Biology Reference
In-Depth Information
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.
Besides, the latter enzyme should be optimally active at pH 6.7-6.8 and cannot be in-
hibited by sodium, calcium, or glucose. Such β-galactosidases are still highly desired.
Only several enzymes optimally hydrolyzing lactose at low temperatures have been
characterized till now [1-14], however, none of them have been produced on the com-
mercial scale. 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].
Additionally, in order to make progress in cheaper production of β-D-galactosidases
of industrial interest, high effi ciency yeast expression systems must be taken into con-
sideration. On the other hand extracellular production must occur to allow easy and fast
isolation of target protein. There are several studies in literature related to the extracel-
lular production of the
Aspergillus niger
β-galactosidase by recombinant
Saccharomyces
cerevisiae
strains [15-19], although this enzyme is mainly interesting for lactose hy-
drolysis in acid whey, because of their acidic pH optimum as well as their activity
at elevated temperatures. The
S. cerevisiae
expression system was also used for the
production of
K. lactis
β-D-galactosidase, the protein of outstanding biotechnological
interest in the food industry but in this case the enzyme production was not strictly
extracellular. The β-galactosidase was released into the culture medium after osmotic
shock of the recombinant
S. cerevisiae
osmotic-remedial thermosensitive-autolytic
mutants [20, 21]. To improve the secretion of the
K. lactis
β-D-galactosidase, cyto-
solic in origin, the hybrid protein from this enzyme and its
A. niger
homologue, that
is naturally extracellular, was constructed. The hybrid protein was active and secreted
by recombinant
K. lactis
strain, but the amount of extracellular enzyme still remained
low [22]. Yeast species especially designated for the production of extracellular pro-
teins are for example
Pichia pastoris
or
Hansenula polymorpha
. There is only one re-
cently published example of an extracellular β-galactosidase production system using
P. pastoris
as a host, however, it concerns thermostable enzyme from
Alicyclobacillus
acidocaldarius
[23].
The
S. cerevisiae
is usually the fi rst choice for industrial processes involving al-
coholic fermentation but this yeast is unable to metabolize lactose and, therefore, the
lactose consuming yeast,
K. fragilis
, has been used in most industrial plants produc-
ing ethanol from whey [24]. The engineering of
S. cerevisiae
for lactose utilization
has been addressed over the past 20 years by different strategies [25]. However, most
recombinant strains obtained displayed no ideal characteristics (such as slow growth,
genetic instability, or problems derived from the use of glucose/galactose mixtures) or
were ineffective for ethanol production [24, 26, 27]. There is only one published ex-
ample of effi cient ethanol production with a recombinant
S. cerevisiae
strain express-
ing the
LAC4
(β-galactosidase) and
LAC12
(
lactose permease
) genes of
K. lactis
[28].
Hence, there is still a need for
S. cerevisiae
strains producing new β-galactosidases
which may appear to be an interesting alternative for the production of ethanol from
lactose-based feedstock.
