Biocatalysis and Agricultural Biotechnology 24 (2020) 101532

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Biocatalysis and Agricultural Biotechnology

The production of β-mannanase from Kitasatospora sp. strain using

submerged fermentation: Purification, characterization and its potential in
mannooligosaccharides production
Yopi a, b, *, Nanik Rahmani a, Siti Amanah a, Pugoh Santoso a, Puspita Lisdiyanti a
a Research Center for Biotechnology, Indonesian Institute of Sciences, Komplek Cibinong Science Center CSC-LIPI, Jl. Raya Bogor Km.46, Cibinong, West Java, 16911,
Indonesia
b Research Center and Human Resource Development, National Standardization Agency (BSN), Gedung I BPPT, Jl. H. M Thamrin No. 8, Kebun Sirih, Jakarta Pusat,

10340, Indonesia

ARTICLE INFO ABSTRACT

Keywords: Actinomycetes have been identified as one of the most diverse groups of microorganisms that play a vital role
Kitasatospora sp. in the production of enzymes and nutraceuticals. In addition, prior studies on the wild type strain of Ki-
β-mannanase
tasatospora sp. emphasized on its ability to exhibit high β-mannanase activity. This study aimed to purify,
Mannan polymer
characterize and evaluate the potential of this strain in the production of mannooligosaccharides using man-
Mannooligosakarida
Submerged fermentation
nan polymer. The enzyme was produced by submerged fermentation of a medium contains locus bean gum as
a carbon source. The crude mannanase was subjected to polyethylene glycol precipitation and ion exchange
chromatography. The completion of the purification process was confirmed by SDS-PAGE and purified of en-
zyme characterization were investigated, analysis hydrolysis product was conducted by TLC. The enzymes ex-
hibited the activity of 37.0 U/mL. A purification factor of 1.4-fold was achieved with specific activity of 6.3
U/mg. An increase of activity was recorded from 15.0 U/mL and 4.4 U/mL to 19.3 U/mL and 6.3 U/mL. In
addition, the total protein decreased from 338.5 mg/mL to 45.7 mg/mL. The purified β-mannanase has the
molecular weight was approximately 37.0 kDa with optimal activity at pH 6.0 and 60 °C and relatively stabil-
ity at a pH variety of 6.0–9.0, retaining > 90% activity. This product was capable of hydrolysing various man-
nan polymers (porang potato, palm sugar fruit, coconut cake, palm cernel cake) and other commercial mannan
(LBG, β-mannan, konjac, ivory nut), subsequently producing various sizes of mannoligosaccharides and man-
nose potential for food and feed industry.

1. Introduction portant representatives of this class. The mannan, an important of the

The second most abundant polymers present in nature after cellu-
lose is hemicellulose (Harris and Stone, 2009), and it is predicted to
contribute for one third of the total plant component (Chaikumpollert
et al., 2004). Hemicellulose is a complex group of heterogeneous
polymers and represents one of the major sources of renewable or-
ganic matter (Moreira and Filho, 2008). Mannan and heteromannan
as part of hemicelluloses in plant tissue and in the wall of higher
plants are polysaccharides that are widely distributed in nature
(Capek et al., 2000; Moreira and Filho (2008); Scheller and Ulvskov
(2010)). Furthermore, hetero-1,4-β-D-mannans is one of the most im-
hemicelluloses family, can be classified in four subfamilies, such as
linear mannan, glucomannan, galactomannan, and galactoglucoman-
nan (Petkowicz et al., 2001). Linear mannans are presents in a aloe
vera, ivory nuts, the endosperms of palmae such as, palm sugar fruits;
galactomannan are presents in a locus bean gum, guar gum and tara
gum; whereas glucomnanan are presents in a Amorphopallus spesies,
porang potato ((Moreira and Filho, 2008). Mannanases are the second
most significant enzymes attributed for the hydrolysis of hemicellu-
loases (Chauhan et al., 2012). Moreover, β-mannanase, also known as
1,4-β-d-mannaanase (EC number of 3.2.1.78) is an essential enzyme
required for hydrolysing β-1, 4- linkages in the mannan backbone

* Corresponding author. Research Center and Human Resource Development, National Standardization Agency (BSN), Gedung I BPPT, Jl. H. M Thamrin No. 8,

Kebun Sirih, Jakarta Pusat, 10340, Indonesia.

E-mail address: yopi@bsn.go.id, yopi@bioteknologi.lipi.go.id.

https://doi.org/10.1016/j.bcab.2020.101532
Received 15 October 2019; Received in revised form 31 January 2020; Accepted 5 February 2020
Available online 7 February 2020
1878-8181/© 2020 Elsevier Ltd. All rights reserved.
Yopi et al.
Fig. 1. Production activities of β-mannanase from Kitasatospora sp. on the LBG
substrate on different day's fermentation.
(Olaniyi et al., 2014). This effect is conferred on the abundant man-
nan-rich polymers, including glucomannan, β-1, 4-mannans and galac-
tomannan, in an attempt to produce manno-oligosaccharides
(Songsiriritthigul et al., 2010; Kumagai et al., 2013; Harnpicharnchai
et al., 2016; Rahmani et al., 2017), and a small amount of sugar
monomer (mannose, glucose and galactose) (Olaniyi et al., 2014;
Songsiriritthigul et al., 2010; Dhawan and Kaur, 2007).
Specifically, mannooligosaccharides are well-known to have bioac-
tivity in several applications, including as a dietary supplement to as-
sist in the improvement of growth performance, gut health, and im-
mune responses in marine organisms, (Sang and Fotedar, 2010).
Meanwhile, β-mannanases have been broadly applied in pulp and pa-
per processing (Clarke et al., 2000; Pan et al., 2011), feed (Kong et al.,
2011; Lv et al., 2013), food (Adiguzel et al., 2015), pharmaceuticals,
oil, textile and detergent/laundry industry (Srivastava and Kapoor,
2014). For this purpose, various enzymes have previously been puri-
fied using various methods and characterized from a variety of mi-
crobes, encompassing purification of Streptomyces galbus NR using am-
monium sulfate precipitation, further purification was done by gel fil-
tration followed by ion exchange chromatography (Amany et al.,
2004); Bacillus subtilis WY34 using ion exchange chromatographic and
gel filtration methods (Jiang et al., 2006); Bacillus cereus N1 using
concentrating using ultrafiltration Amicon, anion-exchange column
chromatography of the DEAE-sepharose, and gel filtration technique
of 32/60 Sephacryl S-100 HR (El-Sharouny et al., 2015); and Bacillus
sp. CSB39 using ammonium sulfate precipitation and continued by
Sepharose CL-6B and DEAE Sepharose methods (Regmi et al., 2016).
In addition, studies have shown the strain Kitasatospora sp. to be
an excellent producer of extracellular mannanase (Rahmani et al.,
2017), and its purification was deemed necessary to obtain the most
significant yield, with the highest activity and the greatest possible
purity for the futuristic applications, such as for mannooligosaccha-
rides production. Therefore, the objectives of this present work were
to evaluate the purification and characterization of β-mannanase ob-
tained from Kitasatospora sp., and to also analyse its potential in man-
nooligosaccharide production, using various mannan polymer.
Table 1
Purification scheme of the β- mannanase from Kitasatospora sp.
Purifications steps Volume (ml) β- mannanase activity (U/ml)
β- mannanase crude extract 100 15.0
Concentration using PEG 10 74.2
HiTrap Q FF column 15 19.3
Biocatalysis and Agricultural Biotechnology 24 (2020) 101532
2. Materials and methods
2.1. Strains, materials, and chemicals
Kitasatospora sp. strain was collection of Biotechnology Culture
Collection (BTCC), Indonesian Institute of Sciences (LIPI), and then
preliminary identification of its capability to produce β-mannanase
was evaluated. In addition, the strain was made available under regis-
tration number BTCC B-606, and it was already registered in
Genebank, with accession number KY576672 (Rahmani et al., 2017).
Locust bean gum (LBG) and standard mannose (M1) were supplied
from Sigma-Aldrich (St. Louis, MO, USA), while mannooligosaccha-
rides, encompassing mannobiose (M2), mannotriose (M3), mannote-
traose (M4), mannopentaose (M5), and mannohexaose (M6) were sup-
plied from Megazyme (Wicklow, Ireland).
2.2. Cultivation and enzyme production
Kitasatospora sp. was maintained on ISP2 agar slants at 28 °C, a
medium that was previously optimized with 2% carbon source of
LBG, to promote β-mannanase production. Subsequently, a single
colony was inoculated, and the solution incubated at a temperature of
28 °C and shaken at 190 rpm in an orbital shaker for three days.
Therefore, the culture was inoculated in 300 mL production medium,
with the components optimized as follows: 0.073% peptone, 0.05%
yeast extract, 0.14% (NH4)2SO4, 0.2% KH2PO4,0.03% MgSO47H2O,
0.03% CO(NH2)2, 0.039% CaCl2, 0.0005% FeSO47H2O, 0.098%
MnSO47H2O, 0.00014% ZnSO47H2O, and 0.00037% CoCl2 in 2 L Er-
lenmeyer flask. Meanwhile, fermentation was conducted for seven
days, at an agitation rate of 190 rpm, and 28 °C, using the rotary
shaker (Taitex), where the β-mannanase activity was analyzed by
withdrawing samples every 24 h. Furthermore, the recovery of crude
enzyme supernatant was performed at 13,000 rpm, and 4 °C for
15 min.
2.3. Enzyme assays
β-mannanase activity was performed according to the method of
Miller (1959), using the 3,5-dinitrosalicylic acid (DNS), by measuring
the rate at which the reducing sugar was released, shown by an in-
crease in absorbance at 540 nm. In addition, the reactions contained
0.25 mL of diluted enzyme, 0.25 mL of substrate solution (1% LBG in
50 mM sodium phosphate buffer, pH 6.0). Subsequently, the assay
was examined at 60 °C, exactly 15 min after, and stopped when
0.5 mL of the DNS solution was added, boiled at 100 °C for 15 min,
and then chilled on ice. Furthermore, a blank was prepared absence a
substrate, and processed with similar conditions as in the sample test
tubes. The amount of enzyme needed to hydrolyse mannan resulting
1 μmol of reducing sugar per minute, according to the assay condition
was determined as one unit of enzyme activity.
2.4. Enzyme purification
2.4.1. Polyethylene glycol (PEG) precipitation (Ingham, 1984)
The free crude enzyme filtrates of cells were precipitated using
PEG, at 4 °C for 4 h, while the supernatant was recovered using the
Total protein (mg) Total activity (Units) Specific activity (U/mg) Purification (fold) Yield (%)
338.5 1503.2 4.4 1 100
38.2 741.5 19.4 4.4 49.3
45.7 288.7 6.3 1.4 19.2
2
Yopi et al.
Fig. 2. Elution profile of the β-mannanase obtained from Kitasatospora sp. in HiTrap Q FF anion exchange column chromatography, showing a single activity
peak.
Fig. 3. The molecular weight and zymogram of the crude supernatants, before
and after precipitation by PEG, and purification by anion exchange. STD: mol-
ecular weight marker, lane 1, β-mannanase crude extract Kitasatospora sp.,
lane 2, crude enzyme after precipitation by PEG; lane 3, protein solution from
elution steps.
dialysis tubing cellulose membrane (Sigma-Aldrich) at 4 °C, with the
change of the same buffer four times.
2.4.2. Ion exchange chromatography
The dialyzed enzyme was loaded with a volume sample injection
of 300 μL, and volume fraction of 1 mL, onto a HiTrap Q FF column
(5 × 5 mL) (GE) that was initiated by equilibration with 50 mM
sodium phosphate buffer pH 6.0, using ÄKTAprime plus (GE). Fur-
thermore, the unbound inactive protein was eluted using the starting
buffer, and a linear gradient of 50 mM sodium phosphate buffer main-
tained at pH 6.0, contain 1000 mM NaCl was applied in the elution of
bound protein. These were further measured using spectrophotometer
at 280 nm, while the enzyme activity was evaluated based on the
standard enzyme assay. Moreover, the experiments were conducted
five times, and the pooled fractions of the purified variety summed up
to 15 mL.
3
Biocatalysis and Agricultural Biotechnology 24 (2020) 101532
2.5. Determine of protein concentration of each stage purification
The protein concentrations in the active fractions were assayed by
measuring the absorbance using spectrophotometer at 280 nm with
bovine serum albumin (BSA) standard (Stoscheck, 1990).
2.6. Molecular weight of purified enzyme from Kitasatospora sp.
The molecular weight of β-mannanase was obtained by SDS-PAGE,
which was carried out with 12.5% (w/v) polyacrylamide gel accord-
ing to Laemmli (1970), using low molecular weight standard proteins
as markers. Prior to this, the crude and purified forms were denatured
with sample buffer (100 mM Tris buffer pH 8.0, 10% SDS, 10 mM
mercaptoethanol, 10% glycerol, 0.001% bromophenol blue), at 95 °C
for 5 min. Furthermore, protein band staining was conducted with
coomassie brilliant blue G-250.
Zymogram gel electrophoresis was performed according to
Piwpankaew et al. (2014) with modification by adding 0.5% (w/v)
LBG to the polyacrilamide gel, and the samples were subsequently de-
natured at 70 °C for 20 min before applying to the PAGE. Staining re-
quired incubating the gel in 2.5% Triton-X-100 for 60 min, and then
50 mM sodium phosphate buffer, at pH 6.0 and 35 °C for 1 h. This
was then stained with congo red solution for 30 min, and the process
was terminated by the immersing into a 1000 mM NaCl solution, fol-
lowed by washing with 0.05% acetic acid to create a clearer zone.
2.7. Purified enzyme characterization
2.7.1. Influence and stability of pH on purified β-mannanase activity
Influence of pH on enzyme activity was assayed using LBG sub-
strate and the optimal value was determined using various buffer so-
lutions in pH ranging from 4.0-9.0, encompassing 50 mM sodium ac-
etate (at 4.0–5.0), 50 mM sodium phosphate (6.0–8.0), and 50 mM
glycin-NaOH (9.0) at 30 °C. These were used in the determination of
pH stability for the purified enzyme, through incubation at room tem-
perature for one night. Furthermore, the percentage remaining activi-
ties were measured in relation with the starting values achieved at
0 h.
Yopi et al. Biocatalysis and Agricultural Biotechnology 24 (2020) 101532

Fig. 4. The influence of pH, temperature and stability on enzyme activity. (A) Optimum pH was assayed at 60 °C, using various pH buffers. (B) Its stability on β-
mannanase was assayed by incubation with different buffers (4–9) at room temperature overnight, and the relative activity was evaluated under standard assay
conditions. (C) Optimal temperature was performed at varying temperatures and optimal pH 6.0., while (D) Temperature stability of the purified enzyme was ob-
tained by storing at various temperatures for 1h. Furthermore, all determinations took place in triplicate, demonstrated as mean ± standard deviation.

Fig. 5. Substrate specificity of mannanase.

2.7.2. Influence and stability of temperature on purified β-mannanase
activity
The influence of temperature on the activity of purified enzyme
obtained from Kitasatospora sp. was assayed by evaluation in different
conditions from 30 to 90 °C, using 50 mM sodium phosphate buffer
(pH 6.0). These were then separately incubated for 30, 60, 90,
120 min at 55 °C, 60 °C and 65 °C. Furthermore, the temperature sta-
bility was investigated by comparing the residual with the initial ac-
tivity.
2.7.3. Substrate specificity
The substrate specificity of purified enzyme was assayed by incu-
bating the enzymes in 50 mM sodium phosphate buffer pH 6.0, which
contain 0.5% of each substrate, at 60 °C for 15 min, using the DNS

4
Yopi et al.
Fig. 6. Thin-layer chromatography analysis of (A) LBG, (B) Ivory nut (C) Porang potato, (D) Palm sugar fruits hydrolyzed by the purified β-mannanasefrom Ki-
tasatospora sp. The Incubation times (h) are indicated as Lane 1: standard M1, M2, M3, M4, M5, M6 and lane 2 standard Galactose, lane 3- lane 7 sample hy-
drolyzed hour to 0, 2, 4, 6 and 8; lane 8 purified β-mannanase.
method. In addition, the hydrolytic activity against LBG, β-mannan,
konjac powder, ivory nut, porang potato, palm sugar fruits, coconut
cake, and palm cernel cake were also evaluated.
2.8. Degradation of mannan polymers
The purified β-mananase was applied in the hydrolysis of various
mannan polymers, including palm kernel cake, porang potato, co-
conut cake, palm sugar fruit and commercial forms, encompassing lo-
cus bean gum, konjac, ivory nut, and β-mannan. This process was con-
ducted on each substrate (2% (w/v)) within a 3 mL volume reaction
consisting of 50 mM sodium phosphate buffer (pH 6.0), and 3.0 μg/μL
of purified β-mannanase, and subsequently incubated in a shaker in-
cubator at an agitation rate of 190 rpm, and a temperature of 40 oC
for 8 h. Furthermore, the reaction mixture was collected at 0, 2, 4, 6
and 8 h, and subsequently boiled at 100 °C for 5 min, in an attempt to
terminate the hydrolysis process. Meanwhile, the hydrolysis of gluco
and galacto-mannan substrates via purified β-mannanase was ana-
lyzed with TLC spotting on a Silica gel 60F254 (EMD/Merck
20 × 20 cm, Darmstadt, Germany) according to the methods of
Rahmani et al. (2017) and the standards adopted in this investigation
include M1 to M6.
3. Results and discussion
3.1. The β-mannanase enzyme production pattern by Kitasatospora sp.
The β-mannanase enzyme activities produced by Kitasatospora sp.
were analyzed at the inception of this study, as they were sourced
5
Biocatalysis and Agricultural Biotechnology 24 (2020) 101532
mainly from extracellular and inducible variety (Dhawan and Kaur,
2007). This production process was conducted in a culture medium,
supplemented with LBG as a galactomannan-rich substrate, which has
been widely adopted as a mannanase enzyme inducer (Kote et al.,
2009; Kim et al., 2011). Fig. 1 shows β-mannanase secretion lines, up
to the 72nd hour, and these activities increased to a peak value after
the 7 days’ cultivation period. Therefore, the enzymes produced sub-
sequently were applied in the selection of a suitable incubation time
for Kitasatospora sp., which is important in this assay.
3.2. Purification of β-mannanase
The β-mannanse crude extract was purified using a combination of
PEG precipitations, dialysis tubing, cellulose membrane and chro-
matographic techniques, in order to obtain the homogeneity, and the
summary of these steps are presented in Table 1. This shows that the
first step involves the use of PEG in the precipitation of crude en-
zymes solution for 4 h, followed by desalting through a dialysing tub-
ing cellulose membrane, against a 0.02 M sodium phosphate buffer
pH 6.0, one night, at 4 °C. Therefore, the dialysis enzyme was loaded
onto HiTrap Q FF column for further purification, which produced
one peak, represented as Fig. 2, and then the process of refining a 1.4-
fold required the use of 6.3 U/mg specific activity (Table 1). Hence,
enzyme and specific activity increased from 15.0 U/mL and 4.4 U/mL
in the crude form to 19.3 U/mL and 6.3 U/mL, after the anion ex-
change, respectively. Also, the total protein was observed to have de-
creased from 338.5 mg/mL to 45.7 mg/mL.
Morever, the molecular weight of the crude supernatants, before
and after precipitation by PEG, and purification were analyzed using
the SDS PAGE, which reveals the presence of a single clear band, indi-
Yopi et al.
Fig. 7. Thin-layer chromatography analysis of (A) β-mannan (B) Palm cernel cake, (C) Coconut cake, (D) Konajc hydrolyzed by the purified β-mannanase obtained
from Kitasatospora sp. The incubation times (h) are indicated as Lane 1:standar M1, M2, M3, M4, M5, M6 and lane 2 standard Galactose, lane 3- lane 7 sample hy-
drolyzed hour to 0, 2, 4, 6 and 8; lane 8 purified β-mannanase.
cating β-mannanase activity, with molecular weight estimated to be
approximately 37.0 kDa (Fig. 3). In addition, the zymogram analysis
was conducted with PAGE, possessing 0.1% LBG content, in an at-
tempt to detect the activity and purity of the enzyme obtained from
the crude supernatants, before, and after the treatment. Furthermore,
its mannolytic capacity was obtained through the assessment of the
clear zone identified in the gel after the Congo-red staining which
clearly indicates high hydrolytic activity toward LBG. This result is
very interesting, the single band of protein on the SDS-PAGE is very
thin, but the prominent clear zone in the zymogram was seen very
thick on the same region as that of the single band of protein on the
SDS-PAGE. Similar result was obtained with β-mannanase, where an
estimated size of molecular weight 36.7 kDa, on products obtained
from Bacillus subtilis ATCC 11774 (Aziz et al., 2017). Moreover, the
purified mannanase from Cellulosimicrobium sp. strain HY-13 was ob-
served to have molecular mass of approximately 35 kDa (Kim et al.,
2011), while the product from Streptomyces lividans 66 exhibited val-
ues of about 36 kDa (Arcand et al., 1993). The molecular weight of
mannanase from Kitasatospora sp. was smaller than that of the β-
mannanase from Bacillus subtilis WY34 that have been found to pos-
sess estimated to be of size 39.6 kDa (Jiang et al., 2006).
3.3. Purified enzyme characterization
3.3.1. Influence and stability of pH on purified of β-mannanase activity
The influence of pH on the activity of purified β-mannanase was
conducted by assaying the enzyme activity over a pH variety of 4–9,
as presented in Fig. 4A. This showed that there is a higher propensity
for an enzyme to retain majority of its activity (>60%) at a range of
6–8, with 6.0 as the optimum. The optimum pH of β-mannanase from
6
Biocatalysis and Agricultural Biotechnology 24 (2020) 101532
Kitasatospora sp. was the same as that of β-mannanase from Bacillus
subtilis BE-91 (pH 6.0) (Cheng et al., 2016), Aspergillus terreus (Soni et
al., 2016). Furthermore, the influence of pH on the stability of β-
mannanase is displayed in Fig. 4B, which was obtained through its ex-
posure to pH 4.0 and 5.0. Subsequently, the outcome obtained for
evaluating Kitasatospora sp. was significantly decreased, although the
enzyme was realatively stability at a pH variety of 6.0–9.0 (retain-
ing > 90% of activity), after preincubating at 4 °C for 24 h. In addi-
tion, a similar result was obtained to the purified variety (ManK),
which also exhibited fair stability at 6.0–9.0, using Cellulosimicrobium
sp. strain HY-13 (Kim et al., 2011).
3.3.2. Influence and stability of temperature on purified β-mannanase
activity
The optimum temperature of β-mannanase was examined through
assay at varying reaction temperatures, which range from 30 to 80 °C,
as presented in Fig. 4C. This showed a relative gradual increase in ac-
tivity at up to the maximum value of 60 °C (Fig. 4C) and a decline oc-
curred subsequently. This result was similar to Bacillus subtilis WY34
(Jiang et al., 2006), Aspergillus terreus which as observed to be opti-
mum at 60 °C (Soni et al., 2016). Thermal stability profile of purified
β-mananase obtained from Kitasatospora sp. was presented in Fig. 4D,
where a loss of 50% was attained at 50 °C, after 30 min of incubation.
Moreover, this marked reduction in activity is possibly explained as a
function of denaturation at 55 °C, 60 °C (Fig. 4B).
3.3.3. Substrate specificity
This study involved the use of various mannan polymers, including
linear mannan, and glucomannan including ivory nuts and coconut
kernel (copra), and also galactomannan, encompassing LBG and glu-
Yopi et al.
comannan is konjac (Chauhan et al., 2012). In addition, the purified
enzyme obtained from Kitasatospora sp. were better able to digest
LBG, in contrast with other substrate of natural products that were as-
sayed under similar standard conditions. This relative activity on vari-
ous mannan polymers is presented in Fig. 5., where a high value
(100%) on LBG (galactomannan) was observed with palm sugar fruits
(68.2%) and palm kernel cake (51.1%). However, the outcome was
considerably weaker with glucomannan konjac powder (19.1%), po-
rang potato (17.1%), and coconut cake (15.3%), and lowest values
were recorded with palm kernel sugar fruits (2.9%) and β-mannan
(5.5%). Furthermore, the most significant activity on linier mannan,
e.g., ivory nut was 178.3%. In contrast to the β-mannanase from Ki-
tasatospora sp., Cheng et al. (2016) reported β-mannanase from Bacil-
lus subtilis BE-91 exhibited the highest activity with konjac glucoman-
nan (100%), slightly hydrolyzed ivory nut (32.7%) and β-mannan
(46.2%). Soni et al. (2016) also reported mannanase from Aspergillus
terreus exhibited the highest activity with konjac mannan 100%, LBG
50%, and Ivory nut 17.56%.
3.4. Hydrolysis properties
The product of hydrolysis for purified β-mannanase obtained from
Kitasatospora sp. was analyzed using TLC, which confirmed it to be an
endo-mannanase, based on its capacity to efficiently and randomly act
on polymers with higher molecular weight, containing over six man-
nose monomers. In addition, it was established that the activity on
various cheap and commercial polymers produced a variation in sizes
of mannoligosaccharides (M2, M3, M4, M5, M6) and mannose (M1).
LBG, ivory nut, porang potato and palm sugar fruits were observed
to further produce mannose and mannooligosaccharidees (Fig. 6),
while β-mannan, palm kernel and coconut cake, as well as konjac
merely created varying sizes of mannooligosaccharides without form-
ing mannose (Fig. 7). In addition, different patterns were observed
with the hydrolysis of linear mannan, including (1) Ivory nut pro-
duced majorly M1, M4 and M5 (Fig. 6B); (2) β-mannan generated M2
to M5, although M6 was also seen (Fig. 7A); while (3) coconut cake
exhibited mainly M2, although M3 and M4 were also identified (Fig.
7C). Meanwhile, the hydrolysis of galactomannan LBG readily pro-
duced M1 to M6, with M1 to M3 as the major products (Fig. 6A).
These results is similar to the hydrolysis of LBG by Bacillus subtilis
WY34 with mainly produced M1-M6 from 6 h until 36 h of hydrolysis
(Jiang et al., 2006). Whereas, hydrolysis of LBG by Penicillium occita-
nis produced M3 and M4 as a main products (Blibech et al., 2011), hy-
drolysis of LBG by Aspergillus terreus mainly produced M2-M6 (Soni et
al., 2016) and hydrolysis of LBG by Bacillus sp. CSB39 just produced
M2 (Regmi et al., 2016). Hydrolysis of palm kernel cake formed M2-
M6, with M3 and M4 as the chief component (Fig. 7B). While, hy-
drolysing of porang potato lead to the formation of M1 to M4, with
M1 and M3 being the most significant (Fig. 6C). Furthermore, palm
sugar fruits produced M1 to M5, with the major product of M1 (Fig.
6D). Hydrolysis of glucomannan konjac produced of M3 and M4 after
a 6–8 h process of hydrolysis (Fig. 7D). The results shown that man-
nanase obtained from Kitasatospora sp., possesses a high level of cat-
alytic activity, with broad range substrate specificity, thus the en-
hanced propensity of its application in the production of prebiotic
mannooligosaccharides.
4. Conclusions
Based on the results obtained, the β-mannanase activity obtained
from Kitasatospora sp. by submerged fermentation, using LBG as the
prime carbon source is 37.0 U/mL. The use of two step purification of
its crude supernatant allowed the achievement a 1.4-fold purification
with 6.3 U/mg of specific activity, whereas the enzyme and specific
activity were observed to increase from 15.0 U/mL and 4.4 U/mL in
7
Biocatalysis and Agricultural Biotechnology 24 (2020) 101532
the crude form to 74.2 U/mL and 19.4 U/mg after precipitation by
PEG600 and to become 19.3 U/mL and 6.3 U/mg, respectively, after
anion exchange, and there was a marked decline in the total protein
from 338.5 mg to 45.7 mg. The purified β-mannanase has the molecu-
lar weight was approximately 37.0 kDa with optimal activity at pH
6.0 and 60 °C. Meanwhile, the β-mannanase was relatively stability at
a pH variety of 6.0–9.0, retaining > 90% activity after preincubation
at 4 °C for 24 h. Finally, the characteristic enzyme properties of high
catalytic activity with broad range substrate specificity are identified
as indices for successful application in the generation of prebiotic
MOS, while the β-mannanase produced has promising utility in
mannnooligosaccharides production.
Acknowledgements
The authors are grateful to Dr. Katsuhiko Ando and Dr. Tomohiko
Tamura from National Institute and Technology Evaluation (NITE),
Japan, as well as Dr. Yantyati Widyastuti and Dr. Shanti Ratnakomala
from Research Center for Biotechnology, Indonesian Institute of Sci-
ences, Indonesia, and Biotechnology Culture Collection (BTCC) for
providing the actinomycetes strains. This work was supported by a
DIPA LIPI research grant.
References
Adiguzel, A., Nadaroglu, H., Adiguzel, G., 2015. Purification and characterization of β-
mannanase from Bacillus pumilus(M27) and its applications in some fruit juices. J.
Food Sci. Technol. 52 (8), 5292–5298.
Amany, L., Kansoh, Zeinat, A., Nagieb, 2004. Xylanase and Mannanase enzymes from
Streptomyces galbus NR and their use in biobleaching of softwood kraft pulp. Antonie
Leeuwenhoek 85, 103–114.
Arcand, N., Kluepfel, D., Paradis, F.W., Morosoli, R., Shareck, F., 1993. β-Mannanase of
Streptomyces lividans 66: cloning and DNA sequence of the manA gene and
characterization of the enzyme. Biochem. J. 290, 857–863.
Aziz, N.F.H.A., Sahar, A., Hui, S.N., Tan, J.S., Pongsathon, P., Mohamad, H.A.B., Yew, J.
T., Tan, J.S., 2017. Purification of β -mannanase derived from Bacillus subtilis ATCC
11774 using ionic liquid as adjuvant in aqueous two-phase system. J. Chromatogr. B
1055–1056. https://doi.org/10.1016/j.jchromb.2017.04.029.
Blibech, M., Fatma, C., Fatma, B., Ilyes, D., Raoudha, E.G., Semia, E.C., 2011.
Production of manno-oligosaccharides from locust bean gum using immobilized
Penicilliumoccitanis mannanase. J. Mol. Catal. B Enzym. 73, 111–115.
Capek, P., Kubackova, M., Alfoldi, J., Billsics, L., Lisková, D., Kákoniová, D., 2000.
Galactomannan from the secondary cell wall of Picea abies L. Karst. Carbohydr. Res.
329 (3), 635–645. https://doi.org/10.1016/s0008-6215(00)00210-x.
Chaikumpollert, O., Pawadee, M., Krisda, S., 2004. Structural elucidation of
hemicelluloses from Vetiver grass. Carbohydr. Polym. 57 (2), 191–196. https://doi.
org/10.1016/j.carbpol.2004.04.011.
Chauhan, P.S., Neena, P., Prince, S., Naveen, G., 2012. Mannanases: microbial sources,
production, properties and potential biotechnological applications. Appl. Microbiol.
Biotechnol. 93 (5), 1817–1830. https://doi.org/10.1007/s00253-012-3887-5.
Cheng, L., Shengwen, D., Xiangyuan, F., Ke, Z., Qi, Y., Zhengchu, L., 2016. Purification
and Characterization of a Thermostable β-Mannanase from Bacillus Subtilis BE-91:
Potential Application in Inflammatory Diseases, vol. 2016. Hindawi Publishing
Corporation BioMed Research International, p. 7 Article ID 6380147. https://doi.
org/10.1155/2016/6380147.
Clarke, J.H., Davidson, K., Rixon, J.E., Halstead, J.R., Fransen, M.P., Gilbert, H.J.,
Hazlewood, G.P., 2000. A comparison of enzyme-aided bleaching of softwood paper
pulp using combinations of xylanase, mannanase and alpha-galactosidase. Appl.
Microbiol. Biotechnol. 53 (6), 661–667.
Dhawan, S., Kaur, J., 2007. Microbial mannanases: an overview of production and
applications. Crit. Rev. Biotechnol. 27 (4), 197–216.
El-Sharouny, E.E., Nabil, M.K., El-Toukhy, El-Sersy, N.A., El-Aal El-Gayar, A.A., 2015.
Optimization and purification of mannanase produced by an alkaliphilic-
thermotolerant Bacillus cereus N1 isolated from BaniSalama Lake in Wadi El-Natron.
Biotechnol. Biotechnol. Equip. 29 (2), 315–323. https://doi.org/10.1080/
13102818.2014.995932.
Harnpicharnchai, P., Waraporn, P., Wanwisa, T., Warasirin, S., Kittapong, S.T., Pennapa,
M., Sutipa, T., 2016. Production of high activity Aspergillus niger BCC4525 β-
mannanase in Pichia pastoris and its application for mannooligosaccharides
production from biomass hydrolysis. Biosci. Biotechnol. Biochem. 80 (12), 2298–
2305.
Harris, P.J., Stone, B.A., 2009. Chemistry and molecular organization of plant cell walls.
In: Biomass Recalcitrance: Deconstructing the Plant Cell Wall for Bioenergy. pp. 61–
93.
Ingham, K.C., 1984. Protein precipitation with polyethylene glycol. Methods Enzymol.
104, 351–356. https://doi.org/10.1016/s0076-6879(84)04101-x.
Jiang, Z., Yun, W., Daoyi, L., Lite, L., Pingping, C.I., 2006. High-level production,
purification and characterization of a thermostable b-mannanase from the newly
Yopi et al.
isolated Bacillus subtilis WY34. Carbohydr. Polym. 66, 88–96.
Kim, D.Y., Su-Jin, H., Hyun, J.L., Yi-Joon, K., Dong-Ha, S., Young, H.R., Kwang-Hee, S.,
Ho-Yong, P., 2011. A highly active endo-β-1,4-mannanase produced by
Cellulosimicrobium sp. strain HY-13, a hemicellulolytic bacterium in the gut of
Eiseniafetida. Enzym. Microb. Technol. 48, 365–370.
Kong, C., Lee, J.H., Adeola, O., 2011. Supplementation of β-mannanase to starter and
grower diets for broilers. Can. J. Anim. Sci. 91 (3), 389–397.
Kote, N.V., Patil, A.G.G., Mulimani, V.H., 2009. Optimization of the production of
thermostable endo-β-1,4 mannanase from a newly isolated Aspergillus niger gr and
Aspergillus flavus gr. Appl. Biochem. Biotechnol. 152, 213–223.
Kumagai, Y., Kayoko, K., Misugi, U., Tadashi, H., 2013. Effect of the binding of bivalent
ion to the calcium-binding site responsible forthe thermal stability of
actinomycetemannanase: potential use in production of functional
mannooligosaccharides. J. Mol. Catal. B Enzym. 94, 63–68.
Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature 227 (5259), 680–685.
Lv, J.N., Chen, Y.Q., Guo, X.J., Piao, X.S., Cao, Y.H., Dong, B., 2013. Effects of
supplementation of corn-soybean meal diets on performance and nutrient
digestibility in growing pigs. AJAS (Asian-Australas. J. Anim. Sci.) 26 (4), 579–587.
Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing
sugar. Anal. Chem. 31 (3), 426–428. https://doi.org/10.1021/ac60147a030.
Moreira, L.R.S., Filho, E.X.F., 2008. An overview of mannan structure and mannan-
degrading enzyme system. Appl. Microbiol. Biotechnol. 79 (165). https://doi.org/
10.1007/s00253-008-1423-4.
Olaniyi, O.O., Arotupin, D.J., Akinyele, B.J., Bamidele, O.S., 2014. Kinetic properties of
purified β-mannanase from Penicilliumitaliticum. Br. Microbiol. Res. J. 4, 1092–1104.
Pan, X., Zhou, J., Tian, A., 2011. High level expression of a truncated β-mannanase from
alkaliphilic Bacillus sp. N16-5 in Kluyveromycescicerisporus. Biotechnol. Lett. 33 (3),
565–570.
Petkowicz, C.L., Reicher, F., Chanzy, H., Taravel, F.R., Vuong, R., 2001. Linear mannan
in the endosperm of Schizolobium amazonicum. Carbohydr. Polym. 44 (2), 107–112.
https://doi.org/10.1016/S0144-8617(00)00212-5.
Biocatalysis and Agricultural Biotechnology 24 (2020) 101532
Piwpankaew, Y., Supa, S., Sunee, N., Thu-Ha, N., Dietmar, H., Suttipun, K., 2014.
Cloning, secretory expression and characterization of recombinant β-mannanase
from Bacillus circulans NT 6. 7 SpringerPlus 3 (430), 1–8. http://www.springerplus.
com/content/3/1/430.
Rahmani, N., Kashiwagi, N., JaeMin, L., Niimi-Nakamura, S., Hana, M., Prihardi, K.,
Puspita, L., Yopi, Bambang, P., Ogino, C., Kondo, A., 2017. Mannan endo-1,4-β-
mannosidase from Kitasatospora sp. isolated in Indonesia and its potential for
mannnooligosaccharide production from mannan polymers. Amb. Express 7 (100).
https://doi.org/10.1186/s13568-017-0401-6.
Regmi, S., Pradeep, G.C., Yun, H.C., Yoon, S.C., Ji, E.C., Seung, S.C., Jin, C.Y., 2016. A
multi-tolerant low molecular weight mannanase from Bacillus sp. CSB39 and its
compatibility as an industrial biocatalyst. Enzym. Microb. Technol. 93, 76–85.
https://doi.org/10.1016/j.enzmictec.2016.06.018.
Sang, H.M., Fotedar, R., 2010. Effects of mannan oligosaccharide dietary
supplementation on performances of the tropical spiny lobsters juvenile
(Panulirusornatus, Fabricius 1798). Fish Shellfish Immunol. 28 (3), 483–489.
Scheller, Ulvskow, 2010. Hemicelluloses. Annu. Rev. Plant Biol. 61, 263–289.
Songsiriritthigul, C., Buranabanyat, B., Haltrich, D., Yamabhai, M., 2010. Efficient
recombinant expression and secretion of a thermostable GH26 mannanendo-1,4-β-
mannosidase from Bacillus licheniformis in Escherichia coli. Microb. Cell Factories 9,
20.
Soni, H., Rawat, H.K., Plestchke, B.I., Kango, N., 2016. Purification and characterization
of β-mannanase from Aspergillus terreus and its applicability in depolymerization of
mannans and saccharification of lignocellulosic biomass. 3 Biotech 6, 136. https://
doi.org/10.1007/s13205-016-0454-2.
Srivastava, P.K., Kapoor, M., 2014. Cost-effective endo-mannanase from Bacillus sp.
CFR1601 and its application in generation of oligosaccharides from guar gum and
as detergent additive. Prep. Biochem. Biotechnol. 44 (4).
Stoscheck, C.M., 1990. Quantitation of protein. Methods Enzymol. 182, 50–69.