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Thermostable Recombinant β‑(1→4)-Mannanase from C.
thermocellum: Biochemical Characterization and Manno-
Oligosaccharides Production
Arabinda Ghosh,
†
Ana SofiaLuís,
‡
Joana L. A. Brá
s,
‡
Carlos M. G. A. Fontes,
‡
and Arun Goyal*
,†
†
Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati-781 039, Assam, India
‡
CIISA-Faculdade de Medicina Veterinaria, Avenida da Universidade Té
cnica, 1300-477 Lisbon, Portugal
*
SSupporting Information
ABSTRACT: Functional attributes of a thermostable β-(1→4)-mannanase were investigated from Clostridium thermocellum
ATCC 27405. Its sequence comparison the exhibited highest similarity with Man26B of C. thermocellum F1. The full length
CtManf and truncated CtManT were cloned in the pET28a(+) vector and expressed in E. coli BL21(DE3) cells, exhibiting 53
kDa and 38 kDa proteins, respectively. On the basis of the substrate specificity and hydrolyzed product profile, CtManf and
CtManT were classified as β-(1→4)-mannanase. A 1.5 fold higher activity of both enzymes was observed by Ca2+ and Mg2+ salts.
Plausible mannanase activity of CtManf was revealed by the classical hydrolysis pattern of carob galactomannan and the release of
manno-oligosaccharides. Notably highest protein concentrations of CtManf and CtManT were achieved in tryptone yeast extract
(TY) medium, as compared with other defined media. Both CtManf and CtManT displayed stability at 60 and 50 °C,
respectively, and Ca2+ ions imparted higher thermostability, resisting their melting up to 100 °C.
KEYWORDS: Man26B, carob galactomannan, manno-oligosaccharides, thermostability
■INTRODUCTION
Plant cell wall is mainly composed of complex structural
polysaccharides like celluloses and hemicelluloses. Polysacchar-
ides of the primary cell wall are cellulose, hemicelluloses such as
xyloglucans, mannans, galactomannans, glucomannans, lami-
narin, glucuronoarabinoxylans, and arabinoxylan, etc. Mannans
are the polysaccharides with a backbone chain of β-(1 →4)-
linked mannose units. They constitute a major portion of
hemicelluloses in hardwoods. The major distribution of
mannan in combination with galactose and glucose units in
plant hemicellulose reservoir is abundant in nature. Carob
galactomannan (from the Ceratonia siliqua plant) contain β-
(1→4)-D-mannan backbone (78%) and galactose as α-(1→6)-
linked (22%) single units, where as guar gum (from endosperm
of guar seeds) backbone is a linear chain of β-(1→4)-linked
mannose residues to which galactose residues are (1→6)-linked
at every second mannose, forming short side-branches.
1
Glucomannan (from Amorphophallus konjac) is a water-soluble
polysaccharide that is considered a dietary fiber. The
component sugars in konjac glucomannan are β-(1→4)-linked
D-mannose and D-glucose residues in a molar ratio of 1.6:1 and
branched chain composed of β-(1→6)-linked D-glucosyl units.
2
β-D-Mannanase [endo β-(1→4)-mannan mannohydrolase,
E.C. 3.2.1.78] hydrolyzes β-(1→4)-D-mannopyranosyl linkages
within the main chain of mannans, glucomannans, galacto-
mannans, and galactoglucomannans.
3
Mannanases have been
listed within glycoside hydrolase (GH) families viz. GH26,
GH5, and GH113 in carbohydrate-active enzyme database
(http://www.cazy.org/Glycoside-Hydrolases.html) based on
sequence similarity.
4
β-D-Mannanases belong to families GH5,
GH26, GH113 and display a (β/α)8barrel-shaped protein
folding pattern, and the acid−base-assisted catalysis via a
double displacement mechanism involving a covalent glycosyl-
enzyme intermediate.
5,6
The mechanism of glycosidic bond
cleavage is found conserved within these families. These are the
characteristic patterns of clan GH-A protein families and helped
β-D-mannanases of families GH5, GH26, and GH113 to group
into this clan.
5,6
Due to the retaining double-displacement
mechanism, these enzymes can perform transglycosylation.
Transglycosylation may lead to the synthesis of new glycosides
or oligosaccharides longer than the original substrate. GH5 and
GH113 mannanases have been described as able to catalyze
transglycosylation reactions,
5,6
while to date no evidence of
transglycosylation has been reported for GH26 mannanases.
7
The benefit of employing novel enzymes for specific
industrial processes is well-recognized with the discovery of
β-mannanases. β-Mannanases (EC 3.2.1.78) hydrolyze man-
nan-based hemicelluloses and liberate short β-(1→4)-manno-
oligosaccharides, which can be further hydrolyzed to mannose
by β-mannosidases (EC 3.2.1.25). There are currently around
50 β-mannanase gene sequences in families 5 and 26 GHs from
various microbial origins. The family 26 Glycoside Hydrolase
(GH26) mannanase has narrow substrate specificity hydro-
lyzing (1→4)-β-D-linkages in mannans, galacto-mannans,
glucomannans, and galactoglucomannans but does not show
activity against β-glycan chain of soluble cellulose derivatives.
8
Several studies exhibited the presence of distinct types of
mannanases (GH5A, GH5B, GH5C, GH26A, GH26B, and
GH26C) expressed on the cell surface of Cellvibrio japonicas
9
Received: July 16, 2013
Revised: November 13, 2013
Accepted: November 13, 2013
Published: November 13, 2013
Article
pubs.acs.org/JAFC
© 2013 American Chemical Society 12333 dx.doi.org/10.1021/jf403111g |J. Agric. Food Chem. 2013, 61, 12333−12344
having different substrate specificity. GH5 mannanase exhibits
some activity for cellulosic substrates.
10
By contrast, Man26B
displays canonical endomannanase activity and linked to the
cell membrane via ∼70residuelinkersequenceofC.
japonicas.
10
Thus, Man26B gets enough space via the linker
sequence to adsorb on the natural substrates galactomannan
and glucomannan than other surface-expressed mannanases.
Man26B rapidly generates large amounts of mannose, even in
the early stages of galactomannan or manno-oligosaccharide
hydrolysis. But Man26A displayed a typical endo-β-(1→4)-
bond cleavage activity against small manno-oligosaccharides,
hydrolyzing mannotriose approximately, 10000 times more
efficiently than Man26B.
10
There are distinct differences in
topology of the substrate-binding cleft and substrate specificity
among the mannnases (Man26A, Man26B, and Man26C)
within GH26 family.
5
There are several reports of manno-oligosaccharides syn-
thesis by the utilization of mannanases from manno-configured
substrates.
10,11
Though the manno-oligosaccharides are indi-
gestible inside the human gut, their potential role as dietary
fiber and prebiotics were attributed in various studies.
12
It was
evident from earlier reports that the efficient prebiotics role of
manno-oligosaccharides that supports the growth of human
intestinal beneficial microflora viz. Bifidobacteria and Lactoba-
cilli.
13,14
In addition, manno-oligosaccharides can prevent the
probability of high blood pressure and higher intestinal
absorption of fatty acid substance from a high fat diet.
15
Clostridium thermocellum expresses a large number of
hemicellulases in its multienzyme complex, targeting various
hemicellulosic components such as mannans and xylans,
removes the hemicellulosic polysaccharides exposing the
cellulose microfibrils and uses it as primary carbon and energy
sources and releases soluble sugars.
16
The carbohydrate binding
modules (CBMs) are the noncatalytic modules known to help
or bring the catalytic modules in close proximity to its
substrates, and also some CBMs are known to stabilize the
enzyme (catalytic module) structure and increase its thermo-
stability.
17,18
The majority of C. thermocellum cellulosomal
enzymes display rather complex multimodular architectures
containing CBMs either at the N- or C- terminal domain.
These CBMs potentiate the interaction of the multifunctional
complex with the diversity of polysaccharides in the plant cell
wall by directing the appended catalytic domains to their target
substrates.
16
The CBMs may be found to contain up to 200
amino acids and can be found attached as single, double, or
triple domains in one protein, located at both the C- or N-
terminal within the parental protein.
19
A family 35 carbohy-
drate binding module (CBM35) often appended with
mannanase that binds to the galactose-decorated mannanas
and facilitates their efficient hydrolysis.
20
There are few reports
of Man26B functions explored earlier from Paenibacillus sp.
BME14,
21
C. japonicas,
22
C. thermocellum strain F1,
23
and
Bacillus licheniformis DSM13.
24
In the present report, we
studied the molecular and biochemical characterization of
family 26 glycoside hydrolase (GH26) mannanase B (Man26B)
from C. thermocellum ATCC 27405. Its potential in manno-
oligosaccharide production by the hydrolysis of the manno-
configured substrate was analyzed.
■MATERIALS AND METHODS
Bacterial Strains and Plasmid. The genomic DNA of C.
thermocellum ATCC 27405 was a gift from Professor Carlos Fontes,
Faculdade de Medicina Veterinaria, Lisbon, Portugal. Escherichia coli
DH5αcells were used for cloning, and E. coli BL21 (DE3) was used as
the expression host. The plasmids used for cloning and expression
were pET-28a (+). All the above-mentioned items were procured from
Novagen (Madison, WI).
Fine Chemicals and Natural and Synthetic Substrates for
Enzyme Assay. Mannose, xylose, glucose, galactose, EDTA, and
NaOH solution (50%, w/v), lichenan (from Cetraria islandica) were
procured from Sigma Chemical Company (St. Louis, MO). Carob
galactomannan, konjac glucomannan, locust bean galactomannan, guar
galactomannan, ivory nut mannan, β-(1→4)-mannnan, barley β-
glucan, rye arabinoxylan, xyloglucan, mannobiose, and mannotriose
were procured from Megazyme International, Ireland. Carboxy
methylcellulose (CMC), hydroxyethyl cellulose, Avicel (microcrystal-
line cellulose) and synthetic pNP-glycosides like pNP-β-mannopyrano-
side, pNP-α-mannopyranoside, glucuronoxylan, and polygalactouronic
acid were purchased from Sigma Chemical Company.
Sequence Analysis. Two mannanase genes (locus name:
Cthe_0032 and Cthe_2811) (http://www.cazy.org/GH26_bacteria.
html) belonging to family 26 glycoside hydrolase (GH26), were
identified in the native host C. thermocellum ATCC 27405 (16S rDNA
sequence ID: CP000568, http://www.straininfo.net/strainPassport.
action?page=34&cultureId=40680). Mannanase encoding ORF region
was identified using the protein sequence (gene bank protein accession
ABN51273.1, locus name: Cthe_0032) of C. thermocellum ATCC
27405 in tBLASTn (http://blast.ncbi.nlm.nih.gov/Blast.cgi) tool. This
sequence was used to design the desired oligonucleotide primers for
amplification of full length CtManf (CBM35-CtManT) and truncated
catalytic module CtManT (nucleotide accession: CP000568.1). The
sequence for amplification was devoid of signal peptide and dockerin
(Doc I). The protein sequence of CtManT was analyzed for the type
of enzyme synthesized using multiple sequence alignment using
MULTALIN (http://multalin.toulouse.inra.fr/multalin/cgi-bin/
multalin.pl) and viewed in the ESPript (http://espript.ibcp.fr/
ESPript/ESPript/) tool. Multiple sequence alignment was performed
using the different types of mannanase (Man26A and Man26B)
sequences of glycoside hydrolase family 26 (GH26) from Paenibacillus
sp BME14,
21
Cellvibrio japonicas,
22
Clostridium thermocellum strain
F1,
23
Bacillus licheniformis DSM13,
24
Cellulomonas fimi,
25
Rhodother-
mus marinus,
26
Bacillus sp JAMB750,
27
Paenibacillus polymyxa GS01.
28
Evaluation of the functional property of this protein sequence was
performed using InterProScan (http://www.ebi.ac.uk/Tools/pfa/
iprscan/), and the molecular architecture of the entire protein
sequence to be cloned was drawn.
Gene Amplification and Cloning. The ORF region encoding full
length CtManf (CBM35-CtManT) containing family 35 carbohydrate
binding module (nucleotide accession: CP000568.1) at the N-terminal
and the family 26 glycoside hydrolase (GH26), a mannanase B
(Man26B) catalytic module, CtManT (nucleotide accession:
CP000568.1) were amplified from the genomic DNA of C.
thermocellum ATCC 27405, using two oligonucleotide primers having
NheI and XhoI restriction sites. The 50 μL PCR reaction mixture
contained Mg2+ ions (2.5 mM), dNTPs (0.2 mM), primers (1.5 μM),
1.0 μL of Taq DNA polymerase (1 μL of 1 Unit/μL), and 1 μLof
genomic DNA (10 ng) of C. thermocellum ATCC 27405 and PCR-
grade water (Sigma Chemical Company). The oligonuclecotide
primers used for amplifying CtManT were forward 5′-cacgctagcgca-
tattcccttcctg-3′and reverse 5′-cacctcgagttagctaaagtatattttg-3′.The
oligonuclecotide primers for CtManf used were: forward 5′-cacgctagc-
gcatattcccttcctg-3′and 5′-cacctcgagttaaagttcatccaagctgc-3′. The PCR
amplification cycles used were denaturation at 94 °C for 4 min
followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 55
°C for 60 s and extension at 72 °C for 2 min, and final extension at 72
°C for 10 min. The amplified products were run on 0.8% agarose gel
and purified by a gel extraction kit (Qiagen). The PCR-amplified
CtManf and catalytic CtManT were cloned into NheI/XhoI digested
pET-28a (+) expression vector containing kanamycin as a resistant
marker, resulting in cloned plasmids pManfand pManGH26,
respectively. The E. coli DH5αcells were transformed with above
recombinant plasmids. These transformed cells were grown on LB
agar plates,
29
supplemented with kanamycin (50 μgmL
−1)at37°C
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf403111g |J. Agric. Food Chem. 2013, 61, 12333−1234412334
for growth of recombinant clones. The positive clones were selected
by restriction digestion analysis of the recombinant plasmids.
Expression and Purification of CtManf and CtManT. E. coli
BL-21(DE3) (Novagen) cells were transformed for expression of
CtManf and CtManT as described elsewhere.
29
The cells were grown
in LB medium containing kanamycin (50 μgmL
−1)at37°C with 180
rpm to the midexponential phase (A600 nm ≈0.6). Then the cells were
induced with 1.0 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG)
and incubated at 24 °C with 180 rpm for 24 h for the hyper-expression
of recombinant proteins. The cells were harvested at 9000g, and the
resulting pellet was resuspended in 50 mM sodium phosphate buffer
pH 7.0, containing 1 mM phenylmethanesulfonyl fluoride (PMSF).
Then the cells were sonicated (Sonics, Vibra cell) on ice for 16 min (9
s on/9 s offpulse, 30% amplitude) and again centrifuged using the
centrifuge (Sigma, 4K15) at 19000gat 4 °C for 20 min. The cell free
supernatant containing the soluble protein was purified by
immobilized metal ion chromatography (IMAC).
29
The recombinant
proteins containing CtManf and CtManT appended by the His6tag
were purified in a single step using 1 mL HiTrap chelating columns
(GE Healthcare) as recommended by the manufacturer. The purity
and molecular mass of recombinant CtManf and CtManT were verified
by SDS−PAGE.
30
Enzyme Assays of CtManf and CtMant with Natural and
Synthetic Substrates. The enzyme activity of CtManf and CtManT
was determined by using natural substrates, such as carob
galactomannan, locust bean galactomannan, konjac glucomannan,
guar galactomannan, ivory nut mannan, β-(1→4)-mannan (an
insoluble polysaccharide prepared from carob galactomannan pre-
treated with Aspergillus niger mannanase and subsequently debranched
to a high extent containing mannose (97%) and galactose (3%),
according to the manufacturer Megazyme International, Ireland),
barley β-glucan, lichenan, carboxymethyl cellulose, hydroxyethyl
cellulose, Avicel, rye arabinoxylan, glucuronoxylan, arabinogalactan,
and polygalactouronic acid (PGA) at (1%, w v−1) in 50 mM sodium
phosphate buffer (pH 7.0) and by measuring the reducing sugar
released, as described previously.
31,32
In both cases, 100 μL of reaction
mixture contained 1.0 (%, w v−1) substrate, 10 μL of enzyme (CtManf,
0.16 mg mL−1and CtManT, 0.15 mg mL−1). One-hundred microliter
reaction mixtures for both the enzymes were incubated at 50 °C for 10
min separately with separate polysaccharides. In the case of Avicel, the
reaction mixture was incubated for 60 min under shaking conditions.
The resulting reducing sugars viz. mannose, xylose, glucose, and
galactose concentrations were measured by the absorbance at A500nm,
using a spectrophotometer (Varian, Cary 100 Bio), and standard
curves were prepared from standard mannose, xylose, glucose, and
galactose (Sigma Chemical Company). The assays were carried out in
triplicate.
A wide range of pH was chosen, ranging from pH 3.0 to 8.0 as β-
mannanse displays optimum range within this range.
33
For studying
the optimum pH profile of CtManf and CtManT, 50 mM sodium
phosphate buffer, pH 3.0−8.0, was used for the enzyme assays that
employed 1.0 (%, w v−1) carob galactomannan at 50 °C. The optimum
temperature study was carried out within the range from 10 to 100 °C
at their respective optimum pH, using 1.0 (%, w v−1)carob
galactomannan.
After the verification of substrate specificity, the enzyme assays for
CtManf and CtManT were performed using 50 mM sodium phosphate
buffer at their optimum pH and temperature to analyze the kinetic
parameters viz. Km,Vmax,k
cat, and kcat/Km. One unit of enzyme activity
was determined as the release of 1 μmole of mannose per minute. The
assays involving synthetic substrates pNP-β-D-mannopyranoside and
pNP-α-D-mannopyranoside were performed as reported earlier by Bey
et al. (2011).
34
Zymogram Study and Activity Staining. Zymogram study of
recombinant CtManf and CtManT were investigated by using 0.5% (w
v−1) carob galactomannan as the substrate incorporated in 12% (w
v−1) SDS−PAGE. Ten micrograms of each of purified CtManf and
CtManT by IMAC was mixed with 1×the sample buffer (62.5 mM
Tris-Cl, pH 6.8, 20% v v−1glycerol, 2% w v−1SDS, and 0.005% w v−1
bromophenol blue)
30
without β-mercaptoethanol
35
were loaded on
the gel. After the completion of electrophoresis, the gels were
incubated in 2.5% (v v−1) of TritonX 100 at 25 °C for 1 h followed by
1 h incubation in 50 mM sodium phosphate buffer, pH 7.0. Then the
gels were incubated in preheated 50 mM sodium phosphate buffer
(pH 6.5) at 55 °C for 30 min and then stained with 0.1% (w v−1)
congo red for 45 min, as described by Aboul-Enein et al. (2010).
35
After congo red staining, the gels were counter-stained with 1 N HCl,
as described elsewhere.
36
Effect of Metal Ions, Chaotropic Agents, And Detergent on
Enzyme Activity. The effects of different metal cations, chaotropic
agents, and detergent on the activity of CtManf and CtManT were
determined. The enzyme activity of both CtManf and CtManT was
determined in the presence of various metal salts, such as Ni2+ (NiSO4·
6H2O), Zn2+ (ZnSO4·7H2O), Cu2+ (CuSO4·5H2O), Co2+ (CoCl2·
6H2O), Mn2+ (MnCl2·4H2O), Al3+ (AlCl3·6H2O), or Ca2+ (CaCl2·
2H2O), chaotropic agents like disodium EDTA, EGTA, urea, or
guanidine hydrochloride and detergent such as SDS. The assays of
CtManf and CtManT were performed at 60 and 50 °C, respectively,
using 50 mM sodium phosphate buffer, of pH 6.9 and pH 6.5,
respectively. One-hundred microliters of the reaction mixture
containing carob galactomannan (1%, w v−1) and metal salt at
concentrations (up to 80 mM) or SDS (up to 20 mM) were incubated
for 10 min, and a control sample in the absence of the additive was
also run. The assays were performed in triplicates. Both enzymes were
incubated with EDTA and urea for 1 h, before measuring the residual
activity. The enzyme activity was determined, as described earlier.
Thin-Layer Chromatography of Hydrolyzed Products by
CtManf. The qualitative analysis of hydrolyzed products by the
reaction of CtManf on carob galactomannan was performed by thin-
layer chromatography (TLC) on silica gel-coated aluminum foil (TLC
Silica gel 60 F254 20 ×20 cm, Merck) for detecting sugars. The enzyme
CtManf (10 μL and 0.16 mg mL−1) with 1% (w v−1) carob
galactomannan in 100 μL reaction mixtures were incubated at
optimized temperature 60 °C and optimized pH 6.9, for time intervals
of 1, 4, 8, 16, and 24 h. The reaction products were boiled for 2 min to
stop enzymatic hydrolysis and then centrifuged at 13000gfor 5 min.
6
Then 0.2 μL of sample as well as of standard solutions (1.0 mg mL−1)
were loaded on the TLC plate and kept in the developing chamber
saturated with the developing solution (mobile phase), which
consisted of acetic acid−n-propanol−water−acetonitrile
(4:10:11:14).
37
Mannose and oligosaccharides (mannobiose and
mannotriose) were used as standards. At the end of the run, migrated
sugars were visualized by immersing the TLC plate in a visualizing
solution (sulphuric acid/methanol 5:95, v v−1;α-napthol 5.0%, w v−1).
The TLC plates were then dried at 80 °C for 20 min. The migrated
reaction products (sugars) appeared as spots on the TLC plate.
HPAEC Analysis of Polysaccharide Hydrolysis by CtManf.
CtManf (10 μL and 0.16 mg mL−1) with 1% (w v−1) carob
galactomannan in 100 μL of reaction mixtures were incubated, at
optimal conditions of 60 °C, and pH 6.9 for 1, 4, 8, 16 and 24 h. These
reaction mixtures were treated with 2 volumes (200 μL) of absolute
ethanol to precipitate the remaining nonreacted polysaccharides
(substrates) and then centrifuged at 13000gat 4 °C for 10 min.
The supernatant containing the liberated sugar was transferred to
another microcentrifuge tube, and the ethanol was removed by
evaporation. The supernatant (50 μL) was diluted to 500 μL by adding
ultrapure (Milli-Q, Millipore) water and filtered through a syringe
filter using a 0.2 μm membrane. The liberated sugars were analyzed by
high-pressure anion-exchange chromatography (HPAEC), using an
ion chromatography system (Dionex, ICS-3000). From the filtered
500 μL, 25 μL of sample (liberated sugars) was run on CARBOPACK
PA-200 column (150 ×3 mm, Dionex), attached with CarboPac
PA200 guard column (30 ×3 mm, Dionex) with borate and amino
trap columns which removed impurities and provided high resolution.
The instrument (Dionex, ICS-3000) was kept at a constant
temperature of 30 °C during the analysis, and the flow rate was
maintained at 0.3 mL min−1. The elution of liberated sugars released
due to enzyme reaction was carried out with 100 mM sodium
hydroxide using a pulsed amperometric detector (PAD). Ten
micrograms per milliliter of D-mannose, mannobiose, and mannotriose
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf403111g |J. Agric. Food Chem. 2013, 61, 12333−1234412335
were used as standards. The solutions of standards were also filtered
through a 0.2 μm membrane before loading onto the column. A
standard curve was prepared by using a mixture of standards
(mannose, mannobiose, and mannotriose) from 10 mg mL−1stock
solutions. Quantitative analysis enzyme-catalyzed hydrolysis products
were determined from the peak intensity of the released products.
Production of CtManf and CtManT in Different Media. Media
screening was performed in order to obtain higher production of
recombinant CtManf and CtManT. Four different media were
screened, as listed in Table 4. All four media LB, 5xLB, Terrific
Broth (TB), and TY media were prepared, as previously described by
Tripathi et al. (2009).
38
LB medium (100 mL) was prepared in a 250
mL flask by weighing constitutents in (%, w/v) were tryptone, 1.0;
yeast extract, 0.5; and NaCl, 1.0.
38
A 100 mL 5xLB medium was
prepared by using (%, w/v) tryptone, 5.0; yeast extract, 2.5; NaCl, 2.5;
glycerol, 1.0.
38
Terrific broth (TB) 100 mL was composed of
constituents in (%, w/v) pancreatic digest of casein, 1.2; yeast extract,
2.4; dipotassium phosphate, 0.94; monopotassium phosphate, 0.22;
and glycerol, 0.4.
38
The components of 100 mL TY medium used in
(%, w/v) were tryptone, 2.68; yeast extract, 2.14; monopotassium
phosphate, 0.54; diammonium hydrogen phosphate, 0.16; magnesium
sulfate, 0.12; NaCl, 0.85; and glycerol, 0.1.
38
Initially, the seed culture
was prepared by inoculating respective E. coli BL21 cells, harboring
recombinant plasmids CtManf and CtManT in 5 mL LB medium
supplemented with 50 μgmL
−1kanamycin and incubated at 37 °C and
180 rpm for overnight. Each 250 mL culture flasks of four media
containing 100 mL medium supplemented with 50 μgmL
−1
kanamycin were inoculated with 1 mL of seed culture. The cells
were grown at 37 °C, 180 rpm up to the mid exponential phase
(A600 nm ≈0.6) followed by induction with 1.0 mM isopropyl-1-thio-β-
D-galactopyranoside (IPTG) for hyper-expression of recombinant
proteins at 24 °C with 200 rpm for 24 h. Dry cell weight of the
bacterial cell was measured, as described by Black (1996).
39
The 10
mL culture broth was centrifuged at 9000gfor 15 min and the
supernatant discarded. The resulting pellet washed with distilled water
3 times followed by centrifugation at 9000gfor 15 min in each wash.
The cell pellet was dried at 60 °C for 16 h, and the dry cell weight was
measured by weighing.
39
The cells were harvested by centrifuging at
9000gat 4 °C for 20 min, and the resulting cell pellet was resuspended
in 50 mM sodium phosphate buffer pH 7.0 containing 1 mM
phenylmethanesulfonyl fluoride (PMSF). The cell suspensions were
sonicated (Vibra cell, Sonics) on ice for 16 min (9 s on/9 s offpulse,
30% amplitude) and then centrifuged at 19000gat 4 °C for 20 min.
The recombinant proteins were purified by a single step, using
immobilized metal ion affinity chromatography (IMAC) on HiTrap
chelating columns (GE Healthcare), as mentioned earlier.
29
The
concentration of IMAC-purified recombinant proteins were deter-
mined by the Bradford method.
40
Thermostability study and protein melting analysis of
CtManf and CtManT. The ability of CtManf and CtManT to retain
its enzymatic activity at a higher temperature was studied. Both
CtManf and CtManT (30 μL each from the stock of 0.16 mg mL−1and
0.15 mg mL−1, respectively) were incubated at temperatures from 10
to 110 °C for 1 h. After the incubation, the enzyme activity was
determined by taking 10 μLofCtManf and CtManT separately in a
100 μL reaction mixture containing 1% (w v−1) carob galactomannan
in 50 mM sodium phosphate buffer of pH 6.9 and pH 6.5, respectively.
The protein melting curves were generated by subjecting CtManf and
CtManT to various temperatures and measuring the change in the
absorbance at 280 nm by a UV−visible spectrophotometer (Varian,
Cary 100-Bio), following the method of Dvortsov et al.
18
The purified
CtManf and CtManT at protein concentration of 0.3 mg mL−1in 50
mM MES [2-(N-morpholino) ethanesulfonic acid] buffer, pH 7.0,
were used. The absorbance at 280 nm was measured at different
temperatures, varying from 40 to 100 °C using a peltier temperature
controller. The protein solutions (1 mL, 0.3 mg mL−1)ofCtManf and
CtManT were kept at the particular temperature for 10 min to attain
the equilibrium. A similar experiment was carried out with the addition
of 10 mM CaCl2in the 1 mL enzyme (0.3 mg mL−1) solution, and the
temperature was then varied. The experiment was repeated with the
addition of CaCl2and EDTA to 1 mL of enzyme solution (0.3 mg
mL−1) containing equimolar concentrations of 10 mM, and finally the
change in absorbance at 280 nm was measured. A curve of relative
derivative absorption coefficient (first derivative coefficient) versus
temperature was plotted, as described earlier by Dvortsov et al.
(2009).
18
■RESULTS
Sequence Analysis of CtManf. The molecular architec-
ture of full length derivative of mannanase CtManf displayed an
appended family 35 carbohydrate binding module (CtCBM35)
at its N-terminal end and catalytic CtManT (Man26B) at its C
terminal end (Figure S1of the Supporting Information). Amino
acid sequence analysis using InterProScan revealed that CtManf
has two distinct modules: from regions 1 to 134, a noncatalytic
carbohydrate binding module family 35 (CBM35) and from
135 to 478, a glycoside hydrolase family 26 (GH26). CtManT
found the most similar 50% sequence homology, (UniProt id:
Q9F1T9) to mannanase 26B of C. thermocellum strain F1
(CtF1Man26B), Bacillus licheniformis DSM13 (BlMan26B)
(33.080%, UniProt id: Q65MP4), Paenibacillus sp. BME-14
(PsMan26B) (37%, UniProt id: C6KL35), and Cellvibrio
japonicas (CjMan26B) (35%, UniProt id: Q840B9). Sequence
homology was found less pronounced, while comparing with
mannanase 26A of Cellulomonas fimi (Cf Man26A) (28%,
UniProt id: Q9XCV5), and Bacillus sp. JAMB750 (BsMan26A)
(26%, UniProt id: Q2ACI1). These results were similarly
analyzed from the phylogenetic tree where CtManT was most
closely related with Man26B of the C. thermocellum strain F1
(Figure 1). Multiple sequence alignment of CtManT displayed
a unique identifier sequence that was a highly conserved
aromatic amino-acid-rich region with the consensus sequence
WFWWG within all the ManGH26 family (Figure S2 of the
Supporting Information, highlighted in the box), as also
similarly stated by Xiaoyu et al. (2010).
21
Cloning and Expression of CtManf and CtManT. The
open-reading frames of CtManT and CtManf amplified by
Figure 1. Phylogenetic tree showing the comparative study of our
query CtManT (highlighted in red box) with two different types of
GH26 mannanase (Man26A and Man26B) of representative members
and their appearance during evolution based on sequence similarity.
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polymerase chain reaction resulted in 1449 bp and 1029 bp
sequences, respectively. The amplified DNAs were cloned into
the pET-28a (+) expression vector and then transformed the E.
coli DH5αcells using the cloned plasmids. The positive clones
of CtManf and CtManT were identified by digestion of resulted
recombinant plasmids with NheI-XhoI restriction enzymes. The
E. coli BL-21 (DE3) cells were transformed with recombinant
plasmids, harboring CtManf and CtManT. The positive clones
were screened by growing individual colonies in LB medium
and after, inducing with 1 mM IPTG and further incubating at
24 °C for 24 h. The expressed proteins were analyzed by SDS−
PAGE. The two Clostridial recombinant His6-tagged proteins
were purified by immobilized metal ion affinity chromatography
(IMAC) from the cell-free extracts to homogeneity. The SDS−
PAGE analysis of purified CtManf and CtManT displayed
molecular sizes of 53 kDa and 38 kDa, respectively (Figure 2,
panels A and B). Both the recombinant proteins expressed as
soluble proteins.
Specificity and Kinetic Parameters of CtManf and
CtManT with Natural Substrates. The optimum pH and
temperature for CtManf were 6.9 and 60 °C, respectively, and
for CtManT were 6.5 and 50 °C, respectively. The substrate
specificities of CtManf and CtManT with natural substrates
were determined at optimized pH and temperature. The
enzyme activities with natural substrates are displayed in Table
1. It is conspicuous from the Table 1 that both CtManf and
CtManT have specificity for galactomannan, and the highest
enzyme activity was achieved with carob galactomannan 97.0 ±
5.0 units mg−1and 91.0 ±4.0 units mg−1, respectively. Both
displayed activity in decreasing order with locust bean
galactomannan, konjac glucomannan, and guar galactomannan.
Both CtManf and CtManT with insoluble polysaccharide ivory
nut mannan displayed a biphasic hydrolysis pattern (Figure S3
of the Supporting Information), where rapid hydrolysis of the
substrate occurred up to 15 min of incubation followed by
slower hydrolysis (Figure S3 of the Supporting Information).
These enzymes perhaps acted on the amorphous sites
(hydrolyzable region) of the substrate during rapid hydrolysis
in the first phase and then accessed the crystalline sites
(tougher region) in the second phase. Similar results were
reported by Mizutani et al. (2012).
41
The enzyme activities of
both enzymes with insoluble ivory nut mannan and β-(1→4)-
mannan from the first phase were calculated (Table 1). CtManf
displayed approximately, two times higher activity than
CtManT with both the substrates (Table 1). The specific
activity of CtManf was 50.0 units mg−1, whereas CtManT was
26.5 units mg−1with ivory nut mannan and with β-(1→4)-
Figure 2. Zymogram study using 12% SDS−PAGE (A) CtManf (panel 1: purified protein, 2: congo red staining, and 3: 1 N HCl counter staining)
and (B) CtManT (panel 1: purified protein, 2: congo red staining, and 3: 1 N HCl counter staining).
Table 1. Substrate Specificity of CtManf and CtManT from
C. thermocellum
substrate
(1%, w v−1)specific activity CtManf
(units mg−1)specific activity CtManT
(units mg−1)
carob
galactomannan 97.0 ±5.0 91.0 ±4.0
locust bean
galactomannan 85.4 ±6.0 83.1 ±5.0
konjac
glucomannan 81.0 ±3.0 79.8 ±4.0
guar galactomannan 47.6 ±3.0 38.7 ±4.0
ivory nut mannan 50.0 ±2.0 26.5 ±0.9
mannan 40.0 ±1.0 21.2 ±2.0
barley-β-glucan 2.94 ±0.2 1.74 ±0.1
lichnan 1.92 ±0.8 1.22 ±0.2
carboxymethyl
cellulose 1.09 ±0.5 0.9 ±0.05
hydroxyethyl
cellulose 0.87 ±0.03 0.47 ±0.03
Avicel 0.39 ±0.02 0.26 ±0.03
xyloglucan 1.5 ±0.5 1.0 ±0.3
rye arabinoxylan NA NA
glucuronoxylan NA NA
arabinogalactan NA NA
polygalactouronic
acid NA NA
Values are in mean ±SD (n= 3). NA = no activity was determined.
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mannan the enzyme activities were 40.0 units mg−1and 21.2
units mg−1, respectively (Table 1). Therefore, the above results
indicated that CtCBM35 plays a role in potentiating the
enzyme activity of the full length enzyme CtManf in
hydrolyzing the insoluble substrates. A similar comment was
stated earlier by Mizutani et al. (2012) for CBM32 appended to
GH5 mannanase from C. thermocellum.
41
Both CtManf and CtManT displayed low but significant
activity against barley β-glucan, lichenan, carboxymethyl
cellulose, hydroxyethyl cellulose, Avicel and xyloglucan, where-
as no activity was observed with arabinogalactan, rye
arabinoxylan, glucuronoxylan, and polygalactouronic acid
(Table 1). The kinetic properties and catalytic efficiency of
both the enzymes were determined with the natural substrates
(Table 2). CtManf and CtManT displayed turnover numbers
(kcat) of 698 and 684 min−1, respectively, and catalytic
efficiencies (kcat/Km) of 4.1 ×102and 3.9 ×102min−1mg−1
mL, respectively, with carob galactomannan. Both the enzymes
CtManf and CtManT efficiently acted on insoluble ivory nut
mannan, showing catalytic efficiencies (kcat/Km) of 3.4 ×102
and 2.4 ×102min−1mg−1mL and with β-(1→4)-mannan, 3.1
×102and 2.2 ×102min−1mg−1mL, respectively (Table 2).
The present results showed that CtManf gave approximately
1.1-fold higher activity against carob galactomannan and
approximately 2-fold higher activity against insoluble ivory
nut mannan and β-(1→4)-mannan than the catalytic CtManT.
Similar results were reported earlier, where enhanced activity in
the presence of a carbohydrate binding domain (CBD) in
Man26A (mannanase A from family GH26) was observed by
Halstead et al. (1999).
8
The presence of CBD at the N-terminal
of Man26A enhanced turnover of carob galactomannan by 1.1-
fold and 2-fold against insoluble ivory nut mannan.
8
Specificity and Kinetic Parameters CtManf and
CtManT with Synthetic Substrates. Both CtManf and
CtManT did not show any activity against pNP-β-D-
mannopyranoside and with pNP-α-D-mannopyranoside. On
the basis of the enzyme activity of CtManf and CtManT against
natural as well as synthetic substrates, it was evident that both
these enzymes are predominantly endo-β-D-mannanase. The
enzymes specifically cleaved the β-(1→4)-glycosidic linkages
between mannopyranosyl residues.
Zymogram Study of CtManf and CtManT. Separate
SDS−PAGE gels were used in the zymogram study to show the
active bands of CtManf and CtManT against carob
galactomannan (Figure 2, panels A and B). CtManf displayed
an active band around 53 kDa and CtManT around 38 kDa,
congo red staining, and counter staining with 1 N HCl (Figure
2, panels A and B). Both the enzymes displayed homogeneous
bands and a clear zone of activity with carob galactomannan.
Mannan endo-β-(1→4)-mannanase activity was detected as
clear zones against red (after staining with Congo red) and blue
background (after counter stained with 1 N HCl). The results
clearly indicated that both of these enzymes have manno-
configured substrate specificity.
Effects of Metal Ions and Chemical Agents on CtManf
and CtManT. The enzymatic activity of CtManf and CtManT
significantly increased by 1.5-fold in the presence of low
concentrations of Ca2+ (10 mM) and Mg2+ (15 mM) (Table 3).
Both CtManf and CtManT retained moderate activities in the
presence of 10 mM Mn2+ (80% and 60%), 8 mM Ni2+ (80%
and 80%), 30 mM Co2+ (70% and 70%), and 10 mM Zn2+
(70% and 60%) salts, respectively. The enzyme activities were
adversely affected by low concentrations of Cu2+ (5 mM) or
Al3+ (6 mM) salts, and CtManf lost 80% and CtManT lost 90%
of the activity at the mentioned concentrations of Cu2+ and Al3+
salts (Table 3). The enzyme activity of both the catalytic
modules decreased to more than 80% in the presence of EDTA
(8 mM) or 10 mM EGTA (Table 3). The presence of SDS (10
mM) CtManf lost 94% enzyme activity, while CtManT almost
completely lost the activity. The decrease in activity in the
presence of EDTA indicated that Ca2+ ions may be essential for
Table 2. Kinetic Properties and Catalytic Efficiencies of CtManf and CtManT from C. thermocellum ATCC 27405
Km(mg mL−1)kcat (min−1)kcat/Km(min−1mg−1mL)
substrate CtManf CtMan T CtManf CtMan T CtManf CtMan T
natural substrates
carob galactromannan 1.8 ±0.2 1.6 ±0.2 737 634 4.1 ×1023.9 ×102
locust bean galactomannan 1.5 ±0.1 1.4 ±0.4 590 520 3.9 ×1023.7 ×102
konjac glucomannan 1.5 ±0.3 1.4 ±0.2 510 462 3.4 ×1023.3 ×102
guar galactomannan 1.2 ±0.2 1.1 ±0.2 320 283 2.6 ×1022.5 ×102
ivory nut mannan 0.9 ±0.1 0.8 ±0.2 310 199 3.4 ×1022.4 ×102
mannanan 0.9 ±0.2 0.7 ±0.1 283 159 3.1 ×1022.2 ×102
synthetic substrates
pNP-β-D-manno- pyranoside ND ND ND ND ND ND
pNP-α-D-manno- pyranoside ND ND ND ND ND ND
Values are in mean ±SD (n= 3). ND = not detected.
Table 3. Effects of Metal Ions and Other Agents on CtManf
and CtManT from C. thermocellum ATCC 27405
relative activity (%)
ions/reagents concentration (mm) CtManf CtManT
control
a
−100 100
Ca2+ 10 150 150
Mg2+ 15 150 150
Mn2+ 10 80 60
Ni2+ 88080
Co+30 70 70
Zn2+ 10 70 60
Cu2+ 52010
Al3+ 62010
EDTA 8 20 20
EGTA 10 20 20
SDS 8 6 2
urea 4 ×103(4 M) 10 5
GnHCl 100 2 1
a
No additives were added in the control, and the activity was taken as
100%.
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enzyme activity as EDTA and specifically binds and chelates the
calcium ions in a 1:1 molar ratio.
36
The enzyme activity
drastically reduced with chaotropic agents such as guanidine
hydrochloride (GnHCl) and urea at higher concentrations. The
enzymatic activity of CtManf decreased by 90% at 100 mM
GnHCl, whereas CtManT lost 95% activity at the same
concentration of GnHCl. In contrast, much higher concen-
tration of urea (4 M) was required for complete diminution
(98%−99%) of enzyme activities of CtManf and CtManT.
Analysis of Polysaccharide Hydrolysis Products by
TLC. The analysis of recombinant CtManf hydrolyzed products
of carob galactomannan by TLC is displayed in Figure 3A.
Time-dependent hydrolysis of carob galactomannan after 10
min of reaction displayed the release of mannose (dp 1), but
after 1 h of consecutive hydrolysis there was a little release of
mannobiose (dp 2) and mannotriose (dp 3) (Figure 3 A).
Standards of mono and oligosaccharides (M1: mannose, M2:
mannobiose, and M3: mannotriose) were run in parallel to
compare the released sugars (Figure 3A). Up to 8 h of
incubation with CtManf, carob galactomannan was not
completely hydrolyzed, but after 16 h, a large amount of
mannose and comparable amounts of mannobiose and
mannotriose were released (Figure 3A). Complete hydrolysis
of carob galactomannan was achieved after 24 h of incubation,
where the maximum amount of mannose, mannobiose, and
mannotriose were produced (Figure 3A). Apart from the
appearance of dp 1, dp 2, and dp 3, there were two other higher
oligosaccharide (dp 4 and dp 5) spots observed and displayed
similar appearance throughout the hydrolysis process (Figure
3A). CtManf displayed a typical endoacting bond cleavage
during mannobiose and mannotriose hydrolysis, releasing
principally mannose and mannobiose (Figure 3, panels B and
C, respectively). CtManf was unable to hydrolyze mannobiose
up to 1 h, but after 4 h, complete hydrolysis of mannobiose
occurred, leaving only a mannose spot on the TLC plate
(Figure 3B). Whereas, the hydrolysis mannotriose by CtManf
after 4 h released predominantly mannose and a trace amount
of mannobiose. The mannobiose spot disappeared completely
after 8 h, leaving only the mannose spot (Figure 3C). Thus
CtManf cleaved β-(1→4) bonds of these manno-oligosacchar-
ides elegantly. Therefore, based on the significant role in
specifically cleaving the β-(1→4) bond, this enzyme was
classified and named as endo-β-(1→4)-mannanase.
HPAEC Analysis of Enzyme Reaction Products.
Qualitative and quantitative analysis of CtManf hydrolyzed
products of carob galactomannan were monitored by HPAEC-
PAD. Time-dependent hydrolysis of carob galactomannan by
CtManf is displayed in Figure 4. The peak intensities of
standards are displayed in Figure 4A. After 1 h of CtManf
treatment of carob galactomannan, the prominent peaks of
mannose at 3.4 min, mannobiose at 4.06 min, and mannotriose
at 4.96 min were observed with concentrations 2.12, 0.73, and
0.76 mg mL−1, respectively (Figure 4B). Mannobiose peak was
more prominent than mannotriose after 4 h of carob
galactomannan hydrolysis, while the mannose intensity
increased continuously (Figure 4C). The concentrations were
determined as 2.23 mg mL−1mannose, 1.12 mg mL−1
mannobiose, and 0.80 mg mL−1mannotriose after 4 h of
incubation. CtManf was able to hydrolyze carob galactomannan
to a greater extent after 8 h, and the products obtained were
mannose (3.16 mg mL−1), mannobiose (1.81 mg mL−1), and
mannotriose (0.90 mg mL−1), with much higher peak
intensities (Figure 4D). After a 16 h incubation, the
mannobiose and mannotriose concentrations increased to 2.1
mg mL−1and 1.1 mg mL−1, respectively. This increase was
approximately 1.17 fold and 1.3 fold, respectively, for
mannobiose and mannotriose (Figure 4E), as compared with
that obtained after 8 h hydrolysis of carob galactomannan. The
complete hydrolysis of carob galactomannan by CtManf was
observed after 24 h of enzymatic reaction, where mannose,
mannobiose, and mannotriose concentrations of 3.6 mg mL−1,
2.3 mg mL−1, and 1.4 mg mL−1, respectively, were obtained
(Figure 4F). All the concentrations were determined from the
regression equation of mannose, mannobiose, and mannotriose
standard curves. Therefore, CtManf quite effectively hydrolyzed
10 mg mL−1(1%, w v−1) carob galactomannan and released a
maximum after 24 h, yielding 36% mannose, 23% mannobiose,
and 14% mannotriose. The overall results quite interestingly
described the performance of CtManf in the releasing of
manno-oligosaccharides from carob galactomannan, which can
be scaled up for commercial production.
Production of Enzymes (CtManf and CtManT) in
Different Media. The highest concentration of recombinant
proteins were obtained from a 100 mL flask containing TY and
TB media, followed by LB and 5xLB media after 24 h of
incubation. TY medium achieved the highest cell densities of 31
and 30 g L−1dry cell weight, respectively, for CtManf and
CtManT. The protein concentration of CtManf and CtManT
Figure 3. Thin layer chromatography analysis of hydrolysis products
from (A) carob galactomannan, (B) mannobiose, and (C)
mannotriose by CtManf. (A) Carob galactoniannan (1%, w v−1) was
incubated with CtManf for 10 min to 24 h, (B) mannobiose (1 mg
mL−1) was incubated with CtManf for 10 min to 4 h, and (C)
mannotriose (1 mg mL−1) was incubated with CtManf for 10 min to 8
h. Samples were taken in intervals, and hydrolysates were analyzed by
TLC (standards used M1: mannose, M2: mannobiose, and M3:
mannotriose).
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after sonication and purification by IMAC obtained was 910
and 880 mg L−1, respectively. Similar results were reported by
Tripathi et al. (2009),
38
where TY medium gave the highest cell
density of E. coli cells achieved, 1.12 g L−1, and the recombinant
dengue protein was expressed at a concentration of 10.37 mg
L−1.
38
CtManf and CtManT gave less DCW with TB medium
28 g L−1and 26.9 g L−1, respectively, as compared with TY
medium. The purified protein concentrations from TB medium
obtained were 500 mg L−1and 410 mg L−1, respectively, for
CtManf and CtManT. The dry cell weight (DCW) from 100
mL LB medium obtained 21 and 20 g L−1for CtManf and
CtManT, respectively. The concentrations of purified protein
obtained from LB medium were 160 mg L−1for CtManf and
150 mg L−1for CtManT. These results are similar to those of
the earlier report of Tripathi et al. (2009).
37
The lowest growth
of cell mass of E. coli BL21 (DE3) cells were observed for both
enzymes in 5xLB medium. The cell densities obtained were
12.0 and 10 g L−1, respectively, for CtManf and CtManT. The
recombinant CtManf and CtManT proteins after purification
obtained were 300 mg L−1and 280 mg L−1in 5xLB medium,
respectively. Therefore, it can be suggested that productivity of
recombinant mannanase and cell mass can be improved by
altering and optimizing media components.
Thermostability Study and Protein Melting Analysis
of CtManf and CtManT. Thermostability study displayed
stability of CtManf and CtManT at higher temperatures (Figure
5 A). CtManf remained stable up to 60 °C, retaining 100%
activity for 1 h. The enzyme activity of CtManf decreased after
60 °C and left with 20% at 100 °C. CtManf and CtManT
retained more than 55% and 30% enzyme activity at 80 °C.
CtManT was stable up to 50 °C and lost 90% at 100 °C.
Therefore, CtManf was more thermostable than CtManT. The
results indicated that the higher thermostability of CtManf
could be due to the presence of the carbohydrate binding
module, CtCBM35. Protein stability was also observed with
protein melting curve analysis. The full-length CtManf showed
two separate melting peaks at 50 and 80 °C (Figure 5 B),
whereas CtManT displayed a single melting peak at around 80
°C (Figure 5C). This suggested that the peak at 50 °C
corresponded to noncatalytic CtCBM35 and the peak at 80 °C
to the catalytic module CtManT and that the two modules are
melting independently (Figure 5B). The presence of Ca2+ ions
(10 mM) caused significant changes in CtManf, as well as in
CtManT protein-melting profiles. The peak for CtManT shifted
toward higher a temperature (i.e., from 80 to 100 °C), but the
peak corresponding to CtCBM35 in CtManf was masked in the
presence of Ca2+ ions (Figure 5, panels B and C). On addition
of EDTA (10 mM) to the enzyme−substrate reaction mixture
containing Ca2+ (10 mM), the melting peaks shifted back to the
original temperature of 80 °C of catalytic CtManT and
CtCBM35 (Figure 5, panels B and C, small dotted lines).
Therefore, from both thermostability and protein melting study
Figure 4. HPAEC-PAD analysis of hydrolyzed products of carob galactomannan by CtManf. (A) Elution patterns of standards used mannose (3.4
min), mannobiose (4.06 min), and mannotriose (4.96 min). Elution pattern of mannose, mannobiose, and mannotriose from carob galactomannan
(l%, w v−1) treated by CtManf after (B) 1, (C) 4, (D) 8, (E) 16, and (F) 24 h.
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of CtManf and CtManT, it could be concluded that both these
enzymes are thermostable and Ca2+ ions provide significant
thermal stability.
■DISCUSSION
The molecular architecture of full length CtManf from C.
thermocellum ATCC 27405 displayed a modular structure.
Sequence homology of the catalytic module CtManT identified
as Man26B, showed highest similarity with that of the C.
thermocellum F1 strain.
21
The Man26B enzymes have subtle
differences with Man26A enzymes in topology of substrate
binding and functional property of substrate hydrolysis as
previously described by Hogg et al. (2003) in a study of
Man26A and Man26B from C. japonicas.
22
Both CtManf and
CtManT preferred higher degrees of catalysis against carob
galactomannan than locust bean galactomannan and konjac
glucomannan, while similar observations were stated earlier by
Xiaoyu et al. (2010) and Kurokawa et al. (2001) for Man26B
isolated from Paenibacillus sp. BME-14 and C. thermocellum
strain F1, respectively.
21,23
It is worth mentioning here about
the wide range of substrate specificity of both CtManf and
CtManT. Both these enzymes were able to hydrolyze β-(1→4)-
gluco based substrates viz. barley β-glucan, lichenan, carbox-
ymethyl cellulose, hydroxyethyl cellulose, Avicel, and xyloglu-
can. ManA from Thermoanaerobacterium polysaccharolyticum
similarly showed both mannanase and endoglucanase activ-
ities.
42
Low activities of CtManf and CtManT against ivory nut
mannan and β-(1→4) may be attributed to the crystalline
nature of the substrate, which greatly inhibits the access of
enzymes for catalysis. Interestingly, CtManf exhibited 2-fold
higher activities against insoluble ivory nut mannan and β-(1→
4)-mannan, as compared to CtManT. By comparing the
hydrolyzing capacity of CtManf and CtManT against different
mannans, it was concluded that the CtCBM35 of C.
thermocellum ATCC 27405 GH26 mannanase played an
important role in the degradation of insoluble ivory nut
mannan and β-(1→4) (Table 2). The apparent biphasic action
of CtManf and CtManT against ivory nut mannan and β-(1→
4)-mannan was quite distinguishable and thus suggested that
both these enzymes preferred to attack the amorphous region
of the substrate in the early reaction stage and the crystalline
region later. This might give a lucid idea about the substrates
that consist of two distinct regions, which was more resistant
toward CtManT action. Similar, types of observations were
reported by Mizutani et al. (2012),
41
while comparing the role
of Man26A from C. thermocellum against insoluble substrate
hydrolysis. Higher degree of hydrolyzing capacity of CtManf
than CtManT was attributed to the appended N-terminal
CtCBM35 domain which facilitated the increased catalysis by
concentrating the catalytic module in the vicinity of the
substrate. CtCBM35 helped in prolonged binding of the
substrate and decreased its resistance to the catalytic attack by
CtManT. As an instance CBM32 from C. thermocellum ATCC
27405 has recently been shown to improve hydrolysis of
insoluble substrates by mannanase of GH5.
38
Moreover, higher
activity of CtManf at a higher temperature was attributed to the
presence of N-terminal carbohydrate binding domain
(CtCBM35) than catalytic CtManT. Similarly in an earlier
report, it was suggested by Xiaoyu et al. (2009),
21
the appended
carbohydrate binding domain in Man26B of Paenibacillus sp.
BME-14 potentiated in higher activity against locust bean gum
than the lone catalytic module.
21
The presence of CtCBM35 in
CtManf played a unique role in higher hydrolysis of soluble
carob galactomannan as compared with the catalytic CtManT.
Both enzymes exhibited significant turn over against other
soluble as well as insoluble substrates. It was reported
previously that carob galactomannan composed of 78%
mannose formed the β-(1→4)-mannan backbone, while
galactose contributes 22%.
1
Each β-(1→4)-mannan is sub-
stituted by the α-(1→6)-galactose side chain. The enzymes
randomly hydrolyze β-(1→4)-linkages in diverse substrates,
such as galactomannans and glucomannan. Both CtManf and
CtManT did not show any activity against synthetic substrates
pNP-β-mannopyranoside and pNP-α-mannopyranoside. There-
fore, it is evident that both CtManf and CtManT are endo-
acting enzymes and are endo-β-(1→4)-mannanases (endo-
Man26B) from C. thermocellum ATCC 27405. The perform-
ances of CtManf and CtManT were investigated under the
influence of salts, chaotropic agents, and detergent. Both
CtManf and CtManT are metalloenzymes, and Ca2+ and Mg2+
ions act as cofactors for these enzymes. The enzyme activities
of CtManf and CtManT increased significantly by 1.5-fold in
the presence of Ca2+ and Mg2+ salts, which suggested that these
ions are required as cofactors. However, the enzyme activity
was unaffected by lower concentrations of Mn2+,Ni
2+,Co
+, and
Zn2+ and were able to retain their moderate activities. The
enzyme activities of CtManf and CtManT were completely
inhibited by lower concentrations of Cu2+ and Al3+. Similar
observation was reported earlier by Yoshikawa et al. (2009),
where a noncompetitive type of inhibition imposed by Cu2+
ions by binding at different sites other than the α-glucosidase
active center or enzyme−substrate complex.
43
In the presence
of a low concentration of chelating agents such as EDTA or
Figure 5. Thermal stability analysis of (A) CtManf and CtManT from
10 to 110 °C. Protein-melting analysis of displaying normal melting
curve without any additives, in the presence of 10 mM Ca2+ ions and
in the presence of 10 mM Ca2+ ions and 10 mM EDTA of (B) CtManf
and (C) CtManT.
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf403111g |J. Agric. Food Chem. 2013, 61, 12333−1234412341
EGTA, the enzyme activity sharply decreased. The enzyme
activity of CtManf and CtManT was adversely affected at the
low concentration of SDS. The higher concentration urea and
lower concentration of guanidine hydrochloride was required to
inactivate the enzymes.
CtManf-catalyzed substrate hydrolysis products were ana-
lyzed by TLC and HPAEC. It was apparent from TLC that
CtManf released mannotriose, mannobiose, and mannose in
prolonged hydrolysis of carob galactomannan. But, in the
earlier stages, the amount of mannobiose was less, as compared
to mannotriose and mannose. After complete hydrolysis of
carob galactomannan, CtManf was able to release mannose,
mannobiose, and mannotriose. The salient feature of CtManf
catalysis involved only β-(1→4)-bond cleavage, when man-
nobiose and mannotriose were used as substrates and liberated
mainly mannose as the main product. The release of large
amounts of mannose at early stages of enzymatic reaction by
CtManf was commonly seen against carob galactomannan and
manno-oligosaccharides. In an earlier report by Hogg et al.
(2003), the release of mannose at the early stage of hydrolysis
of manno-configured substrates and oligosaccharides is
characteristic of a typical Man26B mannanase.
22
HPAEC
analysis corroborated the results of TLC analysis of hydrolysis
of carob galactomannan products released by CtManf. The
results of HPAEC showed that CtManf exclusively cleaves
carob galactomannan into mannotriose, mannobiose, and
mannose. It was apparent from TLC and HPAEC analyses
that CtManf was able to hydrolyze only the β-(1→4) bond
cleavage and had potential to produce manno-oligosaccharides
from carob galactomannan. Thus effective β-(1→4)-mannanase
from C. thermocellum ATCC 27405 may be exploited for higher
production of manno-oligosaccharides, especially for the
controlled synthesis of mannobiose and mannotriose. Kurakake
et al. (2006) reported the synthesis of manno-oligosaccharides
from guar gum by utilizing of β-mannanase from Penicillium
oxalicum SO.
44
Media composition plays a significant role in production of
recombinant proteins.
38
Use of chemically defined medium is a
common practice in producing recombinant proteins.
45−47
The
recombinant CtManf and CtManT showed the highest cell
density and concentration of protein in TY medium. In TB
medium, moderate cell densities and protein production were
observed. In LB medium, moderate cell density was achieved
with low protein concentration. The 5xLB medium did not
support the growth due to higher concentrations of yeast
extract, tryptone, and sodium chloride, and as a result, the
lowest protein concentration was achieved. Similar effects of
media were stated earlier, while producing recombinant dengue
protein in E. coli.
38
The rich source of tryptone, yeast extract,
and phosphate salts facilitated to achieve highest cell densities
in TY media as compared to other chemically defined media
used. Yeast extract is a known source of trace components and
can relieve cellular stress responses such as the production of
proteases during synthesis of recombinant protein in E. coli.
38
Higher concentration of phosphate is important for attaining
high cell densities of E. coli, as the lower concentrations of
phosphate limits the growth.
37
The phosphate salts in the
medium provided buffering capacity against pH fluctuations,
which adversely affects the metabolic activity of cells.
38
The low
cell densities and lower production of recombinant proteins in
LB and 5xLB medium were due to a lack of buffering capacity.
Protein stability while functioning at higher temperature is a
major concern in industry. A temperature stability study of
CtManf and CtManT showed that after 1 h of incubation at 60
and 50 °C, respectively, 100% activity was retained. But they
have retained around 10% of enzymatic activity at 100 °C.
When compared with recombinant ManB from Bacillus
licheniforms DSM13,
24
the recombinant Man26B from C.
thermocellum ATCC 27405 was thermally more stable at higher
temperatures. The protein melting phenomenon of recombi-
nant CtManf and CtManT was analyzed to study their
thermostability. Protein-melting curves of full length CtManf
showed that the catalytic module CtManT and carbohydrate
binding module CtCBM35 melt independently of each other.
The protein-melting peaks of CtManT and CtCBM35 shifted to
higher temperature in the presence of Ca2+ ions. However, on
addition of equimolar concentration of EDTA to the solutions
of CtManf and CtManT, the melting temperature peaks shifted
back to the original positions. The shift of peak to a higher
temperature in the presence of Ca2+ ions might be due to the
reason that Ca2+ ions provide stability to the protein structure
by inducing electrostatic interactions with amino acids, as
reported by Noorbatcha et al. (2012).
48
The electrostatic
interactions imparted by Ca2+ ions in bound protein resulted in
less hydrogen bonds and higher number of salt bridges as
compared to nonbonded proteins.
48
Because of the higher
fluctuations in the backbone of protein at higher temperature,
the number of hydrogen bonds will be destabilized, which
allowed residues in close proximity to calcium ions to form
more numbers of salt bridges in the Ca2+ ion-bound state as
compared with the Ca2+ ion-free state.
48
Thus, the binding by
Ca2+ ion makes protein more conformationally stable at higher
temperature.
47
The shifting back of melting peaks in the
presence of EDTA was due to chelation of calcium ions,
making them unavailable for the enzyme. This is the first report
of cloning and biochemical characterization of a thermostable
Man26B form C. thermocellum ATCC 27405 and its potential
role in manno-oligosaccharide production from manno-
configured substrates.
■ASSOCIATED CONTENT
*
SSupporting Information
Molecular architecture of full length CtManf of C. thermocellum
ATCC 27405; multiple sequence alignment of CtManT with
Man26A from Cellulomonas fimi; and biphasic hydrolysis
pattern of insoluble ivory nut mannan. This material is available
free of charge via the Internet at http://pubs.acs.org.
■AUTHOR INFORMATION
Corresponding Author
*E-mail: arungoyl@iitg.ernet.in. Tel: (361) 258 2208. Fax:
(361) 269 0762.
Notes
The authors declare no competing financial interest.
■ACKNOWLEDGMENTS
A.G. is supported by a scholarship from University Grants
Commission (UGC), New Delhi, India. The research work in
part was supported by a Cutting-edge Research Enhancement
and Scientific Training (CREST) Fellowship from the
Department of Biotechnology, Ministry of Science and
Technology to Arun Goyal.
Journal of Agricultural and Food Chemistry Article
dx.doi.org/10.1021/jf403111g |J. Agric. Food Chem. 2013, 61, 12333−1234412342
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