-cost energy bio-industrial processes in biotechnology are highly desired This has led to increased interest in the production of cold adapted enzymes. One class of such enzymes includes cold-adapted β-D-galactosidases (EC 3.2.1.23) that can find many applications in industrial biotechnology. These enzymes are capable of hydrolyzing 1,4-β-D-galactoside linkages and can sometimes catalyse the synthesis of oligosaccharides. Commercially available β-galactosidase preparations (e.g. Lactozym – Novo Nordisk, Maxilact – DSM Food Specialties) applied for lactose hydrolysis contain Kluyveromyces lactis β-galactosidase naturally intracellularly biosynthesized by K. lactis strains This enzyme is optimally active at approximately 50°C and displays low activity at 20°C while an ideal enzyme for treating milk should work well at 4–8°C. The β-galactosidases were obtained from different microbial sources, including those from Arthrobacter sp. [1,2,7,8,12], Arthrobacter psychrolactophilus [9,13]Carnobacterium piscicola [3], Planococcus sp. [4,14], Pseudoalteromonas haloplanktis [5], and Pseudoalteromonas sp. [10,11]
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