Skip to main content

Xylanopectinolytic enzymes by marine actinomycetes from sediments of Sarena Kecil, North Sulawesi: high potential to produce galacturonic acid and xylooligosaccharides from raw biomass



Actinomycetes isolated from marine habitats are known to have the potential for novel enzymes that are beneficial in the industry. In-depth knowledge is necessary given the variety of this bacterial group in Indonesia and the lack of published research. Actinomycetes isolates (BLH 5-14) obtained from marine sediments of Sarena Kecil, Bitung, North Sulawesi, Indonesia, showed an ability to produce pectinase and xylanase that have equal or even higher potential for pectic-oligosaccharides (POS) and xylooligosaccharides (XOS) production from raw biomass than from commercial substrates. This study's objective was to characterize both enzymes to learn more for future research and development.


Pectinase had the highest activity on the 6th day (1.44±0.08 U/mL) at the optimum pH of 8.0 and optimum temperature of 50 °C. Xylanase had the maximum activity on the 6th day (4.33±0.03 U/mL) at optimum pH 6.0 and optimum temperature 60 °C. Hydrolysis and thin layer chromatography also showed that pectinase was able to produce monosaccharides such as galacturonic acid (P1), and xylanase was able to yield oligosaccharides such as xylotriose (X3), xylotetraose (X4), and xylopentaose (X5). BLH 5–14 identified as the genus Streptomyces based on the 16S rDNA sequences and the closely related species Streptomyces tendae (99,78%).


In the eco-friendly paper bleaching industry, Streptomyces tendae has demonstrated the potential to create enzymes with properties that can be active in a wide range of pH levels. The oligosaccharides have the potential as prebiotics or dietary supplements with anti-cancer properties. Further research is needed to optimize the production, purification, and development of the application of pectinase and xylanase enzymes produced by Actinomycetes isolates.


Oligosaccharides are short carbohydrate polymer chains composed of 2 to 10 monosaccharides. As nutrients for the growth of beneficial microbes in the intestines, oligosaccharides are typically found in the fiber structure of plants and have the potential to serve as prebiotics. Because these oligosaccharides generally cannot be broken down by human enzymes, they can pass through the gut intact. The prevention of harmful bacterial growth, improved mineral absorption, and enhanced gut immunity are a few benefits for humans. Malto-oligosaccharides from starch, fructo-oligosaccharides from sucrose, pectic-oligosaccharides from pectin, and xylo-oligosaccharides from xylan are a few examples of oligosaccharides. These latter two are promising targets for potential prebiotic sources [1, 2].

Oligosaccharides produced by the partial hydrolysis of pectin are known as pectic-oligosaccharides (POS). The hydrolysis process can result in smaller units with different polymerization stages that come from the complex structure of the backbone sides of galacturonic acid and the chain sides of rhamnose and neutral sugars. Because of their anti-cancer, anti-bacterial, and antioxidant characteristics, pectin and POS are utilized in the biomedical sector as dietary fiber and treatments for conditions including ulcers, colon cancer, and diarrhea [3,4,5]. Research by Wilkowska et al. in 2019 [6] shows the effect of larger-size POS on the growth of the human gut microbiota and inhibition of pathogen growth. POS function as a modulator for immunometabolism in macrophages was studied by Hu et al. in 2021 [7].

On the other side, the hydrolysis reaction of xylan can yield xylo-oligosaccharides (XOS), which are known to be stable in acidic environments. Since 1990, xylo-oligosaccharides have been developed and sold as a food supplement in Japan due to their health benefits, such as anti-tumor and anti-inflammation [8, 9]. One of the newest reports by Abdo et al. in 2021 [10] shows XOS ability to improve gut health in hamsters by reducing plasma cholesterol levels and changing sterols composition. The positive impact of XOS consumption and nutrition as a prebiotic was also demonstrated on human intestinal health through the growth of lactic acid bacteria and Bifidobacterium spp. in studies by Lin et al. [11] and Alvarez et al. [12].

The production methodology of oligosaccharides can be chemical by heat and acid treatment or enzymatic, employing pectinase and xylanase enzymes to degrade the product. There has been an investigation regarding pectinase and xylanase enzymes from marine Actinomycetes. Endo-β-1,4-xylanases from Kitasatospora sp. and Streptomyces variabilis are used in studies by Rahmani et al. [13, 14] to show that they may produce xylooligosaccharides from sugarcane bagasse and beechwood substrate, respectively. Screening results of marine Actinomycetes from Visakhapatnam coast, India, carried out by Yugandhar et al. [15] against 52 isolates, succeeded in obtaining one potential isolate in producing the optimal pectinase enzyme at pH 6.0 media. However, there has not been a research report prior on combined pectinase and xylanase enzymes from marine Actinomycetes isolates. Publications on microorganisms that can produce both enzymes, including the bacteria Bacillus amyloliquefaciens, Bacillus pumilus, Streptomyces sp. from terrestrial habitats, and the fungus Mucor sp. [16,17,18,19].

The lack of research is a significant factor with the increasing need for a mixture of pectinase and xylanase, especially for utilizing waste biomass and industrial base materials containing xylan and pectin substrates. Beyond the benefit of oligosaccharide production, there are many other applications for these two enzymes. The capacity to substitute chemicals in the paper-bleaching process, enhance the extraction and clarification of fruit juices containing hemicellulose and pectin, such as apple and pineapple, and hemp fiber preparation for use in the textile industry are some of these few examples [16, 17, 20,21,22].

Based on the results of previous research in 2021, screening of 21 Actinomycetes isolates from marine sediments and sponges at Sulawesi and Lampung marine ecosystems in Indonesia. One candidate of Actinomycetes (BLH 5-14) originated from the marine sediments of Sarena Kecil, Bitung City, North Sulawesi, was chosen, and shows potential as a producer of pectinase and xylanase enzymes, with clear zones of 3.6 cm and 3.2 cm on double-layered agar media, respectively. Therefore, further research is needed to explore the potential utilization and characterization of pectinase and xylanase enzymes from Indonesian marine sediments Actinomycetes isolates (BLH 5-14) and their oligosaccharides production using raw biomass.


Microorganism, materials, and chemicals

Actinomycetes (BLH 5-14) are isolated from the marine sediments of Sarena Kecil, Bitung, North Sulawesi, Indonesia. The primary materials used in this research include yeast-malt culture medium with the addition of artificial seawater and pectin from citrus peel [Sigma-Aldrich; St. Louis, MO, USA] or xylan from beechwood [Himedia; Kennett Square, PA, USA] as the glucose substitute. The experiment uses the highest quality and grade of reagents, chemicals, and standards.

Enzyme production

The enzyme production was carried out based on the method by Rahmani et al. [14], using the marine yeast-malt medium, with the addition of 3% (w/v) Marine ART SF-1, 2% (w/v) commercial pectin substrate from orange peel for pectinase culture, and 2% (w/v) commercial xylan substrate from beechwood for xylanase culture. The production stages consist of the rejuvenation process of BLH 5-14 isolates on a yeast-malt agar medium and cultivation on the 4th day at 28 °C, followed by the pre-culture process on a liquid marine yeast-malt medium that incubates for three days at 28 °C, 190 rpm, and the culture process on liquid marine yeast-malt medium without glucose with the addition of pectin or xylan substrate, that incubates at 28 °C, 190 rpm, for seven days. Samples were taken once every 24 h, and the results were separated by centrifugation at 4 °C for 20 min, 12,000 rpm. The supernatant was stored at 4 °C for further analysis, while the cell pellets were freeze-dried for three days to constant weight.

Enzyme activity, protein concentration and growth curve

The growth curve was made by measuring the enzyme activity according to the method by Miller [23] and Rahmani et al. [24] on the results of crude enzymes from 0 to 168 h of enzyme production with 3 replications each. The protein concentration during the enzyme production process was carried out according to the BCA Protein Assay Kit [Pierce] protocol, with a standard curve made using bovine serum albumin (BSA) at a concentration of 0.0 - 2.0 mg/mL. The dry weight measurement of pure cells was obtained from freeze-dry results.

Enzyme activity assay

The enzymatic reaction was based on the method by Rahmani et al. [24], which was carried out by mixing 250 μL of substrate solution with 250 μL of crude enzyme solution at 30 °C for 15 min. Another test tube containing a mixture of 250 μL of the substrate and 250 μL of milli-Q was also reacted as a control at the same time with a blank tube containing 500 μL of the buffer. Dinitrosalicylic acid (DNS) solution of 750 μL was added, and the reaction was heated at 100 °C for 10 min. The reaction tube was then cooled in ice water for 10 min before the optical density (OD) could be measured using a spectrophotometer at a wavelength of 540 nm. The quantity of enzyme needed to produce 1 μmol of reducing sugar every minute under the reaction variables was referred to as an enzyme activity unit (U/mL).

Enzyme characterization

Enzyme characterization was carried out to calculate optimum pH, optimum temperature, and the influence of metal ions and chemical compounds on enzyme activity. Enzyme activity was measured according to the method by Miller et al. [23]. The combination of pH tested consisted of 50 mM sodium citrate buffer (pH 3.0 - 5.0), 50 mM sodium acetate buffer (pH 4.0 - 6.0), 50 mM sodium phosphate buffer (pH 6.0 - 8.0), 50 mM Tris-HCl buffer (pH 7.0 - 9.0), and 50 mM Glycine-NaOH buffer (pH 8.0 - 10.0). The temperatures were tested from 30 °C to 90 °C. The metal ions solution includes KCl, CaCl2, MnCl2.4H2O, ZnCl2, FeSO4.7H2O, MgSO4.7H2O, CuSO4, dan HgCl2 (5 mM) [Sigma-Aldrich; St. Louis, MO, USA], while the chemical compound includes Triton X-100 [Merck; Jakarta, Indonesia], EDTA [Sigma-Aldrich; St. Louis, MO, USA], PEG-6000 [Merck; Jakarta, Indonesia], methanol [Emsure, ACS; Darmstadt, Germany], ethanol [Emsure, ACS; Darmstadt, Germany], sodium dodecyl sulfate (SDS) [Sigma-Aldrich; St. Louis, MO, USA], and isopropanol [Emsure, ACS; Darmstadt, Germany] (5%).

Molecular weight analysis with SDS PAGE and zymogram

Running gel and stacking gel were made according to the method of Laemmli [25] and Yopi et al. [26]. Sample preparation consisted of a mixture of 10 μL crude enzyme and 10 μL loading buffer. SDS PAGE and zymogram settings are 200 W, 120 V, and 50 mA for 100 min. SDS PAGE staining using coomassie brilliant blue G250 solution for one night and de-staining for 1 h. Staining of the zymogram was carried out in stages using Triton X-100 (2.5%), milli-Q, incubation in a 50 mM buffer with optimum pH and optimum temperature, Lugol dye for pectin and congo red dye for xylan, and de-staining with 1M NaCl solution and 0.5% (v/v) acetic acid solution.

Thin layer chromatography on hydrolysis products of pectinase and xylanase

The hydrolysis reaction was done based on the method by Rahmani et al. [27] by mixing 1% (w/v) pectin and xylan substrates in a 50 mM buffer at optimum pH with crude pectinase and xylanase enzymes (1:1). The substrates used for pectinase include commercial substrates of pectin from apples and orange peels, as well as biomass substrates of apple peels, orange peels, and cacao peel. The substrates used for xylanase include commercial substrates of xylan from beechwood, bagasse biomass, palm kernel cake, and xylan extracts from corn cobs and tobacco plants. The entire substrate mixture was then incubated at 40 °C with reaction sampling carried out at 0, 1, 4, 24, 48, and 72 h. The samples were heated at 90 °C for 10 min. The results were separated by centrifugation for 20 min at 4 °C, 12,000 rpm. Thin layer chromatography (TLC) was performed on silica gel paper (20x10 cm) with a mixture of butanol, acetic acid glacial, and milli-Q (2:1:1) as the liquid phase, and diphenylamine acetone phosphoric acid (DAP) as spray solution.

Molecular identification of isolate BLH 5-14

The DNA extraction steps were carried out according to the protocol on the Wizard Genomic DNA Purification Kit [Promega]. The DNA extraction results then entered the PCR stage using the EmeraldAmp GT PCR Master Mix with a total volume of 100 μL. The primers used were 9F (5’ AGRGTTTGATCMTGGCTCAG 3’) and 1510R (5’ TACGGYTACCTTGTTAYGACTT 3’) with cycles according to the method by Hayat et al. [28]. The sequencing process was carried out with the assistance of the Apical Scientific Laboratory, Selangor, Malaysia, and mediated by PT. Genetics Science, Tangerang, Banten, Indonesia. The sequences were then analyzed using the FinchTV application, DNA Baser Assembler, Bioedit, and NCBI DNA Blast. The phylogenetic tree was created using MEGA X.


Enzyme production, protein concentration and growth curve

Fig. 1 shows that enzyme activity, protein concentration, and total dry weight of cells in pectinase and xylanase production cultures continued to increase until day 6th and decreased on day 7th. The highest pectinase enzyme activity was 1.44±0.08 U/mL, the protein was 0.33 mg/mL, and dry cell weight was 0.0567 g, while the optimum xylanase enzyme activity was 4.33±0.03 U/mL, protein of 0.32 mg/mL, and dry cell weight of 0.1147 g.

Fig. 1
figure 1

Production curve of pectinase (A) and xylanase (B) for 7 days culture production. Sampling was done every 24 h. Enzyme activity, protein concentration, and dry cell weight was calculated

Enzyme characterization

The results of enzyme characterization for optimum pH of pectinase and xylanase are in Fig. 2A and B. The pectinase enzyme showed the highest activity in sodium phosphate buffer pH 8.0 at 5.08±0.17 U/mL, and xylanase enzyme showed the highest activity at sodium acetate buffer pH 6.0 at 3.58±0.01 U/mL. The activity of the pectinase enzyme in Fig. 2C increased to a temperature of 50 °C by 5.08±0.17 U/mL and then decreased to a temperature of 90 °C. At the same time, the activity of the xylanase enzyme still increased to a temperature of 60 °C by 6.22±0.04 U/mL.

Fig. 2
figure 2

Effect of different buffers, pH (A-B), and temperatures (C) on pectinase and xylanase activity. A Effect of buffers and pH on pectinase. B Effect of buffers and pH on xylanase. The reaction for optimum pH characterization was done under the same condition reactions at 30°C for 15 min, while the reaction for optimum temperature characterization was done using optimum pH for 15 min reaction time

The results in Table 1 show that pectinase and xylanase have a drastic decrease in activity by Hg2+ ions with activity values of 0.00±0.08 U/mL (0%) and 0.65±0.13 U/mL (11%), respectively. On the other hand, the addition of K+, Mn2+, and Fe2+ ions result in increased activity. The pectinase enzyme activity values for K+, Mn2+, and Fe2+ ions were 6.70±0.01 U/mL (132%), 7.57±0.27 U/mL (149%), and 10.62±0.09 U/mL (209%), while in xylanase it was 8.39±0.12 U/mL (135%), 13.62±0.04 U/mL (219%), and 13.38±0.14 U/mL (215%), consecutively. Characterization of chemical compounds showed the highest activity inhibition by SDS for both enzymes, followed by isopropanol, methanol, and ethanol.

Table 1 Influence of metal ions and chemical compounds on pectinase and xylanase enzymes

Enzyme molecular weight analysis

SDS PAGE and zymogram analysis on the pectinase enzyme did not produce clear enzyme bands. In contrast, the SDS PAGE and zymogram on the xylanase enzyme in Fig. 3 show the separation of enzyme bands from xylanase culture samples, with the size between 34.8 and 25 kDa, respectively.

Fig. 3
figure 3

SDS PAGE (A) and zymogram (B) of xylanase crude enzyme. M, molecular weight marker; lane 0-7: culture supernatant sampling of enzyme production day 0-7; K-, negative control

Thin layer chromatography hydrolysis product analysis

Based on Fig. 4 shows the oligosaccharide product of pectinase in the form of galacturonic acid (P1) from the biomass of apple peels and orange peels from the 1st hour. Fig. 5 shows the results of oligosaccharide products of xylanase in the form of xylotriose (X3), xylotetraose (X4), and xylopentaose (X5) on all substrates from the 1st hour, with xylotriose starting to become depleted at 48 h. Production of XOS can be seen similarly between raw biomass and commercial substrates of beechwood xylan.

Fig. 4
figure 4

TLC Analysis of hydrolysis products from various pectin commercial substrates and biomass (A-E). A Pectin from apple. B Pectin from citrus peel. C Biomass apple peel. D Biomass citrus peel. E Biomass cacao; M, Standards; P1, Galacturonic acid; P2, Digalacturonic acid; P3, Trigalacturonic acid

Fig. 5
figure 5

TLC Analysis of hydrolysis products from various xylan commercial substrates and biomass (A-E). A Xylan from Beechwood. B Bagasse. C Palm kernel cake. D Corn Cob. E Tobacco; M, Standards; X1, Xylose; X2, Xylobiose; X3, Xylotriose; X4, Xylotetraose; X5, Xylopentaose

16S rDNA Molecular identification

Based on the results of sequence analysis using NCBI BLAST and the obtained phylogenetic tree in Fig. 6, the Actinomycetes isolate (BLH 5-14) belongs to the genus Streptomyces, with the closest species being Streptomyces tendae strain NBRC 12822 (99.78%).

Fig. 6
figure 6

Neighbor-joining phylogenetic tree of genus Streptomyces and BLH 5-14 based on 16S rDNA analysis. Bootstrap values based on 1000 replicates are shown at the branch nodes. Actinospica robiniae was used as an outgroup


Streptomyces tendae (Ettlinger et al., 1958) was 99.78% similar to BLH-14 isolate. This species was first isolated from soil in Tendae, France. Its characteristics are known to grow in a wide pH range (pH 5.0-12.0) and NaCl concentration of 0-10% [29, 30]. Research by Abdulkhair & Aghuthaymi [31] demonstrated the ability of this species to produce pectinase enzymes and utilize xylose carbon sources.

The production and growth curves obtained from BLH-14 isolates showed similar results with other species of the genus Streptomyces in previous studies, namely Streptomyces coeliflavus GIAL86 from Meyghan Salt Lake in Iran and Streptomyces actuosus A-151 in Taiwan [32, 33]. The optimum day range is generally found from day 5th to day 7th, with the highest value obtained at the beginning of the stationary phase. Proteins and enzymes produced in the culture process are known to be in the growth associate group, which will increase and decrease along with the metabolic rate of microorganisms in the culture [34].

The process of characterizing the optimum pH and temperature also showed results that followed previous studies by Kuhad et al. [35] and Nascimento et al. [36]. Both enzymes can remain active at a temperature of 30°C to 70°C (relative activity >50%) and a pH range of 3.0-10.0. These show the potential of BLH 5-14 isolate to be used in the paper bleaching process, replacing compounds such as chlorine and NaOH that can pollute the environment. Pectinase and xylanase enzymes are essential in cutting xylan bonds with polysaccharides, as well as the degradation of pectin on paper during the bleaching process at alkaline pH conditions [37, 38].

The influence of metal ions and chemical compounds on pectinase and xylanase enzymes tends to increase along with the higher concentration of compounds in the reaction solution [39]. Metal ion compounds can form interactions with carboxyl or sulfhydryl groups on proteins, causing disruption of protein structure or helping to increase reaction activity. The inhibitory nature of the Hg2+ ion is known to be the result of the interaction with the sulfhydryl group on pectinase and xylanase [40]. Sodium dodecyl sulfate (SDS) acts as a surfactant, causing the denaturation of protein structures along with the disruption of hydrophobic bonds in enzymes [41, 42].

Xylanase enzyme from isolate BLH 5-14 showed decreased activity value due to the administration of the Ethylenediaminetetraacetic acid (EDTA) compound. This result indicates that this enzyme requires metal ions for the reaction process because EDTA acts as a chelating agent which tends to react and attract metal ion compounds in solution [43]. On the other hand, xylanase usually does not respond significantly to the administration of organic alcohol solutions. According to Amobonye et al. [42], this may indicate the presence of a coil-like structure in a higher ratio of protein which tends to be stable in organic solutions, which can be beneficial in industries involving alcohol organic solutions. Some examples include the bioethanol production process, the production of alcoholic beverages, and the process of dissolving non-polar substrates [42, 44, 45].

The analysis of the enzyme molecular size using SDS PAGE and zymograms showed a difference between pectinase and xylanase. Generally, the molecular weight of pectinase from Actinomycetes is in the 35-50 kDa range, with pectate lyase and polygalacturonase types from Actinomadura keratinilytica and Streptomyces coelicolor [46, 47]. The results of the xylanase molecular size are in the 20-50 kDa molecular weight size range of the Streptomyces genus [14]. The presence of the two size enzyme molecules may indicate the presence of 2 types of a xylanase enzyme family by Actinomycetes isolates (BLH 5-14), namely GH11 that generally <30 kDa, and GH10 that >30 kDa [14, 48, 49].

Xylanase enzymes from GH10 and GH11 tend to work synergistically, with GH11 producing large hydrolysis products, and with the help of GH10, can be degraded further into smaller xylan molecules. Results of larger-sized xylooligosaccharide molecules produced as a result of this reaction indicate the endo-type cleavage mechanism [48, 50]. On the other hand, pectinase produces monosaccharide products as the smallest unit, which implies that the pectinase in this study has an Exo type of cleavage. In general, this product can benefit industries with a demand for the direct production of D-galacturonic acid [51, 52]. One example is a dietary supplement in the health industry that can reduce intestinal inflammation and prevent the development of cancer-causing tumor cells [53].


The results and data from a series of studies show that Actinomycetes isolates from marine sediments of Indonesia identified as Streptomyces tendae can produce pectinase and xylanase enzymes. Both show properties susceptible to a wide range of pH and temperature, along with the distinct influence of chemical and metal ion compounds. In the storage process, this characterization procedure can be utilized as a reference, particularly to maintain or even boost the anticipated enzyme activity in large-scale production. In addition to using waste and being cost-effective, oligosaccharide products made from biomass waste, such as galacturonic acid and xylooligosaccharides, are currently a market target due to their numerous applications in the biomedical industry. Several studies regarding in vitro and in vivo assays, as well as purification methods to extract POS and XOS from the fermentation process, have been conducted in previous studies. As a result, there is a greater probability of developing this isolate to produce POS and XOS that will be beneficial and accessible to a larger community.

Availability of data and materials

The datasets used and analyzed during this research are available from the corresponding author upon reasonable request.







Dinitrosalicylic acid


Optical density


Sodium dodecyl sulfate


Sodium dodecyl-sulfate polyacrylamide gel electrophoresis


Thin layer chromatography


Diphenylamine acetone phosphoric acid


National Centre for Biotechnology Information


Basic Local Alignment Search Tool


Glycoside Hydrolases


Ethylenediaminetetraacetic acid


  1. Gullón B, Gómez B, Martínez-Sabajanes M, Yáñez R, Parajo JC, Alonso JL (2013) Pectic oligosaccharides: Manufacture and functional properties. Trends Food Sci Technol 30:153–161

    Article  Google Scholar 

  2. Ibrahim OO (2018) Functional oligosaccharide: Chemical structure, manufacturing, health benefits, applications and regulations. J Food Chem Nanotechnol 4(4):65–76

    Article  Google Scholar 

  3. Míguez B, Gómez B, Gullón P, Gullón B, Alonso JL (2016) Pectic oligosaccharides and other emerging prebiotics. In: Rao V, Rao LG (eds) Probiotics and Prebiotics in Human Nutrition and Health. IntechOpen, London, pp 301–330

    Google Scholar 

  4. Tan H, Chen W, Liu Q, Yang G, Li K (2018) Pectin oligosaccharides ameliorate colon cancer by regulating oxidative stress- and inflammation- activated signaling pathways. Front Immunol 9(1504):1–13

    Google Scholar 

  5. Zhu R, Zhang X, Wang Y, Zhang L, Wang C, Hu F, Ning C, Chen G (2019) Pectin oligosaccharides from hawthorn (Crataegus pinnatifida Bunge. Var. major): Molecular characterization and potential antiglycation activities. Food Chem 286:129–135

    Article  Google Scholar 

  6. Wilkowska A, Nowak A, Antczak-Chrobot A, Motyl I, Czyżowska A, Paliwoda A (2019) Structurally different pectic oligosaccharides produced from apple pomace and their biological activity in vitro. Foods 8(365):1–22

    Google Scholar 

  7. Hu H, Zhang S, Pan S (2021) Characterization of citrus pectin oligosaccharides and their microbial metabolites as modulators of immunometabolism on macrophages. J Agric Food Chem 69:8403–8414

    Article  Google Scholar 

  8. Mäkeläinen H, Juntunen M, Hasselwander O (2009) Prebiotic potential of xylo-oligosaccharides. In: Charalampopoulos D, Rastall RA (eds) Prebiotics and Probiotics Science and Technology. Springer, New York, pp 245–258

    Chapter  Google Scholar 

  9. Chen Y, Xie Y, Ajuwon KM, Zhong R, Li T, Chen L, Zhang H, Beckers Y, Everaert N (2021) Xylo-oligosaccharides, preparation, and application to human and animal health: a review. Front Nutr 8(731930):1–10

    Google Scholar 

  10. Abdo AAA, Zhang C, Lin Y, Liang X, Kaddour B, Wu Q, Li X, Fan G, Yang R, Teng C, Xu Y, Li W (2021) Xylo-oligosaccharides ameliorate high cholesterol diet induced hypercholesterolemia and modulate sterol excretion and gut microbiota in hamsters. J Funct Foods 77(104334):1–9

    Google Scholar 

  11. Lin S-H, Chou L-M, Chien Y-W, Chang J-S, Lin C-I (2016) Prebiotic effects of xylooligosaccharides on the improvement of microbiota balance in human subjects. Gastroenterol Res Pract 2016(5789232):1–6

    Google Scholar 

  12. Álvarez C, González A, Ballesteros I, Gullón B, Negro MJ (2023) In vitro assessment of the prebiotic potential of xylooligosaccharides from barley straw. Foods 12(83):1–14

    Google Scholar 

  13. Rahmani N, Kahar P, Lisdiyanti P, Lee J, Yopi, Prasetya B, Ogino C, Kondo A (2018) GH-10 and GH-11 endo-1,4-β-xylanase from Kitasatospora sp. produce xylose and xylooligosaccharides from sugarcane bagasse with no xylose inhibition. Bioresour Technol 272:315–325

    Article  Google Scholar 

  14. Rahmani N, Apriliana P, Jannah AM, Ratnakomala S, Lisdiyanti P, Hermiyati E, Prasetya B, Yopi. (2019) Endo-xylanase enzyme from marine actinomycetes and its potential for xylooligosaccharide production. IOP Conf Series: Earth Environ Sci 251(2019):1–8

    Google Scholar 

  15. Yugandhar NM, Sushma C, Beena C, Prudhvi V, Vasudha G (2019) Optimizing of cultural conditions for the production of pectinolytic enzyme from marine actinomycetes by submerged fermentation along with statistical approach. Int J Curr Innov Adv Res 2(3):29–43

    Google Scholar 

  16. Bhardwaj N, Kumar B, Verma P (2019) A detailed overview of xylanases: An emerging biomolecule for current and future prospective. Bioresour Bioprocess 6:1–36

    Article  Google Scholar 

  17. Singh A, Varghese LM, Battan B, Patra AK, Mandhan RP, Mahajan R (2019) Eco-friendly scouring of ramie fibers using crude xylano-pectinolytic enzymes for textile purpose. Environ Sci Pollut Res 27(6):6701–6710

  18. Hassan SS, Tiwari BK, Williams GA, Jaiswal V (2020) Bioprocessing of brewers’ spent grain for production of xylanopectinolytic enzymes by Mucor sp. Bioresour Technol Rep 9:1–10

    Google Scholar 

  19. Nawawi MH, Ismail KI, Sa’ad N, Mohamad R, Tahir PM, Asa’ari AZ, Saad WZ (2022) Optimisation of xylanase-pectinase cocktail production with Bacillus amyloliquefaciens ADI2 using a low-cost substrate via statistical strategy. Fermentation 8(119):1–17

    Google Scholar 

  20. Praveen KG, Suneetha V (2015) Efficacy of pectinase purified from Bacillus VIT sun-2 and in combination with xylanase and cellulose for the yield and clarification improvement of various culinary juices from South India for pharma and health benefits. Int J PharmTech Res 7(3):448–452

    Google Scholar 

  21. Sharma D, Agrawal S, Yadav RD, Mahajan R (2017) Improved efficacy of ultrafiltered xylanase-pectinase concoction in biobleaching of plywood waste soda pulp. Biotech 7(2):1–7

    Google Scholar 

  22. Cano ME, García-Martin A, Morales PC, Wojtusik M, Santos VE, Kovensky J, Ladero J (2020) Production of oligosaccharides from agrofood wastes. Fermentation 6:1–27

    Article  Google Scholar 

  23. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31(3):426–428

    Article  Google Scholar 

  24. Rahmani N, Kahar P, Lisdiyanti P, Hermiati E, Lee J, Yopi, Prasetya B, Ogino C, Kondo A (2018) Xylanase and feruloyl esterase from actinomycetes cultur could enhance sugarcane bagasse hydrolysis in the production of fermentable sugars. Biosci Biotechnol Biochem 82(5):904–915

    Article  Google Scholar 

  25. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–658

    Article  Google Scholar 

  26. Yopi, Rahmani N, Amanah S, Santoso P, Lisdiyanti P (2020) The production of β-mannanase from Kitasatospora sp. strain using submerged fermentation: Purification, characterization and its potential in mannooligosaccharides production. Biocatal Agric Biotechnol 24:1–8

    Article  Google Scholar 

  27. Rahmani N, Kashiwagi N, Lee J, Niimi-Nakamura S, Matsumoto H, Kahar P, Lisdiyanti P, Yopi, Prasetya B, Ogino C, Kondo A (2017) Mannan endo-1,4-β-mannosidase from Kitasatospora sp. isolated in Indonesia and its potensial for production of manooligosaccharides from mannan polymers. AMP Express 7(100):1–11

    Google Scholar 

  28. Hayat R, Sheirdil RA, Iftikhar-ul-Hassan M, Ahmed I (2013) Characterization and identification of compost bacteria based on 16S rRNA gene sequencing. Ann Microbiol 63:905–912

    Article  Google Scholar 

  29. Shirling EB, Gottlieb D (1968) Cooperative description of type cultures of Streptomyces: II Species description from first study. Int J Syst Bacteriol 18(2):69–189

    Article  Google Scholar 

  30. Eftekharivash L, Hamedi J (2020) Genome sequence and annotation of Streptomyces tendae UTMC 3329, acid and alkaline tolerant actinobacterium. Iran J Microbiol 12(4):343–352

    Google Scholar 

  31. Abdulkhair WM, Alghuthaymi MA (2016) Double inhibitory effect of extracellular protein of marine Streptomyces tendae against different strains of MRSA. Der Pharm Lett 8(9):1–10

    Google Scholar 

  32. Wang S-L, Yen Y-H, Shih I-L, Chang AC, Chang W-T, Wu WC, Chai Y-D (2003) Production of xylanase from rice bran by Streptomyces actuoses A-151. Enzym Microb Technol 33:917–925

    Article  Google Scholar 

  33. Salehghamari E, Nasrollahzadeh Z, Tahmaseb M, Amoozegar MA (2019) Pectinase enzyme from Streptomyces coelicoflacus GIAL86 isolated from Meyghan Salt Lake, Arak, Iran. Int J Aquatic Biol 7(2):106–111

    Google Scholar 

  34. Sakthiselvan P, Meenambiga SS, Madhumathi R (2019) Kinetics studies on cell growth. In: Vikas B, Fasullo M (eds) Cell growth. IntechOpen, London

    Google Scholar 

  35. Kuhad RC, Kapoor M, Rustagi R (2004) Enhanced production of an alkaline pectinase from Streptomyces sp. RCK-SC by whole-cell immobilization and solid-state cultivation. World J Microbiol Biotechnol 20(3):257–263

    Article  Google Scholar 

  36. Nascimento RP, Reis AD, Girio F, Pereira N Jr, Bon EP, Coelho RR (2020) A thermotolerant xylan-degrading enzyme is produced by Streptomyces malaysiensis AMT-3 using by products from the food industry. Environ Sci 63:1–12

    Google Scholar 

  37. Ahlawat S, Battan B, Dhiman SS, Sharma J, Mandhan RP (2007) Production of thermostable pectinase and xylanae for their potential application in bleaching of kraft pulp. J Ind Microbiol Biotechnol 34:763–770

    Article  Google Scholar 

  38. Bhagat DD, Dudhagara PR, Desai PV (2016) Statistical approach for pectinase production by Bacillus firmus SDB9 and evaluation of pectino-xylanolytic enzymes for pretreatment of kraft pulp. J Microbiol Biotechnol Food Sci 5(5):396–306

    Article  Google Scholar 

  39. Anggraini DP, Roosdiana A, Prasetyawan S, Mardiana D (2013) Pengaruh ion-ion logam terhadap aktivitas pektinase dari Aspergillus niger pada penjernihan sari buah jambu. Natural 2(1):66–72

    Article  Google Scholar 

  40. Prejanò M, Alberto ME, Russo N, Toscano M, Marino T (2020) The effects of the metal ion substitution into the active site of metalloenzymes: a theoretical insight on some selected casaes. Catalysts 10(1038):1–28

    Google Scholar 

  41. Bhardwaj N, Verma VK, Chaturvedi V, Verma P (2020) Cloning, expression and characterization of a thermos-alkali-stable xylanase from Aspergillus oryzae LC1 in Escherichia coli BL21 (DE3). Protein Expr Purif 168(105551):1–11

    Google Scholar 

  42. Amobonye A, Bhagwat P, Singh S, Pillai S (2021) Beauveria bassiana xylanase: Characterization and wastepaper deinking potential of a novel glycosyl hydrolase from an endophytic fungal entomopathogen. J Fungi 7(688):1–18

    Google Scholar 

  43. Ghoshal G, Banerjee UC, Shivhare US (2015) Utilization of agrowaste and xylanase production in solid state fermentation. J Biochem Technol 6(3):1013–1024

    Google Scholar 

  44. Okonji RE, Itakorode BO, Ovumedia JO, Adedeji OS (2019) Purification and biochemical characterization of pectinase produced by Aspergillus fumigatus isolated from soil of decomposing plant materials. J Appl Biol Biotechnol 7(3):1–8

    Article  Google Scholar 

  45. Liu C, Zhang L, Tan L, Liu Y, Tian W, Ma L (2021) Immobilized crosslinked pectinase preparation on porous ZSM-5 eolites as reusable biocatalysts for ultra-efficient hydrolysis of β-glycosidic bonds. Front Chem 9(677868):1–13

    Google Scholar 

  46. Xiao Z, Boyd J, Grosse S, Beauchemin M, Coupe E, Lau PCK (2008) Mining Xanthomonas and Streptomyces genomes for new pectinase-encoding sequences and their heterologous expression in Escherichia coli. Appl Microbiol Biotechnol 78:973–981

    Article  Google Scholar 

  47. Saoudi B, Habbeche A, Kerouaz B, Haberra S, Romdhane ZB, Tichati L, Boudelaa M, Belghith H, Gargouri A, Ladjama A (2015) Purification and characterization of a new thermoalkaliphilic pectate lyase from Actinomadura keratinilytica Cpt20. Process Biochem 50:2259–2266.

  48. Yagi H, Takehara R, Tamaki A, Teramoto K, Tsutsui S, Kaneko S (2019) Functional characterization of the GH10 and GH11 xylanases from Streptomyces olivaceoviridis E-86 provide insights into the advantage of GH11 xylanase in catalyzing biomass degradation. J Appl Glycosci 66:29–35

    Article  Google Scholar 

  49. Li Y, Zhang X, Lu C, Lu P, Yin C, Ye Z, Huang Z (2022) Identification and characterization of novel endo-β-1,4-xylaase from Streptomyces sp. T7 and its application in xylo-oligosaccharides production. Molecules 27:1–13

    Google Scholar 

  50. Alvarez TM, Goldbeck R, dos Santos CR, Paixão DAA, Gonçalves TA, Cairo JPLF, Almeida RF, de Oliveira Pereira I, Jackson G, Cota J, Büchli F, Citadini AP, Ruller R, Polo CC, de Neto MO, Murakami MT, Squina FM (2013) Development and biotechnological application of a novel endoxylanase family GH10 identified from sugarcane soil metagenome. PLoS One 8(7):1–14

    Article  Google Scholar 

  51. Combo AMM, Aquedo M, Goffin D, Wathelet B, Paquot M (2012) Enzymatic production of pectin oligosaccharides from polygalacturonic acid with commercial pectinase preparations. Food Bioprod Process 90:588–595

    Article  Google Scholar 

  52. Yuan P, Meng K, Shi P, Luo H, Huang H, Tu T, Yang P, Yao B (2012) An alkaline-active and alkali-stable pectate lyase from Streptomyces sp. S27 with potential in textile industry. J Industr Microbiol Biotechnol 39:909–915

    Article  Google Scholar 

  53. Abari AH, Rourani HA, Ghasemi SN, Kim H, Kim Y-G (2021) Investigation of antioxidant and anticancer activities of unsaturated oligo-galacturonic acids produced by pectinase of Streptomyces hydrogenans YAM1. Sci Rep 11(8941):1–9

    Google Scholar 

Download references


Acknowledgment and sincere gratitude for everyone involved in this research, especially the Carbohydrate-related Enzymes Research Group, Research Center for Applied Microbiology, at the National Research and Innovation Agency, Indonesia, who has allowed and supported this research.


The funding for all the material used in this research was from INSINAS (Intensif Riset Sistem Inovasi Nasional), Kementerian Riset dan Teknologi/Badan Riset dan Inovasi Nasional, the year 2021. Grant No 15/E1/KPT/2021 P.I Dr. Eng Nanik Rahmani.

Author information

Authors and Affiliations



HN performed the main experimental part of the work, wrote the manuscript, interpreted the data, and revised the manuscript. NR designed the experiments, provided advice throughout the research, and substantively revised the manuscript. WM and YP designed the experiments, provided guidance throughout the work, and substantively revised the manuscript. AA, PL, and SR isolated, characterized, and provided the strains. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Nanik Rahmani.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

All authors approve the manuscript for publication.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Nadhifah, H., Rahmani, N., Mangunwardoyo, W. et al. Xylanopectinolytic enzymes by marine actinomycetes from sediments of Sarena Kecil, North Sulawesi: high potential to produce galacturonic acid and xylooligosaccharides from raw biomass. J Genet Eng Biotechnol 21, 31 (2023).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: