Biology Reference
In-Depth Information
Chapter 11
Antarctic
Arthrobacter
Sp. 32c
Piotr Hildebrandt, Marta Wanarska, and Józef Kur
INTRODUCTION
The 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 bioreme-
diation processes with simultaneous bio-ethanol production.
In 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
, purifi ed and characterized. 27 mg of β-D-galactosidase was purifi ed 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 purifi ed enzyme from 1 l of
P. pastoris
culture for the AOX1
and a constitutive system, respectively. The enzyme was purifi ed to electrophoretic
homogeneity by using either one step- or a fast two step-procedure including protein
precipitation and affi nity chromatography. The enzyme was found to be active as a ho-
motrimeric 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.
The properties of
Arthrobacter
sp. 32cβ-D-galactosidase suggest that this enzyme
could be useful for low-cost, industrial conversion of lactose into galactose and glu-
cose in milk products and could be an interesting alternative for the production of
ethanol from lactose-based feedstock.
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 fi nd many applications in industrial biotechnology. These enzymes are ca-
pable of hydrolyzing 1,4-β-D-galactoside linkages and can sometimes catalyze the
synthesis of oligosaccharides. The production of lactose-free milk and synthetic oli-
gosaccharides like lactulose are only examples of this cutting edge enzyme class ap-
plication.
Currently, commercially available β-galactosidase preparations (e.g., Lactozym-
Novo Nordisk, MaxilactDSM Food Specialties) applied for lactose hydrolysis contain
