Biochemical Engineering Journal 76 (2013) 17–24

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Biochemical Engineering Journal

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Regular article

␤-Galactosidase over-production by a mig1 mutant of Kluyveromyces

marxianus KM for efficient hydrolysis of lactose
Hai-Xiang Zhou, Jin-Li Xu, Zhe Chi, Guang-Lei Liu, Zhen-Ming Chi ∗
UNESCO Chinese Center of Marine Biotechnology, Ocean University of China, Yushan Road, No. 5, Qingdao, China

a r t i c l e i n f o a b s t r a c t

Article history: In this study, the MIG1 gene in the industrial yeast Kluyveromyces marxianus KM was disrupted by inte-
Received 3 July 2012 grating the HPT gene (encoding hygromycin B phosphotransferase) into ORF (Open Reading Frame)
Received in revised form 2 April 2013 of the MIG1 gene. The disruptant (mig1 mutant) KM-15 obtained could grow in the media containing
Accepted 5 April 2013
hygromycin or 2-deoxy-d-glucose, respectively. ␤-Galactosidase production and expression of its gene in
Available online 13 April 2013
the disruptant KM-15 were significantly enhanced compared to those in K. marxianus KM. This confirmed
that Mig1, the transcriptional repressor, indeed regulated expression of the gene and biosynthesis of the
Keywords:
enzyme in K. marxianus KM. Under the optimal conditions, the ␤-galactosidase activity of 121.0 U/mL
MIG1 gene
Kluyveromyces marxianus
was produced by the disruptant KM-15 within 60 h during 2-L fermentation. 99.2% of lactose in 9.0% of
␤-Galactosidase lactose solution, 99.0% of lactose in 12.0% of whey powder suspension and 99.3% of lactose in the milk
Biocatalysis were hydrolyzed by the crude ␤-galactosidase within 3 h, 4 h and 3 h, respectively. The end products of
Metabolic engineering the lactose hydrolysis were only glucose and galactose.
Biomass © 2013 Elsevier B.V. All rights reserved.

1. Introduction
Kluyveromyces marxianus is phylogenetically related to Saccha-
romyces cerevisiae, and is a sister species to Kluyveromyces lactis
[1,2]. The important feature of K. marxianus is the capacity to assimi-
late lactose and inulin and to use these two sugars as carbon sources
[3]. As K. marxianus has the capacity to assimilate lactose and inulin;
an extremely rapid growth rate, short generation time, thermo-
tolerance, and a high secretory capacity, it has been widely used
in biotechnology [4]. The yeast also has achieved GRAS (gener-
ally regarded as safe) and QPS (qualified presumption of safety)
[3], meaning that it can be safely used in the biotechnology. ␤-
Galactosidase (EC 3.2.1.23), commonly known as lactase, catalyzes
the hydrolysis of lactose, producing a mixture of glucose and galac-
tose [5]. The ␤-galactosidase produced by K. marxianus has been
purified and characterized and the gene encoding ␤-galactosidase
in K. marxianus has been cloned and characterized [6,7]. It was
found that the ␤-galactosidase produced by K. marxianus is intracel-
lular [8]. The ␤-galactosidase produced can be used for hydrolysis
and removal of lactose in milk and whey, for treatment for alleviat-
ing symptoms of lactose intolerance in human. It also can be utilized
for production of ethanol, galacto-oligosaccharides and galactose
[9]. However, it has been well known that glucose repression occurs
in this yeast and Mig1, the transcriptional repressor is found to
∗ Corresponding author. Tel.: +86 532 82032266; fax: +86 532 82032266.
E-mail address: zhenming@sdu.edu.cn (Z.-M. Chi).
play an important role in glucose repression [10]. Mig1, the main
effector in glucose repression, is a C2 H2 zinc finger protein that is
able to bind the promoters of a variety of genes repressed by glu-
cose. Snf1 complex encoded by SNF1 gene is a protein kinase [10].
When the glucose concentration in the medium is very low or there
is no glucose in the growth medium, the Snf1 complex is phos-
phorylated by another unknown kinase in yeast cells. In this case,
the Snf1 complex is active and can catalyze the phosphorylation
of Mig1. The phosphorylated Mig1 complex fails in binding to the
promoters of the genes repressed by glucose and is translocated to
cytoplasm, leading to the active transcription of the genes. There-
fore, the Mig1 protein is a central component for glucose repression
in yeasts. When the glucose concentration in the medium is very
high, the Snf1 complex is dephosphorylated and inactive. So Mig1
complex cannot be phosphorylated and bind to the promoters of
the genes repressed by glucose [10,11]. So far, the genes similar to
MIG1 have been cloned in the yeast K. marxianus and other yeasts
and in some filamentous fungi [12]. A number of mutants with
mutations which act synergistically with mig1 to relieve glucose
repression of invertase gene SUC2 and to suppress the effect of a
snf1 mutation on invertase expression or on the growth on glu-
coneogenic carbon sources have been isolated from S. cerevisiae
[10]. In our previous study [13], it has been confirmed that Mig1,
the transcriptional repressor, indeed regulates expression of the
genes and production of the extracellular enzymes in Saccharomy-
copsis fibuligera A11. K. marxianus KM is an industrial yeast, can
produce 4140 U/g of ␤-galactosidase and has been widely used
for ␤-galactosidase production on a large scale in China [14]. In

1369-703X/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.bej.2013.04.010
18 H.-X. Zhou et al. / Biochemical Engineering Journal 76 (2013) 17–24
this study, in order to know if Mig1, the transcriptional repres-
sor regulates expression of the gene encoding ␤-galactosidase and
production of the enzyme, the MIG1 gene in K. marxianus KM was
disrupted and effects of the disruption of the MIG1 gene on the
enzyme production, gene expression, and glucose repression were
examined. Finally, ␤-galactosidase produced by the disruptant was
used to effectively hydrolyze lactose from different sources.
2. Materials and methods
2.1. The strains and media
K. marxianus KM, which is being widely used for ␤-galactosidase
production on large scale in China [14] was used in this study. As
described below, the disruptant KM-15 was obtained by disrup-
tion of the MIG1 gene in K. marxianus KM. The cultivation medium
for the yeast growth was YPD medium containing 20 g/L glucose,
20 g/L polypeptone, 10 g/L yeast extract. The lactose medium for
␤-galactosidase production was as follows: 40 g/L lactose, 20 g/L
polypeptone, 10 g/L yeast extract, 10.0 mM of MnCl2 , pH 7.0. The
whey medium for ␤-galactosidase production was as follows:
100 g/L whey powder, 10 g/L polypeptone, 15 g/L yeast extract,
10 mM of MnCl2 , pH 7.0. The Escherichia coli strain used in this
study was DH5a [F− endA1 hsdR17 (rK− /mK+ 110) supE44 thi−k−
recA1 gyrlacU(80-lacZM15)] and was grown in 5.0 ml of Luria broth
(LB) at 37 ◦ C overnight [15]. YNB medium contained 6.7 g/L yeast
nitrogen base without amino acids, 10 g/L (NH4 )2 SO4 , pH 7.0.
2.2. Plasmids
The plasmid pCAMBIA 1381 carrying the bacterial hygromycin
B phosphotransferase (HPT) gene was kindly supplied by Dr. Lining
Zhang in Canada [16]. pMD19-T simple for cloning of PCR products
was purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China).
2.3. Whey powder
The whey powder (lactose content was 77 g/L) used in this study
was purchased at Jinan Biotechnology Company, China.
2.4. Construction of the knock-out vector for disruption of the
MIG1 gene
Construction of the knock-out vector was described as follows
(Fig. 1) [17]. The promoter of the MIG1 gene was PCR amplified
from the genomic DNA of K. marxianus KM using primers H1 which
added a 5 XhoI site, and H2 which shared the 5 -sequence with the
HPT gene specific primer H3 used in the subsequent PCR amplifica-
tion (Table 1). The terminator of the MIG1 gene was PCR amplified
from the genomic DNA of K. marxianus KM, using the primers H6,
which added a 5 XhoI site, and H5, which shared the 3 -sequence
with the HPT gene specific primer H4 used in the subsequent PCR
amplification (Table 1). The primers H1 and H2 and H5 and H6 were
designed according to the MIG1 gene sequence (accession number:
Z50018). The HPT gene (1026 bp) was amplified from the plasmid
pCAMBIA 1381 carrying the HPT gene using the primers H3, which
shared the 3 -sequence with the primer H2, and H4, which shared
the 5 -sequence with the primer H5. The primers H3 and H4 were
designed according to the HPT gene sequence (accession number:
AF234302) (Table 1). The PCR reaction was performed in a total vol-
ume of 50 ␮L PCR mixture containing 5 ␮L of 10× La PCR buffer II
(Mg2+ Plus), 8 ␮L of 2.5 mM dNTPs, 1 ␮L of 20 ␮M each primer, 2 ␮L
of 10 ng/mL of the genomic DNA, 22.5 ␮L of sterile deionized water
and 0.5 ␮L of U/␮L TaKaRa LaTaq. The conditions for the PCR ampli-
fication were as follows: initial denaturation at 94 ◦ C for 5 min,
denaturation at 94 ◦ C for 40 s, annealing temperature at 55 ◦ C for
40 s, extension at 72 ◦ C for 40 s, final extension at 72 ◦ C for 5 min.
PCR was run for 30 cycles and the PCR cycler was GeneAmp PCR
System 2400 (Perkin-Elmer, Waltham, MA, USA). The PCR products
from the three PCR amplifications were mixed, denatured, allowed
to anneal using the shared sequence of the primers and subjected
to a fusion PCR amplification, using the flanking primers H1 (5 ) and
H6 (3 ) as described above. The amplification products were sep-
arated by electrophoresis on a 0.8% agarose gel and the expected
1377 kb (the promoter of the MIG1 gene::HPT gene::the terminator
of the MIG1 gene) fragments were extracted using TaKaRa Agarose
Gel DNA Fragment Recovery Kit Ver.2.0 (Invitrogen), and sequenced
to verify the correct assembly of the insert (Fig. 1). The recov-
ered fragments were ligated into the plasmid pMD19-T simple and
the resulting plasmid carrying the fragments was designated as
pMD19/kmMIG1-HPT (Fig. 1).
2.5. Transformation and selection
The plasmid pMD19/kmMIG1-HPT obtained was digested with
the enzyme XhoI. The recovered fragments carrying the promoter
of the MIG1 gene::HPT gene::the terminator of the MIG1 gene were
transformed into the cells of K. marxianus KM by lithium acetate
methods [18]. The transformants were spread on the YPD plates
supplemented with 100 ␮g/mL of hygromycin and X-gal and grown
at 28 ◦ C for 96 h. The untransformed K. marxianus KM was used as
a control. The blue transformant colonies, which could grow on
the YPD plates supplemented with 100 ␮g/mL of hygromycin were
regarded as the positive transformants. One of the blue transfor-
mant (disruptant) colonies was named disruptant KM-15 and used
in the subsequent investigation.
2.6. Confirmation of the glucose-derepression
In order to confirm that the disruptant KM-15 is a glucose-
derepression mutant, K. marxianus KM and the disruptant KM-15
were grown on the YPD plates supplemented with 2-deoxy-d-
glucose or X-gal at 28 ◦ C for three days.
2.7. Confirmation of the disrupted MIG1 gene
The genomic DNAs isolated from the yeast strain K. marxianus
KM and the positive disruptant KM-15 were used as the tem-
plate for PCR. The DNA fragments (600 bp) containing the whole
sequence of the promoter of the MIG1 gene and the partial sequence
of 5 -end of the HPT gene were PCR amplified using the primers H1
and HY1 (Table 1 and Fig. 1) and the DNA fragments (585 bp) con-
taining the whole sequence of the terminator of the MIG1 gene and
the partial sequence of 3 -end of the HPT gene were PCR amplified
using the primers H6 and HY2 (Table 1 and Fig. 1). PCR reactions
and conditions were performed as described above and the PCR
products obtained were separated in agarose gel. The sizes of the
PCR products were estimated using the Automated Documentation
and Analysis System (Gene-Genius, USA). The PCR products were
sequenced by Shanghai Sangon Company (Shanghai, China).
2.8. Preparation of the enzyme
One loop of the cells of the yeast strain K. marxianus KM and
the disruptant KM-15 was transferred to 50 mL of YPD medium in
250-mL flask and the culture was aerobically cultivated for 24 h.
One milliliter of the cell culture (at 600 nm absorbance = 3.0) was
transferred to the lactose medium for ␤-galactosidase production
and the culture was grown aerobically at 28 ◦ C and 180 rpm for 2
days. As ␤-galactosidase produced by K. marxianus KM and the dis-
ruptant KM-15 was intracellular, the cultures were centrifuged at
8000 × g and 4 ◦ C for 10 min and the pellets obtained were washed
 H.-X. Zhou et al. / Biochemical Engineering Journal 76 (2013) 17–24 19

Fig. 1. Construction of the knock-out vector for disruption of the MIG1 gene in K. marxianus KM.

with sterile distilled water three times by centrifugation at 8000 × g
and 4 ◦ C for 10 min and the washed cells were used as the crude ␤-
galactosidase preparation. The washed cells were mixed with 1 g of
glass beads (diameter was 0.5 mm, bought from Sigma, USA). The
cells were shaken in a Merckenschlager cell homogenizer (Braun-
Melsungen, Germany) with CO2 cooling for 7 min. The disrupted
cells were used as the crude ␤-galactosidase preparations.
2.9. Determination of ˇ-galactosidase
The washed cells obtained above were suspended in 4% acetate
ether and the suspension was incubated at 4 ◦ C for 4 h. Then, 5.0 mL
of 0.1 M phosphate buffer (pH 7.0) was added to the suspension and
the new cell suspension was further incubated at 4 ◦ C for 4 h. The
mixture containing 0.5 mL of the new cell suspension and 0.5 mL of

Table 1
The primers used in this study.

Primer Sequence

H1 5 -CTCGAGCCATAAGCATCCGAAGAATAGAAAAGTCGA-3 (Both the underlined and bold bases encode XhoI site)
H2 5 -GTGAGTTCAGGCTTTTTCATACCTATTCTATACTCGTCGTCG-3 (The underlined base are the shared bases)
H3 5 -GAGTATAGAATAGGTATGAAAAAGCCTGAA-3 (The underlined and bold bases are the shared bases)
H4 5 -GTAGCTGCAGTCAGTGCTTCAGTGTTCTATTTCTTTGCCCTCGGACGAGTGC-3 (the underlined and bold bases are the shared bases)
H5 5 -GCACTCGTCCGAGGGCAAAGAAATAGAACACTGAAGCACTGACTGCAGCTAC-3 (the underlined and bold bases are the shared bases)
H6 5- CTCGAGACTAGTAATTATGTCCAGTAAGTAGGTTGTG-3 (Both the underlined and bold bases encode XhoI site)
HY1 5 -AACCCGCTCGTCTGGCTAAGATCGGC-3
HY2 5 -ACGCGGATTTCGGCTCCAACAATGTC-3
K26S 5 -CGAGTGAAGCGGCAAAAGC-3
K26X 5 -CCAAGGAACATAGACAAGGACCAG-3
KLS 5 -TTGGGCTTTTGCGTTGTTTG-3
KLX 5 -TCCATTTCTTTGCCGTTTCC-3
20 H.-X. Zhou et al. / Biochemical Engineering Journal 76 (2013) 17–24
4 mg/mL of o-nitrophenyl-␤-d-galactopyranoside (ONPG) in 0.1 M
phosphate buffer (pH 7.0) was incubated at 40 ◦ C for 10 min. The
␤-galactosidase activity in the mixture was inactivated by adding
2 mL of Na2 CO3 (0.5 M) solution to the mixture. The o-nitrophenol
(ONP) released was quantitatively measured by reading the OD
value of the mixture at 420 nm using the spectrophotometer. The
same mixture containing the new cell suspension that was heated
at 100 ◦ C for 5 min was used as the control and the ONP from Sigma
served as the standard. One unit of ␤-galactosidase activity was
defined as the amount of ␤-galactosidase producing 1 ␮M ONP per
min under the conditions used in this study.
2.10. Measurement of cell dry weight
The yeast cells from 10.0 mL of the culture were harvested
and washed three times with distilled water by centrifugation at
5000 × g and 4 ◦ C for 10 min. Then, the cells in the tube were dried
at 100 ◦ C until the cell dry weight was constant.
2.11. Fluorescent real-time PCR
The seed cultures were prepared as described above. One
milliliter of the seed culture (OD600nm = 3.0) was inoculated into
the lactose medium for ␤-galactosidase production and the culture
was grown aerobically at 28 ◦ C and 180 rpm for 48 h. The cultures
were centrifuged at 8000 × g and 4 ◦ C for 10 min and the pellets
obtained were used as the samples for total RNA isolation. Total
RNA was purified by a RNAprep pure Tissue Kit (TIANGEN, China).
Reverse transcription was performed using PrimeScript RT reagent
Kit (TaKaRa, Japan) according to the manufacturer’s protocol. The
fluorescent real-time RT-PCR assay was carried out according to the
methods described by Liu et al. [13]. The primers KLS and KLX were
designed according to the ␤-galactosidase gene sequence (GenBank
accession No. AY526090) (Table 1). The primers K26S and K26X
were designed according to the 26S rDNA gene sequence (GenBank
accession No. AB617981) (Table 1). The relative expression quantity
was calculated using the formula RATE = 2−DDCt . The sample data
obtained from the real-time PCR analysis were subjected to One-
way Analysis of Variance (ANOVA) [15]. P values were calculated by
Student’s t-test (n = 3). P values less than 0.05 were considered sta-
tistically significant. Statistical analysis was performed using SPSS
11.5 for Windows (SPSS Inc., Chicago, USA).
2.12. ˇ-Galactosidase production and cell growth by the
disruptant KM-15 in the 2-I fermentor
The seed cultures were prepared as described above. The fer-
mentation was carried out in a Biostat B2 2-L fermentor (B. Braun,
Germany). Fifty milliliters of the seed culture was transferred into
1500 mL of the whey powder medium. The fermentation was per-
formed under the conditions of the agitation speed of 180 rpm, the
aeration rate of 8 L/min, the temperature of 28 ◦ C, and the fermen-
tation period of 84 h. Samples were taken approximately every 12 h
to determine cell growth and ␤-galactosidase activity as described
above.
2.13. Determination of total sugar in the fermented media
The mixture containing 200 ␮L of the sample and 1.0 mL of 2 M
HCl was heated at 100 ◦ C for 30 min. After cooling, the mixture was
neutralized by adding 1.0 mL of 2 M NaOH. The total reducing sugar
in the hydrolysate was determined by the Nelson–Somogyi method
[19].
2.14. Hydrolysis of lactose
Time course of lactose hydrolysis was obtained by incubation of
the mixture containing 600 ␮L of 300 g/L of lactose solution (dis-
solved in 0.1 M phosphate buffer, pH 7.0), 500 ␮L of the treated
yeast cells (final ␤-galactosidase activity was 440 U/g of lactose),
and 900 ␮L of 0.1 M phosphate buffer, pH 7.0 at 40 ◦ C for 5 h, and
lactose concentration in the mixture was determined as described
below each 1.0 h until no more lactose content was changed. Time
course of lactose hydrolysis in the whey powder was obtained by
incubation of the mixture containing 600 ␮L of 400 g/L of the whey
powder (suspended in 0.1 M phosphate buffer, pH 7.0), 500 ␮L of
the treated yeast cells (final ␤-galactosidase activity was 450 U/g of
lactose), and 900 ␮L of 0.1 M phosphate buffer, pH 7.0 at 40 ◦ C for
4 h. Lactose in the mixture was determined using the same methods
as described below. Time course of lactose hydrolysis in the treated
milk (lactose content was 46.5 g/L) was acquired by incubation of
the mixture containing 10.0 mL of the treated milk and 12.5 mg of
the treated yeast cells (final ␤-galactosidase activity was 268.8 U/g
of lactose) at pH 7.0 and 40 ◦ C for 3 h. Lactose in the mixture was
determined using the same methods as described below.
2.15. Analysis of lactose in the whey powder and in the milk
Lactose content in the whey powder and in the milk was deter-
mined according to the methods described by Teles et al. [20].
Lactose in the standard solutions with different concentrations
(0 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 0.75 mg/mL and 1 mg/mL) of lac-
tose was also determined using the same methods as described
above, and lactose content in the whey powder was calculated from
the standard curve of lactose.
2.16. Thin layer chromatography
The end products of lactose or whey powder hydrolysis or milk
after the incubation at 40 ◦ C were withdrawn and identified to
ascertain the extent of hydrolysis by ascending thin layer chro-
matography (Silica gel 60, MERCK, Germany) with the solvent
system of butanol:absolute ethanol:water (5:3:2 by volume) and
a detection reagent comprising 20 g/L diphenylamine in acetone-
20 g/L aniline in acetone-850 g/L phosphoric acid (5:5:1 by volume).
The reaction mixtures in which the crude ␤-galactosidase was inac-
tivated prior to addition by heating at 100 ◦ C for 10 min were used
as the controls.
3. Results and discussion
3.1. Confirmation of glucose repression
The previous studies have shown that production of many extra-
cellular enzymes by yeasts was repressed by glucose available in the
medium [10]. However, it is still unknown if the ␤-galactosidase
produced by the industrial yeast K. marxianus KM is repressed
by glucose available in the medium. In order to know if glu-
cose in the medium affects ␤-galactosidase production by the
industrial yeast K. marxianus KM used in this study, effects of differ-
ent concentrations of glucose on ␤-galactosidase production were
examined. The results in Fig. 2 indicated that as the glucose concen-
trations were increased from 0 to 20 g/L, ␤-galactosidase activity
was increased continuously. However, as the glucose concentra-
tions were increased from 30 to 80 g/L, ␤-galactosidase activity
was decreased continuously. This confirmed that ␤-galactosidase
production by K. marxianus KM was indeed sensitive to high con-
centration of glucose available in the media. However, in our
previous studies [21,22], it was found that repression of invertase
 H.-X. Zhou et al. / Biochemical Engineering Journal 76 (2013) 17–24 21

4
Beta-galactosidase (U/mg) 3.5
3
2.5
2
1.5
1
0.5
0
0 2 4 6 8
Concentration of glucose (g/100ml)

Fig. 2. Effects of different concentrations of glucose on ␤-galactosidase biosynthesis

by K. marxianus KM. The yeast strain was grown in the YNB medium supplemented
with different concentrations of glucose at 28 ◦ C for three days and ␤-galactosidase
activity was determined as described in Section 2. Data are given as means ± SD,
n = 3.

secretion in Saccharomyces sp. W4 occurs at glucose concentra-

tions higher than 20 g/L while repression of invertase secretion
in Schizosaccharomyces pombe occurs at glucose concentrations
Fig. 4. Cell growth of the disruptant KM-15 on the YPD medium containing 2-deoxy-
higher than 10 g/L. d-glucose and X-gal (A1) and no growth of K. maxiaxum KM on this medium (A2);
cell growth of the disruptant KM-15 on the YPD medium containing hygromycin and
3.2. Disruption of the MIG1 gene X-gal (B1) and no growth of K. maxiaxum KM on this medium (B2). The cultivation
temperature and time were 28 ◦ C and 3 days, respectively.
The results in Fig. 2 showed that glucose repression occurred
partial sequence of 5 -end of the HPT gene (data not shown) and
in this yeast and the KmMIG1 gene encoding the transcriptional
after PCR products were amplified from the genomic DNA of the
repressor has been cloned [12]. To determine the role of KmMig1
disruptant KM-15 using the primers H6 and HY2, the PCR products
in the regulation of the ␤-galactosidase production and its gene
(585 bp) (Lane 3 in Fig. 5) contained the whole sequence of the ter-
expression in the industrial yeast K. marxianus KM used in this
minator and the partial sequence of 3 -end of the HPT gene (data
study, we disrupted the KmMIG1 gene as described in Section 2
not shown). In contrast, after PCR products were amplified from
and in Fig. 1. After determination of ␤-galactosidase activity from
the genomic DNA of K. marxianus KM using the same primers, no
different disruptants and K. marxianus KM, it was found that all
PCR products were obtained (Lanes 1 and 4 in Fig. 5). These results
the disruptants could produce more ␤-galactosidase activity than
demonstrated that the HPT gene indeed had been inserted into the
K. marxianus KM (Fig. 3). One of the disruptants could produce the
ORF so that the disruptant KM-15 obtained could grow on the plate
highest amount of ␤-galactosidase and named disruptant KM-15
with hygromycin (Fig. 4A1).
(Fig. 3). It was found that the positive disruptant KM-15 obtained
could grow on both the plate with hygromycin and that with 2-
deoxy-d-glucose (Fig. 4A1 and B1) while K. marxianus KM could 3.3. Effects of disruption of KmMIG1 gene on ˇ-galactosidase
not grow on either of them (Fig. 4A2 and B2), suggesting that the production and its genes expression
HPT gene has been expressed and glucose-derepression occurred
in the disruptant KM-15. It also can be seen from the results in As stated above, K. marxianus KM can produce high level of
Fig. 4A1 and B1 that the colonies of the disruptant KM-15 were ␤-galactosidase. Therefore, we examined the effects of KmMIG1
blue when they grew on the plates with X-gal, indicating that the
disruptant KM-15 could produce ␤-galactosidase. After PCR prod-
ucts were amplified from the genomic DNA of the disruptant KM-15
using the primers H1 and HY1, the PCR products (600 bp) (Lane 2
in Fig. 5) contained the whole sequence of the promoter and the

7
Beta-galactosidase (U/mg)

1
Fig. 5. PCR products amplified from the genomic DNAs of the disruptant KM-15 and
0 K. maxiaxum KM. Lanes M1: DNA markers (sizes of bands from top to bottom were
KM 2 6 8 9 15 35 37 44 49 56 59 67 69 74 78 2 kb. 1 kb, 0.75 kb, 0.5 kb, 0.25 kb and 0.1 kb); Lane 1: No PCR products amplified
Different yeast strains from the genomic DNA of K. maxiaxum KM using the primers H1 and HY1; Lane 2:
PCR products amplified from the genomic DNA of the disruptant KM-15 using the
Fig. 3. ␤-Galactosidase activity of the different disruptants and their parent strain K. primers H1 and HY1; Lane 3: PCR products amplified from the genomic DNA of the
marxianus KM. The medium was the lactose medium for ␤-galactosidase production disruptant KM-15 using the primers H6 and HY2; Lane 4: No PCR products amplified
and the yeast strains were aerobically cultivated at 28 ◦ C for three days. Data are from the genomic DNA of K. maxiaxum KM using the primers H6 and HY2; The DNA
given as means ± SD, n = 3. sequences of the primers H1, H6, HY1 and HY2 are shown in Table 1.
22 H.-X. Zhou et al. / Biochemical Engineering Journal 76 (2013) 17–24

8 P=0.0144
250% KM
7 KM-15

Beta-galactosidase activity (U/mg)

6 200%

Relative transcription level

5
150%
4

3 100%

2
50%
1

0 0%
KM KM-15 1
β-Galactosidase

A1 A2

Fig. 6. Effects of KmMIG1 disruption on ␤-galactosidase biosynthesis (A1) and transcript levels of ␤-galactosidase gene (A2). The medium was the lactose medium for
␤-galactosidase production and the yeast strains were grown aerobically at 28 ◦ C and 180 rpm for 48 h. Data are given as means ± SD, n = 3.

disruption in this pathway in K. marxianus KM. The results in the solid ␤-galactosidase preparation was gained (Fig. 7B). After the
Fig. 6A1 showed that the specific ␤-galactosidase activity in ␤-galactosidase activity in the lyophilized solid sample was deter-
the cells of the disruptant KM-15 reached 7.2 U per mg of cell mined as described above, it was found that the ␤-galactosidase
dry weight within 48 h of the cultivation, while the specific ␤- activity was 10,440 U/g (data not shown). As shown in other
galactosidase activity in the cells of its wild type strain KM reached research [14], K. marxianus KM, the parent strain of the disruptant
only 3.4 U per mg of cell dry weight under the same conditions. KM-15, is industrial yeast, can produce 4140 U/g of ␤-galactosidase
After ANOVA analysis, it was found that there were significant and has been widely used for ␤-galactosidase production on a large
differences (P = 0.015) of the specific ␤-galactosidase activity in scale in Chinese food industries. This meant that ␤-galactosidase
the cells of the wild type strain KM and its disruptant KM-15. yields produced by the disruptant KM-15 were much higher than
This meant that the production of the intracellular enzyme was those produced by K. marxianus KM. Therefore, the disruptant
derepressed and the ␤-galactosidase production in the cells of the KM-15 mutant obtained in this study had highly potential appli-
disruptant KM-15 was greatly enhanced after the KmMIG1 gene cation in food biotechnology and other fields. After the effect of
had been disrupted by the HPT gene. After analysis of transcript oscillating dissolved oxygen tension (DOT) on the metabolism of
level of the corresponding gene using real-time PCR, it was found K. marxianus was investigated, it was found that oscillations at
that the expression of ␤-galactosidase gene was also significantly
increased from 100% in the cells of K. marxianus KM to 205.5%
in the cells of the disruptant KM-15 (Fig. 6A2). This strongly
demonstrated that the expression of the gene encoding the intra-
cellular enzyme in the disruptant KM-15 was derepressed at the
transcript level. In our previous study [10], it also was found that
the production of all the extracellular enzymes was derepressed
after the SfMIG1 gene in the disruptant A11-c of S. fibuligera A11
had been disrupted by the HPT gene and the expression of all the
genes encoding the extracellular enzymes in the disruptant A11-c
was derepressed at the transcriptional level. This suggests that dis-
ruption of the MIG1 gene encoding the transcriptional repressor is
one way to greatly enhance enzyme production in industrial fungi.
In contrast, glucose repression of the K. lactis invertase gene KIINV1
does not require Mig1p [23]. However, after disruption of the
KmMIG1 gene, the cell growth (OD600nm value of the culture was
32) of the disruptant KM-15 was negatively influenced compared
to that (OD600nm value of the culture was 35) of its parent strain
(data not shown). The similar phenomenon (OD600nm value of the
culture of the wild type strain was 2.2 while OD600nm value of the
culture of the mig1 mutant was 1.8) also happened in the mig1
mutant of S. fibuligera A11 [13]. However, it is still unknown why
the cell growth of the disruptant KM-15 was negatively influenced.

3.4. ˇ-Galactosidase production and cell growth during the 2-l

fermentation and preparation of the solid ˇ-galactosidase

During ␤-galactosidase production and cell growth in the
2-l fermentor, the disruptant KM-15 produced 121.8 U/mL of ␤-
galactosidase activity within 60 h when cell growth (cell dry weight
was 12.3 mg/mL) reached the end of log phase (Fig. 7A) while K.
marxianus KM only produced 60.6 U/mL under the same condi-
tions (data not shown). After the washed cells were lyophilized,
Fig. 7. The time course of ␤-galactosidase production (), cell growth () and
changes in total sugar () by the disruptant KM-15 during 2-l fermentation (A)
and the solid ␤-galactosidase preparation obtained from the washed cells (B). The
medium was the whey medium for ␤-galactosidase production and the fermenta-
tion conditions were the agitation speed of 180 rpm, the aeration rate of 8 l/min,
the temperature of 28 ◦ C, and the fermentation period of 84 h. Data are given as
means ± SD, n = 3.
 H.-X. Zhou et al. / Biochemical Engineering Journal 76 (2013) 17–24 23

(A) 110
100
Hydrolysis degree (%)

90

80
70
60
50
0 1 2 3 4 5 6
Time (h)

(B) 110

100
Hydrolysis degree (%)

90
Fig. 9. Thin layer chromatography of hydrolysis products of lactose and
whey powder. 1: lactose + the active ␤-galactosidase; 2: lactose + the inacti-
80 vated ␤-galactosidase; 3: lactose; 4: glucose; 5: galactose; 6: whey powder; 7:
whey powder + the inactivated ␤-galactosidase; 8: whey powder + the active ␤-
70 galactosidase, 9: milk, 10: milk + the inactivated ␤-galactosidase, 11: milk + the
active ␤-galactosidase.
60

50
0 1 2 3 4 5 440 U/g of lactose was found to be the most suitable for lactose
Time (h) hydrolysis (data not shown). The results in Fig. 8A showed that
99.2% of lactose in 90 g/L of lactose solution was hydrolyzed within
(C) 110 3 h while the data in Fig. 8B and C revealed that 99% of lactose in
100 120 g/L of the whey powder suspension was hydrolyzed within 4 h
and 99.3% of lactose in the milk was hydrolyzed within 3 h. After
Hydrolysis degree (%)

90
80
70
60
50
40
30
0 0.5 1 1.5 2 2.5 3 3.5
Time (h)
Fig. 8. The time course of lactose hydrolysis in the lactose solution (90 g/L lactose)
(A), in the whey powder suspension (120 g/L) (B) and in the milk (C). Data are given
as means ± SD, n = 3.
the shortest period tested (i.e., 300 s) resulted in higher specific
and volumetric ␤-galactosidase activity (2800 U/g and 31.7 U/mL,
respectively) [24]. When K. marxianus CBS 6556 was grown in
concentrated cheese whey in the bioreactor, resulting in a higher ␤-
galactosidase production (11 U/mL and 900 U/g) [25]. The mutant of
G. pullulans 17-1 could produce 48.1 U/mL of total ␤-galactosidase
activity including 17.2 U/mL of extracellular ␤-galactosidase activ-
ity within 144 h in the same 2-L fermentor [26]. This demonstrated
that the disruptant KM-15 mutant obtained in this study over-
produced very high level of ␤-galactosidase activity from the whey
medium within the short period. The results in Fig. 7A also indi-
cated that when the ␤-galactosidase activity reached 121.8 U/mL,
only 12 g/L of total sugar was left in the fermented medium, indicat-
ing that most of sugar in the medium was sued for ␤-galactosidase
production and cell growth.
3.5. Hydrolysis of lactose
Our results showed that 90 g/L of lactose in the hydrolysis sys-
tem was most suitable for lactose hydrolysis (data not shown). After
effects of different amount of ␤-galactosidase on lactose hydrolysis,
permeabilization of alginate-immobilized yeast cells, the lactose
hydrolysis rate was increased and ethanol fermentation was pre-
vented, allowing 99.5% of milk whey lactose to be hydrolyzed at
30 ◦ C for 30 h [27]. Over 77.1% of lactose in the whey powder (50 g/L)
was hydrolyzed in the presence of the ␤-alactosidase activity of
280 U/g of lactose within 9 h while over 77.1% of lactose in the
whey powder (the concentration was 50 g/L) was hydrolyzed by
the ␤-galactosidase produced by the mutant NTG-133 within 9 h
[26]. It has been reported that the optimum conditions for lactose
hydrolysis in whey by the commercial lactase were lactose con-
centration of 150 g/L, temperature of 40 ◦ C, reaction time of 3 h,
and lactase dosage of 90 U/g. Under these conditions, the hydroly-
sis degree was 68% [28]. After K. marxianus MTCC 1389 was grown
in the whey containing 44 g/L lactose for 36 h, lactose concentra-
tion dropped drastically to 0.4 from 44 g/L [29]. The hydrolysis
rate of lactose in milk could reach 70% by addition of the liq-
uid lactase from the K. fragilis cells, which was disrupted by low
concentration of ␳-hydroxybenzoate [30]. Moreover, 99.5% of milk
whey lactose was hydrolyzed by a new ␤-galactosidase based bio-
catalyst consisting of whole K. lactis cells entrapped in calcium
alginate beads at 30 ◦ C for 30 h [27]. This demonstrated that the
mig1 mutant obtained in this study could efficiently hydrolyze lac-
tose and lactose in the whey powder and in the milk within very
short time.
3.6. Hydrolysis products
The hydrolysis products of lactose, whey powder and milk by the
crude ␤-galactosidase were analyzed by thin layer chromatography
(TLC). Only mono-saccharides were detected after the hydrolysis
(Fig. 9). This meant that the crude ␤-galactosidase indeed effec-
tively converted lactose and lactose in whey powder and in the
milk into glucose and galactose.
24 H.-X. Zhou et al. / Biochemical Engineering Journal 76 (2013) 17–24
4. Conclusions
After the MIG1 gene in the industrial yeast K. marxianus KM was
disrupted, the disruptant (mig1 mutant) KM-15 obtained could pro-
duce more ␤-galactosidase than K. marxianus KM and expression of
its gene in the disruptant KM-15 were significantly enhanced com-
pared to that in K. marxianus KM. Under the optimal conditions, the
␤-galactosidase activity of 121.0 U/mL was produced by the disrup-
tant KM-15 within 60 h during 2-L fermentation. 99.2% of lactose in
90 g/L of lactose solution, 99.0% of lactose in 120 g/L of whey pow-
der suspension and 99.3% of lactose in the milk were hydrolyzed
by the crude ␤-galactosidase within 3 h, 4 h and 3 h, respectively.
The end products of the lactose hydrolysis were only glucose and
galactose.
Acknowledgments
This work was supported by Grant 31070029 from National Nat-
ural Science Foundation of China, and State Oceanic Administration
People’s Republic of China for providing financial support to carry
out this work. The Grant No. 201005032.
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