A new cold-adapted beta-D-galactosidase from the Antarctic Arthrobacter sp. 32c - gene cloning, overexpression, purification and properties

Abstract

Background: The development of a new cold-active beta-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.

Results: In this article, we present a new beta-D-galactosidase as a candidate to be applied in the above mentioned biotechnological processes. The gene encoding this beta-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 beta-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 degrees C, 60% of the maximum activity of the enzyme was determined at 25 degrees C and 15% of the maximum activity was detected at 0 degrees C.

Conclusion: The properties of Arthrobacter sp. 32cbeta-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.

Figure 1

Phylogenetic analysis of the Arthrobacter sp. 32c 16S rDNA sequence (A) and Arthrobacter sp. 32c β-D-galactosidase gene sequence (B). Sequences were aligned using the sequence analysis softwares: ClustalX 1.5 b and Gene-Doc 2.1.000. Phylogenetic trees were reconstructed with the PHYLIP COMPUTER PROGRAM PACKAGE, using the neighbour-joining method with genetic distances computed by using Kimura's 2-parameter mode. The scale bar indicates a genetic distance. The number shown next to each node indicates the percentage bootstrap value of 100 replicates.

Figure 2

SDS-PAGE analysis of the expression and purification steps of the Arthrobacter sp. 32c β-D-galactosidase expressed by E. coli host (A), P. pastoris GS115 pPICZαA-32cβ-gal methanol induced variant (B) and P. pastoris GS115 pGAPZαA-32cβ-gal constitutive variant (C). Lanes 1 – protein weight marker. Panel A: lane 2 – cell extract after expression, lane 3 – purified β-D-galactosidase after affinity chromatography. Panel B and C: lane 2 – broth after protein expression, lane 3 – protein precipitate, lane 4 – purified β-D-galactosidase after affinity chromatography.

Figure 3

Effect of temperature on activity of recombinant Arthrobacter sp. 32c β-D-galactosidase at pH range from 4.5 to 9.5.