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Thermostable Recombinant β-(1→4)-Mannanase from C. thermocellum: Biochemical Characterization and Manno-Oligosaccharides Production

  • Mahapurusha Srimanta Sankaradeva Viswavidyalaya

Abstract and Figures

Functional attributes of a thermostable β-(1→4)-mannanase were investigated from Clostridium thermocellum ATCC 27405. Its sequence comparison exhibited highest similarity with Man26B of Clostridium thermocellum F1. The full length CtManf and truncated CtManT were cloned in pET28a(+) vector and expressed in E. coli BL21(DE3) cells exhibiting 53 kDa and 38 kDa proteins, respectively. Based on 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°C and 50°C, respectively and Ca2+ ions imparted higher thermostability resisting their melting up to 100°C.
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Thermostable Recombinant β(14)-Mannanase from C.
thermocellum: Biochemical Characterization and Manno-
Oligosaccharides Production
Arabinda Ghosh,
Ana SoaLuís,
Joana L. A. Brá
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-(14)-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 specicity and hydrolyzed product prole, CtManf and
CtManT were classied as β-(14)-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 dened 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
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-
(14)-D-mannan backbone (78%) and galactose as α-(16)-
linked (22%) single units, where as guar gum (from endosperm
of guar seeds) backbone is a linear chain of β-(14)-linked
mannose residues to which galactose residues are (16)-linked
at every second mannose, forming short side-branches.
Glucomannan (from Amorphophallus konjac) is a water-soluble
polysaccharide that is considered a dietary ber. The
component sugars in konjac glucomannan are β-(14)-linked
D-mannose and D-glucose residues in a molar ratio of 1.6:1 and
branched chain composed of β-(16)-linked D-glucosyl units.
β-D-Mannanase [endo β-(14)-mannan mannohydrolase,
E.C.] hydrolyzes β-(14)-D-mannopyranosyl linkages
within the main chain of mannans, glucomannans, galacto-
mannans, and galactoglucomannans.
Mannanases have been
listed within glycoside hydrolase (GH) families viz. GH26,
GH5, and GH113 in carbohydrate-active enzyme database
( based on
sequence similarity.
β-D-Mannanases belong to families GH5,
GH26, GH113 and display a (β/α)8barrel-shaped protein
folding pattern, and the acidbase-assisted catalysis via a
double displacement mechanism involving a covalent glycosyl-
enzyme intermediate.
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.
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,
while to date no evidence of
transglycosylation has been reported for GH26 mannanases.
The benet of employing novel enzymes for specic
industrial processes is well-recognized with the discovery of
β-mannanases. β-Mannanases (EC hydrolyze man-
nan-based hemicelluloses and liberate short β-(14)-manno-
oligosaccharides, which can be further hydrolyzed to mannose
by β-mannosidases (EC 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 specicity hydro-
lyzing (14)-β-D-linkages in mannans, galacto-mannans,
glucomannans, and galactoglucomannans but does not show
activity against β-glycan chain of soluble cellulose derivatives.
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
Received: July 16, 2013
Revised: November 13, 2013
Accepted: November 13, 2013
Published: November 13, 2013
© 2013 American Chemical Society 12333 |J. Agric. Food Chem. 2013, 61, 1233312344
having dierent substrate specicity. GH5 mannanase exhibits
some activity for cellulosic substrates.
By contrast, Man26B
displays canonical endomannanase activity and linked to the
cell membrane via 70residuelinkersequenceofC.
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-(14)-
bond cleavage activity against small manno-oligosaccharides,
hydrolyzing mannotriose approximately, 10000 times more
eciently than Man26B.
There are distinct dierences in
topology of the substrate-binding cleft and substrate specicity
among the mannnases (Man26A, Man26B, and Man26C)
within GH26 family.
There are several reports of manno-oligosaccharides syn-
thesis by the utilization of mannanases from manno-congured
Though the manno-oligosaccharides are indi-
gestible inside the human gut, their potential role as dietary
ber and prebiotics were attributed in various studies.
It was
evident from earlier reports that the ecient prebiotics role of
manno-oligosaccharides that supports the growth of human
intestinal benecial microora viz. Bidobacteria and Lactoba-
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.
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 microbrils and uses it as primary carbon and energy
sources and releases soluble sugars.
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-
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
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.
A family 35 carbohy-
drate binding module (CBM35) often appended with
mannanase that binds to the galactose-decorated mannanas
and facilitates their ecient hydrolysis.
There are few reports
of Man26B functions explored earlier from Paenibacillus sp.
C. japonicas,
C. thermocellum strain F1,
Bacillus licheniformis DSM13.
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-
congured substrate was analyzed.
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-(14)-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) (
html) belonging to family 26 glycoside hydrolase (GH26), were
identied in the native host C. thermocellum ATCC 27405 (16S rDNA
sequence ID: CP000568,
action?page=34&cultureId=40680). Mannanase encoding ORF region
was identied using the protein sequence (gene bank protein accession
ABN51273.1, locus name: Cthe_0032) of C. thermocellum ATCC
27405 in tBLASTn ( tool. This
sequence was used to design the desired oligonucleotide primers for
amplication of full length CtManf (CBM35-CtManT) and truncated
catalytic module CtManT (nucleotide accession: CP000568.1). The
sequence for amplication 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 ( and viewed in the ESPript (
ESPript/ESPript/) tool. Multiple sequence alignment was performed
using the dierent types of mannanase (Man26A and Man26B)
sequences of glycoside hydrolase family 26 (GH26) from Paenibacillus
sp BME14,
Cellvibrio japonicas,
Clostridium thermocellum strain
Bacillus licheniformis DSM13,
Cellulomonas mi,
mus marinus,
Bacillus sp JAMB750,
Paenibacillus polymyxa GS01.
Evaluation of the functional property of this protein sequence was
performed using InterProScan (
iprscan/), and the molecular architecture of the entire protein
sequence to be cloned was drawn.
Gene Amplication 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 amplied 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-3and reverse 5-cacctcgagttagctaaagtatattttg-3.The
oligonuclecotide primers for CtManf used were: forward 5-cacgctagc-
gcatattcccttcctg-3and 5-cacctcgagttaaagttcatccaagctgc-3. The PCR
amplication 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 nal extension at 72
°C for 10 min. The amplied products were run on 0.8% agarose gel
and puried by a gel extraction kit (Qiagen). The PCR-amplied
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,
supplemented with kanamycin (50 μgmL
Journal of Agricultural and Food Chemistry Article |J. Agric. Food Chem. 2013, 61, 123331234412334
for growth of recombinant clones. The positive clones were selected
by restriction digestion analysis of the recombinant plasmids.
Expression and Purication of CtManf and CtManT. E. coli
BL-21(DE3) (Novagen) cells were transformed for expression of
CtManf and CtManT as described elsewhere.
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 buer
pH 7.0, containing 1 mM phenylmethanesulfonyl uoride (PMSF).
Then the cells were sonicated (Sonics, Vibra cell) on ice for 16 min (9
s on/9 s opulse, 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 puried by
immobilized metal ion chromatography (IMAC).
The recombinant
proteins containing CtManf and CtManT appended by the His6tag
were puried 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 veried
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-(14)-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 v1) in 50 mM sodium
phosphate buer (pH 7.0) and by measuring the reducing sugar
released, as described previously.
In both cases, 100 μL of reaction
mixture contained 1.0 (%, w v1) substrate, 10 μL of enzyme (CtManf,
0.16 mg mL1and CtManT, 0.15 mg mL1). 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
A wide range of pH was chosen, ranging from pH 3.0 to 8.0 as β-
mannanse displays optimum range within this range.
For studying
the optimum pH prole of CtManf and CtManT, 50 mM sodium
phosphate buer, pH 3.08.0, was used for the enzyme assays that
employed 1.0 (%, w v1) 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 v1)carob
After the verication of substrate specicity, the enzyme assays for
CtManf and CtManT were performed using 50 mM sodium phosphate
buer 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).
Zymogram Study and Activity Staining. Zymogram study of
recombinant CtManf and CtManT were investigated by using 0.5% (w
v1) carob galactomannan as the substrate incorporated in 12% (w
v1) SDSPAGE. Ten micrograms of each of puried CtManf and
CtManT by IMAC was mixed with 1×the sample buer (62.5 mM
Tris-Cl, pH 6.8, 20% v v1glycerol, 2% w v1SDS, and 0.005% w v1
bromophenol blue)
without β-mercaptoethanol
were loaded on
the gel. After the completion of electrophoresis, the gels were
incubated in 2.5% (v v1) of TritonX 100 at 25 °C for 1 h followed by
1 h incubation in 50 mM sodium phosphate buer, pH 7.0. Then the
gels were incubated in preheated 50 mM sodium phosphate buer
(pH 6.5) at 55 °C for 30 min and then stained with 0.1% (w v1)
congo red for 45 min, as described by Aboul-Enein et al. (2010).
After congo red staining, the gels were counter-stained with 1 N HCl,
as described elsewhere.
Eect of Metal Ions, Chaotropic Agents, And Detergent on
Enzyme Activity. The eects of dierent 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 buer, of pH 6.9 and pH 6.5,
respectively. One-hundred microliters of the reaction mixture
containing carob galactomannan (1%, w v1) 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 mL1) with 1% (w v1) 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.
Then 0.2 μL of sample as well as of standard solutions (1.0 mg mL1)
were loaded on the TLC plate and kept in the developing chamber
saturated with the developing solution (mobile phase), which
consisted of acetic acidn-propanolwateracetonitrile
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 v1;α-napthol 5.0%, w v1).
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 mL1) with 1% (w v1) 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 ltered through a syringe
lter 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 ltered
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 ow rate was
maintained at 0.3 mL min1. 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 |J. Agric. Food Chem. 2013, 61, 123331234412335
were used as standards. The solutions of standards were also ltered
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 mL1stock
solutions. Quantitative analysis enzyme-catalyzed hydrolysis products
were determined from the peak intensity of the released products.
Production of CtManf and CtManT in Dierent Media. Media
screening was performed in order to obtain higher production of
recombinant CtManf and CtManT. Four dierent media were
screened, as listed in Table 4. All four media LB, 5xLB, Terric
Broth (TB), and TY media were prepared, as previously described by
Tripathi et al. (2009).
LB medium (100 mL) was prepared in a 250
mL ask by weighing constitutents in (%, w/v) were tryptone, 1.0;
yeast extract, 0.5; and NaCl, 1.0.
A 100 mL 5xLB medium was
prepared by using (%, w/v) tryptone, 5.0; yeast extract, 2.5; NaCl, 2.5;
glycerol, 1.0.
Terric 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.
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.
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 asks of four media
containing 100 mL medium supplemented with 50 μgmL
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).
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.
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 buer pH 7.0 containing 1 mM
phenylmethanesulfonyl uoride (PMSF). The cell suspensions were
sonicated (Vibra cell, Sonics) on ice for 16 min (9 s on/9 s opulse,
30% amplitude) and then centrifuged at 19000gat 4 °C for 20 min.
The recombinant proteins were puried by a single step, using
immobilized metal ion anity chromatography (IMAC) on HiTrap
chelating columns (GE Healthcare), as mentioned earlier.
concentration of IMAC-puried recombinant proteins were deter-
mined by the Bradford method.
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 mL1and
0.15 mg mL1, 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 v1) carob galactomannan
in 50 mM sodium phosphate buer 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 UVvisible spectrophotometer (Varian,
Cary 100-Bio), following the method of Dvortsov et al.
The puried
CtManf and CtManT at protein concentration of 0.3 mg mL1in 50
mM MES [2-(N-morpholino) ethanesulfonic acid] buer, pH 7.0,
were used. The absorbance at 280 nm was measured at dierent
temperatures, varying from 40 to 100 °C using a peltier temperature
controller. The protein solutions (1 mL, 0.3 mg mL1)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 mL1) 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
mL1) containing equimolar concentrations of 10 mM, and nally the
change in absorbance at 280 nm was measured. A curve of relative
derivative absorption coecient (rst derivative coecient) versus
temperature was plotted, as described earlier by Dvortsov et al.
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 mi (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 identier 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).
Cloning and Expression of CtManf and CtManT. The
open-reading frames of CtManT and CtManf amplied by
Figure 1. Phylogenetic tree showing the comparative study of our
query CtManT (highlighted in red box) with two dierent types of
GH26 mannanase (Man26A and Man26B) of representative members
and their appearance during evolution based on sequence similarity.
Journal of Agricultural and Food Chemistry Article |J. Agric. Food Chem. 2013, 61, 123331234412336
polymerase chain reaction resulted in 1449 bp and 1029 bp
sequences, respectively. The amplied 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 identied 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 puried by immobilized metal ion anity chromatography
(IMAC) from the cell-free extracts to homogeneity. The SDS
PAGE analysis of puried 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.
Specicity 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
specicities 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 specicity for galactomannan, and the highest
enzyme activity was achieved with carob galactomannan 97.0 ±
5.0 units mg1and 91.0 ±4.0 units mg1, 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 rst phase and then accessed the crystalline sites
(tougher region) in the second phase. Similar results were
reported by Mizutani et al. (2012).
The enzyme activities of
both enzymes with insoluble ivory nut mannan and β-(14)-
mannan from the rst phase were calculated (Table 1). CtManf
displayed approximately, two times higher activity than
CtManT with both the substrates (Table 1). The specic
activity of CtManf was 50.0 units mg1, whereas CtManT was
26.5 units mg1with ivory nut mannan and with β-(14)-
Figure 2. Zymogram study using 12% SDSPAGE (A) CtManf (panel 1: puried protein, 2: congo red staining, and 3: 1 N HCl counter staining)
and (B) CtManT (panel 1: puried protein, 2: congo red staining, and 3: 1 N HCl counter staining).
Table 1. Substrate Specicity of CtManf and CtManT from
C. thermocellum
(1%, w v1)specic activity CtManf
(units mg1)specic activity CtManT
(units mg1)
galactomannan 97.0 ±5.0 91.0 ±4.0
locust bean
galactomannan 85.4 ±6.0 83.1 ±5.0
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
cellulose 1.09 ±0.5 0.9 ±0.05
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
acid NA NA
Values are in mean ±SD (n= 3). NA = no activity was determined.
Journal of Agricultural and Food Chemistry Article |J. Agric. Food Chem. 2013, 61, 123331234412337
mannan the enzyme activities were 40.0 units mg1and 21.2
units mg1, 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.
Both CtManf and CtManT displayed low but signicant
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 eciency of
both the enzymes were determined with the natural substrates
(Table 2). CtManf and CtManT displayed turnover numbers
(kcat) of 698 and 684 min1, respectively, and catalytic
eciencies (kcat/Km) of 4.1 ×102and 3.9 ×102min1mg1
mL, respectively, with carob galactomannan. Both the enzymes
CtManf and CtManT eciently acted on insoluble ivory nut
mannan, showing catalytic eciencies (kcat/Km) of 3.4 ×102
and 2.4 ×102min1mg1mL and with β-(14)-mannan, 3.1
×102and 2.2 ×102min1mg1mL, 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-(14)-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).
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.
Specicity 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 specically cleaved the β-(14)-glycosidic linkages
between mannopyranosyl residues.
Zymogram Study of CtManf and CtManT. Separate
SDSPAGE 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-(14)-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-
congured substrate specicity.
Eects of Metal Ions and Chemical Agents on CtManf
and CtManT. The enzymatic activity of CtManf and CtManT
signicantly 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 aected 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 Eciencies of CtManf and CtManT from C. thermocellum ATCC 27405
Km(mg mL1)kcat (min1)kcat/Km(min1mg1mL)
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. Eects of Metal Ions and Other Agents on CtManf
and CtManT from C. thermocellum ATCC 27405
relative activity (%)
ions/reagents concentration (mm) CtManf CtManT
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
No additives were added in the control, and the activity was taken as
Journal of Agricultural and Food Chemistry Article |J. Agric. Food Chem. 2013, 61, 123331234412338
enzyme activity as EDTA and specically binds and chelates the
calcium ions in a 1:1 molar ratio.
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-(14) bonds of these manno-oligosacchar-
ides elegantly. Therefore, based on the signicant role in
specically cleaving the β-(14) bond, this enzyme was
classied and named as endo-β-(14)-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 mL1, 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 mL1mannose, 1.12 mg mL1
mannobiose, and 0.80 mg mL1mannotriose 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 mL1), mannobiose (1.81 mg mL1), and
mannotriose (0.90 mg mL1), with much higher peak
intensities (Figure 4D). After a 16 h incubation, the
mannobiose and mannotriose concentrations increased to 2.1
mg mL1and 1.1 mg mL1, 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 mL1,
2.3 mg mL1, and 1.4 mg mL1, respectively, were obtained
(Figure 4F). All the concentrations were determined from the
regression equation of mannose, mannobiose, and mannotriose
standard curves. Therefore, CtManf quite eectively hydrolyzed
10 mg mL1(1%, w v1) 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
Dierent Media. The highest concentration of recombinant
proteins were obtained from a 100 mL ask 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 L1dry 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 v1) was
incubated with CtManf for 10 min to 24 h, (B) mannobiose (1 mg
mL1) was incubated with CtManf for 10 min to 4 h, and (C)
mannotriose (1 mg mL1) 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:
Journal of Agricultural and Food Chemistry Article |J. Agric. Food Chem. 2013, 61, 123331234412339
after sonication and purication by IMAC obtained was 910
and 880 mg L1, respectively. Similar results were reported by
Tripathi et al. (2009),
where TY medium gave the highest cell
density of E. coli cells achieved, 1.12 g L1, and the recombinant
dengue protein was expressed at a concentration of 10.37 mg
CtManf and CtManT gave less DCW with TB medium
28 g L1and 26.9 g L1, respectively, as compared with TY
medium. The puried protein concentrations from TB medium
obtained were 500 mg L1and 410 mg L1, respectively, for
CtManf and CtManT. The dry cell weight (DCW) from 100
mL LB medium obtained 21 and 20 g L1for CtManf and
CtManT, respectively. The concentrations of puried protein
obtained from LB medium were 160 mg L1for CtManf and
150 mg L1for CtManT. These results are similar to those of
the earlier report of Tripathi et al. (2009).
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 L1, respectively, for CtManf and CtManT. The
recombinant CtManf and CtManT proteins after purication
obtained were 300 mg L1and 280 mg L1in 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 signicant changes in CtManf, as well as in
CtManT protein-melting proles. 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 enzymesubstrate 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 v1) treated by CtManf after (B) 1, (C) 4, (D) 8, (E) 16, and (F) 24 h.
Journal of Agricultural and Food Chemistry Article |J. Agric. Food Chem. 2013, 61, 123331234412340
of CtManf and CtManT, it could be concluded that both these
enzymes are thermostable and Ca2+ ions provide signicant
thermal stability.
The molecular architecture of full length CtManf from C.
thermocellum ATCC 27405 displayed a modular structure.
Sequence homology of the catalytic module CtManT identied
as Man26B, showed highest similarity with that of the C.
thermocellum F1 strain.
The Man26B enzymes have subtle
dierences 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.
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.
It is worth mentioning here about
the wide range of substrate specicity of both CtManf and
CtManT. Both these enzymes were able to hydrolyze β-(14)-
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-
Low activities of CtManf and CtManT against ivory nut
mannan and β-(14) 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 dierent
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-(14) (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),
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.
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),
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.
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 signicant turn over against other
soluble as well as insoluble substrates. It was reported
previously that carob galactomannan composed of 78%
mannose formed the β-(14)-mannan backbone, while
galactose contributes 22%.
Each β-(14)-mannan is sub-
stituted by the α-(16)-galactose side chain. The enzymes
randomly hydrolyze β-(14)-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-(14)-mannanases (endo-
Man26B) from C. thermocellum ATCC 27405. The perform-
ances of CtManf and CtManT were investigated under the
inuence 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 signicantly 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 unaected by lower concentrations of Mn2+,Ni
+, 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 dierent sites other than the α-glucosidase
active center or enzymesubstrate complex.
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 |J. Agric. Food Chem. 2013, 61, 123331234412341
EGTA, the enzyme activity sharply decreased. The enzyme
activity of CtManf and CtManT was adversely aected 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-(14)-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-congured substrates and oligosaccharides is
characteristic of a typical Man26B mannanase.
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-(14) bond
cleavage and had potential to produce manno-oligosaccharides
from carob galactomannan. Thus eective β-(14)-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.
Media composition plays a signicant role in production of
recombinant proteins.
Use of chemically dened medium is a
common practice in producing recombinant proteins.
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 eects of
media were stated earlier, while producing recombinant dengue
protein in E. coli.
The rich source of tryptone, yeast extract,
and phosphate salts facilitated to achieve highest cell densities
in TY media as compared to other chemically dened 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.
Higher concentration of phosphate is important for attaining
high cell densities of E. coli, as the lower concentrations of
phosphate limits the growth.
The phosphate salts in the
medium provided buering capacity against pH uctuations,
which adversely aects the metabolic activity of cells.
The low
cell densities and lower production of recombinant proteins in
LB and 5xLB medium were due to a lack of buering 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,
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).
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.
Because of the higher
uctuations 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.
Thus, the binding by
Ca2+ ion makes protein more conformationally stable at higher
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 rst 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-
congured substrates.
SSupporting Information
Molecular architecture of full length CtManf of C. thermocellum
ATCC 27405; multiple sequence alignment of CtManT with
Man26A from Cellulomonas mi; and biphasic hydrolysis
pattern of insoluble ivory nut mannan. This material is available
free of charge via the Internet at
Corresponding Author
*E-mail: Tel: (361) 258 2208. Fax:
(361) 269 0762.
The authors declare no competing nancial interest.
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 Scientic Training (CREST) Fellowship from the
Department of Biotechnology, Ministry of Science and
Technology to Arun Goyal.
Journal of Agricultural and Food Chemistry Article |J. Agric. Food Chem. 2013, 61, 123331234412342
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... Further research accessibility. Similar substrate preferences have previously been reported for recombinant β-1,4-mannanases from Clostridium thermocellum (Ghosh et al. 2013). ManH1 was not active on BG or CMC, this indicates that ManH1 exhibits a stringent specificity for mannan substrates containing mixed β-1,4 and α-1,6 glycosidic linkages and unable to cleave cellulosic substrates with either only β-1,4 (CMC) or mixed β-1,3 − 1,4 (BG) glycosidic linkages. ...
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β-mannanase catalyzes the hydrolysis of mannans β-1,4-mannosidic linkages to produce industrially relevant oligosaccharides. These enzymes have numerous important applications in the detergent, food, and feed industries, particularly those that are resistant to harsh environmental conditions such as salts and heat. While, moderately salt-tolerant β-mannanases are already reported, existence of a high halotolerant β-mannanase is still elusive. This study aims to report the first purification and characterization of ManH1, an extremely halotolerant β-mannanase from the halotolerant B. velezensis strain H1. Electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS) analysis revealed a single major peak with a molecular mass of 37.8 kDa demonstrating its purity. The purified enzyme showed a good thermostability as no activity was lost after a 48 h incubation under optimal conditions of 50 °C and pH 5.5. The enzyme’s salt activation nature was revealed when its maximum activity was obtained in the presence of 4 M NaCl, it doubled compared to the no-salt condition. Moreover, NaCl strengthens its resistance to thermal denaturation, as its melting temperature (Tm) increased steadily with increasing NaCl concentrations reaching 75.5 °C in the presence of 2.5 M NaCl. The Km and Vmax values were 5.63 mg/mL and 333.33 µmol/min/mL, respectively, using carob galactomannan (CG) as a substrate. The enzyme showed a significant ability to produce manno-oligosaccharides (MOS) from lignocellulosic biomass releasing 13 mg/mL of reducing sugars from olive mill wastes (OMW) after 24 h incubation. The results revealed that this enzyme may have significant commercial values for agro-waste treatment, and other potential applications.
... A general base catalyzed process takes place which involves water attacking the anomeric center of the intermediate, yielding the product and releasing the enzyme to its original state [56]. Only GH5 and 113 mannanases have been reported to exhibit transglycosylation activity, while no such reports have been recorded for GH26 mannanases [57,58]. ...
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A growing demand in novel food products for well-being and preventative medicine has attracted global attention on nutraceutical prebiotics. Various plant agro-processes produce large amounts of residual biomass considered “wastes”, which can potentially be used to produce nutraceutical prebiotics, such as manno-oligosaccharides (MOS). MOS can be produced from the degradation of mannan. Mannan has a main backbone consisting of β-1,4-linked mannose residues (which may be interspersed by glucose residues) with galactose substituents. Endo-β-1,4-mannanases cleave the mannan backbone at cleavage sites determined by the substitution pattern and thus give rise to different MOS products. These MOS products serve as prebiotics to stimulate various types of intestinal bacteria and cause them to produce fermentation products in different parts of the gastrointestinal tract which benefit the host. This article reviews recent advances in understanding the exploitation of plant residual biomass via the enzymatic production and characterization of MOS, and the influence of MOS on beneficial gut microbiota and their biological effects (i.e., immune modulation and lipidemic effects) as observed on human and animal health.
... is called as βmannanase, which causes random cleavage of β-mannosidic linkages in mannan backbone (Zhu et al., 2020). Depending on sequence similarity, mannanases from various sources, such as bacteria, fungi, and plants, have been categorized within glycoside hydrolase families 5, 26, 113, and 134 within carbohydrate-active database (Ghosh et al., 2013). ...
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Thermotoga maritima (Tma) contains genes encoding various hyperthermophilic enzymes with great potential for industrial applications. The gene TM1752 in Tma genome has been annotated as cellulase gene encoding protein Cel5B. In this work, the gene TM1752 was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. Interestingly, the purified enzyme exhibited specific activities of 416 and 215 U/mg on substrates galactomannan and carboxy methyl cellulose, which is the highest among thermophilic mannanases. However, the putative enzyme did not show sequence homology with any of the previously reported mannanases; therefore, the enzyme Cel5B was identified as bifunctional mannanase and cellulase and renamed as Man/Cel5B. Man/Cel5B exhibited maximum activity at 85°C and pH 5.5. This enzyme retained more than 50% activity after 5 h of incubation at 85°C, and retained up to 80% activity after incubated for 1 h at pH 5–8. The Km and Vmax of Man/Cel5B were observed to be 4.5 mg/mL galactomannan and 769 U/mg, respectively. Thin layer chromatography depicted that locust bean gum could be efficiently degraded to mannobiose, mannotriose, and mannooligosaccharides by Man/Cel5B. These characteristics suggest that Man/Cel5B has attractive applications for future food, feed, and biofuel industries.
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In this study, an acidophilic GH5 β-mannanase (TaMan5) from Trichoderma asperellum ND-1 was efficiently expressed in Pichia pastoris (a 2.0-fold increase, 67.5 ± 1.95 U/mL). TaMan5 displayed the highest specificity toward locust bean gum (Km = 1.34 mg/mL, Vmax = 749.14 μmol/min/mg) at pH 4.0 and 65°C. Furthermore, TaMan5 displayed remarkable tolerance to acidic environments, retaining over 80% of its original activity at pH 3.0-5.0. The activity of TaMan5 was remarkably decreased by Cu2+, Mn2+, and SDS, while Fe2+/Fe3+ improved the enzyme activity. A thin-layer chromatography (TLC) analysis of the action model showed that TaMan5 could rapidly degrade mannan/MOS into mannobiose without mannose via hydrolysis action as well as transglycosylation. Site-directed mutagenesis results suggested that Glu205, Glu313, and Asp357 of TaMan5 are crucial catalytic residues, with Asp152 playing an auxiliary function. Additionally, TaMan5 and commercial α-galactosidase displayed a remarkable synergistic effect on the degradation of galactomannans. This study provided a novel β-mannanase with ideal characteristics and can be considered a potential candidate for the production of bioactive polysaccharide mannobiose.
One of the emerging non-digestible oligosaccharide prebiotics is β-mannooligosaccharides (β-MOS). β-MOS are β-mannan derived oligosaccharides, they are selectively fermented by gut microbiota, promoting the growth of beneficial microorganisms (probiotics), whereas the growth of enteric pathogens remains unaffected or gets inhibited in their presence, along with production of metabolites such as short-chain fatty acids. β-MOS also exhibit several other bioactive properties and health-promoting effects. Production of β-MOS using the enzymes such as β-mannanases is the most effective and eco-friendly approach. For the application of β-MOS on a large scale, their production needs to be standardized using low-cost substrates, efficient enzymes and optimization of the production conditions. Moreover, for their application, detailed in-vivo and clinical studies are required. For this, a thorough information of various studies in this regard is needed. The current review provides a comprehensive account of the enzymatic production of β-MOS along with an evaluation of their prebiotic and other bioactive properties. Their characterization, structural-functional relationship and in-vivo studies have also been summarized. Research gaps and future prospects have also been discussed, which will help in conducting further research for the commercialization of β-MOS as prebiotics, functional food ingredients and therapeutic agents.
β-Mannanase (EC is an enzyme that cleaves within the backbone of mannan-based polysaccharides at β-1,4-linked D-mannose residues, resulting in the formation of mannooligosaccharides (MOS), which are potential prebiotics. The GH26 β-mannanase KMAN from Klebsiella oxytoca KUB-CW2-3 shares 49–72% amino-acid sequence similarity with β-mannanases from other sources. The crystal structure of KMAN at a resolution of 2.57 Å revealed an open cleft-shaped active site. The enzyme structure is based on a (β/α) 8 -barrel architecture, which is a typical characteristic of clan A glycoside hydrolase enzymes. The putative catalytic residues Glu183 and Glu282 are located on the loop connected to β-strand 4 and at the end of β-strand 7, respectively. KMAN digests linear MOS with a degree of polymerization (DP) of between 4 and 6, with high catalytic efficiency ( k cat / K m ) towards DP6 (2571.26 min ⁻¹ m M ⁻¹ ). The predominant end products from the hydrolysis of locust bean gum, konjac glucomannan and linear MOS are mannobiose and mannotriose. It was observed that KMAN requires at least four binding sites for the binding of substrate molecules and hydrolysis. Molecular docking of mannotriose and galactosyl-mannotetraose to KMAN confirmed its mode of action, which prefers linear substrates to branched substrates.
Galactomannan (GM) in legumes and acetyl-galactoglucomannan (AcGGM) in softwoods are wide-spread β-mannans. Their depolymerization is catalyzed by β-mannanases. We have investigated a cell-surface exposed and galactose-tolerant β-mannanase (BoMan26B) from the abundant gut bacterium Bacteroides ovatus. Glycosidases from the gut microbiota have potential for production of prebiotics, such as dietary saccharides that would promote beneficial bacteria in the gut. BoMan26B was explored for production of potential prebiotics. Using the above β-mannans as substrate we investigated the product profiles using a herein developed new high-resolution anion-exchange chromatography procedure. The produced linear and galactosyl-decorated β-mannan-oligosaccharides (MOS/GMOS) were mainly of degree of polymerization (DP) 2–6, consistent with the glycan-binding subsites of BoMan26B. Some GM and AcGGM products were acetylated. DP 2–6 MOS were produced at a yield of 30 and 33% (w/w) from GM and AcGGM, respectively. In addition, about as much DP 2–6 GMOS were produced, assessed using guar α-galactosidase as analytical aid. Growth studies using the human gut bacteria Bifidobacterium adolescentis ATCC 15703 (acetate producer) and Roseburia hominis DSMZ 6839 (butyrate producer) revealed significant differences in utilization of specific MOS/GMOS. The prebiotic potential of the MOS/GMOS generated by BoMan26B was further underlined by the observation that both bacterial strains produced short-chain fatty acids.
The production of bioenergy from wastes attracts worldwide attention to overcome energy crisis and increasing pollution (Thakur et al., Microbial fermentation and enzyme technology, Taylor and Francis Group, Boca Raton, FL, 257–268, 2020). Lignocellulosic biomass can serve as an alternative source for bioenergy production. Thermostable enzymes can hydrolyze the lignocellulosic biomass and produce reducing sugars, which can be fermented to produce bioethanol by using fermenting microbes. Clostridium thermocellum is a gram-positive, anaerobic and rod-shaped, thermophilic microorganism having great potential applications. It can directly transform lignocellulosic biomass into valuable products such as acetate, ethanol, formate, and lactate. Clostridium thermocellum expresses a multi-enzyme complex bound to scaffoldin proteins called cellulosome that contains cellulolytic, hemicellulolytic, and other carbohydrate degrading enzymes. The thermophilic enzymes possess wide applications in several industries for producing sustainable green products. This chapter evaluates the production and properties of recombinant thermostable cellulases, hemicellulases, and pectinases from C. thermocellum, their structure, and applications in different industrial processes.
A novel and efficient method for manno-oligosaccharides (MOS) production has been proposed by utilizing Gleditsia microphylla galactomannan as the starting material. This co-operative hydrolysis using ferrous chloride (Fe²⁺) and acetic acid (HAc) effectively improved the MOS yield and meanwhile decreased the amount of monosaccharide and the 5-hydroxymethyl-furfural (HMF). The highest yields under the optimum conditions were 46.7% by HAc hydrolysis (5 M HAc at 130 °C for 120 min); 37.3% by Fe²⁺ hydrolysis (0.1 M Fe²⁺ at 150 °C for 120 min); and 51.4% by co-operative hydrolysis (2 M HAc, 0.05 M Fe²⁺ at 160 °C for 10 min). From the changes in the value of M/G (mannose/galactose) ratios, it was deduced that Fe²⁺ predominantly cleaves the main chain, and HAc assists in the breakage of the side chain, thus resulting in the high-efficient co-operative hydrolysis for the production of MOS.
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Phytase is added to animal feeds to improve phosphorus absorption and reduce phosphorus excretion of swine and poultry. It hydrolyses phytate, which is a major form of storage of cereal grains and legumes in commercial animal feeds, into myo-inositol and inorganic phosphate. Adequate thermostability of phytase is necessary for field application as diets for swine and poultry are normally pelleted at high temperatures (60-80°C). In this study, Molecular Dynamics simulation (MD) was used to study the effect of calcium metal ions on the thermostability of Bacillus amyloliquefacience phytase. MD simulations of the enzyme in the calcium-loaded and calcium-free states were performed at 60°C (333 K) and 80°C (353 K) in water as the solvent medium, for duration of 4 ns. Root mean square deviations (RMSD) of calcium-bound residues, backbone atoms, and other secondary structures were found to be lower in the presence of calcium ions at both temperatures. In addition, calcium-loaded enzyme was found to have fewer numbers of hydrogen bonds and salt bridges at both temperatures, yet calcium-loaded enzyme remains more thermostable due to network of electrostatic interactions induced by calcium binding. The binding effect of calcium ions becomes weaker at 80°C and the small increase of binding effect offered by the calcium ions at higher temperatures in not sufficient enough to maintain the activity at this temperature. It is proposed that suitable mutations at the coil region would lead to increase in the stability of the enzyme at high temperatures.
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The microbial deconstruction of the plant cell wall is a key biological process that is of increasing importance with the development of a sustainable biofuel industry. The glycoside hydrolase families GH5 (PaMan5A) and GH26 (PaMan26A) endo-beta-1,4-mannanases from the coprophilic ascomycete Podospora anserina contribute to the enzymatic degradation of lignocellulosic biomass. In this study, P. anserina mannanases were further subjected to detailed comparative analysis of their substrate specificities, active site organization and transglycosylation capacity. Although PaMan5A displays a classical mode of action, PaMan26A revealed an atypical hydrolysis pattern with the release of mannotetraose and mannose from mannopentaose resulting from a predominant binding mode involving the -4 subsite. The crystal structures of PaMan5A and PaMan26A were solved at 1.4Å and 2.85Å resolution, respectively. Analysis of the PaMan26A structure supported strong interaction with substrate at the -4 subsite mediated by two aromatic residues W244 and W245. The PaMan26A structure appended to its family 35 carbohydrate-binding module revealed a short and proline-rich rigid linker that anchored together the catalytic and the binding modules.
The effect of mannooligosaccharides (MOS) obtained from coffee mannan on cecal microbiota and short chain fatty acid production was examined in male Sprague-Dawley rats. The rats were given water and a dietary treatment containing 5% MOS ad libitum for 28 days. The body weight of those fed the MOS diet showed no significant difference compared with rats that consumed the control diet. The consumption of MOS increased the concentration of bifidobacteria (p < 0.05) and the ratio of bifidobacteria to total microbes (p < 0.05). The addition of MOS resulted in a significantly higher (p < 0.05) concentration of short chain fatty acids in the cecal contents compared with the control diet. The concentrations of acetate, propionate and butyrate were higher (p < 0.05) in rats fed the MOS diet compared with the control diet. These results suggest that MOS in the 5% diet promotes bifidobacteria growth and increased production of short chain fatty acids in rats.
Digestibility of mannooligosaccharides obtained from thermal hydrolysis of spent coffee grounds was examined by in vitro digestion method. Mannooligosaccharides were resistant to human salivary a-amylase, artificial gastric juice, porcine pancreatic enzymes and rat intestinal mucous enzymes. Fermentation products of mannooligosaccharides in human large intestine were estimated by in vitro fecal incubation method. Mannooligosaccharides were fermented by human fecal bacteria and the products of fermentation were short chain fatty acids. Acetic, propionic and n-butyric acids were the main short chain fatty acids as end fermentation products. These results suggest that mannooligosaccharides are indigestible saccharides and are converted to short chain fatty acids in human large intestine. The short chain fatty acids are thought to improve the large intestinal environment. Moreover, they are absorbed and utilized by the host as an energy source.
The multi-modular non-cellulosomal endo-1,3(4)-beta-glucanase Lic16A from Clostridium thermocellum contains a so-called X module (denoted as CBMX) near the N terminus of the catalytic module (191-426 aa). Melting of X-module-containing recombinant proteins revealed an independent folding of the module. CBMX was isolated and studied as a separate fragment. It was shown to bind to various insoluble polysaccharides, including xylan, pustulan, chitin, chitosan, yeast cell wall glucan, Avicel and bacterial crystalline cellulose. CBMX thus contains a hitherto unknown carbohydrate-binding module (CBM54). It did not bind soluble polysaccharides on which Lic16A is highly active. Ca2+ ions had effects on the binding, e.g. stimulated complex formation with chitosan, which was observed only in the presence of Ca2+. The highest affinity to CBMX was shown for xylan (binding constant K=3.1x10(4) M(-1)), yeast cell wall glucan (K=1.4x10(5) M(-1)) and chitin (K=3.3.10(5) M(-1) in the presence of Ca2+). Lic16A deletion derivatives lacking CBMX had lower affinity to lichenan and laminarin and a slight decrease in optimum temperature and thermostability. However, the specific activity was not significantly affected.
Enhanced production of recombinant dengue multi-epitope protein in Escherichia coli was achieved by optimization of culture medium. Complex media (Luria Bertani broth, Terrific broth, Super broth, M9 minimal media, five times Luria Bertani broth, semi-defined medium enriched with tryptone and yeast extract, and semi-defined medium enriched with glucose) were evaluated for production of recombinant dengue multi-epitope protein in shake flasks. The recombinant protein was further produced by fed-batch fermentation using 5 L bioreactor. Cells were grown in optimized semi-defined medium, and feeding was carried out with 5X medium and glycerol. When growth reached 14.35 g/L of dry cell weight, culture was induced with 0.5 mM IPTG and further grown for 4 h to reach 18.37 g/L dry cell weight. The recombinant dengue multi-epitope protein was purified from inclusion bodies under denaturing conditions using metal affinity chromatography, which yielded 96.43 mg/L of protein. The purified protein was found to be reactive with dengue-infected human serum samples.
Mannanases can be useful in the food, feed, pulp and paper industries. In this research a Bacillus subtilis strain (named Bs5) which produced high-level beta-mannanase was isolated. Maximum level of beta-mannanase (1231.41 U/ml) was reached when Bacillus subtilis Bs5 was grown on konjac powder as the carbon source for nine hours at 32 degrees C. The beta-mannanase was a typical cold-active enzyme and its optimal temperature of 35 degrees C was the lowest among those of the known mannanases from bacteria. In addition, the optimal pH was 5.0 and much wide pH range from 3.0-8.0 was also observed in the beta-mannanase. These properties make the beta-mannanase more attractive for biotechnological applications. The DNA sequence coding the beta-mannanase was cloned and the open reading frame consisted of 1089 bp encoding 362 amino acids. A phylogenetic tree of the beta-mannanase based on the similarity of amino acid sequences revealed that the beta-mannanase formed a cluster with the beta-mannanases of Bacillus subtilis, which was separated from the mannanases of fungi and other bacteria. The beta-mannanase gene could be expressed in Escherichia coli and the recombinant beta-mannanase was characterized by Western blot. This study provided a new source of carbohydrate hydrolysis enzyme with novel characteristics from Bacillus subtilis.