Overexpression of an Acidic Endo-β-1,3-1,4-glucanase in Transgenic Maize Seed for Direct Utilization in Animal Feed
4-glucanase in Transgenic Maize Seed for Direct Utilization
in Animal Feed. PLoS ONE 8(12): e81993. doi:10.1371/journal.pone.0081993
Overexpression of an Acidic Endo-b-1,3-1,4-glucanase in Transgenic Maize Seed for Direct Utilization in Animal Feed
Yuhong Zhang 0
Xiaolu Xu 0
Xiaojin Zhou 0
Rumei Chen 0
Peilong Yang 0
Qingchang Meng 0
Kun Meng 0
Huiying Luo 0
Jianhua Yuan 0
Bin Yao 0
Wei Zhang 0
M. Lucrecia Alvarez, TGen, United States of America
0 1 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences , Beijing , P. R. China , 2 Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences , Beijing , P. R. China , 3 Institute of Food Crops, Jiangsu Academy of Agricultural Sciences , Nanjing , P. R. China
Background: Incorporation of exogenous glucanase into animal feed is common practice to remove glucan, one of the antinutritional factors, for efficient nutrition absorption. The acidic endo-b-1,3-1,4-glucanase (Bgl7A) from Bispora sp. MEY-1 has excellent properties and represents a potential enzyme supplement to animal feed. Methodology/Principal Findings: Here we successfully developed a transgenic maize producing a high level of Bgl7AM (codon modified Bgl7A) by constructing a recombinant vector driven by the embryo-specific promoter ZM-leg1A. Southern and Western blot analysis indicated the stable integration and specific expression of the transgene in maize seeds over four generations. The b-glucanase activity of the transgenic maize seeds reached up to 779,800 U/kg, about 236-fold higher than that of non-transgenic maize. The b-glucanase derived from the transgenic maize seeds had an optimal pH of 4.0 and was stable at pH 1.0-8.0, which is in agreement with the normal environment of digestive tract. Conclusion/Significance: Our study offers a transgenic maize line that could be directly used in animal feed without any glucanase production, purification and supplementation, consequently simplifying the feed enzyme processing procedure.
. These authors contributed equally to this work.
b-1,3-1,4-D-Glucans (b-glucans) are the main component of
cereal cell walls, particularly in the endosperm cell walls of barley
and other grains . It is composed of b-D-glycosyl residues linked
through irregular b-1,3 and/or b-1,4 glycosidic bonds. Ruminants
can utilize b-glucans through enzyme digestion of rumen
microbes. However, monogastric animals such as pig, poultry,
and fish do not have such enzymes to decompose the b-glucans. By
combining with water, b-glucans increase the viscosity of chyme,
block the intestinal surface partially, and prevent the mixing of
intestinal endogenous digestive juice with the chyme . Thus
bglucan represents one of the intense anti-nutritional factors in
wheat- and barley-based diets .
To overcome these problems, the most common and effective
practice is to add exogenous endoglucanases into animal feed .
Majority of endoglucanases are grouped into glycoside hydrolase
(GH) families 3, 5, 7, 12 and 16, based on the amino acid sequence
and catalytic domain structures (http://www.cazy.org/).
According to the degradation mode against glycosidic linkage,
endoglucanases have been grouped into four main categories:
b-1,3glucanase (laminarinase, EC 22.214.171.124), b-1,4-glucanases (cellulase,
EC 126.96.36.199), b-1,3-1,4-glucanases (lichenase, EC 188.8.131.52), and
b1,3(4)-glucanase (EC 184.108.40.206) . Among them,
b-1,3-1,4glucanase has received significant attention in feed industrial
applications because of their hydrolysis ability against grain-based
glucan and multiple enzymatic functions. b-1,3-1,4-Glucanase is
able to catalyze the hydrolysis of b-glucan into low molecular
weight glucose polymers, thus reducing the hydrophilicity and
viscosity of chyme and eliminating the anti-nutritional negative
effect. Moreover, addition of b-1,3-1,4-glucanase can improve
feed intake, enhance animal production, regulate cecal microbiota
and increase feed conversion ratio . Besides, the hydrolysis
products from glucansglucooligosaccharides may serve as
fermentable dietary fiber-like substrates and positively affect
gastrointestinal tract health .
To date, commercial feed additive b-1,3-1,4-glucanases are
generally from microbial expression systems, commonly Aspergillus
japonicus , Pichia pastoris  and Clostridium thermocellum .
This process is flexible and convenient, but has disadvantages like
Figure 1. Construction of the recombinant vector and the transgenic maize seed. A The recombinant expression vector pHP20754-bgl7Am.
B The chimeric gene cassettes for expression in maize. C Ears and seeds of transgenic maize (T1 and BC3) compared with that of wild-type Zheng58.
high energy consumption, high equipment cost and serious
environmental pollution. Moreover, enzyme addition is a complex
process involving enzyme isolation, purification and
supplementation, which requires more energy and resources. Thus its a good
way to produce feed enzymes (e.g. b-1,3-1,4-glucanase) in
transgenic feed grains directly without any industrial processing.
Transgenic plants are being developed for both commercial and
environmental values. In 2011, the plantation area of transgenic
plants reached about 160 million hectares worldwide and was
distributed in 29 countries; transgenic maize accounted for nearly
one third of the total genetically modified crops . Maize (Zea
mays L.) is the main ingredient of animal feed (nearly 50%), and
represents an ideal bioreactor of feed enzymes because of its
cultivation worldwide. A phytase gene phyA2 from Aspergillus niger
has been successfully overexpressed in maize seeds .
In this study, we developed a genetically stable maize line that
had high b-glucanase activity in the seeds. The
endo-b-1,3-1,4glucanase, Bgl7A, from acidophilic Bispora sp. MEY-1 was selected
due to its excellent properties as feed additive, such as acidic pH
optimum, good thermostability and broad pH stability, highly
resistance to proteases, and broad substrate specificity . The
gene codon was optimized for better expression in maize.
Materials and Methods
Maize Hi-II  was used for genetic transformation as host
variety. The immature embryos, approximately 1.02.0 mm long,
were preserved on N61-100-25 medium  containing 0.2% (w/
v) phytagel (Sigma, St. Louis, MO) for callus induction. The
commercial maize inbred-line Zheng58 was used as recurrent
parent to produce progenies.
Codon optimization of the b-1,3-1,4-glucanase gene
To improve its expression level in transgenic maize, the DNA
sequence of native endo-b-1,3-1,4-glucanase gene bgl7A from
Bispora sp. MEY-1 (Genbank accession No. FJ695140)  was
optimized according to the translationally optimal codon usage of
maize . Codon adaptation index (CAI), optimal codon usage,
GC content and distribution, effective number of codons (Nc),
negative CIS elements, negative repeat elements, and mRNA
structure were used to evaluate the gene sequence (https://www.
codons (,15% frequency) were replaced by high-usage ones
according to the known codon bias of maize . The modified
gene was named bgl7Am that encoded the same amino acid
sequence as bgl7A. The optimized gene was synthesized by
Genscript (Nanjing, China) and cloned into pUC57 vector to
construct the recombinant plasmid pUC57-bgl7Am.
The transformation vector pHP20754 (Fig. 1a) consists of the
corn legumin1A (leg1) promoter ZM-leg1A Pro, signal peptide (SP),
vacuole targeting sequence (VTS) of corn Proaleurain and the
corn leg1 terminator ZM-leg1 Term . The b-glucanase
gene was excised from pUC57-bgl7Am with BamHI and XmaI and
subcloned into pHP20754 to produce the expression construct
pHP20754-bgl7Am, which was further digested with PvuII to
generate the chimeric gene expression cassette (Fig. 1b) for
transformation. The plasmid pHP17042BAR carrying the maize
histone H2B promoter, the maize Ubiquitin 59-UTR intron-1, the
bar gene and the potato protease II (PINII) terminator  was
used as the selectable marker for screening of positive transgenic
plants. The bar gene expression cassette was excised from
pHP17042BAR by digest with HindIII, XhoI and SacI.
Maize transformation, selection and regeneration
The plasmid fragments containing the gene cassette of bgl7Am
and bar, respectively, were mixed at the ratio of 1:1 and adjusted
the concentration to 200 ng/mL. Maize transformation was
carried out with high-velocity tungsten microprojectile (Bio-Rad,
Hercules, CA) wrapped by the DNAs of bgl7Am and bar according
to the method described before . After recovery, embryonic
calli were transferred onto the selective medium supplemented
with bialaphos as the selectable marker. The positively
transformed calli were cultivated in differentiation medium and rooting
medium in succession. Seedlings (T0 plants) were transplanted into
greenhouse and pollinated with the inbred-line Zheng58 to
produce T1 seeds. Seeds were dried on the plant and harvested
3545 days after pollination. Zheng58 was used as recurrent
parent for backcrossing to produce filial generations (T1, BC1,
BC2 and BC3). b-Glucanase activity determination in the kernels
and PCR for the bgl7Am gene of seedlings were used in
combination to screen the transgenic lines.
PCR detection of exogenous gene integration
The specific primers bgl7am-875F
(59-ACGGCAAGGTCATCCAGAACGCGAAGG-39) and 20754-398R
(59TTCCTGGCAAATCACTCGGTGTATC-39) were used for
PCR detection of the positive plants harboring bgl7Am. The gene
actin as control was amplified using primers AC326F
(59ATGTTTCCTGGGATTGCCGAT-39) and AC326R
(59GCATCACAAGCCAGTTTAACC-39). Genomic DNA of the
maize immature leaves was used as PCR templates. The
recombinant plasmid pHP20754-bgl7Am and the genomic DNA
of Zheng58 were used as the positive and negative controls,
Southern blot analysis
Five grams of maize leaves of generations T1 to BC3 were
ground to powder in liquid nitrogen, and the genomic DNA was
extracted with the CTAB method. Genomic DNA of Zheng58 was
used as the negative control. About 50 mg of genomic DNA was
digested by EcoRI and HindIII and then separated on a 0.8% (w/v)
agarose gel. The agarose gel was transferred onto a hybond-N+
nylon membrane (GE Healthcare, Uppsala, Sweden) with a
Trans-Blot SD system followed by UV-crosslinking. A
digoxinlabeled probe containing a 800-bp fragment of bgl7Am was used for
southern-blot hybridization. Immunologic process was conducted
following the instructions of DIG-high prime DNA labeling and
detection starter kit II (Roche, Indianapolis, IN).
Western blot analysis
Five milligrams of lyophilized purified Bgl7A produced in Pichia
pastoris GS115  was used for the production of polyclonal
antibody in rabbits. Recombinant proteins were extracted from
seed meals. Kernels were ground with a high-throughput tissue
homogenizer Geno/Grinder 2010 (SEPX CertiPrep, Metuchen,
To extract protein from seed meals, 30 mg of seed powder were
placed into a 1.5-mL tube containing 300 mL extraction buffer
(50 mM citric acid-Na2HPO4, pH 3.5). The tube was agitated on
a shaker at room temperature for 1 h. After centrifugation at
50006 g for 10 min, the supernatant was incubated with 2-fold
volume of pre-cooled acetone for 30 min, followed by
centrifugation at 14,0006 g for 10 min. The protein precipitate was
dissolved in 30 mL of deionized water, and the protein sample was
divided into two equal parts. One was deglycosylated with
endo-bN-acetylglucosaminidase (Endo H) according to the suppliers
instructions (New England Biolabs, Ipswich, MA), the other
remained intact. Protein extract of purified Bgl7A from P. pastoris
and Zheng58 were used as the positive and negative controls,
respectively. Proteins from the stem, root and leaf of a transgenic
plant of generation BC1 were extracted in the same way and used
for tissue specificity analysis.
Proteins were separated on SDSPAGE (12% acrylamide, 0.4%
acryl-bisacrylamide). and transferred onto PVDF membrane (Pall,
Port Washington, NY). The polyclonal antibody raised in rabbits
was added into the membrane confining liquid for
prehybridization. The goat anti-rabbit IgG labeled with alkaline phosphatase
was used as the secondary antibody. BCIP/NBT kit (Zomanbio,
Beijing, China) was used for color development. To identify the
proteins, bands were excised from the gel and analyzed using
matrix assisted laser desorption/ionization time of flight mass
spectrometry (MALDI-TOF-MS) at Tianjin Biochip Corporation
b-Glucanase activity assay and enzyme characterization
Crude proteins of five randomly selected seeds were extracted
with extraction buffer as described above, and the supernatant was
subject to b-glucanase activity assay. b-Glucanase activity was
determined by measuring the amount of reducing sugar released
from lichenan with the method of 3,5-dinitrosalicylic acid (DNS)
[11,20]. One unit of enzyme activity was defined as the amount of
enzyme required to release 1 mmol of reducing sugar per minute
from 1.0% lichenan in citric acid-Na2HPO4 (50 mM, pH 3.5) at
60uC for 10 min. b-Glucanase activities of generations T1, BC1,
BC2 and BC3 of transgenic maize and Zheng58 were all
evaluated. Each reaction and its control were run in triplicate.
The enzyme properties of Bgl7AM derived from maize was
determined using crude proteins from BC1 seeds as in Luo et al.
(2010). The pH optimum of the protein was determined at 60uC
and pH 1.06.0. The pH stability was determined by measuring
the residual activity under standard conditions (pH 5.0, 60uC and
10 min) after pre-incubation at 37uC and pH 1.09.0 for 1 h. The
optimal temperature was determined at 2580uC at pH 5.0.
Thermal stability of the enzyme was determined by assessing the
residual enzyme activity under standard conditions after
incubation of the enzyme at 70uC for various durations.
Evaluation of anti-inactivation stability over feed
Feed pelleting was carried out with a twin-screw extruder
(DSE25 Extruder Lab-Station Brabender OHG, Duisburg, Germany).
Part of the maize seeds were mixed and extruded at 70uC or 80uC,
respectively. b-Glucanase activities and dry matter content (DM)
values were determined before and after pelleting. Zheng58 seeds
were treated as the non-transgenic control. Stability comparison
was conducted with the b-glucanases derived from transgenic
maize seeds and P. pastoris. Crude Bgl7A derived from P. pastoris
with equal enzyme activity to transgenic maize was added into
Zheng58 seeds, followed by pelleting treatment as described
above. And the b-glucanase activity was detected after pelleting.
One-way analysis of variance (ANOVA) was performed using the
Duncans multiple-range test to compare treatment means.
Significance was defined at P,0.05.
Construction and transformation of transgenic vector
The CAI value and GC content of bgl7A were 0.715 and 49.6%,
respectively. After codon optimization and gene modification, the
CAI value and GC content of bgl7Am was increased to 0.937 and
67.0%, respectively (Figure S1 and S2). These higher values are
better for exogenous gene expression in maize. Furthermore,
effective Nc, negative CIS elements, negative repeat elements, and
mRNA structure of the target gene were also considered in gene
modification (Figure S1, S2 and S3). As a result, native bgl7A and
synthetic bgl7Am shared 82.2% nucleotide sequence identity but
encoded identical amino acid sequences.
The 1221-bp bgl7Am was inserted into the expression vector
pHP20754 between the embryo-specific ZM-leg1A promoter and
ZM-leg1 terminator (Fig. 1b), which is a transcriptionally active
spacer region that allows highly efficient transgene expression. The
positive calli of maize Hi-II were regenerated on bialaphos
medium and identified by PCR.
Plant regeneration and phenotypic evaluation
The regenerated young plants described above showed good
growth in the greenhouse. A total of 27 independent transgenic
lines were obtained. Based on b-glucanase activities of the seeds,
330 seeds of three independent transgenic events (40, 46 and 51-1)
were selected to cultivate in fields and backcross with Zheng58 for
progeny production. As shown in Fig. 1c, the ears and seeds of
generation T1 showed significant phenotypic difference from
Zheng58. This difference was generally subsided in the later
generations because of the successive backcrossing with Zheng58.
Up to transgenic generation BC3, the traits of transgenic maize
were almost the same as that of non-transgenic Zheng58 through
visual observation. The result suggests that the inserted exogenous
gene has no negative impact on the maize seed.
Determination of exogenous gene integration
PCR assay with primers specific for bgl7Am was used to evaluate
the inheritance of transgenic maize. Gene fragments of about
500 bp were detected in the transformation events 40, 46 and 51-1
(Fig. 2a). PCR results of actin gene (,300 bp) indicated the high
quality of genomic DNA (Fig. 2b). To confirm the gene integration
and the copy number of bgl7Am in transgenic plants, the genomic
DNAs of three positive transgenic plants of event 40 were analyzed
by southern blot after restriction digest with EcoRI and HindIII. The
bgl7Am probe was prepared with a 800-bp fragment of the bgl7Am
gene. There is only one EcoRI restriction site located between the
promoter and the bgl7Am gene in the expression cassette of
pHP20754-bgl7Am. A total of three bands of ,3.5, 5.0 and 6.0 kb,
respectively, were detected in the positive lane via EcoRI digest, but
not in non-transgenic Zheng58 (Fig. 2c). There is no HindIII site in
the bgl7Am. While HindIII cut the gene expression cassette twice and
released an internal fragment of 2.5 kb (Fig. 2c). These results
indicate that there are three copies of bgl7Am in event 40.
Evaluation of site-specific expression
To determine the expression efficiency of exogenous Bgl7AM,
proteins were extracted from two BC1 plants of event 40 that had
high b-glucanase activities. In western blot analysis, no band was
detected in the negative control of Zheng58 (Fig. 3a). The positive
control, Bgl7A expressed by P. pastoris, showed a band of about
60 kDa, the same as that reported before . One main band of
,60 kDa was identified on the PVDF membrane after
hybridization with the antibody (Fig. 3a). This molecular weight (60 kDa)
was much higher than the predicted molecular weight (45.3 kDa).
After Endo H treatment, the band had no significant reduction in
molecular weight (Fig. 3a). It suggested that other post-translation
modifications rather than N-glycosylation, such as O-linked
glycosylation, phosphorylation, acetylation or methylation, may
occur in the transgenic maize. The band was verified to be
Bgl7AM through MALDI-TOF-MS analysis (Figure S4). Except
for the seeds, proteins extracted from the root, stem and leaf of the
positive lines had no objective band (Fig. 3b), indicating the tissue
specificity of Bgl7AM. Moreover, Bgl7AM present in seeds are
more convenient for storage, transportation and direct utilization.
Evaluation of seed-derived b-glucanase activity
Positive transgenic plants of transgenic event 40 were selected
for b-glucanase activity assay. Approximately 200400 seeds of
each generation were assessed using the DNS method (Table 1).
Compared with the non-transgenic Zheng58 that had b-glucanase
activity of 3300 U/kg of seeds, T1 seeds (207,800 U/kg) showed
approximately 63-fold activities of Zheng58. Both the maximal
and average activities of BC1, BC2 and BC3 seeds were increased
slightly. The maximal b-glucanase activity of BC3 seeds was up to
779,800 U/kg, which was 236 folds of that of Zheng58. About
47% of the seeds showed over 200,000 U/kg of b-glucanase
activity. The result further confirmed that bgl7Am is genetically
stable over generations in maize.
Characterization of maize seed-derived Bgl7AM
The crude proteins of transgenic BC1 seeds were characterized
(Fig. 4), and compared with Bgl7A of P. pastoris reported before
. Bgl7AM had a pH optimum at 4.0, while Bgl7A exhibited
high activity at pH 1.5, 3.5 and 5.0 (maxima). Both enzymes
remained active at pH 1.08.0. The temperature optimum of
Bgl7AM was 70uC, which was 10uC higher than that of Bgl7A.
Moreover, thermostability of Bgl7AM was improved. After
incubation at 70uC for 15 min, Bgl7AM retained 50% of the
initial activity while Bgl7A remained less than 30%.
Evaluation of anti-inactivation stability in feed pelleting
The b-glucanase activities of Bgl7A (from P. pastoris) and
Bgl7AM (from transgenic maize seeds) were determined after feed
pelleting at 70uC and 80uC, respectively (Table 2). The initial
bglucanase activities in transgenic line or in Zheng 58 by
supplementation of Bgl7A were set to 77,860 U/kg and
85,350 U/kg, respectively. After pelleting at each of the tested
temperatures, Bgl7A lost more activities than Bgl7AM. In
combination with the data of enzyme characterization, Bgl7AM
Number of seeds with b-glucanase activity (U/kg)*
*Five kernels from each ear were randomly selected, pooled, and glucanase activity assayed. One unit of enzyme activity was defined as the amount of enzyme required
to release 1 mmol of reducing sugar per minute from 1.0% lichenan at 60uC for 10 min. U/kg, glucanase units per kilogram of seed.
**The values were means of three replicates6standard deviation.
a,b,c,d,eMeans in the same column not sharing a common superscript are significantly different (P,0.05).
Figure 4. Property comparison of recombinant endo-b-1,3-1,4-glucanase expressed in maize (BGL7AM) and P. pastoris (BGL7A). A
Effect of pH on b-glucanase activity of BGL7AM and BGL7A at 60uC. B pH stability of BGL7AM and BGL7A. After incubation at 37uC for 1 h in buffers
ranging from pH 1.0 to 9.0, the b-glucanase activity was assayed in 100 mM citric acid-Na2HPO4 (pH 5.0) at 60uC. C Temperature-dependent activity
profiles of BGL7AM and BGL7A in 100 mM citric acid-Na2HPO4 (pH 5.0). D Thermostability of BGL7AM and BGL7A pre-incubated at 70uC at pH 5.0.
The aliquots were removed at different time points then measure residual b-glucanase activity at 60uC and pH 5.0. Error bars represent the standard
deviation of triplicate measurements.
b-Glucanase activity (U/kg seeds)**
*Bgl7A was the recombinant protein expressed in P. pastoris; Bgl7AM was the
recombinant protein expressed in transgenic maize seeds. The amino acid
sequences of Bgl7A and Bgl7AM are totally identical.
**The values were means of three replicates6standard deviation.
a,b,cMeans in the same column not sharing a common superscript are
significantly different (P,0.05).
was more excellent than Bgl7A even though they had complete
identical amino acid sequences.
So far several b-glucanase genes have been introduced into
plants for different purposes. Endo-b-1,3-glucanase (laminarinase)
can defend plants against fungal pathogens, introduction of its
coding genes into crops is a plausible strategy to develop durable
resistance against fungal pathogens . Over the last two
decades, transgenic plants harboring endo-b-1,4-glucanase
(cellulase) genes have taken more attention for conversion of cellulosic
biomass into fermentable sugars [22,23]. Production of
recombinant endo-b-1,4-glucanases E1 in transgenic plants have been
reported in Arabidopsis , leaf and root tissues of maize [25,26]
and rice seeds . Endo-b-1,3-1,4-glucanase (lichenase) is an
important enzyme additive in monogastric animal feed to
decompose b-glucan in cereals [3,6,28]. Up to now,
b-1,3-1,4glucanases have been expressed in transgenic barley [29,30] and
potato  for feed purpose. However, maize seed has never been
used for production of endo-b-1,3-1,4-glucanase. Here we
developed a transgenic maize line that overexpressed an
endo-b1,3-1,4-glucanase from Bispora sp. MEY-1 in seeds. Compared
with enzyme production by microbial fermentation and other
transgenic crops, transgenic maize seed has several advantages,
such as low cost production, cultivation worldwide and direct
utilization in animal feed. On the other hand, the genetic
manipulation of maize is more easily. For feed industrys interest,
maize seed as the major feed ingredient represents an ideal
bioreactor to produce feed enzymes.
About 65% of the maize seed produced in China is used as feed.
If maize seeds express sufficient endo-b-1,3-1,4-glucanase, no
supplementation of microbial glucanase will be required. To
achieve high-level expression of bgl7Am in maize seed, several
strategies have been utilized in combination, including a synthetic
gene with preferred maize codons , a strong tissue-specific
promoter, and an excellent transformation receptor with high
competence and regeneration capacity that improves the
transformation efficiency [13,14]. As a result, the average and
maximum glucanase activities in maize seeds without purification
and enrichment were up to 239,300 and 779,800 U/kg seeds,
respectively (Table 1). Previous feeding trials have shown that the
effectiveness of glucanase as a feed additive was maximized at
approximately 30,000 units per kg of diets . Typically maize
grains constitute 50% of the animal diet, thus the transgenic maize
seeds having an average glucanase activity of 239,300 U/kg is
high enough to substitute the glucanase supplement. When the
transgenic maize line developed in this study is propagated in field,
it will enhance the nutritive values of glucan-abundant grains such
as wheat and barley. The development of transgenic maize will not
only reduce the loss of resources and simplify the production
process, but also provide an environmental friendly approach to
Moreover, Bgl7AM has good thermostability and excellent
acidic stability, which are important factors for supplementation to
animal feed. Thermostability is a key index of feed enzyme
because of the high temperature during feed processing. Since
most b-1,3-1,4-glucanases are not stable during coating of feed
pellets (7090uC), selection of a thermostable b-1,3-1,4-glucanase
with high activity is of great interest to the animal feedstuff
industries [8,32,33]. Bgl7AM retains most activities after pelleting
at 80uC. This thermostability allows it to survive the heat
generated from maize pressing into feed pellets and pasteurization.
Similar results that plant-derived enzymes showed better stability
have been reported. [34,35]. This phenomenon might be ascribed
to the different folding patterns and disulphide bond formations in
microbes and plants . Protection from maize seed starch might
be the other cause.
Furthermore, feed enzyme should be stable within the acid
environment of monogastric animals digestive tract, in which the
pH value is lower than pH 3.0 in stomach (pH 1.33.5 for pigs
and pH 2.84.8 for chickens) . An acidic-tolerable b-glucanase
has been isolated from Trichoderma koningii ZJU-T, with optimal
activity at pH 2.0 . Molecular modification approaches have
been employed to enhance the activity of a b-1,3-1,4-glucanase at
acidic pH . Compared with counterparts, Bgl7AM is highly
active and stable within pH 1.04.0, and retains above 90% of its
activity at pH 3.0, the average pH in the animal digestive tract.
This study provides an environment-friendly and low-cost
approach to produce transgenic maize with social and ecological
significance. Its the first report that produces a biologically active
endo-b-1,3-1,4-glucanase in transgenic maize seeds. Approaches
to increase the seed glucanase activities are preceding, including
selection of more transgenic events and application of stronger
promoters. In the future studies, well evaluate its direct
application effectiveness in animal feed by comparison with
traditional feed supplemented with glucanases.
Figure S1 Codon related parameters of wild-type gene
bgl7A and optimized bgl7Am. A Codon adaptation index
(CAI), negative CIS elements, and negative repeat elements of the
bgl7A and bgl7Am. B effective number of codons (Nc) of the bgl7A
Figure S2 Codon usage and GC content of wild-type
gene bgl7A and optimized bgl7Am. A Relative codon
frequency of bgl7A. B Relative codon frequency of bgl7Am. C
GC content and distribution of bgl7A. D GC content and
distribution of bgl7Am. E Percentage of high frequency used
codons of maize in bgl7A. F Percentage of high frequency used
codons of maize in bgl7Am.
Conceived and designed the experiments: PLY BY WZ. Performed the
experiments: XLX YHZ QCM. Analyzed the data: RMC JHY.
Contributed reagents/materials/analysis tools: XJZ KM HYL. Wrote
the paper: YHZ.
1. Buliga GS , Brant DA , Fincher GB ( 1986 ) The sequence statistics and solution conformation of a barley (1,3-1,4)-b-D-glucan . Carbohyd Res 157 : 139 - 156 .
2. Almirall M , Francesch M , Perez-Vendrell AM , Brufau J , Esteve-Garcia E ( 1995 ) The differences in intestinal viscosity produced by barley and b-glucanase alter digesta enzyme activities and ileal nutrient digestibilities more in broiler chicks than in cocks . J Nutr 125 : 947 - 955 .
3. Choct M ( 2006 ) Enzymes for the feed industry: past, present and future . World Poultry Sci J 62 : 5 - 16 .
4. McCarthy T , Hanniffy O , Savage AV , Tuohy MG ( 2003 ) Catalytic properties and mode of action of three endo-b-glucanases from Talaromyces emersonii on soluble b- 1 , 4 - and b- 1 , 3 - 1 , 4 - linked glucans. Int J Biol Macromol 33 : 141 - 148 .
5. Mathlouthi N , Mallet S , Saulnier L , Quemener B , Larbier M ( 2002 ) Effects of xylanase and b-glucanase addition on performance, nutrient digestibility, and physico-chemical conditions in the small intestine contents and caecal microflora of broiler chickens fed a wheat and barley-based diet . Anim Res 51 : 395 - 406 .
6. Kiarie E , Owusu-Asiedu A , Peron A , Simmins PH , Nyachoti CM ( 2012 ) Efficacy of xylanase and b-glucanase blend in mixed grains and grain coproducts-based diets for fattening pigs . Livest Sci 148 : 129 - 133 .
7. Jozefiak D , Rutkowski A , Kaczmarek S , Jensen BB , Engberg RM , et al. ( 2010 ) Effect of b-glucanase and xylanase supplementation of barley- and rye-based diets on caecal microbiota of broiler chickens . Brit Poultry Sci 51 : 546 - 557 .
8. Ribeiro T , Lordelo MMS , Prates JAM , Falcao L , Freire JPB , et al. ( 2012 ) The thermostable b- 1 , 3 - 1 , 4 - glucanase from Clostridium thermocellum improves the nutritive value of highly viscous barley-based diets for broilers . Brit Poultry Sci 53 : 224 - 234 .
9. Flickinger EA , Wolf BW , Garleb KA , Chow J , Leyer GJ , et al. ( 2000 ) Glucosebased oligosaccharides exhibit different in vitro fermentation patterns and affect in vivo apparent nutrient digestibility and microbial populations in dogs . J Nutr 130 : 1267 - 1273 .
10. Grishutin S , Gusakov A , Dzedzyulya E , Sinitsyn A ( 2006 ) A lichenase-like family 12 endo-(1, 4)-b-glucanase from Aspergillus japonicus: study of the substrate specificity and mode of action on b-glucans in comparison with other glycoside hydrolases . Carbohyd Res 341 : 218 - 229 .
11. Luo HY , Yang J , Yang PL , Li J , Huang HQ , et al. ( 2010 ) Gene cloning and expression of a new acidic family 7 endo-b - 1 , 3 - 1 , 4 - glucanase from the acidophilic fungus Bispora sp . MEY-1. Appl Microbiol Biot 85 : 1015 - 1023 .
12. James C ( 2011 ) Global status of commercialized biotech/GM crops: 2011 . ISAAA Brief No . 43. Ithaca, NY.
13. Chen RM , Xue GX , Chen P , Yao B , Yang WZ , et al. ( 2008 ) Transgenic maize plants expressing a fungal phytase gene . Transgenic Res 17 : 633 - 643 .
14. Armstrong C , Green C , Phillips R ( 1991 ) Development and availability of germplasm with high type II culture formation response . Maize Genet Coop Newslett 65 : 92 - 93 .
15. Liu HM , He R , Zhang HY , Huang YB , Tian ML , et al. ( 2010 ) Analysis of synonymous codon usage in Zea mays . Mol Biol Rep 37 : 677 - 684 .
16. Holwerda BC , Padgett HS , Rogers JC ( 1992 ) Proaleurain vacuolar targeting is mediated by short contiguous peptide interactions . Plant Cell 4 : 307 - 318 .
17. Woo YM , Hu DWN , Larkins BA , Jung R ( 2001 ) Genomics analysis of genes expressed in maize endosperm identifies novel seed proteins and clarifies patterns of zein gene expression . Plant Cell 13 : 2297 - 2317 .
18. Yamagata T , Kato H , Kuroda S , Abe S , Davies E ( 2003 ) Uncleaved legumin in developing maize endosperm: identification, accumulation and putative subcellular localization . J Exp Bot 54 : 913 - 922 .
19. Tomes D ( 1995 ) Direct DNA transfer into plant cell via microprojectile bombardment . In: Gamborg O, Philipps G, editors. Plant cell tissue and organ culture: fundamental methods . Berlin: Springer-Verlag. pp. 197 - 213 .
20. Miller GL ( 1959 ) Use of dinitrosalicylic acid reagent for determination of reducing sugar . Anal Chem 31 : 426 - 428 .
21. Sridevi G , Parameswari C , Sabapathi N , Raghupathy V , Veluthambi K ( 2008 ) Combined expression of chitinase and b-1,3-glucanase genes in indica rice (Oryza sativa L.) enhances resistance against Rhizoctonia solani . Plant Sci 175 : 283 - 290 .
22. Taylor LE , Dai Z , Decker SR , Brunecky R , Adney WS , et al. ( 2008 ) Heterologous expression of glycosyl hydrolases in planta: a new departure for biofuels . Trends Biotechnol 26 : 413 - 424 .
23. Venkatesh B , Dale B , Ahmad R , Ransom C , Oehmke J , et al. ( 2007 ) Enhanced conversion of plant biomass into glucose using transgenic rice-produced endoglucanase for cellulosic ethanol . Transgenic Res 16 : 739 - 749 .
24. Ziegler MT , Thomas SR , Danna KJ ( 2000 ) Accumulation of a thermostable endo-1,4-b-D-glucanase in the apoplast of Arabidopsis thaliana leaves . Mol Breeding 6 : 37 - 46 .
25. Biswas GCG , Ransom C , Sticklen M ( 2006 ) Expression of biologically active Acidothermus cellulolyticus endoglucanase in transgenic maize plants . Plant Sci 171 : 617 - 623 .
26. Ransom C , Balan V , Biswas G , Dale B , Crockett E , et al. ( 2007 ) Heterologous Acidothermus cellulolyticus 1,4-b-endoglucanase E1 produced within the corn biomass converts corn stover into glucose . Appl Biochem Biotech 136-140 : 207 - 219 .
27. Zhang Q , Zhang W , Lin CY , Xu XL , Shen ZC ( 2012 ) Expression of an Acidothermus cellulolyticus endoglucanase in transgenic rice seeds . Protein Expres Purif 82 : 279 - 283 .
28. von Wettstein D , Mikhaylenko G , Froseth JA , Kannangara CG ( 2000 ) Improved barley broiler feed with transgenic malt containing heat -stable ( 1 , 3 - 1 , 4 ) -bglucanase . Proc Natl Acad Sci 97 : 13512 - 13517 .
29. Horvath H , Huang J , Wong O , Kohl E , Okita T , et al. ( 2000 ) The production of recombinant proteins in transgenic barley grains . Proc Natl Acad Sci 97 : 1914 - 1919 .
30. Jensen LG , Olsen O , Kops O , Wolf N , Thomsen KK , et al. ( 1996 ) Transgenic barley expressing a protein-engineered , thermostable ( 1 , 3 - 1 , 4 ) -b-glucanase during germination . Proc Natl Acad Sci 93 : 3487 - 3491 .
31. Armstrong JD , Inglis GD , Kawchuk LM , McAllister TA , Leggett F , et al. ( 2002 ) Expression of a Fibrobacter succinogenes 1,3-1,4-b-glucanase in potato (Solanum tuberosum) . Am J Potato Res 79 : 39 - 48 .
32. Hua CW , Yan QJ , Jiang ZQ , Li YN , Katrolia P ( 2010 ) High-level expression of a specific b-1,3-1,4-glucanase from the thermophilic fungus Paecilomyces thermophila in Pichia pastoris . Appl Microbiol Biot 88 : 509 - 518 .
33. Wang JL , Ruan H , Zhang HF , Zhang Q , Zhang HB , et al. ( 2007 ) Characterization of a thermostable and acidic-tolerable b-glucanase from aerobic fungi Trichoderma koningii ZJU-T . J Food Sci 72 : C452 - C456 .
34. Agrawal P , Verma D , Daniell H ( 2011 ) Expression of Trichoderma reesei bmannanase in tobacco chloroplasts and its utilization in lignocellulosic woody biomass hydrolysis . PloS one 6: e29302.
35. Verma D , Kanagaraj A , Jin S , Singh ND , Kolattukudy PE , et al. ( 2010 ) Chloroplast-derived enzyme cocktails hydrolyse lignocellulosic biomass and release fermentable sugars . Plant Biotechnol J 8 : 332 - 350 .
36. Deng F , Wang J , Pu D , Liu K , Zhou L , et al. ( 2009 ) Advance in assessment of direct-fed microorganism . Chin Agric Sci Bull 25 : 7 - 12 .
37. Jia HY , Li YN , Liu YC , Yan QJ , Yang SQ , et al. ( 2012 ) Engineering a thermostable b- 1 , 3 - 1 , 4 - glucanase from Paecilomyces thermophila to improve catalytic efficiency at acidic pH . J Biotechnol 159 : 50 - 55 .