The production of β-mannanase from Kitasatospora sp. strain using submerged fermentation: Purification, characterization and its potential in mannooligosaccharides production

Journal Pre-proof The production of β-mannanase from Kitasatospora sp. strain using submerged fermentation: Purification, characterization and its potential in mannooligosaccharides production Yopi, Nanik Rahmani, Siti Amanah, Pugoh Santoso, Puspita Lisdiyanti PII:

S1878-8181(19)31584-1

DOI:

https://doi.org/10.1016/j.bcab.2020.101532

Reference:

BCAB 101532

To appear in:

Biocatalysis and Agricultural Biotechnology

Received Date: 15 October 2019 Revised Date:

31 January 2020

Accepted Date: 5 February 2020

Please cite this article as: Yopi, , Rahmani, N., Amanah, S., Santoso, P., Lisdiyanti, P., The production of β-mannanase from Kitasatospora sp. strain using submerged fermentation: Purification, characterization and its potential in mannooligosaccharides production, Biocatalysis and Agricultural Biotechnology (2020), doi: https://doi.org/10.1016/j.bcab.2020.101532. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

Palm kernel cake : Galactomannan

Porang potato : Glucomannan

…

Kitasatospora sp.

Crude enzyme supernatant

Purification Characterization

End product

Mannanase producer bacteria

Enzyme production, puriifcation and characterization

Hydrolysis of mannan polymer

The Production of β-Mannanase from Kitasatospora sp. strain using Submerged Fermentation: Purification, Characterization and Its Potential in MannooligosaccharidesProduction

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Yopi12*, Nanik Rahmani1, Siti Amanah1, Pugoh Santoso1, and Puspita Lisdiyanti1

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Research Center for Biotechnology, Indonesian Institute of Sciences,Komplek Cibinong Science Center CSC-LIPI, Jl. Raya Bogor Km.46, Cibinong West Java, 16911, Indonesia 2 Research Center and Human Resource Development, National Standardization Agency (BSN), Gedung I BPPT, Jl. H. M Thamrin No. 8, KebunSirih Jakarta Pusat, 10340, Indonesia * Corresponding author at: Research Center and Human Resource Development, National Standardization Agency (BSN), Gedung I BPPT, Jl. H. M Thamrin No. 8, KebunSirih Jakarta Pusat, 10340, Indonesia

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Abstract

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1. Introduction

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The second most abundant polymers present in nature after cellulose is hemicellulose (Haris and Stone, 2008), 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 organic 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 important representatives of this class. The mannan, an important of the hemicelluloses family, can be classified in four subfamilies, such as linear mannan, glucomannan, galactomannan, and galactoglucomannan (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 thehydrolysis of hemicelluloases (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 hydrolyzing β-1, 4- linkages in the mannan backbone (Olaniyiet al., 2014). This effect is conferred on the abundant mannan-rich polymers, including glucomannan, β-1, 4-mannans and galactomannan, in an attempt to produce manno-oligosaccharides (Songsiriritthigulet al., 2010; Kumagaiet al., 2013; Harnpicharnchaiet al.,

E-mail addresses: [email protected] (Yopi)

Actinomycetes have been identified as one of the most diverse groups of microorganisms that play a vital role in the production of enzymes and nutraceuticals. In addition, prior studies on the wild type strain of Kitasatospora sp. emphasized on its ability to exhibit high β-mannanase activity. This study aimed to purify, characterize and evaluate the potential of this strain in the production of mannooligosaccharides using mannan 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 enzyme characterization were investigated, analysis hydrolysis product was conducted by TLC. The enzymes exhibited 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 oC and relatively stability at a pH variety of 6.0 to 9.0, retaining > 90% activity. This product was capable of hydrolysing various mannan 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 mannose potential for food and feed industry.Keywords:Kitasatospora sp.; β-mannanase; mannan polymer; mannooligosakarida; submerged fermentation

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2016; Rahmaniet al., 2017), and a small amount of sugar monomer (mannose, glucose and galactose) (Olaniyiet al., 2014; Songsiriritthigulet al., 2010; Dhawan and Kaur, 2007). Specifically, mannooligosaccharides are well-known to have bioactivity in several applications, including as a dietary supplement to assist in the improvement of growth performance, gut health, and immune responses in marine organisms, (Sang and Fotedar, 2010). Meanwhile, β-mannanases have been broadly applied in pulp and paper processing (Clarke et al., 2000; Pan et al., 2011), feed (Kong et al., 2011; Lvet al., 2013), food (Adiguzelet al., 2015), pharmaceuticals, oil, textile and detergent/laundry industry (Srivastava and Kapoor, 2014).For this purpose, various enzymes have previously been purified using various methods and characterized from a variety of microbes, encompassing purification of Streptomyces galbus NR using ammonium sulfate precipitation, further purification was done by gel filtration followed by ion exchange chromatography(Amanyet al., 2004);Bacillus subtilis WY34using 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-Sharounyet 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 (Rahmaniet 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 mannooligosaccharides production. Therefore, the objectives of this present work were to evaluate the purification and characterization of β-mannanase obtained from Kitasatosporasp., and to also analyse its potential in mannooligosaccharide production, using various mannan polymer. 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 registration number BTCC B-606, and it was already registered in Genebank, with accession number KY576672 (Rahmaniet al., 2017).Locust bean gum (LBG) and standard mannose (M1) weresupplied from Sigma-Aldrich(St. Louis, MO, USA), while mannooligosaccharides, encompassingmannobiose(M2), mannotriose (M3), mannotetraose (M4),mannopentaose (M5), and mannohexaose (M6) were supplied from Megazyme(Wicklow, Ireland). 2.2. Cultivation and enzyme production Kitasatospora sp. was maintained on ISP2 agar slants at 28 oC, 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 28oC 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 Erlenmeyer flask. Meanwhile, fermentation was conducted for seven days, at an agitation rate of 190 rpm, and 28 oC, using the rotary shaker (Taitex), where the β-mannanase activity was analysed by withdrawing samples every 24 h. Furthermore, the recovery of crude enzyme supernatant was performed at 13,000 rpm, and 4 oC 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 increase 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 50mM sodium phosphate buffer, pH 6.0). Subsequently, the assay was examined at 60oC, exactly 15 min after, and stopped when 0.5 mL of the DNS solution was added, boiled at 100oC 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 oC for 4 hours, while the supernatant was recovered using the dialysis tubing cellulose membrane (Sigma-Aldrich) at 4 oC, 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 uL, and volume fraction of 1 mL, onto a

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HiTrap Q FF column (5 x 5 mL) (GE) that was initiated by equilibration with 50mM sodium phosphate buffer pH 6.0, using ÄKTAprime plus (GE). Furthermore, the unbound inactive protein was eluted using the starting buffer, and a linear gradient of 50mM sodium phosphate buffer maintained at pH 6.0, contain1000mMNaCl 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.

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3. Results and discussion

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3.1. The β-mannanase enzyme production pattern by Kitasatosporasp.

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The β-mannanase enzyme activities produced by Kitasatospora sp. were analyzed at the inception of this study, as they were sourced mainly from extracellular and inducible variety (Dhawan and Kaur, 2007). This production process

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 Kitasatosporasp. The molecular weight of β-mannanase was obtained by SDS-PAGE, which was carried out with 12.5% (w/v) polyacrylamide gel according to Laemmli (1976), using low molecular weight standard proteins as markers. Prior to this, the crude and purified forms were denatured with sample buffer (100mMTrisbuffer pH 8.0, 10% SDS, 10 mMmercaptoethanol, 10% glycerol, 0.001% bromophenol blue), at 95 oC 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 denatured at 70 oC for 20 min before applying to the PAGE. Staining required 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 oC for 1 h. This was then stained with congo red solution for 30 min, and the process was terminated by the immersing into a 1000mMNaCl solution, followed 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 substrate and the optimal value was determined using various buffer solutions in pH ranging from 4.0-9.0, encompassing 50 mM sodium acetate (at 4.0-5.0), 50 mM sodium phosphate (6.0-8.0), and 50 mMglycin-NaOH (9.0) at 30oC. These were used in the determination of pH stability for the purified enzyme, through incubation at room temperature for one night. Furthermore, the percentage remaining activities were measured in relation with the starting values achieved at 0h. 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 oC, using 50 mM sodium phosphate buffer (pH 6.0). These were then separately incubated for 30, 60, 90, 120 min at 55 oC, 60 oC and 65 oC. Furthermore, the temperature stability was investigated by comparing the residual with the initial activity. 2.7.3. Substrate specificity The substrate specificity of purified enzyme was assayed by incubating the enzymes in 50 mM sodium phosphate buffer pH 6.0, which contain 0.5% of each substrate, at 60 oC for 15 min, using the DNS 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, coconut cake, palm sugar fruit and commercial forms, encompassing locus bean gum, konjac, ivory nut, and β-mannan. This process was conducted on each substrate (2% (w/v)) within a 3 mL volume reaction consisting of 50mM sodium phosphate buffer (pH 6.0), and 3.0 ug/uL of purified β-mannanase, and subsequently incubated in a shaker incubator at an agitation rate of 190 rpm, and a temperature of 40 oCfor 8 h. Furthermore, the reaction mixture was collected at 0, 2, 4, 6 and 8 h, and subsequentlyboiled at 100 oC 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 analyzed with TLC spotting on a Silica gel 60F254 (EMD/Merck 20 x 20 cm, Darmstadt, Germany) according to the methods of Rahmaniet al., (2017) and the standards adopted in this investigation include M1 to M6.

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was conducted in a culture medium, supplemented with LBG as a galactomannan-rich substrate, which has been widely adopted as a mannanaseenzyme inducer (Koteet al., 2009; Kim et al., 2011). Fig. 1 shows β-mannanase secretion lines, up to the 72th hour, and these activities increased to a peak value after the 7 days’ cultivation period. Therefore, the enzymes produced subsequently were applied in the selection of a suitable incubation time for Kitasatospora sp., which is important in this assay.

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3.2. Purification of β-mannanase

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The β-mannansecrude extract was purified using a combination of PEG precipitations, dialysis tubing, cellulose membrane and chromatographic 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 enzymes solution for 4 h, followed by desalting through a dialysing tubing cellulose membrane, against a 0.02M sodium phosphate buffer pH 6.0, one night, at 4 oC. 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 exchange, respectively. Also, the total protein was observed to have decreased from 338.5 mg/mL to 45.7 mg/mL.

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Fig. 1. Production activities of β-mannanase from Kitasatospora sp. on the LBG substrate on different day’s fermentation

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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.

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Table 1. Purification scheme of the β-mannanase from Kitasatospora sp.

Purifications steps β-mannanase crude extract Concentration using PEG HiTrap Q FF column

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Volume (ml)

βmannanase activity (U/ml)

Total protein (mg)

Total activity (Units)

Spesific activity (U/mg)

Purification (fold)

Yield (%)

100

15.0

338.5

1503.2

4.4

1

100

10

74.2

38.2

741.5

19.4

4.4

49.3

15

19.3

45.7

288.7

6.3

1.4

19.2

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, indicating 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 attempt to detect the activity and purity of the enzyme obtained from

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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 mannanasefrom Cellulosimicrobium sp. strain HY-13 was observed to have molecular mass of approximately 35 kDa (Kim et al., 2011), while the product from Streptomyces lividans 66 exhibited values of about 36 kDa(Arcandet 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 possess estimated to be of size 39.6 kDa(Jiang et al., 2006).

Fig. 3. The molecular weight and zymogramof the crude supernatants, before and after precipitation by PEG, and purification by anion exchange. STD: molecular weight marker,lane 1, β-mannanasecrude extractKitasatospora sp., lane 2, crude enzyme after precipitation by PEG; lane 3, protein solution from elution steps.

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 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 exposure to pH 4.0 and 5.0. Subsequently, the outcome obtained for evaluating Kitasatospora sp. was significantly decreased, although the enzyme wasrealatively stability at a pH variety of 6.0 to 9.0 (retaining > 90% of activity), after preincubating at 4 oC for 24 h. In addition, 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).

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Fig. 4. The influence of pH, temperature and stability on enzyme activity. (A) OptimumpH was assayed at 60 oC, 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 obtained by storing at various temperatures for one hour. Furthermore, all determinations took place in triplicate, demonstrated as mean ± standard deviation.

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 oC, as presented in Fig. 4C. This showed a relative gradual increase in activity at up to the maximum value of 60 oC (Fig. 4C) and a decline occurred subsequently. This result was similar to Bacillus subtilis WY34 (Jiang et al., 2006), Aspergillus terreuswhich as observed to be optimum at 60 oC(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 oC, after 30 min of incubation. Moreover, this marked reduction in activity is possibly explained as a function of denaturation at 55 oC, 60 oC (Fig. 4B). 3.3.3. Substrate specificity This study involved the use of various mannan polymers, including linear mannan, and glucomannanincluding ivory nuts and coconut kernel (copra), and also galactomannan, encompassing LBG and glucomannan is konjac(Chauhan et al., 2012). In addition, the purifiedenzyme obtained from Kitasatospora sp. were better able to digest LBG, in contrast with other substrate of natural products that were assayed under similar standard conditions. This relative activity on various 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 glucomannankonjac powder (19.1%), porang 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 Kitasatospora sp., Cheng et al., (2016) reportedβ-mannanase fromBacillus subtilis BE-91 exhibited the highest activity with konjac glucomannan (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 withkonjac mannan 100%, LBG 50%, and Ivory nut 17.56%.

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Fig. 5. Substrate specificity of mannanase

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 mannose 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 forming mannose (Fig. 7). In addition, different patterns were observed with the hydrolysis of linear mannan, including (1) Ivory nut produced majorly M1, M4 and M5 (Fig. 6B); (2) βmannan 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 produced 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 occitanis produced M3 and M4 as a main products (Blibech et al., 2011), hydrolysis 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 ofpalm kernel cake formed M2-M6, with M3 and M4 as the chief component (Fig. 6B). While, hydrolysing 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 mannanase obtained from Kitasatospora sp., possesses a high level of catalytic activity, with broad range substrate specificity, thus the enhanced propensity of its application in the production of prebiotic mannooligosaccharides.

Fig. 6. Thin-layer chromatography analysis of (A) LBG, (B) Ivory nut (C) Porang potato, (D) Palm sugar fruits hydrolyzed by the purified βmannanasefrom Kitasatospora 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 hydrolyzed hour to 0, 2, 4, 6 and 8; lane 8 purified β-mannanase.

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Fig. 7. Thin-layer chromatography analysis of (A) β-mannan (B) Palm cernel cake, (C) Coconut cake, (D) Konajc hydrolyzed by the purified βmannanase 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 hydrolyzed hour to 0, 2, 4, 6 and 8; lane 8 purified β-mannanase.

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4. Conclusions

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Based on the results obtained, the β-mannanase activity obtained from Kitasatosporasp.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 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 molecular weight was approximately 37.0 kDa with optimal activity at pH 6.0 and 60 oC. Meanwhile, the β-mannanase was relatively stability at a pH variety of 6.0 to 9.0, retaining > 90% activity after preincubation at 4 oC for 24h. 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.

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Acknowledgements

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The authors are grateful to Dr. Katsuhiko Ando and Dr.Tomohiko Tamura from National Institute and Technology Evaluation (NITE), Japan, as well as Dr.YantyatiWidyastuti and Dr. Shanti Ratnakomala from Research Center for Biotechnology, Indonesian Institute of Sciences, Indonesia, and Biotechnology Culture Collection (BTCC) for providing the actinomycetes strains. This work was supported by a LIPI research grant.

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Adiguzel, A., Nadaroglu, H., and Adiguzel, G., 2015. Purification and characterization of β-mannanase from Bacillus pumilus(M27) and its applications in some fruit juices. J.ofFood Sci and Tech. 52 (8), 5292–5298.

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Fig. 1. Production activities of β-mannanase from Kitasatospora sp. on the LBG substrate on different day’s fermentation 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 zymogramof the crude supernatants, before and after precipitation by PEG, and purification by anion exchange. STD: molecular weight marker,lane 1, β-mannanasecrude extract Kitasatospora sp., lane 2, crude enzyme after precipitation by PEG; lane 3, protein solution from elution steps. Fig. 4. The influence of pH, temperature and stability on enzyme activity. (A) OptimumpH was assayed at 60 oC, 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 obtained by storing at various temperatures for one hour. Furthermore, all determinations took place in triplicate, demonstrated as mean ± standard deviation. Fig. 5. Substrate specificity of mannanase Fig. 6. Thin-layer chromatography analysis of (A) LBG, (B) Ivory nut (C) Porang potato, (D) Palm sugar fruits hydrolyzed by the purified βmannanase from Kitasatospora 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 hydrolyzed hour to 0, 2, 4, 6 and 8; lane 8 purified β-mannanase. Fig. 7. Thin-layer chromatography analysis of (A) β-mannan (B) Palm cernel cake, (C) Coconut cake, (D) Konajc hydrolyzed by the purified βmannanase 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 hydrolyzed hour to 0, 2, 4, 6 and 8; lane 8 purified β-mannanase.

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HIGHLIGHTS

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 A wild type Kitasatospora sp. strain exhibiting high activity (37.0 U/mL) were obtained from medium

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locus bean gum by a submerged fermentation.

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 A one step chromatographic procedure was employed for purification of the mannanase

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 The mannanase could successfully purified with the molecular mass was approximately 37.0 kDa

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 The enzyme fairly stable at pH range of 6.0 to 9.0 with retained above 90% of its original catalytic

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activity  The enzyme could hydrolysed various mannan polymers and commercial mannan produced mannoligosaccharides and mannose.

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